*Systematic Review* **Vitamin D-Related Risk Factors for Maternal Morbidity and Mortality during Pregnancy: Systematic Review and Meta-Analysis**

**María Morales-Suárez-Varela 1,2, Nazlı Uçar 1, José Miguel Soriano 3, Agustín Llopis-Morales 1, Beth S. Sanford <sup>4</sup> and William B. Grant 5,\***


**Abstract:** Vitamin D deficiency (serum 25-hydroxyvitamin D [25(OH)D] levels <20 ng/mL in serum) is a common health condition among pregnant women, especially in high-risk groups. Evidence has connected vitamin D levels with many health-related problems during pregnancy, including gestational diabetes and preeclampsia. Because of vitamin D's effect on both mother and fetus, we systematically review the association between 25(OH)D level and its health effects. From a total of 143 studies, 43 came from PubMed, 4 from Cochrane, and 96 from EMBASE. After screening, we identified 38 studies as candidates for inclusion. Ultimately, we limited this review to 23 articles originating from 12 countries, written in English or Spanish, and conducted between 2010 and 2022. We conducted this review according to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines and evaluated the quality and strength of the evidence by using the Navigation Guide Systematic Review Methodology (SING). These systematic reviews summarize findings that support vitamin D's role in reducing risks of multiple outcomes and the possible contribution of adequate vitamin D levels to a healthy pregnancy.

**Keywords:** maternal mortality; maternal morbidity; preeclampsia; pregnancy; vitamin D deficiency; supplementation; vitamin D; 25-hydroxyvitamin D

#### **1. Introduction**

Vitamin D is a fat-soluble vitamin critical within the body for many functions, including cell proliferation, differentiation, apoptosis, and immune modulation [1]. Vitamin D is transmitted from the mother to the fetus via the placenta and is fundamental at all stages of embryonic and fetal development, from implantation to general growth, including skeletal maturation and placental function [2–4].

Worldwide, about 1 million people suffer from vitamin D deficiency (serum 25 hydroxyvitamin D [25(OH)D] <20 ng/mL, referring to vitamin D2 and/or D3) [5]. Due to the increased physiological demand for vitamin D during pregnancy, pregnant women are considered a high-risk group for developing vitamin D deficiency (VDD), with prevalence ranging from 51.3% [6] to 100% [7]. VDD in pregnant women increases maternal mortality and morbidity rates. Worldwide, the highest prevalence (>80%) of deficiency in pregnancy was observed among Chinese women (100%) [7] and pregnant Turkish women (95.6%) [8]. In Middle Eastern countries, VDD among pregnant women is an estimated 60–80% [9,10]. Among Iranian pregnant women, studies have reported prevalence rates of

**Citation:** Morales-Suárez-Varela, M.; Uçar, N.; Soriano, J.M.;

Llopis-Morales, A.; Sanford, B.S.; Grant, W.B. Vitamin D-Related Risk Factors for Maternal Morbidity and Mortality during Pregnancy: Systematic Review and Meta-Analysis. *Nutrients* **2022**, *14*, 4124. https://doi.org/10.3390/ nu14194124

Academic Editor: Andrea Fabbri

Received: 14 September 2022 Accepted: 30 September 2022 Published: 4 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

78%, 76%, 70.4%, and 69.2% [11]. The estimated prevalence in pregnant women in the USA and Canada was reported to be 42–72% [12]. In Sweden, a longitudinal study reported 37% of first-trimester pregnant women had 25(OH)D concentrations <20 ng/mL [13], in comparison with 23% of Canadian women [14]. In Mexico, a previous cross-sectional study reported VDD among 61% of women in the third trimester, and 98% of their newborns had vitamin D deficiency [15].

There are a number of reasons why maternal VDD rates are high during pregnancy. One reason is sun avoidance. In the Middle East, that can be due to wearing concealing clothing as well as not going outdoors during the hot summers. Another reason is that diet provides only a small amount of vitamin D, and then only from animal products, including eggs, fish and meat, unless the food is fortified. Thus, low-latitude countries, which have largely plant-based diets, obtain little vitamin D from food. A third reason is that health care providers do not generally recommend enough vitamin D supplementation in general and during pregnancy in particular.

Vitamin D may impact maternal, fetal, and postnatal growth by affecting calcium absorption [16], parathyroid hormone expression [17], phosphate metabolism [18], growth plate function [19], and possibly regulating the insulin-like growth factor axis [20]. VDD during pregnancy has therefore been associated with adverse health outcomes in the mother, including increased risk of preeclampsia, glucose intolerance, gestational diabetes, preterm birth, and hypocalcemia crisis [21], as well as poor fetal skeletal development [22]. Through this review's comprehensive meta-analysis, we aim to determine the effect of vitamin D supplementation in preventing maternal mortality and morbidity.

#### **2. Materials and Methods**

#### *2.1. PICO Strategy*

We used the PICO strategy (Pregnancy, Intake/level, C/D, mOrtality/mOrbidity) to identify potentially relevant studies. In PICO, the question needs to identify the patient or population problem we intend to study, the planned intervention or treatment, the comparison of one intervention with another (if applicable), and the anticipated outcome. Our PICO framework was "Is there more mortality or morbidity in pregnant women with low levels of vitamin D than in those with adequate levels of vitamin D?", in which P is pregnant women, I is a low intake/level of vitamin D, C is adequate intake/level of vitamin D, and O is pregnancy mortality and morbidity.

#### *2.2. Literature Search*

We searched the PubMed, Cochrane and Embase databases; keywords included "pregnancy," "gestation," "vitamin D," "mortality," "morbidity," and "review." First, we performed a literature search to identify publications eligible for inclusion in PubMed, Cochrane and Embase. Keywords included "pregnancy" OR "gestation" AND "vitamin D" AND "review" AND "mortality" OR "morbidity." The search was limited to human subjects and English- and Spanish-language articles published between 2010 and January 2022. We recovered 43 studies from PubMed, 4 from Cochrane, and 96 from Embase, for a total of 143 studies.

Results were screened in a three-stage process based on title, abstract, and full-text review in duplicate by reviewers at each stage. Study selections were compared and discrepancies were resolved by discussion with N.U. and M.M.-S.-V. Duplicates and studies not meeting selection criteria were removed at each round. Search results were uploaded in Mendeley to remove duplicates, and the reference list was entered in Excel for study selection. The initial screening identified 38 candidates, of which 23 articles met inclusion and exclusion criteria.

The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) flowchart (Figure 1) shows the number of articles at each stage of the screening process.

**Figure 1.** Search and screening strategy for candidate studies.

#### *2.3. Study Inclusion/Exclusion Criteria and Data Extraction*

Studies included in this review met the following criteria: reviews, narrative reviews, clinical review, systematic review, and meta-analysis studies to look at the effects of vitamin D on maternal mortality and morbidity. All studies focused on how vitamin D levels in pregnancy related to maternal mortality and morbidity and were longitudinal in nature. Specific inclusion/exclusion criteria were developed, and only published works meeting all criteria were included. The selection criteria were the following:


After we thoroughly assessed the candidate studies, 23 were included in this metaanalysis. We examined articles to tabulate data, which we summarized under the headings of design, location, vitamin D status, and main findings.

#### *2.4. Quality Assessment*

This systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [23,24]. The systematic review has been registered in the International prospective register of systematic reviews (PROSPERO) (CRD42022343174).

In addition, to ensure that studies reflected the most current science and evidencebased practice, the Scottish Intercollegiate Guidelines Network (SIGN) was used to ensure rigorous assessment of study quality, validity, bias, and possible confounding variables [25]. Using SIGN ensures a robust assessment of a study's validity, including key factors such as bias and confounding. SIGN is based on the principles of evidence-based medicine, an approach that ensures using the most up-to-date, reliable, and scientifically solid evidence available in making decisions about a situation being studied [26].

SIGN establishes levels of evidence and recommendations to describe a given study and its results. Levels of evidence are based on study design and the methodological quality of individual studies. Scores are ranked best to worst using 1, 2, 3, and 4, with those scores further ranked with ++, +, and − signs. Grades of recommendation, rated best to worst as A, B, C, and D, are based on the strength of the evidence on which the recommendation is based and do not reflect the recommendation's clinical importance.

#### **3. Results**

#### *3.1. Study Characteristics*

Our search yielded 143 studies; 23 review studies remained after further screening. Studies were published between 2010 and 2022. They used data from Chile [27], Canada [28,29], Spain [30,31], Pakistan [32], Brazil [33,34], the United States [35–42], Germany [43], Iran [44,45], India [46], Puerto Rico [47], Poland [48], and Australia [49]. The chosen studies were analyzed according to design, location, vitamin D concentration and supplementation, major findings, and SIGN scores. Table 1 summarizes the study characteristics.

Overall, most included studies were deemed high quality. With the SIGN scores, the 23 review articles could be regarded as good quality. The risk of bias was generally low, with at least 75% of judgments assessed as low risk for four domains: blinding of participants, personnel and outcome assessment, selective reporting, and other biases.

#### *3.2. Review and Meta-Analysis Studies*

Twenty-three reviews reported analyses for vitamin D supplementation [27–49]. Vitamin D supplementation is is protective for pregnant women, having a significant effect on the incidence of preeclampsia [27,28,30,33,35,44,46] and reduced risk of preterm births [29], prematurity [30], gestational diabetes [31,39,41], and both maternal and infant infections [40].

Through a meta-analysis of observational studies, we suggest that vitamin D supplementation acts as a protective factor for preeclampsia and prematurity [30]. One metaanalysis [30] reviewed cohort studies evaluating the association between vitamin D and prematurity. Four studies used concentrations <75 nmol/L, and six studies considered concentrations <50 nmol/L as vitamin D cutoff points. Results of the subgroup analysis of cohort studies showed a significant association between maternal vitamin D and preterm birth only for concentrations <75 nmol/L: pooled odds ratio (OR) = 1.56; 95% confidence interval (CI), 1.25–1.94; *I* <sup>2</sup> = 70%; *p* = 0.02. For concentrations <50 nmol/L, pooled OR = 1.09; 95% CI, 0.91–1.30; *I* <sup>2</sup> = 91%; *p* < 0.00001.


*Nutrients* **2022**, *14*, 4124

Although studies showing relation between vitamin D and lower

risk of PE are limited, maternal status of vitamin D seems to

influence risk of developing

supplementation

 in women may improve pregnancy outcomes.

 PE. Therefore, vitamin D

3 C

De Souza and Pisani

2020 [33]

Narrative review

 Brazil

 Status and risk of PE





diabetes mellitus; LCPUFA, long-chain RR = relative risk; SGA, small for gestational age.

polyunsaturated

 fatty acid; PE, preeclampsia;

 PTL, preterm labor; PTB, preterm birth; RCT, randomized controlled trial;

In Oh, Keats, and Bhutta [29], the risk for preterm birth may have been reduced by 36% through vitamin D supplementation (average relative risk (RR) = 0.64; 95% CI, 0.40–1.04; studies = 7), though the upper limit of the confidence interval just crossed the line of no effect. In Tabesh and colleagues [45], a significant association was found between VDD and risk of preeclampsia; however, significant between-study heterogeneity was found (*I* <sup>2</sup> = 52.7%; *p =* 0.03). In Palacios and colleagues [47], data from original studies involving 446 women showed probable gestational diabetes risk reduction with vitamin D supplementation in comparison with no intervention or placebo groups (RR = 0.51; 95% CI, 0.27–0.97; moderate-certainty evidence). In addition, vitamin D supplementation probably makes little or no difference in risk of gestational hypertension in comparison with no intervention or placebo (*n* = 1130; RR = 0.78; 95% CI, 0.41–1.49).

#### **4. Discussion**

The most common causes of maternal mortality are severe bleeding, infections/sepsis, embolisms (blockage in the heart or lungs), stroke (can be a blown blood vessel or an embolus), blood pressure disorders (preeclampsia/eclampsia), and gestational diabetes. According to the WHO, the leading causes of maternal mortality (nearly 75%) are severe bleeding, postnatal infections, blood pressure disorders, and complications of labor and delivery [49]. In the US, the top causes of maternal mortality are hemorrhage, cardiovascular complications, infections/sepsis, embolism (pulmonary embolism or other embolisms), cerebrovascular accidents, and noncardiovascular medical conditions such as gestational diabetes mellitus (GDM) [51].

In this review, we aimed to evaluate the current evidence on the effect of vitamin D and vitamin D supplementation on maternal mortality and morbidity. Interventions yielded significant effects, albeit with sparse evidence in some areas.

In recent years, scientific interest has increasingly focused on the consequences of VDD on pregnant women, in particular, the impact of its deficiency on adverse maternal health outcomes. With all significant effects taken together, vitamin D supplementation was associated with a reduced risk of maternal mortality and morbidity-related outcomes.

Many observational studies did report that vitamin D levels were associated with adverse maternal, fetal, and neonatal outcomes, including increased risk of developing preeclampsia, preterm labor, gestational diabetes, being small for gestational age, low birth weight, an increased rate of Cesarean delivery, and infertility [50]. Since vitamin D RCTs have not yielded much useful information regarding the role and requirements for vitamin D for many health outcomes while observational studies have [52], results from observational studies are highlighted in the following paragraphs.

#### *4.1. Hemorrhage*

Low maternal 25(OH)D concentrations have been found to be associated with an increased risk of postpartum hemorrhage. An observational study from Taiwan involving 600 pregnant women with 25(OH)D concentrations measured in the 36th week of pregnancy found that 25(OH)D below 30 ng/mL was associated with a factor of four-to-five increased risk of postpartum hemorrhage [53].

#### *4.2. Gestational Diabetes*

In a meta-analysis of 31 observational trials, low vitamin D levels increased the risk of gestational diabetes by 49 (OR = 1.49; 95% CI, 1.18–1.89) [54]. Another meta-analysis of 24 observational studies showed similar results [22]. Observational studies also have shown VDD in pregnancy increases the risk of preeclampsia and that vitamin D supplementation, with or without calcium, may reduce that risk [55].

#### *4.3. Pulmonary Embolism or Other Embolism*

The incidence of pulmonary embolism, a common cause of maternal mortality, has been found in many studies to be increased in a state of maternal VDD, and the risk decreased with supplementation [27,28,30,33,35,37,42–46]. Other studies concluded pulmonary embolism was not influenced by supplementation [31] or that the connection was unclear [34].

#### *4.4. Preterm Birth Risk*

Reports have conflicting findings on the role of vitamin D in reducing preterm birth risk. Some studies identified the association of VDD and the inflammatory response with premature rupture of the amniotic membrane and preterm delivery [56,57]. Pooled analysis of four randomized controlled trials in that study showed no significant effect of vitamin D supplementation in preventing preterm birth. Other publications have reported alterations in the cervicovaginal fluid content of vitamin D and vitamin D binding protein as biomarkers of vaginal inflammation and preterm birth risk several weeks before delivery [58]. A review published in 2017 reported that based on 6 vitamin D RCTs, vitamin D supplementation could significantly reduce the risk of preterm birth (pooled RR = 0.57 (95% CI, 0.36–0.91)) and from 18 observational studies that maternal 25(OH)D <20 ng/mL was associated with a pooled OR = 1.25 (95% CI, 1.13–1.38) [59]. The best observational study on preterm delivery to date was conducted at the Medical University of South Carolina [60]. A total of 1064 consecutive pregnant women were enrolled at their first prenatal visit around the 12th to 14th week of pregnancy. The participants included 488 whites, 395 African Americans, 117 Hispanics, 19 Asians, and 29 multiple or other ethnicities. Their serum 25(OH)D concentration was measured, and they were given bottles of 5000 IU vitamin D3 and counseled on how to achieve 25(OH)D >40 ng/mL. Achieved 25(OH)D was also measured during pregnancy. Those who achieved >40 ng/mL had a 62% lower risk of PTB compared to those <20 ng/mL (*p* < 0.0001). There was no effect of race/ethnicity on the outcomes.

The journal literature on vitamin D and maternal mortality is relatively limited. However, there is a reasonable body of literature on the role of vitamin D in reducing the risk of adverse pregnancy and birth outcomes for both the developing fetus and mother, e.g., [61]. The review by Wagner et al. [62] outlines important findings regarding complications, including preterm birth, preeclampsia, and gestational diabetes, as well as adverse effects that appear in early childhood, such as asthma and neurological development. This review also points out that vitamin D regulates gene expression through DNA methylation, which has profound effects on fetal development and life after birth. They point out that pregnant women should supplement with 4000–5000 IU/d vitamin D3 and achieve 25(OH)D concentrations >40 ng/mL.

#### **5. Strengths and Limitations**

An important strength of this review is that it presents an overview of reviews of the effect of vitamin D on the risk of many risk factors for maternal morbidity and mortality during pregnancy. Table 1 can serve as a starting point for those wanting to know the results to date and can help guide future research efforts. The studies included here show significant methodological differences, including mixed ethnicities and genetic reservoirs, countries, times and conditions of vitamin D evaluation, and different brands and qualities of vitamin D supplements among studies. Those factors all contributed to the heterogeneity of the included studies. A limitation is that we may not have been able to access all publications on the relationship between vitamin D and maternal mortality and morbidity during pregnancy because we limited our analysis to studies published in English and Spanish and available through the PubMed, Cochrane, and Embase databases.

#### **6. Conclusions**

Our meta-analysis showed evidence to support vitamin D supplementation as a costeffective public health strategy to minimize adverse maternal health outcomes. Whenever possible, supplementation should be based on initial vitamin D serum levels with the intent to obtain and maintain optimal levels of a minimum of 40 ng/mL throughout

the pregnancy for maximum impact [61]. In venues where testing is not affordable or convenient, innovative evidence-based technologies such as the *Vitamin D Deficiency Risk Assessment Quiz* (beta) and the *Vitamin D\*Calculator* can aid providers of prenatal care in assessing individual VDD risk and calculating an individualized evidence-based loading and maintenance doses based on target optimal blood levels of 40 ng/mL, respectively (GrassrootsHealth.net, accessed 15 September 2022) In light of the results of the present review, further studies should be conducted. Randomized, controlled, blinded vitamin D supplementation trials must be conducted with pregnant women using standard nutrient physiological design criteria to ensure homogeneity of study design [62], including vitamin D levels (baseline and at time of birth) for all participants, to facilitate future systematic review and meta-analyses, In addition, RCT design may not be ideal for vitamin D outcomes studies because vitamin D intake is difficult to quantify from other sources, as well as lack of compliance, which can lead to unclear study results. Alternatively, they could be observational studies with vitamin D supplementation as done at the Medical University of South Carolina [59]. These studies must include large enough sample sizes to permit evaluating the prevalence of maternal mortality and morbidity.

**Author Contributions:** Conceptualization: W.B.G. Methodology: N.U., W.B.G., J.M.S., A.L.-M. and M.M.-S.-V. Writing—original draft preparation: N.U., W.B.G. and M.M.-S.-V.; writing—review and editing: N.U., W.B.G., J.M.S., A.L.-M., B.S.S. and M.M.-S.-V. All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** W.B.G. receives funding from Bio-Tech Pharmacal Inc. (Fayetteville, AR, USA). The other authors declare no conflict of interest.

#### **References**


## *Review* **Targeting the Platelet-Activating Factor Receptor (PAF-R): Antithrombotic and Anti-Atherosclerotic Nutrients**

**Rajendran Harishkumar 1,2, Sakshi Hans 1,2, Janelle E. Stanton 1,2, Andreas M. Grabrucker 2,3,4, Ronan Lordan 5,6,7,\* and Ioannis Zabetakis 1,2,3,\***


**Abstract:** Platelet-activating factor (PAF) is a lipid mediator that interacts with its receptor (PAF-R) to carry out cell signalling. However, under certain conditions the binding of PAF to PAF-R leads to the activation of pro-inflammatory and prothrombotic pathways that have been implicated in the onset and development of atherosclerotic cardiovascular diseases (CVD) and inflammatory diseases. Over the past four decades, research has focused on the identification and development of PAF-R antagonists that target these inflammatory diseases. Research has also shown that dietary factors such as polar lipids, polyphenols, and other nutrient constituents may affect PAF metabolism and PAF-R function through various mechanisms. In this review we focus on the inhibition of PAF-R and how this may contribute to reducing cardiovascular disease risk. We conclude that further development of PAF-R inhibitors and human studies are required to investigate how modulation of the PAF-R may prevent the development of atherosclerotic cardiovascular disease and may lead to the development of novel therapeutics.

**Keywords:** platelet-activating factor; platelet-activating factor receptor; polar lipids; antithrombotic activity; inflammation; atherosclerosis

#### **1. Introduction**

Atherosclerotic cardiovascular diseases (CVD) are the leading cause of morbidity and mortality globally [1]. Various factors contribute to the development of atherosclerosis, but evidence in recent decades has demonstrated that nutrition plays a pivotal role in the prevention of atherosclerosis and other chronic inflammatory conditions including diabetes and obesity [2,3]. Hence there is a requirement to research the effects of diets and food components on cardiovascular health.

Atherosclerosis is a progressive inflammatory disease responsible for the development of atherothrombotic complications including myocardial infarction, peripheral artery disease, and ischaemic or transient stroke among other cardiac manifestations [4,5]. Atherosclerosis develops through several steps including endothelial dysfunction followed by the deposition of lipids in the intima, which accumulate in the lining of blood vessels. These lipids are then engulfed by macrophages, which eventually undergo apoptosis forming foam cells and a necrotic core that leads to the development of the characteristic lesions or fatty streaks in blood vessels. Erosion of these lesions or plaques causes microruptures that

**Citation:** Harishkumar, R.; Hans, S.; Stanton, J.E.; Grabrucker, A.M.; Lordan, R.; Zabetakis, I. Targeting the Platelet-Activating Factor Receptor (PAF-R): Antithrombotic and Anti-Atherosclerotic Nutrients. *Nutrients* **2022**, *14*, 4414. https:// doi.org/10.3390/nu14204414

Academic Editor: Hayato Tada

Received: 12 September 2022 Accepted: 18 October 2022 Published: 20 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

activate platelets causing fibrin netting and platelet aggregates to form on the inner walls of arteries, thus leading to the narrowing of blood vessels affecting blood supply [6]. With time, the lumen may narrow and erode further causing plaque rupture, leading to a major cardiovascular event such as myocardial infarction or stroke. The main mechanistic events that lead to these events are characterised by persistent low-grade inflammation [5].

However, inflammation is a necessary physiological response of the innate immune system, and its main role is to maintain a constant internal environment despite being subjected to constantly changing environmental pressures. These can include mechanical, physical, chemical, infectious, immunological, or reactive natural adverse events. The inflammatory response seeks to diminish and/or minimize the agents that causes tissue damage, promote adequate wound healing, and restore tissue homeostasis. However, if the inflammatory response fails to resolve owing to the persistence of the triggering factors or poor restoration of the original tissue, a prolonged underlying inflammatory process arises, leading to increased tissue dysfunction and adverse effects. At the molecular and cellular level, it has been postulated that endothelial dysfunction leading to systemic inflammation appears to be the primary underlying mechanistic factor in the onset and progression of atherosclerosis [7]. Endothelial dysfunction is often defined by an inflammatory microenvironment that acts on leukocytes and endothelial cells via interactions with other immune cells such as T lymphocytes, mast cells, dendritic cells (DC), and platelets [8].

Platelets play a key role in the onset and development of atherosclerosis [9–13]. Platelets also orchestrate the development of obstructive thrombi in the latter stages of the atherosclerotic process in response to plaque rupture through the sequential processes of haemostatic responses to vascular injury such as initiation, extension, and stabilization [14]. Each of these stages contains pro-haemostatic molecular mechanisms, in balance with antihaemostatic processes, which restrict the reaction to the damage site and prevent inappropriate vascular occlusion. The molecular players involved in the initiation process include adhesion molecules, signalling ligands, and their associated platelet surface receptors [15]. Strong inflammatory and prothrombotic mediators such as platelet-activating factor (PAF) play pivotal roles in these processes, particularly in the activation of platelets [16]. Indeed, PAF and its receptor have previously been investigated as a pharmaceutical target for some inflammatory conditions including asthma and sepsis with limited success to date. They have also been implicated in many of the key processes that lead to the development of atherosclerosis. However, researchers over the years have postulated that dietary PAF-R antagonists may affect PAF-related signalling and inflammatory pathways [7,17,18]. This has opened several avenues of research that aim to investigate certain dietary patterns such as the Mediterranean diet, which is thought to offer protection from atherosclerotic cardiovascular disease and other inflammatory diseases due to a high concentration of these compounds in the diet [18,19]. In this review, we examine the role of various nutrients and their effects on PAF and its receptor PAF-R and how attenuating this inflammatory and thrombotic pathway may contribute to atherosclerosis prevention via altering one's diet. It is also important to recognise that while this review largely focusses on the relationship between PAF and the PAF-R, there are also ongoing developments in cardiovascular research relating to the metabolic enzymes of PAF, which have been discussed at length elsewhere [7,20].

#### **2. Platelet-Activating Factor (PAF) and PAF-Receptor (PAF-R)**

PAF (1*-O*-alkyl-2-acetyl-s*n*-glycero-3-phosphocholine) is a phospholipid mediator that functions through the PAF-receptor (PAF-R). PAF was discovered when Ig-E sensitised basophils of rabbits were challenged with antigen stimuli [21]. In physiology, PAF is an important signalling molecule in the renal, cardiovascular, immune, and reproductive systems. However, PAF is not just one molecule; there happens to be a family of PAFlike lipids (PAFLL) or PAF-like moieties, which all have varying degrees affinity with the PAF-R leading to various levels of potency [22]. The classic PAF molecule has an alkyl ether linkage at the *sn*-1 position, a characteristic acetyl group at the *sn*-2 position, and a

phosphocholine group at the *sn*-3 position of the glycerol backbone [23]. The most potent PAF molecules contains a 16:0 at the *sn*-1 position, but may also have 18:0, 17:0, and 18:1 on the alkyl ether-linked side chain leading to varying degrees of affinity for the PAF-R and as a consequence, varying degrees of biological activity [7,24]. PAF is known to carry out its biological activities at concentrations as low as 10−<sup>12</sup> M and almost always by 10−<sup>9</sup> M as an intercellular messenger [25] and it carries out its functions in a autocrine, juxtracrine, and paracrine manner [26,27]. The history of the elucidation of the PAF structure and developments in the field has recently been reviewed [28].

The PAF-R is expressed by cells in various tissues, including the lungs, spleen, heart, kidneys, skeletal muscle, and in blood cells as shown in Figure 1 [24]. Therefore, it is also unsurprising that PAF-R signaling is implicated in many physiological processes [28]. There is an abundance of phospholipids in the brain and central nervous system (CNS) [29], where the PAF-R is expressed by various parts of the CNS including the spinal cord, substantia niagra, hypothalamus, hippocampus, frontal cortex, nucleus accumbens, cortex, cerebellum, cerebellar hemisphere, basal ganglia, and the amygdala [30]. Notably, PAF is also synthesised by neuronal tissue and its signaling is associated with neurotrophic effects [31]. Indeed, permeability of the blood-brain barrier (BBB) increases via PAF-R dependent mechanisms, consequent to calcium (Ca2+) influx, increased nitric oxide levels, and alterations to proteins that regulate intercellular gaps in the BBB *in vivo* [32].

**Figure 1.** Bulk gene expression for the platelet-activating factor receptor (PAF-R) encoded by the gene *PTAFR* in various human tissues using data from the Genotype-Tissue Expression (GTEx) Project [30]. The expression data is shown in transcripts per million (TPM) with the plots showing the median and the 25th and 75th percentiles. Dots indicate outliers, which are above or below 1.5 times the interquartile range.

PAF-R signalling also plays a prominent role in reproductive biology, including ovulation, fertilisation, preimplantation, and parturition in women. In men, PAF is present in spermatozoa and is thought to be involved in sperm motility and in the induction of acrosome reactions [33–38]. PAF and PAF-R is also a known physiological mediator of healthy cardiovascular function via modulating inflammatory signaling, platelet function, and blood pressure [39–41]. As the name suggests, PAF is a platelet activator via binding to the PAF-R in the normal response to injury [13]. PAF-R binding by PAF induces platelet shape change and the release of platelet granules via stimulation of the phosphatidylinositol cycle and intracellular Ca2+ mobilization. Serotonin and platelet factor 4 are secreted, along

with arachidonic acid and other bioactive lipids, including PAF, which mediate platelet aggregation [13,42,43].

While we are still learning about the roles of PAF and PAF-R in physiology, PAF is mostly known for its role as an inflammatory messenger that passes signals to cell types such as platelets, neutrophils, endothelial cells, macrophages, and lymphocytes [7]. PAF is involved in multiple communicable and non-communicable diseases through excessive binding with the PAF-R. Some studies have shown that PAF mediates metastasis in tumour cells. For example, PAF triggers human melanoma cells via stimulating the phosphorylation of cAMP-responsive element (CRE)-binding protein (CREB) and activating transcription factor-1(ATF-1). This signal transduction leads to the overexpression of major effectors involved in tumour growth, angiogenesis, and malignant progressions such as MMPs, STAT-3, and NF-*κ*B [44]. PAF also affects other pathological processes including increased vascular permeability, hypotension, ulcerogenesis, bronchoconstriction triggering airway hyperresponsiveness, and platelet degranulation. PAF has also been implicated in septic shock, asthma, ischemia/ reperfusion injury, pancreatitis, inflammatory bowel disease, and rhinitis [45]. PAF-R activation has also been reported to manifest in communicable diseases. For example, PAF-R activation causes increased thrombocytopenia, haemoconcentration, increased systemic levels of cytokines, and lethality in wild-type mice compared with PAF-R-silenced mice in a model of dengue fever [46]. PAF is implicated in other infectious diseases characterised by inflammation including human immunodeficiency virus (HIV) [47] and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [48,49]. Considering the vast pathological functions of PAF and its receptor, many investigations have focused on preventing PAF from binding to the PAF-R with the aim of reducing prothrombotic and proinflammatory signalling.

Structurally, the PAF-R is a seven transmembrane G-protein coupled receptor encoded by the *PTAFR* gene. The gene locus has been identified in humans as chromosome 1p35-p34.5. Human and guinea pig PAF receptors are single polypeptides with 342 amino acids; rat and mouse PAF receptors omit one amino acid in the third extracellular loop. Despite various findings to the contrary, it is presently believed that a single receptor subtype mediates all PAF's actions in humans and is generally located on the plasma membrane, endomembrane, nucleus, and nuclear envelope [18,50]. The gene, *PTAFR*, is tightly regulated by two distinct promotors that are involved in the transcriptional regulation, consequently there are two alternatively spliced transcripts that differ in their untranslated regions. The first, transcript 1, is widely expressed in tissues regulated by inflammation, predominantly in leukocytes, macrophages, eosinophils, and monocytes. The second, transcript 2, is found in organs such as the heart, kidney, lung, and spleen and its expression can be influenced by oestrogen, thyroid hormone T3, retinoic acid, transforming growth factor-β (TGF-β), tumour necrosis factor-α (TNF-α), interferon-γ and others. The second transcript is not thought to be expressed by hematopoietic cells or in the brain [24,51,52]. In a positive feedback manner, PAF may upregulate the expression of its own receptor via transcript 1 through NF-κB signalling [53]. There is also evidence that PAF-R transcription is dependent on activation of the Jak/STAT pathway [51]. The upregulation of PAF synthesis and its degradation is also tightly regulated and has been extensively reviewed [16]. However, there are many aspects of PAF-R (*Ptafr*) expression that have been underexplored including whether it exhibits circadian rhythmicity as some data indicates that it might, along with genes associated with PAF metabolism, including *Pla2g7* (Circadb: Circadian Expression Profiles Data Base. Available online: http://circadb.hogeneschlab.org (accessed 12 October 2022)). Considering the PAF-R is expressed in numerous cells and tissue types there is a lot to left to be explored regarding its function and modulation.

The first binding experiment of PAF was conducted on human platelets in 1982, whereby two distinct binding sites were revealed. The first site had shown higher affinity (Kd value = 37 ± 13 nm) and the other site possessed nearly low affinity toward PAF [54]. To understand the pathophysiological function of PAF, gene modifications were applied in earlier studies. For example, cDNA encoding the PAF-R was isolated from the guinea

pig lung cDNA library and was cloned into *Xenopus laevis* oocytes depicted in Figure 2. In this cloned receptor, several amino acids are highly conserved when compared to other G protein receptors, including aspartic acid (Asp) in the second transmembrane segment, one cysteine (Cys) in both the second and third intracellular loops, and three proline (Pro) in the sixth and seventh segments. The PAF receptor's cytoplasmic tail comprises four serines (Ser) and five threonines (Thr). There is a total of 12 tyrosine (Tyr) residues, with two of them located in the cytoplasmic loops. Asparagine (Asn) residues are found on the receptor's exterior surface and may serve as sites for glycosylated residue attachment [55]. Some other reports stated that cloning of human PAF receptors can be achieved by isolating cDNA from peripheral leukocytes, heart, and EoL-1 eosinophilic leukaemia cells [56]. Figure 1B shows the helical 3D structure of PAF-R (Chain-A) that was obtained from the protein data bank (PDB ID: 5ZKQ) and its bound ligands were removed by UCSF Chimera [57].

**Figure 2.** (**A**) Diagrammatic representation of the platelet-activating factor receptor (PAF-R) with its seven transmembrane domains within the plasma membrane bilayer [Note: PAF-R cloned from guinea pig represented with amino acid residues]; (**B**) Human PAF-R (Chain-A) with selective amino acid residues (PDB ID: 5ZKQ).

#### *PAF and PAF-R Activation in Inflammatory Diseases*

Elevated levels of PAF can be detected in tissues affected by inflammatory diseases [7]. Excessive activation of the PAF-R via PAF and PAF-like lipids (PAF-LL) in inflammatory diseases induces several biological effects including systemic pro-inflammatory, prothrombotic, and pro-proliferative signalling. Indeed, delayed immune responses have also been reported and PAF-R signalling has been implicated in cancer development. Many malignant cells have been shown to overexpress PAF-R [58]. The PAF-R receptor is related to phosphoinositide metabolism via a G-protein that is also linked to phospholipases C and A2. PAF-R stimulation results in the brief synthesis of diacylglycerol, which activates protein kinase C, and inositol triphosphate, which triggers the release of internal calcium reserves [59]. The activation of PAF-R through PAF is represented the Figure 3.

PAF increases tyrosine phosphorylation of several proteins in neutrophils, macrophages, and platelets, as well as nuclear factor kappa B (NF-*k*B) activation and transcription of *c-fos* and c*-jun* in inflammatory cells. PAF can activate the mitogen-activated protein kinase (MAPK) kinase-3, a known activator of p38 MAPK, and the Jak/STAT pathway [59]. Following ligand activation, the PAF-R is degraded through both the proteasome and lysosomal pathways.

**Figure 3.** Mechanism of PAF-R activation and PAF-mediated signalling pathway [59,60]. Abbreviations: cPLA2—cytosolic phospholipase A2; DAG—Diacylglycerol; ER—Endoplasmic reticulum; InP2 —inositol 4,5-bisphosphate; InsP3—inositol 1,4,5-triphosphate; LPCAT—lysophosphatidylcholine acyltransferase; PKC—protein kinase C; PLA2—Phospholipase A2; PLC—Phospholipase C; PtdInP2 phosphatidyl 4,5-bisphosphate; TxA2—thromboxane A2.

While platelet activation leads to aggregation as part of normal haemostatic function, under acute or systemic inflammatory conditions PAF-R activation has been shown to induce various immune and inflammatory pathways [44] that can lead to both acute and chronic conditions. For example, PAF-R activation by PAF induces histamine and prostaglandin D2 release from mast cells [61,62] and it is involved in the chemotaxis of mast cells [63]. PAF has been shown to be a powerful chemoattractant for eosinophils [64,65] and it is responsible for the generation of chemokines and prostaglandins [65–67]. PAF along with leukotriene B4 (LTB4) and matrix metalloproteinase-9 (MMP-9) are involved in the accumulation of eosinophils in asthmatic airways via interleukin-8 (IL-8) stimulation of neutrophils [68]. Indeed, PAF has been shown to promote the recruitment of neutrophils and polymorphonuclear cells to inflammatory sites [7], and these cells can also generate PAF [7,69,70], which is thought to be one of the underlying mechanisms by which conditions such as atherosclerosis may propagate [7,71].

As a consequence of the wide-ranging inflammatory actions of PAF and the PAF-R, pharmaceutical companies and scientists have previously investigated the use of PAF inhibitors and developed pharmaceutical grade products to target these inflammatory pathways. These include products such as Lexipafant [72], Modipafant [73], and Rupatadine [74,75] among others that have previously reviewed [28] for the treatment of asthma, sepsis, and other conditions characterised by PAF-related inflammation. However, a recent study has shown that PAF and PAFLL can mediate nucleotide-binding domain, leucinerich-repeat-containing protein 3 and never in mitosis A-related kinase 7 (NLRP3-NEK7) inflammasome induction in a PAF-R independent manner, which may explain observations

of the ineffectiveness of many PAF-R antagonists [76] including those aforementioned. These findings may lead to further developments in our understanding of the role of PAF in diseases such as cancer and atherosclerosis considering the important role of the inflammasome in these diseases. Pharmaceuticals aside, research has also determined that there are a broad range of naturally occurring PAF-R antagonists present in certain edible plants and foods, which will be discussed in the ensuing sections.

#### **3. Antiplatelet Properties of Nutrients**

Diet has long been associated with the maintenance of health and the prevention of disease. It is well established that healthy dietary patterns, such as the Mediterranean diet and the dietary approaches to stop hypertension (DASH) diet, may offer protection against the development of atherosclerosis and cardiovascular diseases [77,78]. With this knowledge, the functional foods, dietary supplements, and nutraceuticals industries have grown exponentially over the last two decades offering individuals food-derived and natural product derived constituents that may confer health benefits on the consumer [79]. Historically, many cultures turn to food and natural products as a source of healing in times of ill health. These practices are particularly prevalent in areas with indigenous rural communities. The World Health Organization (WHO) defines traditional medicine as "the total of knowledge, skills, and practices based on theories, beliefs, and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as the prevention, diagnosis, improvement, or treatment of physical and mental illness" [80]. Some traditional medicine systems are supported by substantial literature and recordings of theoretical notions and practical abilities; others are passed down verbally from generation to generation. Until now the turn of the 20th century, the majority of the world's population relied on their own traditional medicine to satisfy their primary health care requirements in several regions of the world [81]. Traditional medicine is commonly referred to as "complementary and alternative medicine" when practised outside of its traditional culture [82]. Traditional medicine is most popular and practiced nowadays in China, India, and many African nations among others [83].

In India, the traditional system of medicine (TSM) has been practiced before the adoption of modern medicine by traditional communities to heal any type of illness. These medical practices provide invaluable assistance in the healthcare system for current and future generations. The traditional systems of Indian medicine, presently known as the Indian System of Medicine (ISM), have a very solid conceptual foundation and have been practiced for a very long period. Ayurveda, Siddha, and Unani are three prominent traditional systems practiced in India [84]. Some Indian medicinal plants are reported to have antihyperlipidemic activity and anti-thrombolytic because of their antiplatelet aggregation activity and fibrinolytic activity [85]. Phytocompounds such as cudratricusxanthone A [86], withaferin A [87], and even some of the serine proteases were identified and tend to prevent clot formation [88].

Over the past three decades, there has been considerable research conducted investigating the potential antithrombotic and anti-inflammatory effects of dietary PAF inhibitors. In particular, there has been a focus on polar lipids, mainly phospholipids and sphingolipids, derived from natural sources such as plants and animal products, which exert antithrombotic activities due their inhibition of the PAF-R activation and other platelet agonists [28,89,90]. A recent study found that dietary supplementation with plant extract containing aloe gel, grape juice, green tea extract, etc. reduced platelet sensitivity upon stimulation with PAF [91]. In this study, it is not clear what constituent or combination of constituents are responsible for these observed effects. However, many compounds such as polyphenols, phenolipids, and polar lipids present in these capsule constituents have previously been associated with antiplatelet effects. For example, certain compounds isolated from *Spirulina* (blue-green algae) and other marine algae also possess bioactive properties beneficial to health, including antiplatelet, anti-inflammatory and antioxidant qualities. These qualities have been traced to the glycolipid sulphoquinovosyl diacylglycerol (SQDG) present in photosynthetic plants [92,93].

However, plants are not the only food-derived antithrombotic polar lipids. Fishderived lipids also exhibit inhibitory properties against PAF. Polar lipid fractions isolated from cod (*Gadus morhua*) and salmon (*Salmo salar*) showed platelet inhibitory capabilities, suggesting that the consumption of such lipids could protect against cardiovascular disease [90,94]. Other animal foods, such as dairy products, notably yoghurt, also exhibit inhibitory activities against PAF *in vitro* [95,96]. In humans, intake of yoghurt enriched with polar lipids from olive oil by-products resulted in lower platelet sensitivity against PAF, and reduced low-grade inflammation, assessed by monitoring serum levels of IL-10 and IL-6 [97].

Many compounds derived from traditional herbal remedies also possess potent anti-PAF activity. Curcumin is a spice derived from turmeric and commonly used in Asian cuisines. A 1999 study found that curcumin inhibits platelet aggregation induced by agonists such as PAF, epinephrine, and ADP, via the inhibition of thromboxane production and Ca2+ signalling [98]. Another investigation found that extracts of several species of Malaysian medicinal plants exhibited significant inhibitory activity against PAF [99]. The Korean folk medicinal plant *Alpinia officinarum* is traditionally used to treat gastrointestinal diseases. Diarylheptanoid compounds were isolated from this plant and also showed a high inhibitory effect against platelet aggregation by PAF [45]. Apart from medicinal plants, plants that are commonly found in various diets also possess bioactive compounds with antithrombotic activities with various target mechanisms as listed in a Table 1.

**Table 1.** Comparison of different studies investigating phytocompounds and their antithrombotic activities against PAF andother platelet agonists.






146


**Abbreviations:** ADP, adenosine diphosphate; AA, Arachidonic acid; COX2, Cyclooxygenase-2; HUVEC, human umbilical vein endothelial cells; IC50, 50% inhibitory concentration; 6-keto-PGF1α, 6-keto prostaglandin F1α; MUFA, Monounsaturated fatty acids; NL, neutral lipids; OBL, *Ocimum basilicum* L; PAF, Platelet activating factor; PGE2- Prostaglandin E2; PL, polar lipids; PUFA, Polyunsaturated fatty acids; TL, Total lipids; TXB2, Thromboxane B2; TRAP, Thrombin receptor activator peptide.

#### **4. Antiplatelet Properties of Polar Lipids**

Polar lipids are amphipathic in nature, possessing both a hydrophilic head group and a hydrophobic tail. Polar lipids are key structural components of cellular membranes, and they play a role in signaling cascades with membrane proteins [128]. Polar lipids are mostly phospholipids and sphingolipids. In contrast, neutral lipids are non-polar and hydrophobic. Neutral lipids include triacylglycerols, cholesterols, waxes, fatty acids, and esters [129]. Polar lipids have been identified as PAF inhibitors that interact and inhibit the PAF-R through various mechanisms, both direct and indirect, as previously reviewed [7]. In contrast, neutral lipids mostly do not exhibit potent antiplatelet activities [130]. In the following sections we discuss the existing evidence involving *in vitro*, *in vivo*, and *ex vivo* studies that investigate the potential anti-PAF properties of polar lipids.

#### *4.1. In Vitro Studies of Platelet-Activating Factor Receptor (PAF-R) Antagonists*

Several *in vitro* studies have been published that reported that polar lipids exhibit antiplatelet properties likely mediated by interactions between the PAF-R. These polar lipids tend to be mostly researched in foods of animal origin, particularly dairy and marine sources. In dairy, it has been reported that the beneficial properties of polar lipids may be altered or enhanced by fermentation of the dairy product. Fermented dairy products, such as yoghurt and cheeses have also been noted for their high inhibitory activity against PAF and other agonists. Many fermented foods that are traditionally part of the Mediterranean diet are rich in omega-3 polyunsaturated fatty acids that support cardiovascular health [131]. Cheeses made from goat's or sheep's milk are an important part of the Greek diet. For example, the traditional Greek cheeses Kefalotyri and Ladotyri have strong inhibitory activity against PAF-induced platelet aggregation [96]. Certain bacterial cultures, such as *Lactobacillus acidophilus* and *Streptococcus thermophilus* can increase the bioactivity of ovine yoghurt milk and alters its anti-thrombotic activity in presence of PAF [132]. These starter cultures are capable of producing and altering bioactive polar lipids by some mechanism, possibly by producing antimicrobial peptides known as bacteriocins which can alter the fatty acid composition. The bacterium *L. acidophilus* has been shown to reduce PAF-induced inflammatory response in human intestinal cells [133]. A similar investigation [134] found that fermentation increases the antithrombotic properties of bovine dairy and plant-based dairy alternative drinks. Homemade dairy alternatives prepared from almond, coconut and rice and bovine dairy milk showed significantly higher antiplatelet activity against PAF, in comparison to their non-fermented counterparts, with the rice-based drink displaying the strongest inhibitory activity.

Other sources of polar lipids include marine sources such as fish and algae [135]. Marine omega-3 PUFA are derived from fish, krill, and roe (fish eggs) and possesses significant antiplatelet activity [136], which may be more bioavailable in polar lipid forms. Polar lipid fractions isolated from codfish (*Gadus morhua*) showed platelet inhibitory capabilities, suggesting that consumption of such lipids could protect against cardiovascular disease [94]. Significant quantities of unused fish by-products by-catch and are generated from the fishing industry, including salmon heads, herring heads and off cuts, and boarfish. While these by-products and by-catch are conventionally regarded as undesirable, valorisation of their antithrombotic and cardioprotective properties could establish these products as important bioactive functional foods [137]. In a 2019 study, polar lipids derived from bycatch and by-products of these fish were assessed for their antiplatelet activity against various platelet agonists, and they exhibited strong inhibitory activities against PAF, thrombin, collagen, and ADP [89]. Another study focusing on salmon [90] demonstrated the potent *in vitro* antithrombotic effects of a food-grade polar lipid extract (FGE) prepared from salmon (*Salmo salar*) fillets in human platelets, in the presence of the platelet agonists PAF and thrombin. Among the lipid subfractions, phosphatidylcholines (PC) and phosphatidylethanolamines (PE) showed the strongest inhibitory capacity against PAF in human platelets. A later investigation found that salmon cooked *sous vide* at higher temperatures (80 ◦C and above) significantly reduced these antithrombotic properties, along with decreased PUFA content in salmon prepared without brining [138].

Another rich animal source of polar lipids is eggs. Egg yolks are a rich source of sphingomyelin, lysophosphatidylcholine (L-PC), and lyso-phosphatidylethanolamine (L-PE), along with other nutrients including protein, vitamins, and minerals [139,140]. Cagefree, organic, and daily fresh eggs were assessed to determine if their polar lipids exhibited antiplatelet properties. Out of the three varieties, lipid fractions from cage-free eggs showed the highest inhibition against PAF, owing mainly to the polar lipid component of the total lipid fraction [140]. Significant advances in poultry science have led to the natural fortification of eggs to contain higher levels of PUFA. It would be interesting to assess whether PUFA-rich eggs have different polar lipid compositions with even more effective antiplatelet properties considering the other potential cardioprotective effects that have been documented [141].

Overall, it appears that animal sources of polar lipids including dairy, meat, and egg products exhibit antithrombotic effects (Table 2). However, it should be noted that lipids sourced from non-animal sources such as vegetable oils are also known for their cardioprotective and antithrombotic properties, especially olive oil. A 2002 investigation [127] compared the *in vitro* antiplatelet properties of olive oil and other seed oils (sunflower, corn, sesame, and soybean) against PAF. Out of all the polar lipid samples, olive oil was the most bioactive and inhibited both PAF and thrombin in washed rabbit platelets [127]. Indeed, olive oil and related by-products have also been shown to affect PAF metabolism [142].

**Table 2.** Comparison of in vitro studies investigating dairy and marine lipids possessing antithrombotic activity against PAF and other platelet agonists.



**Abbreviations:** ADP, adenosine diphosphate; TNL, total neutral lipids; PAF, platelet-activating factor; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PL, polar lipids; TL, total lipids; TPL, total polar lipids; WRP, washed rabbit platelets.

#### *4.2. Ex Vivo and Human Studies*

*Ex vivo* and human studies are important to conduct to gain an understanding of how polar lipids affect platelet and cardiometabolic homeostasis. Certain populations in which the local diet is rich in omega-3 PUFA, such as the Greenland Eskimos [145] and Mediterranean people [146] exhibit a lower rate of cardiovascular diseases. It has been speculated that dietary components such as polar lipids or PUFA may contribute to the observed benefits of these diets. As aforementioned, marine lipid sources, notably polar lipids and potentially PUFA sourced from oily fish species, exhibit antiplatelet activity. A 2019 crossover study involving healthy human volunteers found that intake of enriched marine oil supplements resulted in reduced platelet and leukocyte activation, among other beneficial effects on immune cell functioning [147]. However, similarly to the *in vitro* studies presented, foods and food derivatives other than marine sources exert antithrombotic effects.

A recent investigation found that intake of yoghurt enriched with polar lipids from olive oil by-products resulted in lower platelet sensitivity against PAF and reduced lowgrade inflammation, which was assessed by monitoring serum levels of IL-10 and IL-6 [97]. Alcoholic beverages are also known to contain anti-inflammatory and antithrombotic properties against PAF and other platelet agonists [148,149]. A crossover study found that the intake of Cabernet Sauvignon red wine and Robola white wine results in decreased postprandial platelet activity against PAF in human platelet-rich plasma (PRP) [150]. In this study, healthy male volunteers were provided with a standardized meal along with portions of either wine, ethanol solution or water, following which plasma samples were obtained at multiple time points. Platelet sensitivity against PAF was significantly affected following the intake of either red or white wine, compared to samples after intake of water in place of wines. Indeed, a related study investigated the consumption of wine and its

effects on PAF metabolism and found that wine beneficially decreases the biosynthesis of PAF [151]. Collectively, these finding contribute to a growing body of literature that indicates there are bioactive constituents including polar lipids in alcoholic beverages such as wine [152] and beer [153]. Results from examples of these *ex vivo* studies are presented in Table 3.

**Table 3.** Studies investigating the *ex vivo* antiplatelet properties of animal lipids and alcoholic beverages.


#### *4.3. PAF Modulation by Micronutrients*

Several dietary micronutrients such as vitamins, trace minerals and elements have exhibited anti-inflammatory, antithrombotic [154,155], and antioxidant functions [156] (Table 4). Among those, carotenoids, one of the main sources of vitamin A, are highly bioactive, with antioxidant, anti-inflammatory, and immunoregulatory properties [156,157]. The other form of vitamin A, retinol is known to affect PAF-R expression [158]. Vitamin E has also been linked to the metabolism of PAF and is capable of regulating platelet function [159]. A deficiency of vitamin E (alpha-tocopherol) was shown to stimulate the biosynthesis of PAF in rat polymorphonuclear leukocytes [160]. A study involving pregnant women found that oral supplementation with alpha-tocopherol inhibits platelet aggregation induced by ADP and PAF, using a range of concentrations from 6.55–500 mg/mL [161]. However, yet another *ex vivo* study in male volunteers found that short-term vitamin E supplementation does not significantly affect platelet function or phospholipase A2 (PLA2) and lyso-PAF activity [162], enzymes involved in PAF metabolism.

Vitamin D is a fat-soluble vitamin that exists in two major forms, namely cholecalciferol (D3) and ergocalciferol (D2). It is typically associated with bone and calcium homeostasis, and the risk of developing diseases such as osteoporosis and rickets [163]. However, vitamin D has diverse physiological functions and is involved in inflammatory and procoagulatory pathways in the body due to its important role in immune function [164]. A randomized study found that vitamin D supplementation can reduce platelet-mediated inflammation and oxidative stress in diabetic patients [165]. Vitamin D can also regulate haemostasis, and its deficiency is associated with increased platelet aggregation in the presence of the agonist ADP [166]. An *in vitro* experiment demonstrated that 25-hydroxyvitamin D, a metabolite of vitamin D, attenuated increased expression of *PTAFR* in a human respiratory epithelial cancer cell line in response to rhinovirus infection [167], indicating that vitamin D might

regulate PAF-R expression. It has also been hypothesized that vitamin D may attenuate PAF signalling in other viral infections via the PAF-R such as in SARS-CoV-2 infection and coronavirus disease 2019 (COVID-19) [168]. Indeed, paricalcitol, a vitamin D analogue, is a known PAF-inhibitor as demonstrated *in vitro* and *in vivo* [169].

Vitamin C is a water-soluble vitamin abundantly found in plant sources such as citrus fruits and leafy vegetables. In addition to its well-documented roles in immune function and wound healing, vitamin C possesses antioxidant and antiplatelet functions [170]. In an *ex vivo* study, the addition of vitamin C effectively halted platelet aggregation and scavenged reactive oxygen species (ROS) in human platelets [171]. Another study found that dietary supplementation with vitamin C prevented the accumulation of PAF-LL agonists and cigarette-smoke-induced platelet adhesion and aggregation [172]. This also has important implications for vitamin C supplementation as a dietary intervention to reduce the risk of cardiovascular disease linked to smoking. These findings are in accordance with studies in rabbits that have shown that vitamin C downregulates PAF and PAF-LL and improves postischemic oxidative and inflammatory responses [173].

**Table 4.** Studies investigating the *in vitro*, *in vivo*, and *ex vivo* antiplatelet properties of micronutrients.


#### **5. Importance of Essential Trace Metals on PAF-R Targets**

Dietary trace metals are principal components and regulators of various metabolic processes in the body. These elements form only 5% of the average human diet and are typically required in doses of 1–100 mg daily in adults [177]. Trace elements such as zinc (Zn), and copper (Cu) have been shown to affect platelet function in health and disease, but these elements may also affect the PAF pathways. Deficiencies in the trace element Se have been shown to upregulate PAF production in human [178] and bovine endothelial cells [174], by enhancing the activity of two important enzymes involved in the remodelling pathway of PAF biosynthesis, PLA2 and lyso-PAF-AT.

Zinc (Zn2+) is a known antioxidant and anti-inflammatory agent [179]. In rat models, zinc deficiency studies have shown a decrease in platelet aggregation and impaired reactivity to agonists, including ADP and thrombin [94,137,138]. Furthermore, recent studies have shown that altered levels of zinc impact platelet reactivity in zinc deficient conditions [180]. Chelation of intracellular zinc can also inhibit the tyrosine phosphorylation cascade, which reduces platelet reactivity and aggregation *in vitro* [181]. In turn, increased dietary zinc increases platelet responses to ADP and thrombin in human plasma [180]. In line with this, zinc supplementation of 50 mg Zn/day demonstrated increased platelet reactivity and serum zinc levels in humans [182]. Zinc supplements have also been shown to decrease oxidative stress and the production of inflammatory cytokines in elderly individuals [179]. The role of zinc in platelet aggregation has, however, not been fully elucidated and some studies also suggest a direct inhibitory role of zinc. It has been suggested that zinc interacts with PAF at the functional receptor site or contiguous site due to its specific inhibition of PAF-induced platelet activation [183]. A further study has shown that zinc levels must be inversely proportional to PAF levels to carry out these inhibitory effects [184]. Additionally, zinc must be present before PAF exposure. This suggests that PAF and receptor binding may be limited by zinc and phospholipid (PAF) interaction [143,144]. This model is supported by zinc's ability to bind to phospholipids in a 2:1–1:1 complex, particularly to the negatively charged phosphate groups [185].

Like zinc, copper is an essential trace metal for the human body. The delicate balance of copper levels in the body is crucial to maintaining terminal oxidation, elimination of free radicals, and iron metabolism [186]. Several studies have shown the effects of altered copper levels on platelet aggregation and thrombin activity. For example, a study using mice subjected to copper deficient diets demonstrated a significant increase in prothrombin time, a parameter used to evaluate blood clotting [187]. This was followed by another study in rats fed a copper-deficient diet (0.3 μg copper/g of diet), which demonstrated impaired platelet adhesion to endothelial cells with an increase in ADP-induced platelet aggregation [176]. However, an *ex vivo* study using blood samples obtained from males found that copper alone, as well as combined with manganese accelerated platelet activation and led to the deformation of erythrocytes [188]. Thus, balanced levels of copper are necessary for healthy platelet activation and aggregation. The relationship between PAF and copper has also been shown to be similar to that of iron in terms of oxidation of lipids and PAF-associated enzymes, whereby the iron-catalysed production of hydroxyl radicals can promptly and conclusively inactivate PAF acetyl hydrolase, which can lead to the prolonged inflammatory effect of PAF. Furthermore, metal-induced oxidative stress and superoxide can activate PAF acetyl hydrolase, increasing PAF levels [149,150]. Trace metals such as copper and iron may indirectly affect PAF signalling through increasing reactive oxygen species and lipid oxidation.

The interplay between trace metals and the PAF/PAF-R pathway has clinical implications. For example, pre-eclampsia is one of many conditions characterized by increased platelet aggregation and superoxide production and has been linked to alterations in trace metal levels, such as a decrease in manganese, copper, and zinc. As such, precautions during pregnancy to ensure balanced levels of essential trace elements are necessary to avoid conditions such as pre-eclampsia [189–194]. Indeed, elevated magnesium (mg) appears to exert protective effects against lesion formation as well as antiarrhythmic and antihypertensive effects [195]. Collectively, these studies show the important of trace metals in PAF biology, but little is known about whether trace metals affect PAF-R expression or function.

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

Although pharmaceutical options exist for PAF-R antagonists, they are sparse, and they are not currently utilized against CVD. However, targeting the inhibition of PAF via the PAF-R through dietary means may be a strategy to reduce the risk of atherosclerosis and CVD by reducing the activities of PAF. In this review, we have presented the *in vitro*, *in vivo*, and human studies that have examined the dietary inhibition of PAF. It appears that dietary PAF inhibitors exert their beneficial effects is through their anti-inflammatory and antithrombotic properties. Indeed, many authors have suggested that the longstanding beneficial effects of the Mediterranean diet may be due to the abundance of PAF inhibitors present in the diet. However, there is still a paucity of research investigating polar lipid consumption in humans. Although outside the scope of this review, there is also significant research in animals and humans demonstrating that polar lipids may be cardioprotective via modulating lipid metabolism. Collectively, these advances in research may lead to the development of dietary interventions or nutraceuticals with the aim to deliver dietary PAF inhibitors. However, there are vast gaps in our knowledge regarding the modulation of PAF-R expression directly in health and disease that requires further investigation.

**Author Contributions:** Conceptualization, R.H., S.H., R.L. and I.Z.; writing—original draft preparation, R.H., S.H., and J.E.S.; writing—review and editing, R.L., R.H., A.M.G. and I.Z.; visualization, R.H., S.H. and R.L.; supervision, I.Z., A.M.G. and R.L.; funding acquisition, I.Z. and R.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** The financial support from Enterprise Ireland (IP20210972) is acknowledged.

**Acknowledgments:** The authors would like to acknowledge the support of the Department of Biological Sciences at the University of Limerick, Enterprise Ireland, and the Perelman School of Medicine at the University of Pennsylvania. Furthermore, we acknowledge the Genotype-Tissue Expression (GTEx) Project and their funders. The data used for the analyses presented in Figure 1 were obtained from the GTEx Portal on 15 October 2022 (https://www.gtexportal.org/home/aboutGTEx). Other figures were created using Biorender.

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

#### **References**

