Hematological and Hemorheological Parameters of Blood Platelets as Biomarkers in Diabetes Mellitus Type 2: A Comprehensive Review

Abstract

Diabetes mellitus type 2 (DM2) is a hypercoagulable state with enhanced platelet (PLT) activation and increased clotting factor production. Simultaneously, the fibrinolytic cell system is inhibited due to the formation of clots with high fibrinolysis resistance. The stages of PLT “activation” have been well characterized microscopically, morphometrically, and nanomechanically using a light microscope, transmission electron microscope (TEM), scanning electron microscope (SEM), and atomic force microscope (AFM). Thrombocytes in an “activated” (procoagulant) state play a central role in two main biological processes: hemostasis and vascular vessel repair. Enhanced PLT reactivity in diabetic patients is considered a “pro-thrombotic” state. PLT hematometric indices are higher in retrospective and prospective studies, such as PLTs (count), MPV (mean platelet volume), PDW (platelet distribution width), PCR (platelet crit), and the PLTs/Ly ratio. The platelet indices MPV and PDW are higher in people with diabetes who have chronic vascular complications, and are statistically significant. PLT parameters/indices are useful biomarkers in the early diagnosis and prognosis of DM2. Precise studies of PLT activation state during DM2 may be useful for new diabetes (DM2) treatment strategies and effective therapeutic agents. Researchers have observed an association between MPV and medications such as insulin, metformin, and sulfonylureas using the blood glucose concentration attached to hemoglobin (HbA1c values) as markers of glycemic control in patients with diabetes. Computational modeling of PLT activation in DM2 is also a controlling factor for thrombocyte distribution and margination in blood vessels, both of which are associated with micro- and macrovascular disease in DM2. PLT-derived microRNAs (miRNAs) are novel molecular biomarkers for the diagnosis and prognosis of DM2, insulin resistance, and diabetes complications. Anti-platelet agents and natural plant products may also be effective in the prevention and secondary treatment of micro- and macrovascular complications in type 2 diabetes mellitus. To determine new ways of diagnosing, treating, predicting, and managing DM2 and its related vascular complications, we propose monitoring a combination of hematological, hemorheological, and hemostatic parameters (indices), which merit future studies.

1. Introduction

In future decades, the number of patients with diabetes mellitus type 2 (DM2) is predicted to increase to over 693 million by 2045 [1]. DM2 will be an epidemic disease in the 21st century, and it is currently a major cause of mortality, morbidity and global health problems in our time. DM2’s relationship to the body’s metabolic syndrome and fat metabolism has been well studied. Human priorities include “quality of life” and the “enhanced physical activity of society”. Our results and conclusions demonstrate the importance of maintaining regular physical activity/exercise and good natural diets for DM2 patients.

In the “area of physical activity/exercise and diet”, original ideas for implementing/applying natural medicines to the clinical diabetology of DM2 would be useful.

In the world of cells and tissues, blood platelets (PLTs, thrombocytes) are closely related to the pathogenesis of DM2 and micro- and macrovascular complications of the disease. Prominent theories in this field include the viscera–portal hypothesis, the ectopic fat hypothesis, and adipose tissue as an endocrine gland [2]. These theories are based on the relationship between observed energy metabolism and beta-oxidation of fats. “Insulation resistance” may be involved when B-oxidation prevails over glycolysis [2].

DM2 affects important hemorheological markers closely related to blood flow [3,4]. Myogenic and endothelial factors, such as basic rheological/biorheological markers, play an essential role in human blood circulation. Diabetes mellitus type 2 can affect the hemorheological markers of erythrocytes, causing a decrease in cell deformability as well as an increase in erythrocyte aggregation and blood viscosity.

Blood platelets (thrombocytes) are produced in the bone marrow and spleen as small (1–2 µm in diameter), anucleate fragments from the cytoplasm of megakaryocytes. Platelets (PLTs) have a smooth surface and a discoid shape. In the peripheral blood, PLTs are involved in thrombogenesis, as confirmed by clot formation modeling both in vivo and in vitro [5].

PLTs contain and synthesize cell growth factors, chemokines, coagulation proteins, two types of secretory granules (alpha granules and “dense” granules), adhesion molecules, etc. [6,7].

In response to various stimuli and/or damages, PLTs undergo morphological and functional changes, known as PLT “activation” [8]. Activated (procoagulant) thrombocytes play a central role in hemostasis and repairing vascular vessels [9,10]. Procoagulant PLTs generate thrombin and develop three-dimensional fibrin matrices during hemostasis/thrombogenesis. Human platelet membrane-derived, small (≤0.1 µm) micro-particles with procoagulant effects are involved in thrombin formation and, thus, thrombotic complications (e.g., attributable to diabetes mellitus type 2 (DM2)) [11,12].

During PLT activation, changes in PLT shape appear first, from discoid to spherical, with actin-rich philopodia (cytoplasmic extrusions) interacting, flattening, and spreading onto surfaces in the final activation phases [13,14,15]. The same authors characterized PLT activation stages morphometrically and nanomechanically by atomic force microscope (AFM) for diagnostic purposes.

In our opinion, DM2 clinical practice should focus its efforts on bridging the gap between scientific research (as a source of updated information on DM2) and DM2 practitioners (as a source of useful information about vascular disease complications).

Currently, several popular hypotheses explain the molecular pathogenesis of DM2 (the viscera–portal theory and ectopic fat) and the endocrine function performed by adipose tissue [2]. The roles of oxidative stress, mitochondrial dysfunctions, and the endoplasmic reticulum “activation” have been fully examined. The shift in energy metabolism—from normal glycolysis to the beta-oxidation of fats—is commonly observed. The beta-oxidation of fats plays a fundamental role in preventing over-glycolysis. It combats insulin resistance/hyperinsulinemia based on defective disulfide bond formation in the insulin receptor, which causes signaling defects (resistance).

This review explores current research topics in diabetology and associated fields by investigating and comparing DM2-associated PLT indices as biomarkers of micro- and macrovascular complications of DM2, thereby elucidating and mitigating vascular disease risks for patients. The present review also aims to clarify platelet parameters as potential biomarkers for early diagnosis and prognosis of DM2.

2. Topics and Results

2.1. Enhanced PLT Reactivity in Diabetic Patients Has Been Considered a “Pro-Thrombotic State”

PLT studies are important for regular treatment, successful management, and improved outcomes of DM2. In retrospective and prospective studies, higher values of hematometric indices—namely PLT count, mean platelet volume (MPV), and platelet distribution width (PDW)—are risk indicators for developing diabetes-related vascular complications. Easily identifiable increases in thrombocyte volume indices—MPV, PDW, and PCR (plateletcrit)—during routine automated hematological analysis may be useful early diagnostic and prognostic markers, especially for thrombogenesis in DM2. Researchers have characterized PLTs from patients with DM2 as “highly reactive, playing a key role in the development of diabetic vascular complications” [16,17].

The processes of micro- and macro-clot formation in DM2 are biochemically induced by increased production of different PLT factors—transcription, fibrinogen/fibrin, thromboglobulin, serotonin, P-selectin, d2-dimer, thromboxane A2, etc.—in cases of PLT hyperactivation [16,17,18,19].

2.2. Platelet Signaling, Hyperaggregation, and Abnormalities in Patients with DM2 Play a Crucial Role in Thrombotic (Clot Formation) Complications and Thromboembolism during DM2 Micro- and Macroangiopathies

Targeted studies [16] of these pathological states would be useful for the development of new DM2 treatment strategies and effective medicines (drugs, agents) to reduce PLT hyperactivation during DM2.

From the pathological and pathophysiological points of view, DM2 and related states (obesity, metabolic syndrome, impaired glucose tolerance, insulin resistance, etc.) are closely associated with subclinical (chronic) inflammation. Thus, several inflammatory hematometric biomarkers for PLTs and erythrocytes (RBCs), such as MPV, PDW, MCV, and RDW, are significantly elevated in DM2 compared to data for healthy individuals (controls) [19,20,21,22,23,24]. MPV is significantly elevated in diabetic study groups compared with healthy individuals, suggesting that this hematological index is closely associated with DM2 development [19,21].

Further prospective studies with a larger cohort are necessary to characterize the probable relationship between MPV and metabolic (glycemic) control (concomitant HbA1c values as markers of glycemic control in patients with DM2). Further results could be significant for DM2 treatment, based on the principle of patient-specific healthcare in DM2 management.

2.3. Hyperglycemia Contributes to Elevated PLT Reactivity (Hyperactivation)

Hyperglycemia in DM2 contributes to elevated thrombocyte activity (hyperactivation). Through the direct effects of glucose on PLT membranes, the appearance of “larger” and “giant” PLTs is possible (both with elevated MPV and PDW) [20]. Additionally, hyperglycemia could promote PLT protein glycation [25]. Insulin deficiency could also promote PLT activation. Different bioproducts in the blood of diabetes patients could interfere with hematopoietic cells at the level of bone marrow and the spleen, activating erythropoiesis and megakaryopoiesis simultaneously to produce differently sized erythrocytes and platelets (anisocytosis), including “larger” and “giant” PLTs. The higher MPV indicating larger PLT sizes could suggest “stimulated thrombogenesis” and “activated thrombocytes” in the blood of diabetes patients [18].

2.4. Platelets to Lymphocytes Ratio (PLT/Ly)

The platelets to lymphocytes ratio (PLT/Ly), neutrophils to lymphocytes ratio (Neu/Ly), and complete blood count (CBC) are parameters proposed in routine laboratory practice and are potential inflammatory response markers [10,26]. The platelets to lymphocytes ratio predicts diabetic retinopathy in DM2 [27]. This parameter significantly correlated with DM2 risk in a Chinese cohort study, indicating that a higher PLT/Ly may reduce the risk [28].

The same authors also proposed reference intervals for PLTs (

Table 1. Thrombocytes (platelets—PLTs) reference data.

PLTs—platelets count	140–440 × 109/L
MPV—mean platelet volume (MPV)	7.80–11.0 fL
PDW—platelet distribution width	15.5–30.5%

). In further routine laboratory practice, simultaneous anisocytosis for PLTs and RBCs was determined, improving prognostic biomarkers for DM2.

Two low-cost indices-neutrophil-to-lymphocyte ratio (NLR) and platelets to lymphocytes ratio (PLT/Ly) have been repeatedly assessed for their efficacy as diagnostic and prognostic tools for dry eye disease (DED), diabetes mellitus, and other diseases. Raised levels of NLR and PLT/Ly have been associated with DED. The value of the systemic immune inflammation (SII) index has been used as a prognostic indicator for inflammatory diseases such as DM2 [29].

Studies on DM2 pathological states (at the level of morphological, cytochemical, and ultrastructural methods) are useful for the development of new DM2 treatment medicines and strategies, leading to reduced PLT hyperactivation in the development of DM2 (e.g., treatment of PLT hyperactivity with oleic acid (OA) [8,30].

The Effect of Oleic Acid on PLT Homeostasis

The physiological mechanisms that underline PLT homeostasis under the influence of OA remain unclear. OA has potential antithrombotic effects, suppressing PLT hyperactivation. OA also inhibits PLT aggregation, granule release, and thrombocyte calcium “mobilization”. Additionally, OA suppresses the spread of PLTs in fibrinogen, delaying arterial thrombosis development. New studies on OA as a probable anti-platelet activation/hyperactivation drug of clinical significance for preventing and treating DM2 contribute to our understanding of diabetology [30].

2.5. Hematological/Hematometric Investigations Related to Metabolic/Glycemic Control of DM2

Some research groups have identified the mean platelet volume (MPV) as a cost-effective tool in the primary healthcare of diabetic patients. Researchers have obtained interesting results related to DM metabolic/glycemic control. For instance, as DM2 glycemic control improves, the MPV and HbA1c in patients decrease. By improving PLT activities and functions, diabetic patients’ regular glycemic control may delay vascular complications from DM.

Hematometric laboratory results should be further explored in clinical practice, as well as in establishing computational models of platelets.

PLT indices [25] are correlated with platelet functional status and emerging risk factors for vascular complications in DM2. A prospective study of PLT parameters, including MPV, PDW, and PCT, in diabetic patients and healthy controls was carried out. Blood glucose and HbA1 levels were also measured [25]. Statistical analysis was performed using Student’s t-test and Pearson’s correlation test. The average age of DM2 patients was 53 ± 5.7 years. The mean disease duration was 4.7 ± 2.5 years. MPV, PDW, and PCT were significantly higher in diabetic than in non-diabetic patients [22,23,24,25]. The indices were insignificantly higher in patients with complications. Higher index values in DM2 cases indicated that they could be better, simpler, and more cost-effective indicators for assessing initial vascular complications in DM2 patients.

The MPV in diabetic patients was 11.3 ± 1.0 fL compared to 9 ± 0.6 fL in non-diabetic patients, with a p-value of 0.004 [25]. The mean PDW in diabetic patients was 14.2 ± 2.5 fL, whereas it was 10.7 ± 0.7 fL in healthy individuals (controls). The same authors observed that p-values for MPV and PDW were significant in diabetic patients (p < 0.05). Additionally, a positive statistical Pearson correlation was obtained between MPV and PDW with HbA1c in the diabetic group [16,17,18,19]. The platelet indices MPV and PDW in the diabetic group were higher in patients with chronic vascular complications than in patients without blood vessel injuries, but the data were not statistically significant [25]. Recently, clinicians have noted that MPV significantly increases in diabetic patients during retinopathy development (p = 0.006) [30]. After conducting a correlation analysis, the same authors obtained a positive correlation between HbA1c and PDW, HbA1c and MPV, as well as HbA1c and PCT. PDW levels were independently associated with DM2 diagnosis, while MPV was associated with PLT activation and impaired glucose regulation [31].

Therefore, regular monitoring of all easily acceptable and inexpensive platelet hematological/hematometric indices is clinically important in diagnosing and preventing the micro- and macrovascular complications of DM2, including cardiovascular and brain vascular diseases, and blood hypercoagulation combined with defective fibrinolysis, dysregulated vasodilation, and increased risk of vascular clot occlusion.

PLT anisocytosis (elevated MPV and PDW) combined with RBC anisocytosis (elevated MCV and RDW) may be a predictive tool for serious DM2 complications (inflammation, vascular thromboses, endothelial dysfunction, etc.) [32,33,34,35,36].

The association of increased MPV and PDW, related to impaired glucose regulation in DM2, has been described at different clinical stages of the disease: pre-diabetes, diabetes, and vascular diabetic complications. In the medical literature, increased levels of PDW with elevated MPV are reported to be closely associated with DM2 and its vascular complications [22].

Complex hematologic/hematometric results obtained by different study groups are important in assessing early DM2 diagnosis, prognosis, clinical management, and improved disease outcomes [37].

Conflicting results have also been reported, e.g., on the probable relationships between PLT count and DM development. Some researchers propose “association”, but several studies support “no relationships”. Besides thromboembolic disorders, PLT indices’ association with inflammatory states has also been examined. Further studies on a larger scale must be evaluated.

2.6. Recent Advances in Modeling Diabetic Blood Platelets

2.6.1. Computational Platelet Modeling in DM2

DM2 creates the conditions for complex thrombotic pathologies. Extensive experimental, theoretical, and computational studies have been conducted to determine PLT properties, as well as their interaction and thrombus formation [38]. Advanced multi-scale models were developed to better understand the role of various factors in flow-mediated transport, PLT deposition, coagulation kinetics, and their overall effect on thrombus formation. Blood shear rate is an important factor controlling platelet distribution in blood vessels [22,38,39,40,41].

Chang et al. (2018) simulated blood with hematocrit at 20% (for controlling different blood cell suspensions) [22,42]. The results indicated that “larger platelets” are more likely to migrate toward the vessel wall. They suggested a roughly 6–10% decrease in platelet migration rate when the wall shear rate decreased from 1000 s⁻¹ to 600 s⁻¹.

Weakened platelet migration at lower wall shear rates is probably due to reduced collision between platelets and RBSs [38].

Continuum-based partial differential equation (PDE) models [38] have been used to describe the time and spatial dependence of thrombin generation, fibrin formation, and thrombus growth under various flow conditions. However, thrombosis development simulations in diabetes require the discrete representation of the blood flow at a cellular level in computational models. To present and simulate micro-scale interactions between different types of blood cells, numerous mathematical and computational models have been designed [39,40,41]. Lattice Boltzmann and lattice kinetic Monte Carlo models have been developed to simulate platelet activation and aggregation by treating PLTs as particles under flow [43]. However, applications for these hybrid models have been mostly limited to two dimensions due to model complexity and significant computational costs.

In future modeling systems, a multi-scale approach would facilitate patient-specific simulation of thrombosis/thrombogenesis in different hemodynamic conditions with different pharmacological treatments.

Yazdani and Karniadakis (2016) [44] performed a systematic study of RBC and PLT transport considering different levels of constriction, controlling hematocrit and flow rates. In their work, normal and diabetic RBC and PLT models were employed to simulate normal and diabetic blood flow in rectangular channels. In the numerical framework, four key sub-processes of blood clotting were combined: hydrodynamics, PLT micromechanics, transport of PLT coagulation factors, and registering coagulation reactions. To consider the coagulation cascade, the authors implemented the mathematical model of Anand et al. (2008) [45].

Yazdani and Karniadakis (2016) [46] concluded that higher levels of blood vessel constriction (stenosis) and elevated vessel wall shear rates led to significantly enhanced platelet migration. This model, with modifications, could explain the experimental data of active PLT aggregation in a postvascular stenosis segment of the vessel wall in different pathological states (DM2, atherosclerosis, etc.).

Computational modeling has emerged as a powerful tool for investigating pathological processes in DM2. However, many unexplored areas and different experimental conditions must be targeted in future research on the topic.

In the cellular, tissue, and organ-specific modeling of diabetic blood cells and injured microvasculature (complications such as retinopathy, nephropathy, microangiopathy, etc.), micro-aneurisms should be obtained simultaneously with edemas and/or hemorrhages, directly affecting organ functions [20,41,46,47].

Instead of great advantages in bioinformatics and biomechanics, there is a lack of computational models in the medical scientific literature to connect blood glucose level prediction in diabetic patients with PLT hematological and hemorheological biomarkers [21,22].

2.6.2. Potential Platelet Biomarkers Related to T2DM

Altered structure and architecture, as well as fibrin clot functions, have been reported in patients with DM2, as well as in cases of coronary artery disease (CAD). DM2 unfavorably affects plasma fibrin by lowering its permeability and susceptibility to lysis while simultaneously increasing fibrinogen plasma levels, leading to hyperfibrinogenemia. Specifically, these changes are attributed to fibrinogen and fibrin glycation in diabetic patients.

Platelet activation in DM2 is a well-known modifier of fibrin clot properties. In the presence of activated PLTs, fibrin clots have a specific architecture with local increases in fibrin fiber diameter and density [13]. P-selectin, expressed in alpha-granules of activated platelets in DM2 patients, also increases. The role of vWF in altering plasma fibrin network properties remains to be established, which is closely related to fibrinolysis/thrombolysis [9].

Dynamic changes in platelet shape and morphology progress from an “active” to a “procoagulant state” [48,49].

Correlations were found between MPV as a main biomarker of PLT activation and metabolic status in type 2 diabetes patients [14,21,25,50,51,52]. Important hemogram parameters are MPV, platelet count (PLT), PDW, PCT, HbA1c, and disease duration. Some authors have suggested that RDW is also an important predictor of vascular complications in DM2 [33]. Prospective studies are needed to define and clarify the relationships between hematologic/hematometric indices. MPV and PDW can be easily determined by routine automated hemograms, and these parameters are significantly elevated in diabetic patients compared to healthy individuals. Among diabetic patients, MPV and PDW means are higher in patients with complications (not statistically significant) [25]. Researchers have suggested that monitoring DM2 and, thus, preventing vascular complications, is needed. PDW is also a main PLT index that is significantly higher in diabetic patients than in healthy controls. PLT indices are useful biomarkers for vascular risks in grading DM. These findings suggest that increased PLT activation plays a role in the pathogenesis of DM vascular complications.

Additionally, regular glycemic control improves platelets’ biological activities and functions. Thus, good glycemic control helps in DM2 treatment and management, potentially delaying micro- and macrovascular complications attributable to DM2 as a socially significant disease.

Vascular complications appearing in DM2’s prothrombic/thrombic state are mainly related to risk factors for clot formation. At this DM2 stage, the presence of larger PLTs, as well as “giant platelets” in patients’ blood smears, may provide simple, effortless, and cost-effective diagnostic/prognostic tools for predicting the thrombotic state in DM2 development and other pathological states.

Platelet activation plays an important role in the development of vascular DM2 complications. MPV and PDW pathological levels also increase as precise biomarkers of platelet activation in DM. In their prospective study, Ulutas et al. found a statistically significant correlation between MPV and HbA1c values [50].

In 1979, Hajek et al. were the first to show that PLTs have insulin receptors, with a density of about 500 receptors/thrombocyte [52,53]. In this way, insulin regulates PLT functions directly and has an inhibitory effect on platelet hyperactivity.

Recent studies [54] provide insight into the potential role of platelet hematological/hematometrical parameters (PLT count, MPV, and PDW) as biomarkers in the diagnosis, management, and prognosis of DM2. These simple DM2 biomarkers may play an important role in routine medical practice [52,55].

At present, mean platelet volume (MPV) is a commonly used marker of PLT size and biological activity. Larger PLTs have increased pro-thrombotic potential compared to smaller ones. In addition to routine clinical practice, SEM and AFM microscopies confirm this conclusion [9,13,14].

MPV may indicate bone marrow and spleen thrombocytogenesis and PLT turnover rate. In the scientific literature, MPV is believed to be directly related to PLT aggregation: when the PLT aggregation function weakens, the MPV level decreases. This conclusion relates to optimal DM2 management and other diseases (cardiovascular, brain vascular, etc.), where pro-thrombotic situations correlate with the degree of PLT activation. MPV is reduced in patients with better glycemic control. In states of improved glycemic management, improvements are due to reduced PLT activity and lower MPV.

The platelet distribution width (PDW) is the main hematometric index linked to DM2. PDW demonstrates PLT size diversity (anisocytosis). PDW levels are also significantly elevated in diabetic patients with HbA1c levels higher than 6.5% [25].

2.7. Combined Properties of Diabetic RBC and PLT Indices

Chang et al. [42] successfully combined the properties of diabetic RBC and PLT indices from the scientific literature. They demonstrated that the less deformable RBCs in DM2 reduce the transportation of platelets toward the vessel walls, whereas platelets with a higher mean volume lead to enhanced margination. They also concluded that increasing flow rate or hematocrit enhanced platelet margination.

New contributions to this field are data on PLTs rolling near endothelial cells in vessel walls. PLT adhesion to vessel walls can demonstrate the platelet activation/hyperactivation [22] process in blood vessel wall injury and the coagulation cascade during thrombogenesis (clot formation) (Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5).

Altered plasma levels in coagulation proteins, pro-coagulatory particles, metal ions, lipid composition and metabolism, endothelial morphology and physiology, and degree of PLT activation are important in DM2 [55].

In the last two decades, numerous computational models have been developed to simulate the dynamics of all blood cells (RBCs, WBCs, and PLTs), under static and dynamic (flow) conditions [38]. Cardiovascular diseases and defects in hemostasis must be addressed simultaneously [56].

Benjamin et al. [57] elucidated the precise biological and molecular mechanisms of DM2, which contribute to the MPV size index.

Researchers have observed an association between MPV and several medications used to manage DM2, including insulin, metformin, and sulfonylureas [58]. For instance, metformin protects PLT mitochondria and inhibits mitochondrial hyperpolarization induced by hyperglycemia in diabetic patients. After six months of metformin treatment, significantly lower levels of MPV were observed in patients [58,59]. Lower levels of MPV may be an effective and successful variable in prediction models for DM2.

Furthermore, DM2 complications (micro- and macrovascular) are associated with elevated RDW and PLT/Ly ratios; thus, precise glycemic control is needed [52,55,60].

Clinical diabetology also employs other main hemorheological parameters (such as WBV—whole blood viscosity; ED—erythrocyte deformability; EA—erythrocyte aggregability). Hemorheological disorders in DM2 patients increase simultaneously with unfavorable alterations in glycemic/carbohydrate metabolism and depend on oxidative stress intensity (micro-hemodynamic index OSI—oxidative stress intensity). Micro-hemodynamic indices such as ERDI (relative deformability of erythrocyte membrane), WBV (whole blood viscosity), blood plasma viscosity (BPV), and the erythrocyte aggregation index (EAI) are also discussed in relation to preventing cardiovascular risks and other DM2 vascular complications [57,60,61].

Figure 1. AFM Bruker images (3D topography) of an unfixed blood clot from a DM2 patient. Erythrocytes/echinocytes, fibrin fibers (blue triangles), and “activated” PLTs (orange-brown stained; blue arrows) [13,62].

Figure 1. AFM Bruker images (3D topography) of an unfixed blood clot from a DM2 patient. Erythrocytes/echinocytes, fibrin fibers (blue triangles), and “activated” PLTs (orange-brown stained; blue arrows) [13,62].

Figure 2. AFM Bruker image (3D topography) of a blood clot surface from a healthy donor. Erythrocytes/discocytes (white star), platelets (white arrows), and fibrin fibers (white triangles) [13,62].

Figure 2. AFM Bruker image (3D topography) of a blood clot surface from a healthy donor. Erythrocytes/discocytes (white star), platelets (white arrows), and fibrin fibers (white triangles) [13,62].

Figure 3. AFM Bruker image (3D topography) of a PLT cluster containing “activated” thrombocytes from the blood of a DM2 patient. Abundant thick fibrin fibers are organized in the fibrin network [13,62].

Figure 3. AFM Bruker image (3D topography) of a PLT cluster containing “activated” thrombocytes from the blood of a DM2 patient. Abundant thick fibrin fibers are organized in the fibrin network [13,62].

Figure 4. SEM JSM 6390 image. “Activated PLTs” on the fibrin network of a fixed blood clot from a patient with DM2 (10,000× magnification) [13,62].

Figure 4. SEM JSM 6390 image. “Activated PLTs” on the fibrin network of a fixed blood clot from a patient with DM2 (10,000× magnification) [13,62].

Figure 5. SEM JSM 6390 image. Parts of blood clot surfaces from the blood of a DM2 patient, including erythrocytes with atypical shapes and “activated” PLTs in the fibrin fiber network (5000× magnification) [13,62].

Figure 5. SEM JSM 6390 image. Parts of blood clot surfaces from the blood of a DM2 patient, including erythrocytes with atypical shapes and “activated” PLTs in the fibrin fiber network (5000× magnification) [13,62].

2.8. PLT-Derived microRNAs Are Novel Biomarkers for Early Diagnosis and Prognosis of Type 2 Diabetes Mellitus

PLTs are a major source of microRNAs. Endothelial cells are also enriched with miR-126. PLT-derived microRNA-103b (miR-103b) plays a critical role in regulating glucose homeostasis during DM2. Circulating miR-126 was investigated as a main biomarker of DM2 [62,63,64,65,66].

Recent data suggest that PLTs contain large amounts of miRNAs circulating in bodily fluids. By regulating multiple gene functions, miRNAs attract scientific interest as biomarkers of vascular disease in DM2. Currently, miRNA studies are closely associated with DM2 micro- and macrovascular complications [31,37,64,65,66].

Circulating microRNAs are related to DM2 and predict a special class of miRNA molecules altered during T2DM development. These molecular modifications can be seen as potential biomarkers for the early diagnosis and prognosis of DM2 and related vascular complications. miRNAs are involved in regulating inflammation, insulin resistance, and DM2 development [64]. In pathological conditions such as DM2-associated PLT activation, drug administration (i.e., aspirin) may lead to reduced circulating miR126 levels [65,66].

Sixteen microRNAs (miR-29a-3p, miR-221-3p, miR-126-3p, miR-26a-5p, miR-503-5p, miR-100-5p, miR-101-3p, mIR-103a-3p, miR-122-5p, miR-199a-3p, miR-30b-5p, miR-130a-3p, miR-143-3p, miR-145-5p, miR-19a-3p, and miR-311-3p) meet the criteria of DM2 biomarkers [65]. MicroRNA expression profiling could provide detailed data on the clinical utility of these 16 new miRNAs.

Monitoring DM2-specific microRNAs [62] allows early prediction, diagnosis, and treatment of DM2 and its related vascular complications.

2.9. Limitations

Studies in this field are limited because “the presence of miRNAs” reflects only the presence of PLT-derived, circulating microRNAs and not in situ miRNAs localized in PLT cytoplasms.

To reduce this limitation and minimize related problems, we propose further visualization of miRNAs in “microclasmatosis/microvesicles/clasmatosis/exosomes” and/or different granules in PLT cytoplasms. For this purpose, we applied our staining method to the in situ visualization of RNPs (ribonucleoproteins) [67,68,69]. Cytoplasmic granules, stained positively for RNP, were observed in our previous studies as good indicators of neoplastic severity. During metastasis appearance, abundant empty vacuoles and lack of RNP-stained granules in PLTs were visualized using light microscopes [69,70].

The cytochemical method for RNP staining, applied to diabetic patients’ platelets, could characterize the PLT-proliferative state (“activation”, “hyperactivation”). DM2 management is also limited because the cellular/molecular mechanisms of DM2-induced platelet hyper-responsiveness and anti-platelet drug resistance were unclear and must be explored in further investigations; e.g., the “hyperactivity” of PLT P2Y12 receptors and their pathway plays an important role in PLT “activation” cellular mechanisms [71]. In this case, PLT “hyperactivity” and “high aggregation degree” could be inhibited by opioid peptides. Some key signaling molecules correlating to opioid peptides probably participate in P2Y12 signaling pathways.

2.10. New Pharmacological Strategies Related to Natural Anti-Platelet Agents

Sobol and Watala investigated the role of platelets (PLTs) in diabetes mellitus as related to micro- and macrovascular DM2 complications. The analysis of these results may help develop new pharmacological strategies for reducing PLT activation/hyperactivation levels in DM2 [71,72,73].

“Activated” and “hyperactivated” PLTs in DM2 undergo shape changes and exhibit morphological features such as membrane blebbing, protrusions/pseudopodia, microvesicles, etc. [74]. The dynamic PLT events progress from an “activated” state during transformation to a “procoagulant phase” when diabetic patients’ PLTs are involved in micro- and macrovascular complications.

Scientific contributions, clinical trials, and efforts to improve anti-platelet drugs and create new medicines based on successful applications of combined hematological indices and hemorheological measurements/parameters include PLTs (count); MPV, PDW, PCR, PLT/Ly ratio; and PLT-derived miRNAs as biomarkers in DM2, etc. Our plan to monitor these hematologic, hemorheologic, and hemostatic indices could lead to new treatments and management strategies for thrombotic micro- and macrovascular DM2 complications (Figure 6).

Diabetes mellitus (DM2) is a hypercoagulable state with enhanced PLT activation and increased clotting factor production: prothrombin, thrombin, fibrinogen, factors VII, VIII, Xi, XII, von Willebrand factor, etc. [73,74,75]. Simultaneously, the fibrinolytic cell system is inhibited due to clot formation with high resistance to fibrinolysis. Anti-platelet agents, including natural plant products, can be effective in secondary treatment and in preventing micro- and macrovascular complications in DM2 [8,30,74,76,77,78,79,80,81].

3. Conclusions

Platelet parameters (hemorheological and hematometrical) and PLT/Ly are useful biomarkers for the risk assessment, early diagnosis, and prognosis of DM2. Abnormal rheological properties of RBCs and platelets flowing in the blood are crucial to platelet adhesion and thrombus formation in DM2. Monitoring thrombocyte activity, as well as hematological, hemorheological, and hemostatic indices, can help develop new and effective therapeutic strategies for managing DM2-related thrombotic micro- and macrovascular DM2 complications. For DM2, some natural plant medicine products have the potential for evaluation and implementation in clinical practice.

The tools of microfluidics technology can be used to organize systematic studies of “activated” PLT behavior in DM2 patients with vascular complications. Many DM2 patients with microvascular complications (microangiopathies such as nephropathy, cardiopathy, peripheral vessels angiopathies, etc.) will benefit from these results.