*Review* **Influence of Cardiometabolic Risk Factors on Platelet Function**

#### **Cristina Barale and Isabella Russo \***

Department of Clinical and Biological Sciences, Turin University, 10043 Orbassano (Turin), Italy; cristina.barale@unito.it

**\*** Correspondence: isabella.russo@unito.it

Received: 29 November 2019; Accepted: 16 January 2020; Published: 17 January 2020

**Abstract:** Platelets are key players in the thrombotic processes. The alterations of platelet function due to the occurrence of metabolic disorders contribute to an increased trend to thrombus formation and arterial occlusion, thus playing a major role in the increased risk of atherothrombotic events in patients with cardiometabolic risk factors. Several lines of evidence strongly correlate metabolic disorders such as obesity, a classical condition of insulin resistance, dyslipidemia, and impaired glucose homeostasis with cardiovascular diseases. The presence of these clinical features together with hypertension and disturbed microhemorrheology are responsible for the prothrombotic tendency due, at least partially, to platelet hyperaggregability and hyperactivation. A number of clinical platelet markers are elevated in obese and type 2 diabetes (T2DM) patients, including the mean platelet volume, circulating levels of platelet microparticles, oxidation products, platelet-derived soluble P-selectin and CD40L, thus contributing to an intersection between obesity, inflammation, and thrombosis. In subjects with insulin resistance and T2DM some defects depend on a reduced sensitivity to mediators—such as nitric oxide and prostacyclin—playing a physiological role in the control of platelet aggregability. Furthermore, other alterations occur only in relation to hyperglycemia. In this review, the main cardiometabolic risk factors, all components of metabolic syndrome involved in the prothrombotic tendency, will be taken into account considering some of the mechanisms involved in the alterations of platelet function resulting in platelet hyperactivation.

**Keywords:** adipose tissue; adipokines; hemostasis; insulin resistance; metabolic syndrome; nitric oxide; oxidative stress; platelets; thrombosis

#### **1. Introduction**

Several lines of evidence suggest a strong correlation between metabolic disorders and hemodynamic such as obesity, dyslipidemia, diabetes, hypertension, and cardiovascular (CV) diseases (CVD), with endothelial dysfunction as the initial step toward atherothrombosis (Figure 1). Oxidative stress and a chronic low-grade of inflammation may be considered a "common soil" able to create a feed-forward cycle that can deeply influence the development of a prothrombotic tendency of these metabolic abnormalities.

**Figure 1.** Potential mechanisms linking cardiometabolic disorders and atherothrombotic vascular diseases.

One of the first epidemiological studies showing the causal relationship between obesity and CVD was the Framingham Heart Study [1,2] and other studies have then confirmed that the waist-to-hip ratio (WHR)—a reliable index of central obesity—was the strongest anthropometric predictor of myocardial infarction [3] and stroke [4,5].

Not only the excess of adipose tissue, but also body fat distribution and the impaired adipose tissue function, rather than total fat mass, better predict CV risk [6]. Actually, atherothrombotic events leading to an elevated risk of CV morbidity and mortality are closely associated to central obesity, which confers a higher degree of CV risk than peripheral adiposity [7,8]. In fact, abdominal adiposity may have a causal, unfavorable effect on plasma triglycerides (TGs) and potentially other cardiometabolic risk factors due to a greater ability to release cytokines and free fatty acids involved in the pathogenesis of both atherothrombosis and insulin resistance [9,10].

The increasing prevalence of obesity, especially in Western Countries, has also contributed to significant increases in the prevalence of other important CV risk factors, including dyslipidemia, insulin resistance, and type 2 diabetes mellitus (T2DM). The presence of a clustering of three or more risk factors in the same individual, including abdominal obesity, atherogenic dyslipidemia, high systolic and diastolic blood pressures, and impaired glucose tolerance has been defined by World Health Organization (WHO) as metabolic syndrome (MS) [11], though there is some minor variation in the definition by other health care organizations. Importantly, each of these cardiometabolic disorders contributes to alter hemostatic balance leading to a prothrombotic phenotype [12]. This review will focus on the role of obesity on prothrombotic tendency in patients affected by MS, being adipocytes able to produce and/or release hormones which deeply influence hemostatic balance, platelet function, pro-inflammatory state and oxidative stress.

#### **2. Definition of Metabolic Syndrome**

MS is a multiplex risk factor for atherosclerotic CV disease, with a prevalence of 34% in the general population [13]. However, due to the lacking of a unifying definition, MS can be present in several forms according to the combination of the different components and the exact evaluation of prevalence of MS changes both in United States and in Europe. It has been estimated that at least one quarter of America population is affected by MS and about 84% of them present abdominal obesity on the basis of the criteria indicated by National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III [14–18].

According to the NCEP's ATP III criteria [19], MS is recognized as a condition related to CVD occurring if the patient has three or more of the following: (1) central obesity characterized by waist circumference >102 cm in men and >88 cm in women; (2) fasting blood TGs ≥150 mg/dL and high-density lipoprotein cholesterol (HDL) ≤40 mg/dL in men or ≤50 mg/dL in women; (3) fasting glucose ≥100 mg/dL; and (4) systolic blood pressure ≥130 mmHg and/or diastolic blood pressure ≥85 mmHg. Indeed, elevated high-sensitivity C-reactive protein, increased prothrombotic factors, endothelial dysfunction, microalbuminuria, elevated inflammatory cytokines, decreased adiponectin plasma levels, and alterations in pituitary-adrenal axis could be involved in MS. However, the inclusion of these abnormalities in the classification of MS needs to be confirmed and this continuous score would be more sensitive to small and large changes that do not modify the most recent Joint Interim Statement of the International Diabetes Federation (IDF) Task Force on Epidemiology and Prevention criteria [20].

Despite other definitions also have been proposed [21], all are associated with the presence of central obesity, thus underlining the crucial role of the abdominal adiposity, together with insulin resistance, as causative factor in the pathogenesis of MS. Actually, the condition of insulin resistance represents a significant link among components of MS even if a subject with MS not necessarily is insulin resistant [22]. It is well established that MS is a constellation of cardiometabolic determinants associated with increase not only of CVD but also a three-fold increase in the risk of T2DM [23–25] with significant adverse effects on health-related quality of life [26].

#### **3. Platelets in Hemostasis and Thrombosis**

Platelets are key players in primary hemostasis and thrombus formation. Platelet activation become when platelets come in contact with exposed collagen in the areas of vascular damage and the subsequent morphological and physiological changes help in stable platelet plug formation thus contributing to primary hemostasis. Platelet activation process is mediated by surface exposure of receptors (glycoproteins, GPs) and lipid rafts, which modulate signaling and intracellular trafficking. These include GPIb/V/IX complex, which interacts with von Willebrand factor (vWF), integrin α IIbβ3 (GPIIb/IIIa), which binds vWF and fibrinogen, and GPVI which binds collagen thus ensuring a stable anchorage with subendothelial matrix [27]. Binding of ligands to the GP receptors changes platelet shape as well as triggers the release of platelet granule contents, which lead to the formation of platelet plug.

However, hemostasis or blood coagulation are not the only function of platelets, which are also involved in pathological processes such as chronic inflammation and atherothrombosis. In fact, platelets store cytokines and growth factors in their alpha-, dense granules and lysosomes [28] and the subcellular machinery of the novo protein synthesis involved in the coagulation cascade and inflammatory pathways including interleukin (IL)-1β, plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF; Figure 2).

The atherothrombotic process underlies acute coronary and cerebrovascular events where the activation of inflammatory mechanisms is strictly dependent on interaction among different cell types, such as platelets, leukocytes, and cells of the vascular wall. As extensively reviewed [29–31], once adhered to the damaged vessel wall platelets participate in multiple mechanisms promoting thromboinflammation by releasing storage granules and aggregating to form thrombi. As mentioned, platelet adhesion is influenced by adhesion molecules present in the subendothelial matrix components, such as E-selectin [32], vWF [33], collagen, fibronectin, and by the level of shear stress in the circulation [34]. In this phase, platelets are subjected to a number of physiological and cytoskeletal changes, with release of soluble cytokines, chemokines, growth factors, and the rapid translocation of P-selectin from alpha-granule to plasma membrane. When intracellular Ca++ concentration exceeds a specific threshold, platelets shift from the resting discoid shape to the activated state with the formation of filopodia and lamellipodia. The recruitment of other platelets, their activation, and aggregation are followed by the formation of three-dimensional aggregates for a number of molecular interaction

triggered by thrombin and generation of endogenous factors such as thromboxane (TX)A2 and release of content from storage granules including adenosine 5-diphosphate (ADP), and platelet activating factor (PAF). Stabilization of platelet–platelet interactions is further mediated by the receptor of fibrinogen GPIIb/IIIa. In the primary hemostasis a pivotal role in plug formation is exerted by platelet aggregation with aggregates anchored at site of injury but this clot remains unstable. Clot stabilization characterizes the secondary hemostasis with consolidation of platelet mass through the assembly of coagulation complexes with conversion of soluble fibrinogen into insoluble fibrin by thrombin and platelet retraction. In some pathological settings, a number of factors can impair the normal hemostasis and aberrant thrombus formation has severe pathological consequences, leading to fatal thromboembolism and tissue ischemia of vital organs, ultimately resulting in acute CVD complications, including myocardial infarction, stroke and critical limb ischemia.

**Figure 2.** Biochemical factors involved in the coagulation cascade and the atherosclerotic process released following platelet activation. AA, arachidonic acid; COX, cyclooxygenase; TXA2, thromboxane A2; PDGF, platelet-derived growth factor; TGF-β, transforming growth factor β; EGF, endothelial growth factor; bFGF, fibroblast growth factor; VEGF, vascular endothelial growth factor; IGF, insulin-like growth factor; IL-1β, interleukin-1β; PAI-1, plasminogen activator inhibitor 1; vWF, von Willebrand factor; GP, glycoprotein; PECAM, platelet endothelial cell adhesion molecule; sCD40L, soluble CD40 ligand; sP-selectin, soluble P-selectin; RANTES, regulated on activation, normal T-cell expressed and secreted; MIP-1α, macrophage inflammation protein- 1α; IL-8, interleukin-8; PF4, platelet factor 4; PMPs, platelet-derived microparticles.

In the presence of intact vascular endothelium, the release of prostacyclin (PGI2) and nitric oxide (NO), two major antiaggregants, regulates the balance between pro- and antiaggregants and prevents the formation of thrombus inside the blood vessel [35]. However, in subjects at risk of arterial thrombosis, this key protective pathway is overcome, resulting in uncontrolled platelet activity.

#### **4. Platelet Function Assays**

For the measurement of platelet function there is no gold-standard method showing the real state of "hyper" or "hypo" reactivity that can be used as reliable marker of high risk in disease settings. However, based on the platelet ability to interact with each other as well as with other cells and for peculiar surface expressions it is possible to measure platelet function and activation by using a

number of tools each measuring different aspects of platelet response. Laboratory tests, including light transmission platelet aggregation, lumiaggregometry, impedance aggregometry on whole blood, flow cytometry or enzyme-linked immunoassays (ELISA), are traditionally utilized for the identification of patients with impaired platelet function.

Light transmission aggregometry (LTA), for a long time considered the gold-standard method, is the most widely employed test in clinical hematology to measure the increase in light transmission through a platelet suspension when platelets were stimulated by a specific agonist such as ADP, arachidonic acid (AA), collagen, and epinephrine. LTA allows us to evaluate the tendency of platelets to aggregate and to identify abnormalities such as hyperaggregation [36]. This assay has some major drawbacks: (i) it is relatively non physiological because during the test platelets are stirred under low shear conditions and only form aggregates after the addition of agonists, without mimicking platelet adhesion, activation and aggregation upon vessel wall damage, (ii) the result obtained may be affected by preanalytical and procedural conditions, (iii) not suitable for platelet-rich platelet numbers below <sup>50</sup> <sup>×</sup> 106/mL and lipemic blood sample, and (iv) its reproducibility is poor. Specific guidelines for LTA have been published in order to correctly perform the procedure [36–38].

Platelet aggregation assessed in whole blood (WB) measures the increase of electrical impedance generated by aggregates upon those fixed to two electrodes. As advantages, WB aggregometry evaluates platelet function under more physiological conditions because of the presence of the other blood components, does not require manipulation of the sample, thus avoiding platelet activation, and all platelet subpopulations are present in WB sample [39].

Flow cytometry assay allows the rapid analysis of physical and antigenic properties of platelets, such as presence of platelet aggregates or leukocyte–platelet aggregates, determination of GP receptor expression (i.e., GPIIb/IIIa, GPIb/IX/V, and GPVI), including conformational changes related to the receptor activation (i.e., GPIIb-IIIa), activation markers (CD62P and CD63), and platelet granule secretion (β-thromboglobulin, thrombospondin-1, vWF, fibrinogen, and P-selectin). In WB samples, the use of a double labeling binding allows the identification of platelets, platelet microparticles, or mixed cell aggregates [40–42]. Although flow cytometry has the advantages to require small volume of blood sample, to perform platelet aggregation also in the presence of low platelet counts, and to analyze platelet function and activation in a physiological environment [43], this approach can be affected by preanalytical manipulations and be prone to artifacts [44,45]. ELISA are now the most commonly used assays for the measurement of platelet activation markers TXA2 metabolites (serum TXB2 and urinary 11-dehydro-TXB2) [46] and alpha-granule factors such as β-thromboglobulin, platelet factor (PF)-4, soluble P-selectin (sP-selectin), and soluble CD40 Ligand (sCD40L) [44,45]. The new Point-of-Care devices (i.e., VerifyNow system, Plateletworks, Platelet Function Analyser-100, and Multiplate Electrode Aggregometry) may be useful supplements to the existing well-known platelet function tests and are mainly utilized for monitoring antiplatelet therapies.

#### **5. Platelet Alterations in Central Obesity**

Obesity is a heterogeneous condition and, when located within the abdominal cavity, becomes an independent determinant for cardiometabolic disease causing or exacerbating other cardiovascular and metabolic risk factors, such as dyslipidemia, hypertension, insulin-resistance, and T2DM [47–49]. Apart from metabolic and hemodynamic alterations, central obesity is characterized by a chronic low grade inflammation and systemic oxidative stress that eventually damages the endothelium causing the loss of the endothelium antithrombotic properties. This justifies the assumption of obesity as a pro-thrombotic clinical condition with increased platelet activation and decreased fibrinolysis [50–53], both contributing to atherogenesis and acute atherothrombotic events via increased vascular deposition of platelets and fibrinous products.

Platelets from obese subjects are known as "angrier" because they show a number of abnormalities, which increase platelet aggregability and activation constituting a relevant risk factor for CVD, especially for the development of atherothrombosis [54]. Recently, studies linking proteomic analysis

and aggregation findings have confirmed the presence of alterations in proteins related to platelet signaling [55]. In particular, a higher expression of GPVI, positively correlated with body mass index (BMI), together with higher levels of Src (pTyr418) and tyrosine phosphorylated phospholipase Cγ2, essential for integrin signaling, mechanistically provide possible explanations for platelet hyperreactivity in obesity [55].

Certain adipokines, bioactive peptides secreted by omental adipose tissue, can modulate not only body weight and metabolism but also vascular function [56]. For instance, in the platelet hyperreactivity of obese individuals [57–61], associations with leptin, the satiety hormone produced primarily by the adipose tissue, and adiponectin, an insulin-sensitizing adipokine produced exclusively by adipocytes, have been found. Platelets express the leptin receptor and both leptin and leptin-receptor-deficient mice have been protected from experimental thrombosis [62]. In in vitro experiments with human platelets, leptin alone does not induce platelet aggregation but increases the proaggregating effects of sub-threshold concentrations of ADP and thrombin [63]. A specific pathway in the leptin-induced platelet activation involves Janus kinase 2 (JAK2), phosphatidylinositol 3 kinase (PI3K) and phospholipases Cγ2 and A2, with effects on 3 ,5 -cyclic adenosine monophosphate (cAMP) hydrolysis, GPIIb/IIIa expression, and TX synthesis. Furthermore, independently of other risk factors, high plasma levels of leptin are associated with an increased risk of thrombotic events such as acute myocardial infarction and stroke [63].

Differently from the other secretory products of adipocytes, adiponectin exerts anti-inflammatory effects protecting against thrombosis, insulin-resistance, dyslipidemia, and endothelial dysfunction [55]. Adiponectin, the most abundant secretory protein produced by adipocytes, is synthesized and secreted as a trimer and in multimeric complexes cleaved to forms that are active transducer of signaling [64]. In mouse, adiponectin has been shown to increase fatty acid oxidation, perhaps through the activation of AMP kinase (AMPK). Disruption of adiponectin leads to high-fat diet–induced insulin resistance and levels are low in humans with obesity and insulin resistance while adiponectin levels are increased by insulin-sensitizing peroxisome proliferator-activated receptors (PPAR)γ agonists.

Although adiponectin per se does not influence platelet aggregation [65], antithrombotic actions have been attributed to this adipokine. In particular, adiponectin deficient mice show increased platelet response to the proaggregating agents and thrombosis tendency [66], high adiponectin plasma concentrations are associated with a decreased risk of coronary artery diseases and increased bioavailability of NO [67]. Both hyperleptinemia and hypoadiponectinemia in MS are associated with increases in leukocytes and platelet indices with platelet count, platelet distribution width (PDW), mean platelet volume (MPV) values, and platelets/lymphocyte ratio significantly higher in MS patients than in healthy subjects [68].

#### **6. In Vivo Markers of Platelet Activation in Obesity**

Activated platelets show peculiar features or express certain proteins that are less detectable in resting platelets, thus these factors can be used as markers of platelet activation. Some of these markers are higher in central obesity than in healthy subjects (Table 1).



#### *6.1. Mean Platelet Volume*

Among the in vivo markers of platelet activation in obesity, MPV represents a parameter closely related to platelet hyperactivation [69] and it has been found increased in obese subjects [70,71]. An interventional study carried out on female subjects, showed MPV values significantly higher in the group of obese women, in comparison with the non-obese [70]. A positive correlation was found between not only MPV and BMI but also reduced values of MPV and weight loss. Conversely, in another cross-sectional study on male individuals, it was not observed any significant difference in MPV values between groups with abdominal and without it. However, in the same study, MPV displayed a positive correlation with prothrombin activity [72]. Weight loss after bariatric surgery is also accompanied by a decrease in platelet count and significant changes in MPV, especially 6 months after surgery, corresponding to the period when weight loss was at its maximum [73].

#### *6.2. Arachidonic Acid Metabolites*

TXA2 is an unstable platelet-derived proaggregant agent with persistent biosynthesis in several CVD [74]. Precursor of TX synthesis is AA dissociated from membrane phospholipids following the increased Ca++ intracellular levels and phospholipases activity [75]. A crucial role in TXA2 production is played by the action of the constitutively expressed cyclooxygenase (COX)-1 in platelets and inducible COX-2 in monocytes and other cells in response to inflammatory and mitogenic stimuli. TXA2 has a short half-life and is nonenzymatically hydrolyzed and further converted into stable metabolites excreted in the urine 2,3-dinor-TXB2 and 11-dehydro-TXB2. The urinary excretion of 11-dehydro-TXB2, which represents the more reliable time-integrated index of systemic TXA2 synthetized for 70% by platelets, has been found increased in women with abdominal obesity and higher in women with android obesity than in those with gynoid obesity [76]. Noteworthy, serum TXB2 levels were found lower in insulin sensitive morbidly obese subjects than in the obese subjects and lean subjects, suggesting that reduced platelet activation could play a role in the paradoxical protection of morbidly obese subjects from atherosclerosis, despite the greater levels of leptin and C-reactive protein [77].

Abdominal obesity increases oxidative stress, as demonstrated by the increased levels of lipid peroxidation or protein oxidation products [78]. Indeed, the chronic 'metabolic inflammation' [79], the hallmark of obesity causing insulin resistance and T2DM [80], where the metabolic disorders trigger inflammatory signals, contributes to generate reactive oxygen species (ROS), which influence platelet function by different ways. For instance, isoprostanes are a family of products derived from AA metabolism through ROS-dependent mechanisms.

An oxidation product of AA is 8-iso-prostaglandin F2<sup>α</sup> (PGF2α), an abundant isoprostane involved in platelet aggregation by activating TX receptor in the presence of sub-threshold concentrations of other agonists. The influence of this isoprostane on platelets can be prevented by TXA2 receptor antagonism but is completely independent of COX-1 activity [29]. A positive linear correlation between urinary excretion of 11-dehydro-TXB2 and PGF2<sup>α</sup> underlines the link of platelet activation with oxidative stress [81].

#### *6.3. Soluble P-Selectin*

A pivotal role in the development of vascular complication of atherothrombosis is played by cellular adhesion pathways and selectins are one of the four main adhesion molecule families. Platelets are the major source of P-selectin, a cellular adhesion molecule with procoagulant activity [82] and able to activate leukocyte integrins [83]. The circulating levels of soluble form of P-selectin mirror platelet activation. Stored in the alpha-granules of platelets, in a setting of inflammation P-selectin translocates to the plasma membrane where it can interact with ligands [84] leading to leukocyte-platelet aggregates that promote adhesion and infiltration of inflammatory cells [85–88]. sP-selectin has been associated with adiposity and both clinical and subclinical atherosclerosis [89] and has been shown to predict

atherosclerosis independently of BMI and other CVD risk factors. The enhanced plasma concentrations of P-selectin in overweight and obese insulin resistant subjects [61,90] are reduced after weight loss [61].

#### *6.4. CD40 Ligand*

Activated platelets also release the sCD40L, a trimeric transmembrane protein structurally related to tumor necrosis factor (TNF)-α superfamily. CD40 and its immunomodulating CD40L show dual prothromboting and proinflammatory role further contributing to amplify vascular diseases and atherogenesis [91].

More than 95% of circulating sCD40L derives from platelets, stored in high amounts in cytoplasma in unstimulated platelets, expressed on the platelet surface where it is cleaved to form the soluble trimeric fragment and released within seconds after platelet activation [92]. sCD40L measurement is considered as a platelet-derived marker of cardiovascular risk able to link thrombosis and inflammation [93]. Studies in mice showed that in obesity the genetic or antibody mediated disruption of CD40L signaling ameliorates adipose tissue inflammation and metabolic disorders in insulin resistance [94], thus confirming the role of sCD40L as a platelet-derived marker of the cardiovascular risk able to link thrombosis, inflammation, and altered metabolism [93]. CD40/CD40L interaction is involved in the expression of many proinflammatory and prothrombotic factors, including IL-1, IL-6, IL-8, IL-12, TNF-α, monocyte chemoattractant protein (MCP)-1, and matrix metalloproteinases (MMPs) accelerating the adhesion of monocytes to the vascular endothelium [95–97], promoting a ROS-mediated endothelial injury [98–100] and the rupture of atheromatous plaques [101]. Recent reports have also indicated that patients with acute cerebral ischemia exhibit increased expression of CD40L on platelets and the CD40/CD40L signaling directly modulates cerebral microvascular thrombosis by the mammalian target of rapamycin (mTOR)/S6K signaling pathway activation [102]. Plasma levels of sCD40 are considered reliable markers of in vivo platelet activation and the increased levels found in obesity are reduced by weight loss [61].

#### *6.5. Platelet-Derived Microparticles (PMPs)*

Platelet-derived microparticles (PMPs) are small membrane-bound microparticles with a diameter less than 0.1 micron containing bioactive proteins and genetic material (i.e., mRNAs and microRNAs) able to deeply influence phenotypes and functions of recipient cells promoting the development of pathological states [103]. Platelets, activated by various agonists or exposed to high shear stress [104] or increased oxidative stress [57], produce PMPs and elevated levels of circulating PMPs are associated with most of the cardiovascular risk factors including hypertension, obesity, and dyslipidemia [105], appearing indicative of a poor clinical outcome. In obese non-diabetic subjects, elevated circulating levels of PMPs positively correlate with BMI and waist circumference [106]. Weight reduction, by calorie restriction with or without exercise [106] or after gastrectomy, reduces PMP production. Interestingly, another study has recently shown that PMPs from obese subjects were not different in number if compared with non-obese subjects but, as supported by proteomics data, they showed greater heterogeneity in size and distribution with different levels of proteins relevant to thrombosis and tumorigenesis [107].

#### **7. Contribution of Insulin Resistance on Platelet Dysfunction**

Insulin is a hormone that mediates its action through the insulin receptor (IR) composed of two monomers comprising an extracellular α-subunit and a transmembrane β-subunit [108]. Insulin binding induces IR autophosphorylation at various tyrosine residues, recruitment of IR substrates (IRS), and activation of mitogen-activated protein kinase (MAPK) and PI3K [109]: the activation of these signaling pathways promotes downstream processes involved in blood glucose control [110]. A less than expected response of target organs to insulin leads to a condition of insulin resistance with hyperinsulinemia for a compensatory increased insulin production by pancreatic β-cells. Insulin-resistance is classically referred to metabolic homeostasis characterizing, in most cases, obesity,

impaired glucose tolerance and T2DM [111]. Indeed, insulin resistance involves also the vascular effects of the hormone [112–114] and it is the common soil of a cluster of metabolic, hemodynamic, thrombotic and inflammatory features deeply involved in atherogenesis and CVD [115]. One of the alterations accounting for the association between insulin resistance and vascular diseases is platelet hyperactivation, also explained by the reduced sensitivity to the physiological and pharmacological antiaggregating agents. Platelet membrane shows functional IR with a density similar to that measured in other target cells of insulin action [116]. In platelets from insulin sensitive subjects, the hormone decreases in vitro platelet aggregation stimulated by common platelet agonists such as ADP, thrombin, catecholamines, PAF, collagen, AA, and angiotensin-II [117,118]. Insulin infusion in euglycemic conditions determines: (i) reduced sensitivity to multiple agonists and deposition to collagen [119]; (ii) impaired primary hemostasis under high shear stress [119]; and (iii) reduced TXA2 metabolite synthesis also in T1DM [120]. Through NO increase, insulin induces a rapid increase of the cyclic nucleotides 3 ,5 -cyclic guanosine monophosphate (cGMP) and cAMP with inhibitory effects on platelet aggregation [121]. In conditions of insulin resistance such as central obesity, T2DM with obesity and hypertension, the inhibitory effects of insulin on platelets are impaired [53] (Figure 3). Among the mechanisms involved in the altered insulin actions on platelets, a role is played by the effects on platelets of the abnormal adipokine content in plasma profile of patients with MS and T2DM [122]. In particular, the adipokines resistin, leptin, PAI-1, and retinol binding protein 4 (RBP4) induce insulin resistance in megakaryocytes by interfering with IRS-1 expression with a negative impact on insulin signaling in platelets.

Platelets from obese insulin-resistant individuals are characterized by multi-step defects at level of NO/cGMP/protein kinase cGMP-dependent (PKG) and PGI2/cAMP/protein-kinase cAMP-dependent (PKA) pathways. In particular, platelets show an impaired NO and PGI2 ability to increase, respectively, cGMP and cAMP synthesis and a resistance to cGMP and cAMP themselves to activate their specific kinases PKG and PKA [59,60]. Since the cyclic nucleotides exert their effects on platelets mainly through a reduction of intracellular Ca++ [123], these data are suggestive for the presence of alterations in Ca++ fluxes handling. Actually, elevated cytosolic Ca++ concentrations have been found in insulin-resistance states [124] and this could explain the defective action of cyclic nucleotides on platelet function. Of note, this multistep resistance is not emphasized by the presence of T2DM [125] as well as the presence of T2DM without obesity is not associated with this cluster of platelet abnormalities [125]. However, lifestyle interventions aiming to reduce body weight by diet can modify the prothrombotic tendency in obese insulin resistant individuals. Actually, the altered platelet sensitivity to NO/cGMP/PKG and PGI2/cAMP/PKA pathways in obesity is restored by weight reduction of at least 10% of the initial body weight and this phenomenon is also accompanied by an improvement of insulin resistance and a decrease of markers of inflammation [61] and synthesis of isoprostanes [126]. The central role of the insulin resistance associated with obesity as a pathogenic factor deeply involved in the impairment of the main inhibitory mechanisms of platelet function is confirmed, in the same study, by multiple regression analysis showing the homeostasis model assessment (HOMA) index, a surrogate marker of insulin-resistance, as the parameter more strongly associated with platelet response to the antiaggregating agents. Successful weight loss obtained with drugs, such as the incretin-based therapy, is associated with a significant reduction in TX-dependent platelet activation, possibly mediated, at least in part, by decreased inflammation and lipid peroxidation [127]. In particular, a direct role on platelets by Liraglutide, an analog of the incretin hormone glucagon-like peptide 1 (GLP-1), initially used for the treatment of T2DM and recently introduced as potential weight loss medication, cannot be excluded because Liraglutide has been shown to inhibit platelet activation in animal models [128] and human platelets [129].

**Figure 3.** Relationships between insulin resistance, increased oxidative stress and inflammation in promoting platelet hyperactivation in obesity. AA, arachidonic acid; A-II, angiotensin-II; cAMP, 3 ,5 -cyclic adenosine monophosphate; cGMP, 3 ,5 -cyclic guanosine monophosphate; IR, insulin receptor; MPV, mean volume platelet; NO, nitric oxide; PAF, platelet activating factor; PAR, protease-activated receptor; PGI2, prostaglandin I2; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; ROS, reactive oxygen species; PMP, platelet-derived microparticles; TP, thromboxane receptor; TX, thromboxane.

#### **8. Type 2 Diabetes Mellitus and Alterations of Platelet Function**

Platelets from diabetic patients are more prone to form spontaneous microaggregates [130], to adhesion, to aggregation in response to agonists [131], and to be less sensitive to antiaggregants [132]. Biochemical abnormalities associated with these impairments of platelet function can be detected by elevation of intracellular calcium levels and expression of platelet activation markers including PMPs, which in patients with T2DM can be used as potential predictors of CV outcomes [133].

Indeed, several mechanisms are involved in the hyperactive platelet phenotype in diabetic patients. Among them, hyperglycemia, oxidative stress, and altered shear stress, interconnected with associated metabolic conditions (obesity, dyslipidemia, and subclinical inflammation) promote atherogenesis and the tendency to a prothrombotic status (Figure 4), which in T2DM represents an important risk factor for a first CV event and for worse outcomes after a CV event.

#### *8.1. Hyperglycemia*

Although some abnormalities in platelet function in T2DM depend on the presence of the insulin resistance condition, some defects occur only in T2DM in relation to hyperglycemia. Hyperglycemia, the basic characteristic feature of diabetes, and glycemic variability are predictive determinants of platelet activation [134] and postprandial hyperglycemia is an independent risk factor for cardiovascular complications [135]. Although the underlying pathogenic mechanisms are multiple, factors promoting oxidative stress are unanimously considered to contribute significantly to platelet activation. Of particular interest, in T2DM patients a marked oxidative response is induced by the consumption of high-calorie meals, which in these individuals determines an abnormal and sustained elevation of blood glucose and lipid levels, mainly TGs, defined as postprandial dysmetabolism [136].

**Figure 4.** Biochemical imbalance towards factors promoting endothelial dysfunction and platelet hyperactivation involved in the development of atherothrombotic diseases in the presence of multiple cardiometabolic risk factors. eNOS, endothelial nitric oxide synthase; NFκB, nuclear factor kappa B; PAI-1, plasminogen activator inhibitor-1; AP-1 activator protein-1; NO, nitric oxide; PGI2, prostaglandin I2; EC, endothelial cell; PMPs, platelet-derived microparticles; MS, metabolic syndrome.

Since the entry of glucose into platelets does not depend on insulin, intraplatelet glucose concentration mirrors blood glucose levels, and chronic hyperglycemia has been clearly identified as a causal factor leading to platelet hyperreactivity, as indicated by enhanced aggregation, increased fibrinogen binding, and TX production [137]. Hyperglycemic spikes trigger ischemic cardiovascular complications in T2DM [138–140] and may elicit arterial thrombosis owing to a transient hyperreactivity of platelets to high shear stress, thus contributing to precipitating arterial thrombotic occlusion at stenotic sites [141]. Furthermore, platelet activation due to high glucose exposure in the absence or in the presence of high shear stress conditions is cause of reduced platelet sensitivity to inhibition by aspirin [142–144]. Recently, a reduced acetylation level of the catalytic Ser529 site associated with an incomplete inhibition of COX-1 activity by aspirin has been found in condition of high glucose and diabetes [145], adding another piece of information, which may contribute to explain the residual platelet hyperreactivity observed in diabetes and implying in T2DM the use of effective therapeutic strategies able to prevent hyperglycemia in order to improve also the protective effects of aspirin against the occurrence of CV events.

Platelet hyperreactivity in T2DM is coupled with biochemical evidence of persistently increased TX-dependent platelet activation [137,146] and in the mechanism by which platelets transduce glucose levels into enhanced TX generation a central role is played by the enzyme aldose reductase, the first enzyme of the polyol pathway. The activity of aldose-reductase is significantly enhanced in vascular cells in T2DM and is thought to contribute to vascular complications by increasing oxidative and osmotic stress. Glucose flux through aldose reductase enzyme generates oxidative stress by distinct mechanisms, including nicotinamide adenine dinucleotide phosphate (NADPH) depletion, decrease of glutathione (GSH) levels, and increase of advanced glycation end products (AGEs), thus promoting ROS formation [147]. ROS also play an important role in signaling upon agonist-induced platelet aggregation, inducing changes in intraplatelet Ca++, and acting as second messenger in thrombin- or collagen-activated platelets [148]. The increased oxidative stress derived from hyperglycemia and platelet activation potentiates p38α MAPK/cytosolic phospholipase A2 signaling, which catalyzes AA release and TXA2 production. T2DM with enhanced biosynthesis of TX despite aspirin therapy may have underlying endothelial damage and thromboembolic disease [149]. As mentioned earlier, sCD40 is both marker and mediator of platelet activation and its upregulation is involved in the advanced

stage of cerebrovascular disease and increased risk of CV events in T2DM. The increased TX-dependent platelet activation is also associated with enhanced CD40L release [150].

Although mechanisms underlying the pathogenesis of ischemia/reperfusion injury are particularly complex and multifactorial, there is evidence of interactions between platelet function and ischemia/reperfusion injury, especially in diabetic conditions [151].

Diabetic heart is among the most susceptible to ischemia/reperfusion injury and some cardioprotective strategies are compromised in the presence of diabetes because of several mechanisms, including alteration at the mitochondrial level, altered production of ROS, and impairment of antioxidant capacities at various intracellular and extracellular sites [152].

Interestingly, a recent study has shown that the infusion of platelets from healthy subjects in rat isolated hearts exerts cardioprotective effects by reducing infarct size [153] with a mechanism that depends on the platelet capacity to activate cardiac sphingosine-1-phosphate (S1P) receptors and extracellular signal-regulated kinase (ERK)/PI3K/protein kinase C (PKC) pathways. However, platelets from poorly controlled T2DM subjects, as mirrored by high values of glycated hemoglobin (HbA1c), lost their cardioprotective effects, released less S1P, and a positive correlation between infarct size and the amount of ROS produced by diabetic platelets was found [153].

High glucose levels were also found to cause in platelets loss of function and damage to mitochondria, mitochondrial membrane potential dissipation, cytochrome c release, caspase-3 activation, and a subgroup of platelets can undergo apoptosis [154]. Enhanced rate of platelet apoptosis can lead to generation of PMPs that carry thrombotic mediators by providing a new prothrombotic interface for the deposition of fibrin and other blood cells [155].

In addition to up-regulated pro-aggregatory stimuli, platelets from diabetic individuals show reduced sensitivity to the antiaggregating insulin, NO, and PGI2 [58]. Since some antiplatelet effects of aspirin are related to increased platelet NO synthesis [144] and preservation of NO from its inactivation [156], an impaired platelet sensitivity to NO signaling may account, at least partially, for less protective aspirin effects against thrombotic events in T2DM.

However, the superoxide-mediated impairment of NO effects on platelet function following hyperglycemia can be corrected by acute aggressive glycemic control [157]. Platelet exposure to high glucose also influences the biophysical state of platelet membrane components and changes in fluidity owing to glycation or acetylation of membrane proteins contribute to the intensified intraplatelet Ca++ mobilization [158]. High cytosolic Ca++ levels deeply influence the procoagulant state of platelet aggregates inducing externalization of phosphatidylserine and thus accelerating the membrane-dependent reactions of blood coagulation [159].

#### *8.2. Oxidative Stress*

As mentioned, superoxide radicals have a strong effect for activating platelets and in T2DM oxidative stress is increased for the imbalance between ROS production and antioxidant defenses. High concentrations of ROS influence platelet function by different mechanisms, including decreased NO bioavailability, calcium mobilization abnormalities, over-expression of membrane glycoproteins, and isoprostane formation. A major source of platelet ROS is the enzyme NADPH oxidase (Nox), as demonstrated in platelets from patients affected by chronic granulomatous disease, a rare primary immunodeficiency, that show very low ROS generation and, in the most frequent form, the deficiency of Nox2 subunits. Nox2 is expressed by platelets and its increased activity has been shown to be correlated with platelet activation, isoprostane formation and/or NO inhibition [160,161]. Nox2 activation, platelet recruitment, and isoprostane levels are parallelly increased in diabetic patients and these could be cause of reduced efficacy of aspirin [162].

Oxidation reactions are elevated in patients with T2DM and significantly contribute to form isoprostanes, which are produced from AA through a non-enzymatic process of lipid peroxidation, catalyzed by oxygen-free radicals on cell membranes [163]. Since structurally similar to prostaglandins, once released isoprostanes activate the same receptors. 8-iso-PGF2<sup>α</sup> influences some aspects of platelet

function such as adhesive reactions and activation by low concentrations of other agonists [134]. In poorly controlled diabetes, plasma levels of 8-iso-PGF2<sup>α</sup> are increased and correlate with impaired glycemic control and enhanced lipid peroxidation, thus providing a biochemical link between impaired glycemic control and persistent platelet activation [164].

#### *8.3. Shear Stress*

The increased tendency of platelets from diabetics to aggregate is tightly regulated not only by the diabetic milieu but also by complex conditions of flow dynamics. Thrombotic complications are deeply influenced by the effects of hemodynamic environment at the site of vessel injury or plaque rupture blood on endothelial cells constantly exposed to multiple physical forces generated by the movement of blood. In normal conditions, the physiological shear stress-induced endothelial release of NO and PGI2 does not allow platelets to adhere to the vessel wall [165,166]. In response to abnormal blood flow endothelial cells can modify their shape, function and gene expression, which, in turn, affect platelets, whose adhesiveness and activation change. High shear rates (>1000 s−1) promote platelet aggregation critically modulated by vWF, endogenously present in the subendothelial matrix or absorbed onto injured tissue components exposed to plasma [167], and subjected to conformational changes that determine vWF self-association and vWF fiber formation [168]. Furthermore, vWF activation also requires the formation of disulfide bridges from free thiols [169], this reaction depends on ROS [170] whose levels, as known, are increased in T2DM. Further studies also showed that in T2DM hyperglycemia causes membrane lipid peroxidation and osmotic fragility in red blood cells [171] leading to increase extracellular hemoglobin which directly affects the GPIbα-vWF interaction [172]. In particular, increased platelet adhesion, and microthrombi formation on fibrin(ogen), extracellular matrix, and collagen at high shear stress in the presence of free hemoglobin (≥50 mg/dL) were found. These may have implications on the shear stress-induced platelet aggregability explaining, at least in part, the increased platelet aggregation in whole blood from T2DM patients. Taking into account that T2DM patients show higher plasma concentrations of vWF, correlated with HbA1c and chronic hyperglycemia, we can suppose that the occurrence of a disturbed microhemorrheology in a diabetic environment, characterized also by elevated ROS levels, contributes to exacerbate the prothrombotic phenotype.

#### **9. Role of Dyslipidemia in the Impaired Platelet Reactivity**

Dyslipidemia is recognized as an independent risk factor for coronary artery and peripheral vascular disease. In this association a major role is exerted by the effects of accumulation of plasma oxidized lipids on platelet function suggesting a potential causative role for dyslipidemia in the promotion of platelet hyperreactivity in CVD [173,174]. Cholesterol accumulation in plasma membrane alters membrane structure with effects on signaling via surface receptors. Indeed, the mechanisms by which dyslipidemia promotes platelet activity and thrombosis in vivo are multiple also for the heterogeneous nature of lipoproteins.

Platelets become sensitive to a wide spectrum of interactions after low-density lipoprotein cholesterol (LDL) binding to the specific receptor on the platelet membrane: in their native form, LDLs alone do not induce platelet aggregation but increase platelet response to proaggregants; if oxidized, LDLs induce platelet aggregation also in the absence of agonist [175].

The capability of oxidized-LDL (oxLDL) particles to stimulate generation of ROS by lectin-like oxLDL receptor-1 (LOX-1) binding, a major receptor for uptake of oxLDL in endothelial cells, is one the mechanisms involved in the reduced NO bioavailability at all stages of atherosclerosis through the increases in Nox, nuclear factor kappa B (NF-κB), and mitochondrial enzymes involved in oxidative signaling [176,177]. As known, loss and/or impaired NO action can induce platelet activation, and in disease states such as hypercholesterolemia and diabetes, where ROS production is increased, a dysregulated NO metabolism becomes a critical determinant of platelet function. Indeed, diseases like hypercholesterolemia, where high levels of LDL are often accompanied by increased oxLDL, platelet hyperactivity could also depend on hyporesponsiveness to NO-related pathways [61,178–180]. In fact, platelets from patients with primary hypercholesterolemia, if compared with healthy controls, show higher aggregability to ADP, collagen, AA, higher ROS production, reduced sensitivity to NO, and increased activation of the proaggregant PI3K/Akt and MAPK/ERK-2 pathways. In the same individuals, platelet exposure to GLP-1, an incretin hormone with effects depending on GLP-1 influence on NO-signaling [129], does not exert any of its antiplatelet actions [178]. In this phenomenon, a role could be played by oxLDL ability to generate Nox2-derived ROS through a CD36-PKC pathway with inhibition of cGMP signaling [181], a key protective pathway activated by NO that, if overcome, results in increased platelet activation.

Platelets from patients with hypercholesterolemia show hyperaggregability, increased fibrinogen binding and surface expression of CD62P, increased production of TXA2 and superoxide anion, whereas plasma derived from the same patients contains increased concentrations of platelet activation markers, such as soluble sCD-40L, PF-4, sP-selectin, and β-thromboglobulin [182–184]. Many of these impaired platelet parameters of platelet aggregation and activation are corrected by lipid-lowering treatments.

In vitro and in vivo studies show that statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and the most relevant drugs used to lower serum cholesterol levels, due to their pleiotropic effects decrease subclinical inflammation, oxidative stress, endothelial dysfunction, platelet aggregation, and activation [185–189], improving platelet sensitivity to NO [178], and aspirin [184], but not to GLP-1 [178]. The causes of the enhanced platelet hyperaggregability and the defective GLP-1 actions in dyslipidemia can be multifactorial, although the strong correlation with LDL underlines the role of cholesterol as a major determinant of platelet hyperreactivity with a putative role also in the impaired response to GLP-1. The modulating effects of GLP-1 on platelet function might have protective roles on the cardiovascular system, thus suggesting that a reduced and/or impaired action of GLP-1 on platelets could be involved in the platelet hyperreactivity described in metabolic disorders such as diabetes [190,191] and dyslipidemia [192,193].

Although statins represent important tools for primary and secondary prevention of CV events in hypercholesterolemia, only a low percentage of patients reach a predefined LDL target thus justifying the development of new approaches to lipid modification. At this purpose, the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) to reduce plasma LDL is a new approach for the treatment of hypercholesterolemia because it allows us to address the unmet clinical needs of achieving goal LDL levels for the majority of patients with high CV risk. PCSK9 is a major regulator of LDL levels as it promotes the degradation of hepatic LDL receptors, thus its inhibition causes an increase of LDL receptor activity and more circulating LDL is removed [194].

It has been recently shown that in primary hypercholesterolemia the in vivo treatment with PCSK9 inhibitors, beyond their lipid-lowering action, had important inhibitory effects on platelet aggregation and activation [129]. Given the activating direct effect of PCSK9 on platelets [195] and the relationship between PCSK9 and higher platelet reactivity [196–198], it is plausible that PCSK9 can directly influence platelet reactivity, thus PCSK9 inhibitors also would reduce the direct PCSK9 stimulatory effects on platelets.

The typical dyslipidemia in patients with T2DM and/or the MS is characterized by increased plasma TG concentration and low HDL concentration. In such context, the presence of small, dense LDL, more prone to oxidation, leads to a mixed atherogenic dyslipidemia.

Even though LDLs affect platelet function by modulating platelet activity more strongly than hypertriglyceridemia [173,199], there is evidence that TG-rich particles can directly activate platelets [200].

HDL has been shown to mediate various antithrombotic effects [201]. The infusions of reconstituted HDL decreased platelet activation in diabetic subjects [202]; on the contrary, in condition of impaired delivery of cholesterol by HDL from plasma and peripheral tissues, marked increases of platelet activation and thrombosis have been found [203].

#### **10. Hypertension and Platelets**

Arterial hypertension is one of the most important worldwide public-health challenges because of its high frequency and a leading preventable cause of premature death. Indeed, hypertension is a multifactorial disease, often clustering with other components of metabolic syndrome such as obesity, dyslipidemia, and insulin-resistance [204] and platelet activation is deeply involved in at least half of deaths due to heart disease and stroke [205–207].

Changes in the biochemical and functional profile of plasma membrane of platelets from hypertensive subjects are suggestive of platelet activation [208]. The increase of shear forces due to elevated blood pressure, especially adjacent to the endothelium, can promote platelet activation and degranulation [209].

Impaired NO availability, increased oxidative stress, altered Ca++ metabolism [210], and membrane permeability [211] are some of platelet abnormalities observed in hypertensive patients.

Vascular and endothelial dysfunction are linked to arterial hypertension and may result in a greater propensity for platelets to cause thrombosis. Actually, given the central role of endothelial cells in avoiding platelet adhesion and maintaining normal platelet function, the presence of dysfunctional endothelium would promote platelet adhesion and activation.

An important consequence of endothelial dysfunction is the reduced bioavailability of NO, a key molecule for CV health. This may be a consequence of the endothelial nitric oxide synthase (eNOS) polymorphisms [212], reduced NO production or increased breakdown of NO by ROS [213,214]. In particular, the uncoupled state of eNOS leads to a decrease in NO synthesis and increase in ROS production. The quenching of NO by superoxide anions contributes to impaired vascular smooth muscle cell response [215] and relaxation [216]. Platelets express both the constitutive eNOS, and inducible NOS (iNOS) with distinct molecular structure and characteristics [217]. The constitutive, calcium-dependent eNOS is responsible for NO production in platelets, which in turn inhibits platelet activation and aggregation by increasing cGMP levels [218]. Increase in intraplatelet Ca++ [219] and decrease of NO bioavailability [210] could explain, at least partially, the higher platelet aggregation observed in hypertension.

Stimulated platelets release vascular endothelial growth factor (VEGF) [220], one of the most potent angiogenic factors, and elevated VEGF levels have been found in patients with atherosclerotic risk factors, including hypertension [221–223]. The association between sP-selectin and VEGF levels corroborates the hypothesis that platelets are likely to be a relevant source of VEGF in hypertension; in this setting, aspirin inhibits the agonist-induced platelet aggregation and also VEGF release [224].

Intracellular Ca++ and Na<sup>+</sup> contents can modify membrane fluidity and microviscosity that, in turn, can influence receptor functions or enzyme activities [225]. Indeed, arterial hypertension is characterized by a number of structural and functional alterations of the cell membrane including changes in membrane permeability, signal transduction, ion transport, receptor functions, because of plasma membrane differential composition, which in turn might disturb the asymmetry of the platelet plasma membrane [226,227]. Recently, structural and biochemical abnormalities in the platelet membrane from hypertensive subjects have been confirmed by studies showing an overexpression of the epithelial sodium channel [228] involved in the regulation of extracellular fluid volume and blood pressure and dispensable in platelets for migration, alpha- and dense-granule secretion and platelet collagen activation [211].

#### **11. Conclusions**

Platelets are key players in the thrombotic process in patients with metabolic abnormalities associated with increased risk of CVD.

This review provides an overview of changes in platelet function occurring in metabolic and hemodynamic disorders mainly characterizing the MS, all with an impact on the risk of CV morbidity and mortality owing to atherothrombotic events. Many of impairments in platelets converge on oxidative stress with release of oxidation products, which have a causal link to platelet hyperaggregability and hyperactivation. The excess of adipose tissue of the trunk and/or abdomen has a strong impact on vascular complications, through the production of PMPs and mediators with paracrine and endocrine actions, which influence platelet response. Platelet indices and biomarkers of platelet activation may have useful clinical value through the whole journey of cardiometabolic diseases for prediction and risk assessment of thrombotic risk. Different methodological approaches for platelet (dys) function investigations are now available and each-one based on different operating principles. However, few assays are able to assess "all in one device" platelet aggregation and activation pathways and standardization and quality controls are still limited despite several efforts.

Insulin resistance, a condition frequently associated with obesity, with or without hyperglycemia, dyslipidemia, and hypertension alters a number of distinct aspects of hemostasis responsible for platelets more prone to aggregate to agonists and less responsive to platelet inhibitors. However, weight reduction is a powerful measure to restore a physiological platelet function in obese subjects.

**Author Contributions:** I.R. conceptualized the work; I.R. and C.B. provided the resources for this work; I.R. did the original draft preparation; I.R. and C.B. reviewed and edited the text; C.B. drew the figures; I.R. lead the funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **The "Janus Face" of Platelets in Cancer**

#### **Maria Valeria Catani \*, Isabella Savini, Valentina Tullio and Valeria Gasperi \***

Department of Experimental Medicine, Tor Vergata University of Rome, 00133 Rome, Italy; savini@uniroma2.it (I.S.); valentinatullio.nu@gmail.com (V.T.)

**\*** Correspondence: catani@uniroma2.it (M.V.C.); gasperi@med.uniroma2.it (V.G.); Tel.: +39-06-72596465 (M.V.C.); +39-06-72596465 (V.G.)

Received: 11 December 2019; Accepted: 22 January 2020; Published: 25 January 2020

**Abstract:** Besides their vital role in hemostasis and thrombosis, platelets are also recognized to be involved in cancer, where they play an unexpected central role: They actively influence cancer cell behavior, but, on the other hand, platelet physiology and phenotype are impacted by tumor cells. The existence of this platelet-cancer loop is supported by a large number of experimental and human studies reporting an association between alterations in platelet number and functions and cancer, often in a way dependent on patient, cancer type and treatment. Herein, we shall report on an update on platelet-cancer relationships, with a particular emphasis on how platelets might exert either a protective or a deleterious action in all steps of cancer progression. To this end, we will describe the impact of (i) platelet count, (ii) bioactive molecules secreted upon platelet activation, and (iii) microvesicle-derived miRNAs on cancer behavior. Potential explanations of conflicting results are also reported: Both intrinsic (heterogeneity in platelet-derived bioactive molecules with either inhibitory or stimulatory properties; features of cancer cell types, such as aggressiveness and/or tumour stage) and extrinsic (heterogeneous characteristics of cancer patients, study design and sample preparation) factors, together with other confounding elements, contribute to "the Janus face" of platelets in cancer. Given the difficulty to establish the univocal role of platelets in a tumor, a better understanding of their exact contribution is warranted, in order to identify an efficient therapeutic strategy for cancer management, as well as for better prevention, screening and risk assessment protocols.

**Keywords:** microvesicles; miRNAs; paraneoplastic thrombocytosis and thrombocytopenia; platelet activation; platelet-derived bioactive molecules; platelet-tumor crosstalk

#### **1. Introduction**

Platelets were described for the first time in 1882, when the Italian pathologist Giulio Bizzozero identified in the blood vessels "very thin platelets, disc-shaped, with parallel surfaces or rarely lens-shaped structures, round or oval and with a diameter 2–3 times smaller than the diameter of the red cells ... " that "when they are circulating in the blood stream of a living animal a small injury to the vessel wall, or contact with a foreign body is sufficient for them to become viscous, to adhere to one another and so form a white thrombus" [1]. Platelets, indeed, are small, anucleated cytoplasmic fragments derived from large (30–100 μm) progenitor cells, the megakaryocytes, formed in bone marrow, lungs and blood [2–5], which are vitally involved in thrombosis and hemostasis [6]. Nevertheless, besides this primary function, platelets are also recognized to affect immune and inflammatory responses, thus participating in regulation of biological mechanisms underlying a broad range of human disorders. A large body of experimental and clinical evidences, indeed, shows that platelet activation and dysfunction are implicated in diabetes, cardiovascular disease, chronic back pain, sepsis, Alzheimer's disease, multiple sclerosis, psychiatric disorders and other central and peripheral pathological conditions [7–11]. In this context, the unexpected central role of platelets in cancer biology

is noteworthy: They actively influence cancer cell behavior, but, on the other hand, platelet physiology and phenotype are impacted by tumor cells [12]. Indeed, a large number of experimental and human investigations support the hypothesis that tumor cells are able to modulate the RNA profile, number and activity of platelets that, once "educated", would regulate the tumor microenvironment and progression in a way dependent on the patient, cancer type and treatment. Nonetheless, the exact molecular mechanisms underlying this platelet-cancer loop are not yet well defined, often due to contradictory data.

Therefore, based on this background, the present review will focus on platelet-cancer crosstalk and their mutual impact, especially considering how platelets might exert either a protective or a deleterious role in all steps of cancer progression.

#### **2. Paraneoplastic Thrombocytosis and Thrombocytopenia in Cancer**

In healthy subjects, platelet count ranges from 150,000 to 450,000/μL, with age-, sex-, race- and genetic background-specific reference intervals [13,14]. A fine-tuned control of both platelet number and function exists, being ensured by the delicate balance among their (i) production, (ii) maintenance in the circulation (average life span of 8–10 days) and (iii) clearance of senescent cells (via hepatic and splenic macrophages, as well by apoptosis) [15–17].

In the light of the central roles played by platelets in a multitude of biological events, abnormalities in their number that often accompany various pathologies are clearly relevant, and this also applies to cancer.

Although, with some inter-individual variations, a platelet count of <sup>≥</sup>450 <sup>×</sup> 109/L is a generally accepted value used to identify a clinically significant thrombocytosis [18], which has a multitude of potential etiologies. In particular, it can be classified as (i) primary thrombocytosis, when it occurs as the result of genetic or chronic myeloid disorders [19–21] or (ii) secondary or reactive thrombocytosis, when it occurs as a comorbidity of another underlying disease independent of a vascular event, including cancer. In the latter case, the pathology is called paraneoplastic thrombocytosis [22,23]. According to the clinical evidence that patients with a high platelet count have a higher risk to develop venous thromboembolism (VTE) [24], cancer patients frequently show activated coagulation pathways, resulting in a four-fold increase in thrombosis risk [25].

The first evidence of paraneoplastic thrombocytosis dates back to 1964, when Levin and Conley found that, among their hospitalized cancer patients, at least 40% had thrombocytosis [26]. Since then, an ever-growing body of studies has reported a significant association between thrombocytosis and solid tumors, with a range of thrombocytosis incidence at initial diagnosis of 4–55% [27–31]. This evidence may assume clinical implications, if we consider that a large amount of retrospective and meta-analysis studies point out to the correlation among higher platelet count and tumor progression, advanced-stage disease, vascular thromboembolic complications and poor survival in patients with different solid tumors, such as esophageal cancer, bladder cancer, inflammatory breast cancer and epithelial ovarian cancers (see Table 1) [28,31–38].

Conversely, such a correlation has not been found by other authors [29,39–41], or it appears strictly dependent on inflammatory components, as described by a recent retrospective study of 3654 patients with stage I–III breast cancer, of whom 6.5% had a diagnosis of Inflammatory Breast Cancer (IBC), the most aggressive form of breast tumors [37]. What emerged from this study is that thrombocytosis, more prevalent in IBC patients, correlated with poor overall survival in these subjects, but not in non-IBC individuals [37].

It must not be overlooked that some investigations reporting a correlation among platelet count, metastasis and shortened survival had some limitations, such as a low platelet count threshold (<<sup>200</sup> <sup>×</sup> <sup>10</sup>9/L) not clinically correct to define a patient as having real thrombocytosis [33,34,37,42]. In addition, heterogeneity in sample size, clinical stages, treatment and follow-up, smoking history and inclusion/exclusion criteria may make it difficult to establish a univocal association between thrombocytosis and poor prognosis in cancer patients.





CI: confidence interval; CRP: C-reactive protein; HR: hazard ratio; OR: odds ratio; PLT: platelet count.1 With respect to the cut-offvalues set by the authors.

However, available literature data suggest that thrombocytosis is a paraneoplastic event not depending on elongation of platelet half-life survival [44], but on increased thrombopoietin (TPO)-dependent thrombopoiesis, together with the action of inflammatory cancer-derived cytokines. TPO is normally produced and secreted by the liver, kidney and bone marrow at a fixed rate and it promotes megakaryocyte growth and platelet generation, by binding to its receptor c-MPL and triggering activation of the Janus kinase (JAK)/signal transducer and activator of the transcription (STAT) pathway [45]. It is well documented that increased circulating TPO levels might be one of the mechanisms accounting for cancer-related thrombocytosis, as demonstrated by elevated TPO levels in plasma of cancer patients with a high platelet count [46–48]. Two different and complementary mechanisms have been proposed, both encompassing tumors that represent a TPO source per se and, moreover, secrete factors targeting hepatic TPO synthesis. In particular, it has been reported that certain cancer cells, besides expressing TPO receptors on their surface [49], are also able to produce and release functional TPO [47,50], thus contributing to the rise in blood TPO levels. Additionally, cancer cells release a plethora of humoral factors and cytokines, and some of them have been shown to upregulate hepatic TPO biosynthesis; this is the case of the pleiotropic cytokine interleukin (IL)-6, a major mediator of inflammation and activator of STAT3 [51], whose deregulated overexpression has been associated with tumor progression [28,52–55]. Both IL-6 and its receptors (IL-6R and sIL-6R) are, indeed, upregulated in tumors [56–61] and their increased content in plasma of cancer patients correlates with a poor diagnosis [28,56,61,62], thus indicating clinical utility of IL-6 as a biomarker or therapeutic target in cancer management. An elegant model proposed by several authors suggests that IL-6 plays a crucial role in inducing cancer-related thrombocytosis, via up-regulation of hepatic TPO transcription [28,53–55]. In particular, this molecular model hypothesizes that cancer cells release large amounts of IL-6 that, in turn, determines complex chains of events (i.e., an increase in platelet count, tumor growth and metastasis) reinforcing themselves through a feed-forward loop. This hypothesis has been confirmed by the study of Stone and co-workers [28], who analyzed 619 patients with epithelial ovarian cancer and of whom 30% had thrombocytosis at the time of initial diagnosis. The researchers found that TPO and IL-6 levels were high in patients who had thrombocytosis, as compared with those who did not, and that an increase in IL-6 levels positively correlated with plasma TPO levels and thrombocytosis, while negatively correlating with patient survival. Further proofs of the crucial role of IL-6 in paraneoplastic thrombocytosis have also been provided by molecular/genetic and pharmacological experiments: Silencing of *Il-6* and *tpo* genes fully abrogated thrombocytosis in murine ovarian cancer, and siltuximab (humanized anti-IL-6 antibody) significantly reduced tumor growth and platelet count, both in murine and human ovarian cancers [28].

Other circulating factors released by cancer cells and known to stimulate thrombopoiesis and megakaryopoiesis are granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), whose blood levels are increased in cancer patients with thrombocytosis [63].

A more in-depth analysis of basal cytokine profile in 81 newly diagnosed IBC patients revealed that patients with thrombocytosis, although not differing in IL-6 levels with respect to IBC subjects without thrombocytosis, showed a positive correlation between serum levels of Growth-Regulated Oncogene (GRO) and Transforming Growth Factor (TGF)-β and IBC-related thrombocytosis [37]. In this context, it should be underlined that both cancer cells and activated platelets are able to release GRO and TGF-β [64–66], thus suggesting that the observed increase in their content might be a consequence rather than a cause of thrombocytosis. In addition, the study has several limitations, above all the lowering of the thrombocytosis threshold from 450 to 300 <sup>×</sup> 109/L. Therefore, more studies are needed to establish a real relationship between these two cytokines and platelets in the context of tumor biology.

While thrombocytosis is more frequently reported to be associated with increased mortality, some findings also suggest the presence of cancer-related thrombocytopenia. For example, a strong trend toward increased mortality has been found in thrombocytopenic patients (hazard ratio (HR) = 1.50, but without reaching statistical significance) [43], although it is conceivable that thrombocytopenia

might be a surrogate for general debility and/or other clinical factors, such as possible sepsis and hematological abnormalities that could contribute to overall mortality.

Thrombocytopenia is a frequent complication in solid tumors [67]. The degree and incidence of this disease depends on the type of malignancy, tumor stage and treatment approach [68]. It has also been described as a complement of local cancer recurrence and may be considered a paraneoplastic syndrome [69] Some tumors can alter the platelet count below 100 <sup>×</sup> <sup>10</sup>9/L, leading to thrombocytopenia, and therefore cancer patients have a high risk of hemorrhagic complications [68]. The first evidence of low platelet count and bleeding episodes in patients with malignancies came from Gaydos in 1962: He demonstrated that bleeding episodes in patients with leukemia were frequently associated with a decreased platelet count [70]. Since then, other studies reported similar bleeding events in solid tumor patients [71,72].

Single nucleotide polymorphisms (SNPs) and mutations in genes encoding for cytokines and transcription factors are both two major causes of thrombocytopenia in solid tumors, including lung, breast, ovary and colorectal cancers [72]. Just as an example, the -31 T > C SNP of the *il-1*β gene was up-regulated in solid tumors associated with thrombocytopenia [73,74]. It is unclear how IL-1β can induce thrombocytopenia in solid tumors, but it is known that -31 T > C SNP can increase susceptibility to thrombocytopenia in these malignancies [72]. A strong association between IL-1β -31 T > C SNP and *Helicobacter pylori* infection has also been reported: The two phenomena collaborate themselves to increase the risk of gastric cancer with hemorrhagic complications [75,76]. IL-6 is also involved in thrombocytopenic mechanisms, as well as in paraneoplastic thrombocytosis. The IL-6 -174 G/C SNP has been reported in many malignancies, including adenocarcinoma, lung, colorectal, gastric and ovarian cancers [73,77,78]. This polymorphism also has been correlated with a poor prognosis, because it can induce antibody production against platelets, increasing the risk of thrombocytopenia [72].

As mentioned before, several transcription factors can be associated with thrombocytopenia, due to their involvement in platelet production. For example, overexpression of GATA3, a member of the GATA family of transcription factors that control maturation of hematopoietic stem cells, can stimulate platelet clearance, worsening the prognosis in breast cancer [79]. Another example is represented by Homeobox (HOA) genes that control proliferation and maturation of hematopoietic stem cells: Hypermethylation of *hoxa11*, for example, increased the incidence of thrombocytopenia and risk of poor prognosis in lung, gastric and breast cancers [80,81]. Although few studies have evaluated genetic changes in the incidence of thrombocytopenia, it seems that investigations on the mechanisms accounting for this phenomenon may be useful for the prevention of bleeding in solid tumors and for choosing the appropriate treatment [72].

Clinical observations of the impact of platelet count on cancer biology have been supported by studies employing platelet-depleted or transgenic mice [82]. A significant reduction in neovascularization has been observed in different transgenic mice-rendered thrombocytopenia [83], with GPIbα/IL4R transgenic mice (lacking the receptor for the von Willebrand factor as well as other adhesive and pro-coagulant proteins) showing the most severe phenotype [83,84]. Furthermore, in a mammary carcinoma murine model, platelet depletion increased the efficacy of the chemotherapy, by favoring drug delivery and tumoricidal action [85]. Thus, currently available animal models of platelet dysfunction may provide a framework for better understanding the molecular mechanisms through which thrombocytosis or thrombocytopenia impact cancer progression.

#### **3. Platelet-Derived Bioactive Compounds**

It is now recognized that tumor cells and platelets strictly influence each other, thus establishing the so-called "platelet-tumor loop". The need for recruiting platelets is supported by several evidences: (i) tumor cells sequester platelets in order to escape themselves from immune system surveillance [86,87]; (ii) platelets associate with cancer emboli, thus prolonging survival of circulating tumor cells and promoting their arrest and adhesion to endothelium for transmigrating to metastatic sites [86,88]; and (iii) platelets secrete a plethora of tumor, angiogenic, growth and permeability factors, which can

regulate tumor growth, epithelial to mesenchymal transition and metastasis (see below). Noteworthy, circulating hyperactivated platelets, as well as exhausted platelets (i.e., with totally or partially depleted granules, as a consequence of previous activation), are commonly found in subjects with different tumor types, concomitant with the high incidence of VTE [89].

In order to grow and develop metastasis, tumor cells concurrently influence platelet behavior by up-regulating synthesis and/or release of several compounds able to promote platelet activation and aggregation [90–92]. For example, when compared to benign tumors, malignant cells show higher generation of thrombin, one of the most potent platelet activators with strong pro-coagulating properties [93], but also able (when bound to thrombomodulin expressed on endothelium) to attenuate the thrombotic cascade [94,95]. Accordingly, blood thrombin concentrations negatively predict both success to surgery/chemotherapy and survival of patients with gynecological tumors [96]. A cohort study enrolling 112 patients with different cancers revealed that, although without association with disease state, rise in thrombin levels were dependent on tumor site, with lung cancers having more significant increases compared to brain and pancreas cancers [97]. Detrimental effects of increased thrombin generation seem to rely on its ability in promoting, in concert with its targets (among them, protease-activated receptor-1 and -4), cancer adhesion to platelets or endothelium via up-regulation of pro-inflammatory cytokines, adhesion molecules, angiogenic factors, and matrix-degrading proteases, thereby dramatically increasing tumor growth, angiogenesis, invasion, and metastasis [98]. Proofs of the "tumor cell-induced platelet aggregation" also come from quantitative analysis of circulating neutrophil elastase (which proteolytically activates integrin αIIbβ3) and serglycin (a pro-apoptotic and half-life cytokine regulating protein) in melanoma patients; while levels of the former were found up-regulated in cancer subjects, content of the latter was low [99], thus indicating the role of activated platelets in promoting cancer progression.

Although data on the molecular mechanisms underlying platelet hyperactivation are still not well-defined and often contradictory, nonetheless they suggest the involvement of some platelet bioactive molecules. Among them, those contained in α-granules play a pivotal role during platelet-cancer crosstalk (Table 2).


Ang-1: angiopoietin-1; EGF: endothelial growth factor; MK: megakaryocytes; NK: natural kill cells; PAI-1: plasminogen activator inhibitor-1; PDGF-BB: Platelet-derived growth factor BB; TF: tumor necrosis factor; TSP-1: thrombospondin-1; VEGF Vascular endothelial growth factor.

Besides P-selectin and other clotting proteins (such as thrombospondin, fibrinogen, fibronectin integrin αIIβ3, integrin αVβ3, factor V, and the von Willebrand factor), α-granules contain growth and pro-angiogenic factors (including Platelet-Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), TGF-β, Epidermal Growth Factor (EGF) and Angiopoetin-1 (Ang-1)), which are secreted following platelet activation and, either directly or indirectly, promote tumorigenesis. For example, besides directly promoting cancer progression and tumor cell extravasation (by facilitating interaction of cancer cells with platelets and endothelium) [109,110], P-selectin may indirectly exacerbate cancer evolution by triggering thrombin generation [111] and rapid monocyte exposure of tissue factor (TF) [100]. The latter protein, primarily involved in activation of the clotting cascade, positively affects tumor growth and metastasis: TF (produced by cancer cells) present in the tumor microenvironment may increase cell survival and/or angiogenesis, while TF present in the bloodstream (deriving from both monocytes and circulating cancer cells) has been shown to enhance thrombosis, tumor growth and metastasis. Several studies showed that P-selectin (either soluble or membrane-bound) changes in cancer patients, although controversial data have been documented. Some clinical studies found high P-selectin levels in cancer patients, with or without correlation to tumor clinical advancement [97,112]; conversely, recent observational and longitudinal studies enrolling patients with heterogeneous cancers found a decrease in platelet surface expression of this protein, which, together with diminished integrin αIIbβ3 exposure, thrombin and collagen receptor responsiveness and monocyte–platelet aggregate formation, correlates with risk of mortality and VTE [113,114]. Nevertheless, a lack of data on platelet activation changes in relation to cancer evolution makes it difficult to clarify whether decreased platelet reactivity is a consequence of continuous pre-activation in patients with poor prognosis or, rather, it represents a cancer-independent event, or even it is the result of a lack of protective effects exerted by activated platelets.

PDGF also has been implicated in tumorigenesis: It acts on tumor cells, thereby favoring proliferation, survival and invasion [104,105], and, in the meanwhile, creates a favorable microenvironment for tumor cells by inducing changes into tumor stroma and promoting blood vessel maturation (especially in advanced stages of angiogenesis) [115]. Accordingly, anti-PDGF drugs significantly inhibit tumor growth and metastasis, although downregulation of PDGF-BB (one of the five isoforms) signaling is associated with tumor cell dissemination and metastasis [106]. In the latter case, a protective role of PDGF-BB has been suggested, since its overexpression, by increasing tumor pericyte content, decreases colorectal and pancreatic cancer growth [116]. However, data on PDGF-BB content in cancer patients are controversial: Concentrations of secreted PDGF-BB, for example, have been found to be significantly high in the serum of colorectal carcinoma patients [117], but low in liver cancer patients with recurrence [118].

Among platelet-derived growth factors/cytokines, VEGF, EGF and Ang-1 play a crucial role in cancer angiogenesis [101–103]. Concerning the pro-angiogenic effect, the mutual influence of platelets and tumor cells also is not fully determined. A recent study performed on twenty-four women with active breast cancer and ten healthy controls showed that breast cancer and its chemotherapeutic treatment influence platelet phenotype, by increasing VEGF release and modulating the response to antiplatelet therapy [119]. Noticeably, an autocrine–paracrine loop, occurring in the bone marrow microenvironment and involving VEGFR-1-dependent megakaryocyte maturation has been documented [120]. Such evidence, together with the finding that tumor-derived IL-6 leads to enhanced megakaryocyte VEGF expression and a higher platelet VEGF load (concomitantly associated with fast tumor growth kinetics and poor diagnosis), strongly suggest a cooperation between platelets and cancer in promoting angiogenesis [121]. Conversely, low EGF levels have been found in cancer subjects with recurrence, and an inverse correlation between its concentrations and survival [118] has been documented in these subjects. Low serum Ang-1 levels, related to poor diagnosis, were also found in subjects with certain types of cancer [118]. On the contrary, patients with lung and ovarian cancer show high Ang-1 concentrations not related to patient survival [122].

In proteomic studies, α-granules also have been shown to contain a plethora of angiogenesis inhibitors, including endostatin, platelet factor-4, thrombospondin-1, α2-macroglobulin, plasminogen activator inhibitor-1 and angiostatin. Therefore, activated platelets can organize regulatory proteins in the α-granules in order to selectively address the release of pro- or anti-angiogenic factors, depending on which different sets of α-granules are segregated [107,108]. Although cancer cells might have the ability to provoke preferential release of pro-angiogenic mediators from platelet α-granules, in order to create a dynamic microenvironment favorable for their growth and survival [107], several experimental and clinical data point out that platelet secretion in the tumorigenic microenvironment might also be oriented towards an anti-angiogenic effect. Such a hypothesis is supported by several findings: (i) significantly higher endostatin levels have been found in hepatocellular carcinoma patients, as well as in gastric cancer subjects [123,124]; (ii) increases in circulating thrombospondin-1 have been found to positively correlate with survival of patients with gynecological and non-small cell lung cancer [125,126]; and (iii) higher levels of angiostatin have been found in serum of prostate cancer patients [127], as well as in urine of patients with epithelial ovarian cancer [128].

Platelets also secrete other factors, which can interfere with all steps of cancer development and metastasis. Serotonin, a monoamine synthesized by enterochromaffin cells in the intestinal mucosa, is largely (about 95%) sequestered by platelets in dense granules, from which it is released in response to various stimuli [12]. A large body of experimental data support both stimulatory and inhibitory properties of serotonin on tumor onset and progression [129–134]. Such different behavior is much likely due to the ability of serotonin to act in a concentration-dependent manner, as well as in its capability to activate distinct signaling pathways, depending on the receptor subtype present at various tumor stages. Coherently, high serotonin levels, correlating with the tumor stage, distant metastases and a poor prognosis, have been found in the serum of patients with different solid tumors [131,135–137], whereas low concentrations have been found in hepatocellular carcinoma patients, who showed recurrence after partial hepatectomy [118]; in untreated breast adenocarcinoma or malignant melanoma patients, where the phenotype (also associated with a high ATP/ADP ratio and index of delta storage pool deficiency) was more marked in regionally spread malignant tumors [138].

Platelets are also important producers of eicosanoids, lipids derived from polyunsaturated fatty acids (PUFAs), through the catalytic action of cyclooxygenase (COX) and lipoxygenase (LOX). Eicosanoids are crucially involved in several pathophysiological conditions [139], and the intersection between changes in certain platelet-derived eicosanoids and cancer appears particularly intriguing. For example, a multi-omics analysis of serum from metastatic melanoma patients revealed a rise in the concentration of 12-hydroxyeicosatraenoic acid (HETE) and 15-HETE eicosanoids, which are respectively produced by platelet 12-LOX and COX-1 [99]. These two platelet-derived eicosanoids have also been shown to exert pro-malignant effects in several cancer types, by activating mitogen and angiogenic pathways [139–142]. Coherently, blockage of COX-1 by aspirin causes the loss of platelet ability to transform human colon carcinoma cells into mesenchymal-like cells [139,143] and the long-term use of low-dose aspirin is associated with a reduction in risk of various cancers [144].

Finally, activated platelets release lysophosphatidic acid (LPA), a bioactive lipid growth factor, which has been shown to promote cell proliferation, survival, migration, tumor cell invasion and reversal of differentiation, through multiple G protein-coupled receptor (LPA1-6) cascades [145]. Several studies have found a relationship between plasma LPA levels and ovarian carcinoma; for example, a meta-analysis, comparing LPA levels in the serum of 980 ovarian cancer patients, 872 benign controls and 668 healthy controls, showed higher LPA plasma levels in the cancer group with respect to the benign and healthy control samples [146], thus suggesting that the raised detection of plasma LPA might be a potential diagnostic biomarker. Nonetheless, this finding is not supported by a recent lipidomic study that did not find any change in the content of this lipid in serum of ovarian cancer patients [147]. Discrepancies in the results can be explained taking into the account that, due to its susceptibility to sample processing procedure (e.g., plasma storage time at room temperature and anticoagulant used for blood drawing), LPA can artificially increase. In the light of this finding, therefore, it appears important to consider these confounding factors, in order to reduce to the minimum potential errors in measuring plasma LPA.

#### **4. Platelet Microvesicle-Derived miRNAs**

Upon activation, platelets release microvesicles (MVs), which are vesicular fragments with a diameter ranging from 0.5 to 1 μm, that express parental antigens (such as P-selectin and integrin αIIbβ3) and contain a plethora of mediators (growth factors, cytokines, inflammatory molecules, mRNAs and miRNAs) able to exert biological effects. These platelet MVs, accounting for 70–90% of all MVs circulating in the bloodstream, contribute to regulation of the tumor microenvironment and cancer-cell interactions [148–150]. Accordingly, cancer patients usually show increased levels of circulating MVs (in a way, that is different depending on tumor type, but it is directly proportional to tumor stage), which may be prognostic for monitoring tumor progression and response to specific therapeutics [151].

Although the role of MVs in cancer progression is multi-faceted and not fully understood, nonetheless it is becoming clear that MVs represent one of crucial determinants in tumor biology. Firstly, surface expression of platelet antigens leads to shedding of MVs displaying pro-coagulant and pro-thrombotic features. Therefore, MVs, together with activated platelets, enhance coagulation (which is further exacerbated by cancer-triggered activation of more platelets), thus playing an additional role in cancer progression [152]. Secondly, MVs are able to enhance angiogenesis: They stimulate the expression of pro-angiogenic molecules [including matrix metalloproteinase (MMP)-9, VEGF, IL-8 and hepatocyte growth factor (HGF)] in tumor cells [153] and drive capillary tube formation by stimulating endothelial cells [27,82,154].

Noteworthy, a bidirectional effect occurs as cancer cells can induce platelet activation and MV release; subsequently, a paracrine positive feedback mechanism is established, since MVs, taken up by cancer cells, potentiate the invasive phenotype through stimulation of migration [148,155]. Interestingly, although different cancer cells are able to induce platelet-derived MV release, only the most aggressive ones are responsive to MV action and, furthermore, only some subsets of MVs can positively feed back to cancer cells [155]. These findings suggest that i) cancer/platelet interplay is complex and strongly dependent on features of tumor cell type and ii) composition of MVs may differ depending on the stimulus given to platelets [155]. These findings may clarify discrepancies observed by authors in studies using different platelet preparations, agonists and cancer cell types. Just as an example, our recent work [156] showed that MVs, once internalized by cancer cells, inhibit migration rather than enhancing invasive properties; this may be explained considering that MV release was induced not by cancer cells but, instead, by a different stimulus (namely, arachidonic acid) that led to MV enrichment of specific bioactive molecules. Therefore, depending on MV composition, the effects on tumors may be completely different. A further point of discrimination may reside in MV internalization, since we found that, albeit taken up, the bioactive molecules delivered to cancer cells had different stability and, thus, exert their action in a time-and concentration-dependent fashion [156].

Among the bioactive molecules contained inside MVs, microRNAs (miRNAs) deserve a particular mention. Human platelets, indeed, contain an abundant repertoire of miRNAs that are released through MVs; depending on the nature of the agonists or stimuli activating platelets, the miRNA content of MVs can vary, but it always mirrors the content found in the platelets from which MVs derive [157]. It is exactly this heterogeneity of composition that may account for the observed differences in terms of molecular targets, mechanisms of action and effects on cancer cells (Table 3) [152]. If, on one side, cancer-promoting effects of MVs have been described (especially related to their content in growth factors, inflammatory cytokines and angiogenic factors), it is also true, on the other hand, that the ability to deliver miRNAs to recipient cells (including cancer cells) suggests a potential tumor-suppressive role.



ND: not determined. ADAM9: ADAM Metallopeptidase Domain 9; ATG4A: autophagy-associated gene 4A; CM: conditioned medium; EPB41L3: Erythrocyte Membrane Protein Band 4.1 Like 3; HAX-1: HS-1-associated protein X-1; β; M-CSF: macrophage colony-stimulating factor; MDR1: multidrug resistance gene; MEF: mouse embryonic fibroblasts; mt-Nd2: Mitochondrial NADH dehydrogenase 2; MV: microvesicle; NF-κB: nuclear factor-κB; Snora75: Small Nucleolar RNA, H/ACA Box 75; STAT5A: signal transducer and activator of transcription 5A; TRAIL: TNF-related apoptosis-inducing ligand; VEGF: vascular endothelial growth factor.

Platelet MVs can, indeed, be viewed as intercellular carriers that transfer inside cells specific molecules able to negatively modulate gene expression, with both positive and negative consequences. In this context, it should be underlined that most of the studies reporting differential effects of specific miRNAs in cancer employed transfection experiments, where miRNA expression was artificially increased and, to the best of our knowledge, only few of them checked the effects in more physiological (delivery of platelet-derived MVs) conditions.

Transfer of miRNAs to target cells has been shown to promote tumor progression [157]: miR-939 delivered by platelet MVs induces, in ovarian cancer cells, epithelial to mesenchymal transition, by down-regulating E-cadherin and up-regulating vimentin expression [163], while miR-223 has been shown to stimulate lung cancer cell invasion, by targeting tumor suppressor EPB41L3 [158]. Besides targeting tumor suppressor genes and oncogenes, several miRNAs enriched in MVs (miR-223, miR-24, miR-27a, miR-155, miR-195, let-7a/b) may also be implicated in therapy resistance. In small-cell lung cancer, miR-24-3p contributes to resistance to combination therapy (etoposide plus cisplatin), by targeting the autophagy-associated gene 4A [164]; other miRNAs that may be involved in drug resistance include miR-130a (which targets the pro-metastatic macrophage colony-stimulating factor (M-CSF)), and miR-27a and miR-451 (which target the multi drug resistance transporter 1) [166,167,172,173].

MVs also deliver angiogenic signals [27]: Transfer of miRNA let-7a or miR-27b in endothelial cells down-regulates the expression of the anti-angiogenic modulator thrombospondin-1, thus enhancing platelet-dependent endothelial tube formation [168,169].

However, what the available literature data suggest is that platelet-derived MVs may support cancer progression and metastatic dissemination at late stages, while it seems likely that they exert tumor suppressive roles at earlier stages. Michael's group found that circulating MVs directly infiltrating lung and colon cancer cells deliver miR-24 that suppress tumor growth; this miRNA localizes to mitochondria where it inhibits mt-Nd2 and Snora75, resulting in mitochondrial dysfunction and induction of apoptotic cell death [165]. Similarly, miR-223 inhibits migration, stimulates anoikis cell death and enhances chemo-sensitivity in different cancer cell types [159–161]. We and others have demonstrated that also platelet-specific miR-126 exert tumor suppressive roles; this miRNA may be a predictor for tumor relapse in postmenopausal breast cancer patients treated with tamoxifen [174], impairs cancer progression through direct repression of MMP-9 [170], and MV-mediated delivery into breast cancer cells induces cell cycle arrest, inhibition of migration and sensitivity to cisplatin [156]. Finally, miR-126 and miR-223 exert antagonistic effects on angiogenesis: miR-126 stimulates VEGF-induced proliferation in endothelial cells [171], while miR-223 exerts an inhibitory effect on formation of new blood vessels, by targeting endothelial β1 integrin [162].

The ability of MVs to acquire distinct roles, depending on their repertoire of proteins and miRNAs, suggest that they may be used as biomarkers with diagnostic and therapeutic implications [175]. For example, plasma levels of platelet MVs, together with VEGF, IL-6 and RANTES, have been found to be increased in patients with stage IV gastric cancer [176]. Elevated amounts of endothelial and platelet MVs (that significantly decreased after chemotherapy) have been found in the plasma of non-small cell lung cancer patients, thus suggesting a predictive role for prognostic clinical outcome [177].

#### **5. Conclusions**

Although some symptoms of cancer, such as breast lumps, are classic "alarm" symptoms, others are ambiguous and more likely caused by other conditions. Accordingly, different studies demonstrated that alterations in platelet number or/and activity often occur in cancer patients. This finding, together with the evidence that platelets may basically affect all steps of tumor development, prompts researchers to carry out more studies for fully understanding the mechanisms underlying cancer-related platelet dysfunction.

Environmental cancer-related stimuli encountered by platelets are intricate, as are the intracellular signaling pathways regulating platelet responses to the stimuli themselves. Moreover, due to

heterogeneity in the cargo of growth factors, cytokines, microRNAs and other bioactive molecules and platelets may potentially release either stimulators or inhibitors in all cancer steps (Figure 1).

**Figure 1.** Schematic representation of the main platelet effects on tumor biology. See text for details. In black: platelet-derived bioactive molecules with positive effects. In blue: platelet-derived compounds with negative effects. In red: platelet-derived compounds with both positive and negative effects. Lines with dot indicate either stimulation or inhibition, depending on the platelet-derived bioactive molecule. 12/15 HETEs: 12 and 15 hydroxyeicosatraenoic acid; Ang-1: Angiopoietin; LPA: lysophosphatidic acid; EGF: Endothelial Growth Factor; P-sel: P-selectin; PDGF: Platelet-Derived Growth Factor; TGF-β: tumor growth factor-β; VEGF: vascular endothelial growth factor.

Accordingly, on one hand, there are a great deal of proofs of deadly interaction between platelets and cancer cells, but, on the other hand, some experimental and clinical data also indicate a protective role. Besides to complexity of platelet signaling in cancer, the scenario is further complicated by other confounding factors extrinsically related to platelets: (i) most of results come from retrospective studies, analyzing a wide range of patients with heterogeneous characteristics, such as age, sex, race, cancer type and stage, as well as treatments not always related to cancer; (ii) some studies have been carried out in cancer patients post-diagnosis and changes in platelets have not been monitored with respect to cancer progression over time, so that it is difficult to establish the exact contribution of platelets and if their changes are the cause or rather a consequence of tumor; and (iii) some of the bioactive compounds, whose plasma concentrations have been shown to be correlated with cancer progression, are also released by other cells, including cancer cells themselves; therefore, the effect on cancer, whether positive or negative, is not necessarily due to platelets.

Other questions still need to be answered: Why do platelet changes occur in some types of cancer and not in others? How does the platelet–cancer relationship change with age, sex and cancer progression over time? Although the heterogeneity and adaptive potential of the tumor features make a "one-size-fits-all" approach for targeting platelet–cancer interactions difficult, a better understanding of the interplay might provide efficient tools for cancer prevention, screening, risk assessment and management.

**Author Contributions:** M.V.C. and V.G. conceptualized the work and did the original draft preparation; V.T. provided the resources for this work and contributed to draft preparation; I.S. reviewed the text; V.G. drew the table and figures. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Platelet Concentrates in Musculoskeletal Medicine**

**Erminia Mariani 1,2,\* and Lia Pulsatelli <sup>1</sup>**


Received: 16 December 2019; Accepted: 6 February 2020; Published: 16 February 2020

**Abstract:** Platelet concentrates (PCs), mostly represented by platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are autologous biological blood-derived products that may combine plasma/platelet-derived bioactive components, together with fibrin-forming protein able to create a natural three-dimensional scaffold. These types of products are safely used in clinical applications due to the autologous-derived source and the minimally invasive application procedure. In this narrative review, we focus on three main topics concerning the use of platelet concentrate for treating musculoskeletal conditions: (a) the different procedures to prepare PCs, (b) the composition of PCs that is related to the type of methodological procedure adopted and (c) the clinical application in musculoskeletal medicine, efficacy and main limits of the different studies.

**Keywords:** platelet-rich plasma; platelet-rich fibrin; preparation; composition; musculoskeletal diseases

#### **1. Introduction**

In the last 10 years, autologous biological blood-derived products have been largely investigated as useful therapeutic tools for treating musculoskeletal conditions (such as osteoarthritis, muscle injuries, tendinopathies and intervertebral disc degeneration) [1–3]. Platelet concentrates (PCs), mostly represented by platelet-rich plasma (PRP) and platelet-rich fibrin (PRF), are included in this type of biology-oriented autologous therapeutic strategy that may combine plasma/platelet-derived bioactive components (cytokines, chemokines, growth-factors and enzymes) with fibrin-forming protein able to create a natural three-dimensional scaffold [4].

This approach allows us to deliver biomolecules released by a concentrated pool of activated platelets to the target tissue site of injury, thus effectively contributing to the modulation of inflammatory process, angiogenesis and immune response, as well as promoting the healing and repair of injured tissues [5,6]. Moreover, biological blood-derived products have been recognized to have antimicrobial effects, such as being able to inhibit and/or to inactivate different bacterial strains [6–8].

The potential clinical application of these biologic products in musculoskeletal medicine relies on their capability of modulating the joint environment and their beneficial role in reducing the local inflammation and promoting cartilage and synovium anabolism [5,9–12].

These types of therapeutic strategies provide advantages in clinical applications due to the autologous-derived source, safety profile, easiness to obtain and the minimally invasive application procedure. On the other hand, clinical efficacy is still controversial, and solid evidence and consensus supporting the therapeutic application are still to be achieved.

Indeed, there are already some issues to be addressed concerning the high variability of platelet concentrate products, which mainly depends on patients' characteristics (age, sex, circadian rhythms and drug regimen) [13–16], as well as on the lack of standardized methods for platelet isolation/collection/activation and on heterogeneity among therapeutic protocols applied in clinical practice.

In this narrative review, we focus on three main challenging topics concerning the use of platelet concentrate for treating musculoskeletal conditions: (a) the different procedures to prepare platelet concentrate, (b) the composition of these products that is mainly related to the type of methodological procedure adopted and (c) the clinical application in musculoskeletal conditions and level of efficacy.

#### *Short History of Platelet Concentrates*

The concept of PRP originally was developed in transfusion medicine. In this field, the PRP term was used in 1954 by Kingsley [17] to identify thrombocyte concentrate for treating patients with severe thrombopenia.

The history of the techniques to obtain blood-derived products for improving tissue healing started in 1970 with the studies of Matras [18] on fibrin glue use in a rat model.

Subsequently, an autologous product termed "platelet–fibrinogen–thrombin mixture" was developed, including, in fibrin glue, a significant concentration of platelets, in order to reinforce the fibrin polymerization [19].

In the following years, the role of platelets in supporting tissue healing was confirmed and clinically demonstrated by using a blood-derived product called "platelet-derived wound healing factors or formula-PDWHF" [20] for treating skin ulcers.

About ten years later, Whitman et al. [21] published a clinical study on the results obtained in oral and maxillofacial surgery by using a "platelet gel" obtained by a gradient density cell separator.

However, the term of PRP in regenerative medicine associated to the notion of platelet growth factors to promote tissue healing was truly introduced by Marx et al. in 1998 [22], in a study that reported the effect of platelet-rich product on bone healing in maxillofacial surgery.

After these publications, the term "PRP" was generically associated with all the multiple formulations of platelet concentrates. Afterward, an end-product characterized by a fibrin matrix denser and more stable than in other PRP formulations was produced and called platelet-rich fibrin matrix (PRFM) or pure platelet-rich fibrin (P-PRF).

In 2001, a different form of platelet concentrates was proposed and identified as leukocyte- and platelet-rich fibrin (L-PRF) [23]. These preparations are organized as a high-density fibrin and were considered as a "second generation" platelet concentrates. This family of platelet concentrates appears to be particularly suitable for oral clinical application.

#### **2. Preparation Procedure**

#### *2.1. Platelet-Rich Plasma*

PRP is obtained from autologous blood by using commercial kits or "in-house techniques", aiming to provide a product characterized by a supra-physiological platelet concentration that can be used as liquid or activated gel form [14,24–26].

Despite the broad spectrum of protocols for PRP preparation, a common sequence of key steps [27,28] can be identified involving peripheral blood drawing from the patients by venipuncture, blood centrifugation to retrieve platelet-enriched fraction and platelet stimulation to release bioactive molecules.

In each of these phases, potential sources of variability may be identified, mainly ascribed to volume of blood samples drawn, type of anticoagulant, centrifugation protocols, material of collection tubes and type of platelet-activating agents [14,24].

The great variability in the different procedures results in a wide heterogeneity among PRP preparations in terms of platelet concentration, presence/absence of leukocytes and erythrocytes, and ultimately in terms of biological potential [14,24].

#### 2.1.1. Anticoagulants

There are multiple choices of anticoagulants (ethylene diamine tetra-acetic acid-EDTA, citrate dextrose-A, tri-sodium citrate and heparin) that are used for blood collection and that can differently affect PRP quality [29].

Lei and colleagues [30] investigated the effect of heparin, citrate, acid citrate dextrose (ACD) and citrate-theophylline-adenosine-dipyridamole (CTAD) on platelet-rich plasma quality, to determine the appropriate anticoagulants for PRP production.

ACD and CTAD appear to be more effective compared to heparin and citrate in maintaining the integrity of platelet structures and in preventing their spontaneous activation. ACD-PRP and CTAD-PRP released more TGF-beta1 and significantly increased the proliferation rate of human marrow stromal cells compared to heparin- and citrate-PRP, thus showing ACD and CTAD appropriate anticoagulants for PRP production [30].

An animal model study, aiming to investigate the influence of sodium citrate and ACD- solution A anticoagulants on cell count and growth factor concentration in pure platelet-rich gel supernatants, reported an increased number of platelets and leukocytes in sodium citrate PRP compared to homologous acid–citrate–dextrose solution A PRP fraction, but no difference concerning growth factor concentration [31].

Another "in vitro" study explored the effects of sodium citrate (SC), EDTA, or anticoagulant ACD- solution A, on PRP characteristics and on mesenchymal stromal cell (MSC) culture [29]. A higher platelet count was observed in blood collected with EDTA, even if an increase of mean platelet volume has been reported after the two centrifugation steps. Conversely, following the centrifugation procedure, platelet yield was higher in SC product. SC and ACD showed similar efficacy in inducing MSC proliferation [29].

These findings support the most frequent use of citrate-based anticoagulants for PRP preparations [29].

A very recent comparative study [32] evaluated the effects of EDTA, heparin sodium (HS) and SC on PRP quality and on bone marrow stem cells' functionality.

Compared to HS and SC, EDTA has been shown to preserve platelet structure, minimize their spontaneous activation and sustain growth factor release for a more extended time.

Overall, these findings underline that also the choice of the best anticoagulant represents an open issue to address for optimizing PRP formulation.

To overcome this criticism, a study published in 2018 described a novel approach of PRP preparation without any additive, named temperature controlled PRP (t-PRP), by which the coagulation was previously inhibited in hypothermic environment. In this study, t-PRP was compared to PRP obtained by ACD-A blood.

Overall, t-PRP showed a more physiologic pH, higher platelet yield, slower release and degradation of growth factors. Furthermore, animal model experiments demonstrated that t-PRP was able to promote wound healing [33].

#### 2.1.2. Isolation Protocols

PRP can be obtained according to two basic protocols designed as plasma-based and buffy-coat-based procedures [14,34]. Plasma-based methods retrieve platelets, while minimizing leukocyte and erythrocyte fractions. For this purpose, a slower and shorter spin regimen is applied in plasma-based protocols. Platelets concentration is usually twofold to threefold increased above baseline whole blood levels (300,000 to 500,000 platelets/μL) [14,34].

Alternatively, the main goal of protocols for buffy-coat systems is to maximize platelet isolation during the centrifugation procedure, by high spin rates and long spin regimens. PRP obtained by this method is characterized by a high platelet recovery, increasing about threefold to eightfold compared to baseline levels (500,000 to 1,500,000 platelets/μL) and by the presence of variable concentrations of leukocytes and erythrocytes [14,34]. This type of PRP preparation is generally called leucocyte-rich PRP (L-PRP).

Specific protocols developed to obtain PRP by using either a commercial device/kit or manual/homemade procedures derive from multiple modifications of these two basic protocols (plasma-based and buffy-coat-based).

Most of commercially available systems produce PRP by buffy-coat-based method [35], and several comparative studies were reported, aiming to analyze different common commercial separation systems, essentially evaluating final PRP products in terms of platelets concentrations and growth factors release [24,35] (Table 1).


 **1.** Centrifugation protocol and composition of platelet-rich plasma (PRP) produced by common commercial PRP

**Table**

As expected, overall findings underlined that commercially buffy-coat-based systems (such as SmartPrep, GPS III and Magellan systems) yield higher concentrations of platelets and leukocytes compared to plasma-based systems (such as ACP and Cascade). Among buffy-coat systems, generally, GPS III preparations demonstrated the highest concentration of platelets and leukocytes [35].

Wide variations of centrifugal force and total centrifugation time among the different common commercial systems were described, respectively, ranging from about 350 to 2000 g, and from 5 to 20 min [35]. The majority of the systems use a dual-spin method; the first centrifugation usually has a lower speed compared to the second one [24,35].

Conflicting results were reported concerning optimal centrifugation rate to maximize platelet concentration, avoiding their activation or damage. Indeed, there is evidence underlining that increasing centrifugation force results in higher platelet concentration [37]. Conversely, other studies reported an inverse relationship between platelet yields and gravitational force [38,39]; furthermore, an elevated centrifugal speed could induce platelet activation [40].

Very recently, Croisè et al. [40] performed a literature review, aiming to check multiple studies focused on PRP protocol optimization. Fourteen included studies were commented upon, and each of them suggested different centrifugation procedures in terms of speed and duration time, number of centrifugations and, consequently, variable platelet concentration enrichments (from no enrichment to about 8.5 times more than peripheral blood). Overall these results underline that, to date, there is no consensus on the optimal centrifugation regimen to obtain a good-quality PRP, in terms of best platelet yields, avoiding structural and/or functional alterations and optimal relative concentration of blood components.

Recently, in order to obtain a standardized PRP formulation, Gato-Calvo et al. [41] developed a novel methodology, defining the optimal content of PRP, based on absolute platelet concentration. This approach allows us to obtain an end-product not influenced by the variability of the donor basal platelet counts, thus improving the reproducibility of PRP effects.

Another source of variability may derive from the material of blood-collection tubes. Some studies have demonstrated that PCs obtained by blood collected in glass or silica-coated tubes presented different buffy-coat morphology, fibrin architecture and platelet/leukocyte distribution in the PC matrix [42]. Furthermore, silica micro-particles may be released by tube walls during centrifugation procedures, entrapped in PC matrix, thus modifying platelet distribution in the end-product [43].

#### 2.1.3. Activation Process

Activation triggers two responses during PRP preparation: the release of the bioactive molecules stored in platelet alpha-granules, and the matrix formation by fibrinogen cleavage [44]. Clot formation entraps released growth factors (GFs), thus enabling bioactive molecules to be delivered and confined at the injured target site.

Activation process may be induced by endogenous and exogenous factors. Among exogenous factors, the most common activators are thrombin, calcium chloride and a mixture of calcium chloride plus thrombin [14,34,45]. Endogenous activation relies on the exposure of native collagen or other coagulation factor (such as adenosine diphosphate-ADP, thrombospondin and platelet-activator factor), spontaneously inducing clot formation at injured site [45].

In general, thrombin triggers a rapid platelet aggregation and stimulates a fast release of GFs [14,34,46]. Calcium chloride and collagen sustain a slower long-term release [34,46,47]. Furthermore, some findings reported that collagen activation results in a lower amount of released GFs compared to thrombin and calcium chloride [47].

A very recent study compared the effects of three different activation factors, thrombin, collagen I and ADP, on PRP quality and on bone marrow stem cells' (BMSCs) functionality. Collagen I-PRP has been shown to induce the most rapid increasing of BMSC number compared to the rate observed with ADP- or thrombin-activated PRP. In addition, BMSC seeded in Collagen-I-activated PRP induced a significantly higher gene expression of osteogenic differentiation markers, osteocalcin and RUNX2, compared to thrombin and ADP. Thrombin induced a rapid and direct GF release, while collagen-I-activated PRP showed a sustained and slow GF release. The lowest total release was observed for ADP-activated PRP [32].

The different kinetic release is a crucial issue that might influence the availability of bioactive molecules, so affecting treatment outcome. Indeed, given GFs' short half-life (from minutes to hours), if they are not promptly used upon platelet release, their degradation may occur before additional receptors, that are involved in the repair process, become available on cell surfaces [34].

Photo-activation has been suggested as an alternative method to trigger platelet activation: a very recent paper [48] described in vitro characterization of platelet photo-activation (polychromatic light source, in the range near-infrared region), in comparison with resting platelets and calcium chloride mediated PRP activation. That study showed that photo-activation of PRP induced a significantly more prolonged release and higher amount of platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF), and transforming growth factor (TGF)-beta than PRP activated with calcium chloride. Future clinical studies should be performed to verify the potential of using the photo-activation approach in PRP formulation.

#### *2.2. Platelet-Rich Fibrin*

This type of PC essentially includes two categories of different preparations organized as a high-density fibrin solid form: leukocyte-poor or pure platelet-rich fibrin (P-PRF) and leukocyte- and platelet-rich fibrin (L-PRF) [25,49]

Concerning P-PRF preparation, there is only one formulation, commercially known as Fibrinet (Platelet Rich Fibrin Matrix-PRFM, Cascade medical, Wayne, NJ, USA,) [25,49]. P-PRF is obtained by a double-centrifugation method analogous to other PRP protocol, but it differs since the clotting phase is a dynamic process occurring during the second centrifugation, after adding CaCl2 [25,49].

L-PRF is a leukocyte-rich product, and compared to PRP, L-PRF preparation is easier and lacks biochemical modifications (no exogenous activation or anticoagulant are required), and unlike PRP, PRF end-products are characteristically organized in tridimensional architecture [25,49].

L-PRF protocol was developed by Choukroun et al. [23] as an open-access technique, based on one-step centrifugation without anticoagulant and blood activators. L-PRF is considered to be a second- generation platelet concentrate [25,50]. Briefly, venous blood collected in glass tube without anticoagulants is centrifuged at low speed, and clot formation is immediately triggered. Three layers become evident after centrifugation: the red blood cells (RBCs) bottom layer, a PRF clot in the middle and the acellular plasma top layer [50].

This procedure allows to harvest almost all the platelets and more than 50% of the leukocytes from the peripheral blood [50]. L-PRF clot appears to be organized in a strong fibrin architecture and presents a specific tridimensional distribution of the platelets and leukocytes [50].

The original open-access experimental method has evolved into a regulated medical device system and is marketed with CE/FDA clearance (Intra-Lock, Boca-Raton, FL, USA). This system is the only certificated L-PRF system available on the market, and it uses the original protocol and devices [51]. This method shows a high efficiency in platelet and leukocyte collection and in leukocyte preservation [25].

Many variations of the original method were proposed, using different centrifuges and/or different protocols. These modifications result in modified-PRF product compared to the original L-PRF.

P-PRF procedure is more expensive and complex compared to L-PRF protocol. Furthermore, this latter procedure allows to simultaneously obtain a large number of end-products [25].

To the best of our knowledge only one paper [52] compared PRFM and PRF products, in terms of growth factor release. In this study, PRFM and PRF were obtained by "home-made" protocols and appeared to have a different kinetic release. PRFM presented an early robust boost of growth factors, while PRF release was more gradual and constant up to 23 days. On the contrary, Lucarelli et al. [53] has shown that Fibrinet PRFM releases elevated levels of growth factors (such as PDGF, TGFβ and

VEGF) in the first 24 h, whereas other growth factors, such as bone morphogenic protein (BMP)-2 and -7 were undetectable.

Conversely, L-PRF products sustained a large growth factor release for up to seven days [50]. Interestingly, BMP-2 was detected in L-PRF releasate strengthening the regenerative potential of this PC [51]. It is hypothesized that the presence of leukocytes may have a relevant impact on the amount and the pattern of the released growth factors, and a potential synergistic effect between leukocytes and platelets has been suggested [25,50,51].

Centrifuge characteristics and centrifugation protocols have been shown to impact fibrin architecture, cellular distribution and growth factor release. Therefore, various PRF preparations could be associated to different biological profile and clinical potential [51]. Up to now, the different PRF preparations are not clearly characterized, and further investigations on the effects of protocol modifications need to be provided.

#### **3. Classification Systems**

The heterogeneity of PC preparation methods can impact on the functional characteristics and on the potential therapeutic efficacy of the final products, giving each PC formulation unique properties. The majority of the studies do not provide a full characterization of the various PC composition, so a reliable comparison among studies still remains a challenging issue [54].

Several classification systems (Table 2) have been developed over the years in attempt to help comparison among studies and to foster standardization of PC preparation process. However currently, no consensus on classification systems has yet been achieved [54].




**Table 2.** *Cont.*

#### **4. Composition**

#### *4.1. Platelets*

The human blood platelet normal concentration ranges from 150,000 to 400,000/μL [61]. There is no consensus on the optimal concentration of platelets in PCs.

Platelet concentration was compared for its healing effect, and different optimal levels were identified for different applications [14,34].

PRP platelet concentration greatly differs in PRP obtained by the various commercial systems.

Plasma-based PRP systems usually contain a platelet concentration between baseline and 3x baseline (less or equal to 750 <sup>×</sup> 10<sup>3</sup> platelets/μL), and they are defined as low-yielding devices (such as ACP, Cascade, Endoret and RegenPrep) [35]. On the other hand, buffy-coat-based systems yield platelet concentration above 3x, ranging from 4x to 6x (greater than 750 <sup>×</sup> <sup>10</sup>3platelets/μL to 1800 <sup>×</sup> 103 platelets/μL). These systems are classified as high-yielded devices that produce PRP (GPS III, SmartPrep and Magellan) [35].

In vitro, in vivo and clinical studies have demonstrated successful results for PRP formulations with both a moderate (2× and 3×) and high platelet concentrations (from 4× to 6×) [14]. In particular, an in vitro study evidenced that the best angiogenic effect of PRP was obtained with 1500 <sup>×</sup> 103 platelets/μL, thus underlining the role of platelet concentrations on the clinical application when the increased angiogenesis contributes to the healing process [14,62].

Platelet concentration greater than 6x (><sup>1800</sup> <sup>×</sup> 103 platelets/μL) may be detrimental or have side effects [63]. In fact, an excessive platelet amount may lead to cellular apoptosis, downregulation and desensitization of growth factor receptors, resulting in a paradoxical inhibitory effect [34].

Another source of variation is the platelet-counting mode. Indeed, it has been reported that, to achieve accurate platelet count, proper sample preparation is required and manual mode in the hematology analyzer is recommended, because automatic mode, allowing the sample to settle, may underestimate the absolute platelet count [34,64].

#### *4.2. Leukocytes*

As previously stated, leukocyte content in PCs depends on PRP preparation procedures.

Plasma-based process reduced leukocyte count up to 22 times the baseline, almost eliminating this cellular fraction. Buffy-coat-based procedures actively concentrate leucocytes from threefold to fivefold the baseline [65]. Furthermore, different buffy-coat methods produce a PRP formulation with different proportions of neutrophils, lymphocytes and monocytes [65]. Indeed, it has recently been reported that different centrifugation regimens, in terms of spin numbers and speed, modified lymphocyte/granulocyte ratio in the final products [66].

The inclusion of leukocytes in PC preparations remains a widely debated concern, as both beneficial and detrimental effects have been suggested.

Deleterious effects are mainly ascribed to leukocyte capacity to release inflammatory cytokines and metallo-proteinases, which can promote pro-inflammatory and catabolic effects on targeted tissue [67–70]. Furthermore, the massive release of reactive oxygen species by neutrophils causes tissue damage, by inhibiting healing process [71,72].

On the other hand, potential beneficial effects rely on leukocyte's role in tissue healing, in regulating inflammatory process [73–75] and in antibacterial activity [76,77] that may switch the inflammatory process toward a regenerative phase.

These potential effects are suggested and corroborated by the following main evidence:


Furthermore, growing evidence on the relevance of leucocyte–platelet interaction and of their relative proportions in PRP preparation has been reported [4,44,50,66,87]. Indeed, leucocyte–platelet interaction may promote biosynthesis of other factors that facilitate the resolution of inflammation, such as lipoxins that are potent anti-inflammatory proteins able to limit neutrophil activation, so promoting the resolution phase of the healing process [44,88,89].

In addition, the interrelationship between platelets, blood cellular components and fibrin may have a key role in proper platelet function and growth factor release [4,50,87], and the relative platelet/leukocyte and lymphocyte/granulocyte ratios might drive the balance between catabolic and anabolic factors [66].

Therefore, future research efforts should not focalize on the concentrations of single PC component but on the optimal relative combination of platelets, leukocytes, growth factors and fibrin within the final preparation for the different clinical application fields.

#### *4.3. Red Blood Cells*

Red Blood Cells (RBCs) can be damaged as a result of high shear force during blood collection or during inadequate centrifugation process, so causing hemolysis with the release of hemoglobin and its degradation products, hemin and iron. The presence of these hemolytic-related products lead to several deleterious effects, such as radical oxygen reactions, endothelial disfunction, vascular endothelium damage, pro-inflammation response and tissue injury [90].

RBC damage also causes the release of migration inhibitory factor (MIF), which has been recognized as a very strong inflammatory cytokine [90]. MIF concentration in whole blood is 1000-fold increased than in plasma. Since leukocytes and platelets have been shown to minimally contribute to MIF concentration, RBCs represent the major reservoir of this factor [91], which is also functionally active [91].

MIF plays a pathophysiological role in promoting and maintaining OA pain [92]. Furthermore, MIF levels in plasma and synovial fluid have been found to be positively correlated to disease severity in knee OA [93]. Blood-induced joint damage has been highlighted by various in vitro studies. In fact, blood exposure results in increased synoviocyte cell death and pro-inflammatory mediator production [94], induction of chondrocyte apoptosis and cartilage degradation [95–97].

On the other hand, effects of free heme may be inhibited by its degradation or by specific binding proteins. The heme–heme oxygenase (HO) system is formed after HO-mediated heme degradation. Growing evidence support the protective HO system activity and its effector molecules against oxidative and inflammatory responses and cell damage and suggest that the heme-HO system may represents a novel and important target in the control of wound healing [98–100].

Even if RBC content is reduced or absent in PC preparations, the detrimental effect of RBCs should be addressed for optimization of PC performance.

#### *4.4. Growth Factors*

GFs and protein are stored in the platelet alpha-granules and are released by activation of the platelets. Over 300 proteins were identified in the platelet releasate [101].

Multiple pieces of evidence have suggested that platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF) and epidermal growth factor (EGF) are the most crucial factors implicated in tissue repair [102]. PDGF, TGF-beta and VEGF appear to be the most investigated, and the concentration of these GFs is often considered as a marker of PC preparation quality [24,35,102].

In PRP preparations, approximately 70% of platelet growth factors are secreted within the first 10 min following activation, and almost 95% within the first hour [103,104]. Platelets may continue to produce small amounts of growth factors during the residual life span (8–10 days) [103,104]. Conversely, PRF presents a more intense, slow and constant long-term release, up to 5–7 days [50,105].

Together with platelets, leukocytes also contribute to the release of some growth factors, as highlighted by several studies that reported a positive correlation between the amounts of released GFs and the number of leukocytes [35,78,79].

Multiple comparative studies have investigated GF released by PRP obtained by various commercial separation systems. A large heterogeneity in the GF concentrations and kinetic release have been shown when comparing multiple PRP preparations obtained by different commercial separation systems [106].

A recently published review underlined that growth-factor concentrations reported by the different studies appeared to be hardly comparable, due to wide variations of these results, not only among the different systems but also when comparing the same separation systems among the different studies [35]. This variability may be essentially ascribed to two criticisms: the different commercial kits used for growth-factor dosage [35], and the incomplete removal of platelets and erythrocytes that may impact the results [107]. Due to these limitations, a comparison between studies appears to be barely reliable, not allowing consistent evidence-based results concerning growth factor content profile of different PRP preparations.

Furthermore, the great inter-individual variability of GF concentration needs to be taken into consideration [107,108]. A study performed on a large number of OA patients (n = 105) showed a wide individual variation of PRP growth factors, with a coefficient of variation ranging from 5.30 to 78.45. In particular, basic FGF and TGF-beta1 showed, respectively, the highest and the lowest variation [109].

Concerning PRF, different GF releases by different formulations have been shown. Comparing original L-PRF to modified-PRF formulations, conflicting results were reported. Kobayashi et al. [105] demonstrated that significantly higher GF levels were released by advanced-PRF (A-PRF) compared to original L-PRF. On the other hand, Dohan Ehrenfest et al. [51] reported a much stronger release of GFs from original L-PRF than from A-PRF membrane.

Nowadays, the literature data highlights that biological profiles in terms of content, amount and release kinetics associated to different PCs need to be further investigated, in order to better understand GF potentiality of the various PCs in clinical applications.

#### **5. Clinical E**ffi**cacy**

#### *5.1. Osteoarthritis*

Osteoarthritis (OA) is a debilitating osteo-articular disease, triggered by a trauma to the joint, and it is associated with a progressive erosion of articular cartilage, subchondral bone sclerosis, excessive stiffness and pain.

Numerous clinical trials and case series, carried out using PRP administration in patients with OA, supported PRP for the symptomatic effect, reduction of pain, improvement in the degenerative injuries and safety of administration, but they have not reached an univocal consensus.

#### 5.1.1. Knee Osteoarthritis

Knee OA is a chronic disease of joints that is characterized by pain and progressive disabilities, usually developing as the sufferer ages [110]. The most common treatments, both non-pharmacological and pharmacological, show positive outcomes, but their effectiveness is not long-lasting. Thus, surgical knee replacement is often the last chance for the relief of symptoms [111,112]

One of the first PRP studies establishing the safety of intra-articular use of this autologous preparation dates back to 2008 [113] (Table 3).




**Table 3.** *Cont.*

Afterward, different studies demonstrated the positive effects of PRGF/PRP injection, either when used alone or when compared to hyaluronic acid (HA) one, in the knee OA patients [114,115,117,119–140]. These PCs were reported not only to have an effect on clinical symptoms (by decreasing pain and improving function), but also on synovial fluid and protein amounts, as well as on cartilaginous degeneration.

However, a recent study reporting results of a follow-up up to six years does not confirm superiority of PRP [142].

The superiority of PRP was also established by comparison with normal saline (physiological control), as indicated by early improving WOMAC (the Western Ontario and McMaster Universities Osteoarthritis) scores, and maintained up to six months [153,158,163], but slightly decreased afterward, in agreement with the anti-inflammatory action supposed for PRP [172].

Similarly, in a trial including 366 young patients (18–30 years old), positive outcomes were reported after intra-lesional PRP administration [162]. In general, better results were obtained in young patients, with low body mass index [117,122].

PRP was reported as better in terms of clinical improvement compared to oral NSAID administration [161], as synergistic and protective, when added to methylprednisolone [150] and comparable to HA and corticosteroids after three months, superior to both the other treatments in the long-term [149].

Both PRP and HA have a biological origin and may be critical for tissue healing at the beginning of OA development. In in vitro studies, the combination of PRP with HA may display synergistic effects on fibroblast migration [173,174], thus suggesting a better effect of PRP–HA combination than PRP alone [175].

In agreement, a recent randomized clinical trial in mild/moderate knee OA reported better outcomes of the patients treated with PRP–HA combination when compared to PRP (up to three months) or to HA (up to 12 months) groups [144].

Furthermore, the synergy between combined PRP and HA treatment was further investigated and compared with each of them alone and with a placebo, via intra-articular injections in a total of 360 patients with knee osteoarthritis [145], demonstrating significantly reduced pain and decreased immune response, as well as PRP treatment compared with low and high molecular weight HA [143].

Even if clinical studies on PRP–HA combined therapy are limited and there are several peculiar aspects of HA alone (such as molecular weight), of the PRP–HA mix (such as ideal combination and dosage schedule), the preliminary data are worth of being deepened.

The PRP administration schedule in OA knee, widely reported with different numbers of injections, different time intervals and duration, represents a further aspect to be defined.

Patel [153], first compared the effect of one with two PRP injections and showed similarly improved WOMAC scores. A following double-blind placebo-controlled randomized trial demonstrated that the patient group that had undergone three PRP injections presented a better score than groups treated with a single dose of PRP or HA [154].

A clinical efficacy of PRP was also described when PRP was alternatively used at annual intervals or at the request of the patient when the effect ended [123]. Moreover, the administration in two phases foreseeing six doses at weekly intervals, and then a three month suspension and a maintenance dose (three injections at three-month intervals), presented interesting functional improvements [152].

A single administration of very pure PRP offered a significant clinical benefit as one injection of HA [140], and a similar improvement was obtained by a single administration of about 9 mL of PRP [155].

An enlarged delivery approach was also described, firstly for the treatment of severe OA [169] and more recently for the treatment of mild to moderate forms [170,171]. In these studies, the intra-articular injection of PRP was associated with concomitant intraosseous PRP injections into the subchondral bone, obtaining significant results.

A significant improvement of pain and functional scores, as well as decreases of the inflammatory response, were also obtained by the concomitant injection of PRP both intra-articular and in peri-meniscal soft tissue structures, thus widening the PRP effect on pes anserine tendons, bursa, medial collateral ligament and medial meniscus [176].

A systematic review on PRGF [177] reported the efficacy of PRGF in pain improvement, but also pointed out the limits of the included studies that prevented to perform a meta-analysis. The heterogeneity of the primary outcomes, PRGF and HA administration schedules, HA molecular weight, the small number of studies fulfilling the eligibility criteria and the lack of placebo treated group were the main drawbacks.

PRP was described as effective, alternative and superior to HA treatment for long-term improvement of joint function and pain in patient with knee osteoarthritis, mainly in early-moderate disease compared to advanced disease. The limits reported in a narrative review [178], in a recent meta-analyses [179,180] and in a systematic review [181] evidenced the variability of OA severity (K-L I-IV), as well as age, sex and BMI in patients treated in the different studies. In addition, main criticisms concerned the number of injections, optimal dosage of PRP, administration schedule, heterogeneous PRP preparations and formulation discrepancies, absence of published studies supporting specific protocols of injection and lack of indications on the appropriate regimen for different OA severity degrees. The limited size of pooled patients that can under-power the statistical analysis to reach a significant threshold of difference in outcome measures, and the lack of a placebo group shades the evidence of PRP effects.

#### 5.1.2. Hip Osteoarthritis

Although various trials have faced up to the use of on PRP use for knee OA, few studies have focused on the treatment of hip OA with PRP. These studies are summarized in Table 4.


**Table 4.** Evidence of PRP treatment in hip osteoarthritis (reported by year of study and grouped by treatment).

A recent study [188] described the intraosseous infiltration of PRP for the treatment of hip osteoarthritis, in agreement with knee reported ones. Future studies are required to confirm the potential advantage of this new application of PRP.

Meta-analysis results of a randomized clinical trial that compared the effectiveness of PRP versus hyaluronic acid (HA) in hip OA underlined that PRP treatment was related to a significant reduction of VAS at two months. Both PRP and hyaluronic acid appeared to be comparable in terms of functional recovery [189].

The systematic review on the use of ultrasound-guided PRP injections in the treatment of hip osteoarthritis concluded that this route of administration appears to be well tolerated. Furthermore, though the level of evidence is relatively low, PRP treatment may lead to efficacious long-term and clinically significant reduction of pain and functional improvement [190].

Overall, intra-articular injection of PRP in hip OA patients has been demonstrated to be safe and have some efficacy in pain reduction and in functional improvement. When compared with HA, PRP showed to induce a better early pain relief; however, over 12 months, PRP and HA had comparable effects.

Future large-size trials that include a placebo group are needed. These studies should increase the level of evidence for the actual potential efficacy of PRP as an alternative conservative treatment to delay surgery in hip OA patients.

#### 5.1.3. Ankle Osteoarthritis

Osteoarthritis of the ankle is less common than the previously described localization of OA. Data concerning the use of PRP in ankle OA are obtained by case series. Four injections of PRP at weekly intervals induced improvement of function, pain and patient satisfaction [191], and similar improvements in pain and function up to 24 weeks after treatment were obtained after the administration of three injections every two weeks [192].

The limited data show some benefit in short–medium time, demonstrate the safety of the therapy and can be considered to be an alternative to postpone the need for surgery, but the comparisons with other injectable controls are lacking; therefore, no definitive conclusion can be made about the benefit of PRP in ankle OA.

#### *5.2. Tendinopathies*

Tendon tissue is poorly vascularized, and this characteristic is responsible for the limited healing capacity and the lesion irreversibility resulting in tendinopathies, which frequently occur in athletes [193].

#### 5.2.1. Achilles Tendinopathy

Achilles tendinopathy is a painful condition. Physical stress leads to tendon micro-trauma, and the inflammatory and degenerative responses that follow are responsible for local pain, swelling and stiffness [194]. Its treatment is difficult, and sufferers easily relapse due to the poor curative effects of the conservative treatment approach. The reason for PRP application lies in the tendency of the tendinopathy to became chronic after the use of nonsurgical approaches.

The outcomes after PRP administration are variable, and the main results are reported in Table 5.


**Table 5.** Evidence of PRP treatment in Achilles tendinopathy (reported by year of study and grouped by treatment).

Case series for chronic Achilles tendinopathy [195–200,211], retrospective studies [201,204] and prospective studies [208–210,215] have described promising efficacy of PRP treatment with lasting improvements [205].

Other studies did not show a superiority of PRP injection over saline solution [206,207,213] and no differences between patients treated with leukocyte-rich or -poor PRP [208]

Evidence for the efficacy of PRP in Achilles tendinopathy is not in agreement, and despite the important clinical significance, a strong basis for the use of PRP for Achilles tendinopathy was not demonstrated by meta-analyses and a systematic review [216–219].

#### 5.2.2. Lateral Epicondyle Tendinopathy

Lateral epicondyle tendinopathy, also known as "tennis elbow" is a common cause of pain and disability. Symptoms have been attributed to micro-trauma to extensor carpi radialis brevis tendon and the resulting angiofibroblastic tendinosis [220].

Different therapeutic approaches have been used, and steroid injections are considered to be the gold standard. Recently, PRP also became popular in treating this disease, with effects opposite to those of steroids, by stimulating the healing process and down-modulating inflammatory response.

The majority of the studies compared PRP efficacy with steroid one; however, other treatment comparisons have been reported (Table 6).



Initial results have been promising [221,222]. The first randomized controlled trials displayed PRP treatment improvements in function and pain, exceeding the effect of steroid injections up to one [232] and two [233] years

Following trials, comparing PRP treatment with saline [228,229,245], steroid [232–240,245] autologous whole blood [225–227] and bupivacaine [241] showed variable effectiveness in reducing pain and improving function.

Studies showing similar therapeutic effects between PRP and whole blood [225–227] suggest that circulating platelet concentrations are enough for obtaining recovery. However, the limited patient number and the absence of placebo arm make questionable these results.

As far as we know, the results of a multicenter randomized controlled IMPROVE trial are not yet available. The four-arms of lateral epicondylitis treatment will compare PRP, whole blood injection and tendon fenestration, each associated with physical therapy and sham superficial subcutaneous soft tissue injection, plus physical therapy. Expected results should significantly impact clinical practice [249].

Despite the heterogeneity of data, a seven-year retrospective study [250] and several meta-analyses, differing for inclusion criteria are available for evaluation the effectiveness of PRP in the treatment of lateral epicondylitis [251–255].

These reviews demonstrated short-term benefits for corticosteroids, but a long-term effectiveness for PRP in regard to improving functional capacity and alleviating pain. The critical factors identified mostly mirror those evidenced in other anatomical sites. Volume and number of administrations, various treatment combination, lack of standardization for PRP preparation and for exercise protocol, different measures for outcome evaluation and different follow-up times need deeper assessments.

#### 5.2.3. Plantar Fasciopathy

Plantar fasciopathy (PF), also known as "plantar fasciitis", affects the proximal insertion of the plantar fascia in the os calcis, causing pain. Tissue thickening and degenerative structural changes are more common than inflammatory findings, so the "plantar fasciopathy" definition better identifies this disorder [256].

The fascia plays a role of primary importance in the transmission of body weight to the foot while walking and running. Plantar fasciitis is very common in athletes, but can also occur in overweight or obese subjects.

Corticosteroids, autologous blood injection and extracorporeal shock wave therapy (ESWT) represent treatment options that have been used with varying results.

At present, a uniform therapy for the management of Plantar fasciopathy is missing; therefore, many studies have considered PRP to be an intriguing alternative option to favor healing in the plantar fascia without significant risk [257] (Table 7).




#### **Table 7.** *Cont.*

Early cohort studies have described the positive effect of PRP injection on relieving pain [260] and improving function [259], as well as on tissue structure [258] for chronic plantar fasciopathy.

The most recent randomized controlled trials comparing PRP, corticosteroids and normal saline administration describe a similar or a superior effect of PRP compared to corticosteroid injection and normal saline in reducing pain and increasing functional scores for chronic plantar fasciopathy [278,279].

Numerous other studies obtained variable results by the comparison of PRP and corticosteroid treatments: PRP was described as being either able to favor early pain relief and functional improvement [267,275] with prolonged effects [266,269,271,274,278] or to be likewise effective up to six months [265,268,270,272,273,276].

Trials comparing PRP with other treatment options for plantar fasciopathy showed a better initial PRP response but similar effects at six months; when PRP was compared with prolotherapy [281], no significant differences compared to extracorporeal shockwave [280] or plasma injection [263], superior and long-lasting effects compared to KT [282].

The latest systematic reviews and meta-analyses comparing PRP to other therapeutic approaches supported the use of PRP for the lack of complications or side effects [284], but, above all, for its superiority to corticoids, especially in long-term pain relief [285,286]; however, small sample number, study heterogeneities, adverse events and the lack of recording PF recurrence following treatment may decrease reliability of outcome measures.

#### 5.2.4. Patellar Tendinopathy

Inferior pole patellar tendinopathy, generally known as jumper's knee, is mostly common among athletes who engage in sports involving frequent jumping, such as volleyball and basketball, but it is also observed in people who do not carry out sporting activities [287]. The main evidence on PRP treatment in patellar tendinopathy is reported in Table 8.


**Table 8.** Evidence of PRP treatment in patellar tendinopathy (reported by year of study and treatment type).

PRP has been administered in several studies as a biological therapy for patellar tendinopathy, improving pain and MRI tendon structure, and significantly increasing functional outcomes, with long-lasting stable results up to two years, thus improving quality of life [196,199,288–290,303],

Multiple injections were found to be better than a single one for patellar tendinopathy, either in case series [293,294] or in a randomized prospective study [295], but the effect of two repeated injections or one single injection was also reported to be similar [296].

PRP treatment displayed better results than ESWT [299] and physiotherapy [297]. Dry-needling used for PRP administration made recovery faster than dry-needling alone; however, beneficial effects on pain and function only lasted three months, without improvement in QoL [301]. Furthermore, no clinical differences were observed when PRP was administered following other inefficacious treatments [298], or among leukocyte-rich or -poor PRP and saline [302].

Not long ago, no randomized controlled quality studies supported the use of PRP over conservative therapies, except in therapy-resistant cases [293,304]. However, recently, a systematic review [305] and meta-analyses of randomized trials have recommended the use of PRP for the management of patellar tendinopathy, due to its superiority to other nonsurgical therapies [306], in long-term pain relief and improvement in knee function [307]. Even if eccentric exercises seem to be the strategic choice in the short-term, in complexes cases, multiple PRP injections can be considered to be an option [308]. Variability on follow-up length, or its absence, and number of interventions are the main limitations of these studies.

#### *5.3. Muscle Injuries*

The use of PRP for the treatment of muscle injuries raised significant interest in the last years.

Similar to tendon healing, the initial muscle healing begins with an inflammatory response, followed by proliferation and differentiation of cells and tissue remodeling.

Acute hamstring injury is one of the most common muscle injuries affecting athletic patients, causing a decline in competition performance [309,310].

Some studies described positive results after injection of PRP in patients with injured skeletal muscles, and no negative side effects were reported [311,312] (Table 9).


**Table 9.** Evidence of PRP treatment in muscle injuries (reported by year of study and treatment type).

Contrasting results were obtained when PRP was compared to saline [313–315].

In general, an earlier comeback to sports activity, together with lower scores of pain severity and no significant increase of the re-injury risk, has been observed in patients/athletes who have undergone PRP administration, combined with a rehabilitation program, compared to patients treated with a rehabilitation program alone [320–322].

In particular, as a randomized clinical trial, this study showed positive outcomes in the PRP group as concerning convalescence time and returning to play [321].

Despite some favorable results, these studies do not have enough statistical power to support evidence-based adoption of PRP administration for skeletal muscle injury in clinical practice, as recently widely debated [325,326]. In general, current clinical evidence are conflicting, and univocal findings on the efficacy of PRP injections in the treatment of muscle injuries have not been achieved. Therefore, further human studies are strongly required to assess and validate the effectiveness of PRP for skeletal muscle regenerative purposes.

Platelet growth factors, specifically myostatin and TGF-β1, have been shown to have harmful effects to muscle regeneration. Indeed, TGF-beta1 is involved in the regulation of the level of fibrosis during muscle-injury repair, which is an important link in the complete restoration of muscle function [327]. An vitro study [328] demonstrated that platelet-poor plasma (PPP) or PRP with a second spin to remove the platelets induced differentiation of myoblasts into muscle cells.

However, since experimental evidence has not received a large consensus [329,330], further studies are needed to define the exact PPP-growth-factor content, its effect on myogenic precursors and its role on skeletal muscle regeneration. In addition, human clinical trials will be required to further explore the potential beneficial effects of muscle injuries treated with PPP.

These overall findings underline that none of the therapeutic options so far adopted have led to reliable results [325,326]. Even if skeletal muscle tissue exhibits an intrinsic remarkable regenerative potentiality in response to injury, in the case of extended damage, a dysregulated activity of different muscle interstitial cells occurs, resulting in aberration of tissue repair and maladaptive fibrotic scar or adipose tissue infiltration [331]. In this context, the morpho-functional recovery of injured skeletal muscle still remains a scientific challenge, and the identification of strategies that efficaciously improve the endogenous skeletal muscle regenerative mechanisms represents an unmet need.

#### **6. Conclusions and Future Perspectives**

PC use has gained popularity for the treatment of musculoskeletal diseases, even if conflicting results have been reported concerning clinical efficacy. Inconsistencies of clinical results rely on the huge heterogeneity of PC preparations, mainly ascribed to individual characteristics, different preparation protocols and variability in composition, as well as on different methodological limits of the protocols adopted in the clinical studies that have been previously underlined.

In addition to the different critical aspects already considered, the indistinct employment of words to refer to fresh, frozen/thawed or activated preparations increases confusion. Therefore, also a simple aspect such as a classification nomenclature comprehensive of all PCs, with the same characteristics allowing an overall clinical outcome comparison, could contribute to define the clinical use and improve our knowledge of PRP.

Besides being a paramount component of PRP, platelets have been proposed as carriers of pharmacological or biological molecules [332]; therefore, "future" PRP could be implemented with suitable molecules favoring specific biological functions.

The possibility of encapsulating PRP with a combination of HA, gelatin and biodegradable scaffolds displayed interesting results in in vitro studies of bone regeneration [333], and a new delivery system linking fibrinogen with high molecular weight HA (RegenoGel™) (merging the respective regenerative/wound healing properties and viscoelastic characteristics) showed positive outcomes in mild/severe osteoarthritis. In addition, this system can be used as a carrier for microRNA or inhibitory molecules (ADAMTs), allowing the preparation of specifically targeted custom-made devices [334,335].

Encouraging in vitro and in animal model studies has demonstrated that PRP combined with different biomaterials prolonged and improved growth factor release [336]; however, the possibility to translate these engineered biomaterials in the clinical practice to develop novel therapeutic strategies remains a future perspective.

**Author Contributions:** The authors similarly contributed to preparation of the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by "5 per mille funds".

**Conflicts of Interest:** The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **Abbreviations**



#### **References**


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