Regulation of platelet function and thrombosis by omega-3 and omega-6 polyunsaturated fatty acids

Abstract

Thrombosis is the most common underlying pathology responsible for morbidity and mortality in cardiovascular disease (CVD). Platelet adhesion, activation, and aggregation play central roles in hemostasis; however, the same process may also cause thrombosis and vessel occlusion at the site of ruptured atherosclerotic lesions leading to heart attack and stroke. ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) are an essential component of the platelet phospholipid membrane and play a major role in many aspects of platelet function. Dietary supplementation of ω-3 and ω-6 PUFAs has long been used to slow the progression of CVD and to prevent acute cardiovascular events. Despite this, the role of ω-3 and ω-6 PUFAs and their oxylipin metabolites in platelet function remains controversial due to the lack in our understanding of the mechanistic regulation controlling platelet reactivity in vitro and substantial evidence for PUFA regulation of thrombotic events in vivo. In this review, we will outline the role of platelet physiology in hemostasis and the effect of ω-3 and ω-6 PUFAs on platelet function, with special emphasis on in vivo effects on hemostasis and thrombosis due to the role of PUFAs and their bioactive lipids in circulation. Further, recent mechanistic insights and evidence for cardio-protective effects of PUFAs and their bioactive lipids will be discussed.

1. Introduction

Cardiovascular disease (CVD) is the leading cause of death globally. Thrombosis is the most common underlying pathology of ischemic heart disease, ischemic stroke, and venous thromboembolism (VTE), the three major cardiovascular disorders. Platelet adhesion and aggregation at the site of vascular injury are key events required for formation of a platelet plug and the arrest of bleeding (hemostasis). However, this process may also have pathological consequences (i.e., thrombosis). Antiplatelet therapy has been widely used in the prevention and management of occlusive thrombosis in atherosclerotic blood vessels, the main cause of ischemic events.[1–4]

Successful development and wide use of anti-platelet drugs have substantially decreased mortality and morbidity of cardiovascular disease.[5] Aspirin, a cyclooxygenase-1 (COX-1) inhibitor, has been shown to reduce the risk of major cardiovascular events by approximately 25% in primary prevention and to reduce morbidity and mortality rates by up to 50% in patients with acute coronary syndrome, suggesting that oxylipins, the bioactive metabolites of polyunsaturated fatty acids (PUFAs), play an important role in regulation of platelet activation.[2] Upon cellular activation, PUFAs are released from the embedded phospholipid bilayer membrane and are oxygenated by three families of enzymes COX, lipoxygenase (LOX), and cytochrome P450 (CYP) into distinct classes of oxylipins (Figure 1).[6] Oxylipins are potent bioactive lipid mediators but with short half-lives, therefore they are not stored but rather are synthesized de novo from PUFAs by oxygenases in tightly regulated manner.[6] Upon cellular activation, cPLA2 hydrolyzes PUFAs from the lipid membrane generating free PUFAs. Oxylipins can diffuse through the plasma membrane and bind GPCRs in the local environment. Additionally, select oxylipins can activate the transcription factor PPAR.[6] Early studies showed that exogenously added oxylipins could become esterified into membrane phospholipids of cells, but it was uncovered more recently that such esterified oxylipins were formed endogenously by multiple types of cells, including platelets.[7] Despite the proven life-saving clinical benefits of inhibiting platelets by aspirin and other antiplatelet agents, these therapies are associated with an increased risk of bleeding.[8] Thus, the burden of thrombotic complications of CVD remains high.

Figure 1. Biosynthesis of oxylipins.

Upon cellular activation, cPLA2 hydrolyzes polyunsaturated fatty acids (PUFAs) from the lipid membrane generating free PUFAs. Oxygenases (COX, LOX, and CYP) metabolize free PUFAs into distinct oxylipins.

While the health benefits of ω-3 and ω-6 PUFAs have been the focus of many reviews, in the present review we will discuss the effects of the main ω-3 and ω-6 PUFAs and their respective oxylipin metabolites on platelet function and the regulation of thrombosis and hemostasis in vivo.

2. Role of platelets in hemostasis and thrombosis

Platelets are small anucleated cells (1–2 microns in diameter) circulating in the blood, first discovered in 1874 by Osler.[9] Derived from megakaryocytes in bone marrow, platelets circulate on average 7 to 10 days after they enter into blood circulation, maintain a concentration of 150 – 400 × 10⁹/L in healthy humans and are the second most prevalent blood cell after red blood cells.[10] Platelets have long been recognized for their critical role in hemostasis, an important physiological process to aid in the cessation of bleeding.[11, 12] Growing evidence from studies on platelet function over the past few decades has shown that the importance of platelet function goes far beyond their primary role in hemostasis.[10]

Platelets are actively involved in a number of physiologic and pathologic processes, such as inflammation[13], anti-microbial host defense[14], aspects of the immune response[15], tumor growth and metastasis[16, 17], angiogenesis[18], lymphatic vessel development[19], and atherosclerosis.[20] Therefore, platelets are emerging as a hot research topic in many different physiologic and pathologic processes. Under normal circumstances, circulating platelets are in a resting state resembling a discoid shape in the bloodstream but always ready to safeguard the vascular integrity by responding to any disruption of the vascular endothelial lining. In the event of vascular injury, endothelial cells in vessel walls are disrupted and expose subendothelial matrix proteins. This process initiates platelet adhesion and subsequent platelet activation and aggregation at the site of vascular injury, which is a key event required for the formation of the platelet plug (referred to as primary hemostasis) to stop bleeding (Figure 2).[21] Activated platelets also provide procoagulant cell surface membranes to activate the coagulation cascade, a series of enzymatic reactions to generate thrombin ultimately leading to the formation of a fibrin clot (referred to as secondary hemostasis) to seal the vessel blood leakage.[22, 23] Thus, platelets not only play a well-established role in primary hemostasis (platelet adhesion, activation, and aggregation); they also actively contribute to the generation of thrombin, the most potent platelet agonist known to amplify the process of secondary hemostasis.[23]

Figure 2. Role of platelets in hemostasis and thrombosis.

After vascular injury, subendothelial proteins (primarily collagen and VWF) become exposed, initiating platelets in circulation to tether and adhere to the site of vascular injury. The process of platelet adhesion is mediated by the binding of platelet surface GPIb-IX-V receptors to immobilized VWF on collagen on the injured vessel wall. This results in the activation phase in which αIIbβ3 undergoes a conformation change to its active form and the release of the soluble agonists (ADP and TxA2) from platelet dense and α-granules. Platelet aggregation is mediated by αIIbβ3 binding to fibrinogen and/or other ligands in plasma. Simultaneously with platelet aggregation, coagulation is initiated to ensure stable hemostatic clot formation. The process of platelet adhesion and aggregation also leads to the formation of occlusive thrombi which results in vessel occlusion at the site of ruptured plaque in pathological conditions.

The process of platelet interaction with the vessel wall and subendothelial matrix is accomplished through the involvement of many platelet receptors and their corresponding ligands (Figure 2).[24] Major platelet surface receptors include the GPIb-IX-V complex, GPVI, integrins αIIbβ3 (also known as GPIIbIIIa), α2β1, α5β1, α6β1, thrombin receptors PAR1 and PAR4 (protease activated receptors), ADP receptors (P2Y1, P2Y12 and P2X1) and thromboxane A2 receptors (TP).[24] Platelet adhesion: Platelet-vessel wall interaction, the tethering and adhesion of platelets to the injured site, is primarily mediated by platelet surface GPIbα receptor in GPIb-IX-V complex which binds to the A1 domain of von Willebrand factor (VWF) immobilized on collagen upon vascular injury, particularly at high shear stress.[25] Subsequent stable platelet adhesion is further mediated by other integrin ligands with their corresponding ligands such as integrin αIIbβ3 bound to fibronectin and fibrinogen/fibrin, α5β1 to fibronectin or collagen, and α2β1 to collagen.[26] Platelet activation: Following initial tethering and adhesion of circulating resting platelets at the site of vascular injury, platelets rapidly undergo a well-defined signaling cascade (Ca²⁺ influx, degranulation, phosphatidylserine exposure, etc.) that leads to platelet shape change facilitating further platelet activation and platelet granule release.[27] Platelet α-granule (P-selectin[28], VWF[29], fibrinogen (Fg)[28], fibronectin[30], vitronectin[31], multimerin[32], platelet factor 4[33], and many other proteins) and dense granule (adenosine di-phosphate (ADP), polyphosphates and other) contents are secreted and, in turn, amplify the activation process of integrin αIIbβ3 inside-out signaling, leading to further platelet aggregation.[34] GPIba-VWF interaction is also shown to play an important role in platelet adhesion at low shear conditions.[35] Platelet aggregation: Activation of platelets is a critical step for aggregation. Platelet activation results in a conformational change in the αIIbβ3 receptor on the platelet surface making it available to bind to its major ligand fibrinogen or other alternative ligand(s).[24] This process allows for cross-linking to adjacent activated platelets resulting in platelet aggregation and hemostatic plug formation. Despite the dogma of VWF and Fg interactions thought to be required for platelet adhesion and aggregation for decades, platelet aggregation and thrombus formation is still successful in mice lacking von Willebrand factor and fibrinogen, even after depletion of plasma fibronectin.[30] This observation indicates that other known or unknown ligands of αIIbβ3 could mediate platelet aggregation independent of VWF and Fg.[30] Interestingly, αIIbβ3 is indispensable for the platelet aggregation process as no thrombus formation is detected in mice lacking αIIbβ3 under intravital microscopy.

After the initial platelet plug formation, the second mechanism required to stop bleeding is the coagulation cascade via extrinsic or intrinsic pathways. Activation of the coagulation system leads to the generation of thrombin, which converts fibrinogen to fibrin.[23] There are many interactions between these two mechanisms, the initial platelet adhesion and aggregation and coagulation system that lead to clotting. For example, activated platelets accelerate coagulation by providing a negatively charged phosphatidylserine-rich membrane surface that enhances cell-based thrombin generation. Conversely, thrombin generation leads to further platelet activation and is crucial for platelet adhesion and aggregation and polymerized fibrin. Fibrin operates to stabilize the platelet plug or hemostatic clot (physiologic).

Although these are important steps for hemostasis, the same processes can also lead to the development of arterial thrombosis and vessel occlusion when the integrity of the vessel wall is compromised by rupture of an atherosclerotic plaque (Figure 2).[36] Excessive platelet activation, aggregation, and blood coagulation may lead to the formation of occlusive thrombi resulting in severe consequences such as myocardial infarction, ischemic stroke, and pulmonary embolism, which are the predominant causes of morbidity and mortality worldwide. Intravascular thrombosis is the cause of myocardial infarction (MI) and stroke as well as venous thrombosis (VT), which often leads to venous thromboembolism (VTE) and pulmonary embolism (PE).[37] Structurally, arterial and venous thrombi are distinct. Arterial thrombi are platelet-rich thrombi forming at the side of atherosclerotic plaques. Unstable angina and myocardial infarction and ischemic stroke are typically the result of excessive platelet adhesion, aggregation, and vessel occlusion formed at ruptured atherosclerotic lesions under high shear flow rates (arterial: 300–800 s⁻¹ and stenotic vessels 800–10,000 s⁻¹) in coronary and cerebral arteries.[36] Platelets also contribute to the orchestration of venous thrombi consisting mainly of fibrin and red blood cells under low shear rate (20–200 s⁻¹).[35] In venous thrombosis, endothelial activation causes adhesion of platelets and leukocytes. In turn, adhered leukocytes become activated and initiate expression of tissue factor and lead to the activation of the coagulation cascade. Recent studies also indicate the potential role of other factors such as platelets-neutrophils aggregates and neutrophil extracellular trap (NET) in the pathogeneses and development of both arterial and venous thrombosis.[37]

3. Anti-platelet therapy in cardiovascular disease

Platelet activation and aggregation play a central role in arterial thrombus formation, which results in acute thrombotic events, such as myocardial infarction and ischemic stroke. Antiplatelet drugs are the frontline treatment of cardiovascular diseases for both the prevention and treatment of thrombotic events. Current antiplatelet therapies inhibit platelet function by targeting platelet enzymes (phosphodiesterase, cyclooxygenase), receptors (purinergic, prostaglandins, protease-activated receptors, thromboxane), and glycoproteins (αIIbβ3, GPVI, VWF, GPIb).[38, 39] Aspirin is by far the most widely used antiplatelet therapy and operates by irreversibly inhibiting platelet COX-1 to block platelet thromboxane A2 (TXA2); however, it does not prevent platelet activation occurring via various signaling pathways that are independent of TXA2. Therefore, a number of other antiplatelet reagents have been developed to overcome the limitations of aspirin.

More recently, 12-lipoxygenase (12-LOX), an oxygenase predominantly expressed in human platelets, is emerging as a potential anti-platelet target.[40] Earlier studies showed 12-LOX utilizes arachidonic acid (AA) released from the phospholipids as a substrate to form bioactive metabolites 12-(S)-hydroperoxyeicosatetraenoic acid (12-HPETE) and 12-Hydroxyeicosatetraenoic acid (12-HETE) which regulate a number of biological processes such as integrin activation, vascular hypertension, and progression of certain types of cancer.[41, 42] Metabolic products of 12-LOX formed during platelet activation have been shown to play a role in platelet activation, thrombin generation, and granule secretion in vitro and ex vivo suggesting a role of 12-LOX in regulating platelet function, as well as hemostasis and thrombus formation in vivo.[43] Furthermore, using a newly developed highly selective platelet 12-LOX inhibitor, ML355, we demonstrated 12-LOX plays an important role in the regulation of human platelet function in vitro and ex vivo flow conditions.[44, 45] Orally administered 12-LOX inhibitor in mice showed dose-dependent inhibition of thrombus formation while minimally impairing hemostasis in preclinical animal models of thrombosis and hemostasis.[46] Thus, pharmacologically targeting 12-LOX may be a viable anti-platelet approach although future clinical studies are needed to validate this hypothesis.

Other potent novel antithrombotic agents, such as αIIbβ3 inhibitors, have been developed and clinically proven to effectively inhibit platelet aggregation and thrombus formation. However, studies have shown that aggressive inhibition of platelet function by platelet αIIbβ3 inhibitors is associated with increased risk of bleeding and only modestly improved mortality.[47] Despite the effectiveness of current antiplatelet therapies, the irreversibility and continued use of these agents is associated with severe bleeding complications.[48] Therefore, an adequate level of suppression of platelet function is critical to maintaining the balance between hemostasis and pathologic thrombosis to prevent adverse events.

Another potential therapeutic treatment to influence platelet function is PUFAs. Fatty acids are hydrocarbon chains with a carboxyl group at one end and a methyl group at the other. The two most common families of PUFAs are classified as omega-3 (ω-3) and omega-6 (ω-6) based on the location of the last double bond relative to the terminal methyl end of the molecule. Dietary supplementation with ω-3 and ω-6 PUFAs has long been used to slow the progression of CVD and to prevent acute cardiovascular events. Furthermore, alteration of the ratio of ω-3 and ω-6 in diets is associated with the pathogenesis of cardiovascular disease and its complications.[49, 50] While the cardioprotective benefits of ω-3 and ω-6 PUFAs remains debatable, ω-3 and ω-6 PUFA supplementation is widely used in both the primary and secondary prevention of CVD.

4. The effects of PUFAs on platelet function

The platelet membrane, like other mammalian cell membranes, is a lipid bilayer composed of an outer leaflet that contains cholinephospholipids, primarily phosphatidylcholine and sphingomyelin, and an inner leaflet comprised of the negatively charged aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS).[51, 52] The platelet phospholipid membrane plays a major role in many aspects of platelet function.[53] Asymmetric distribution of the membrane phospholipids is well regulated by a number of calcium and adenosine triphosphate (ATP) dependent enzymes.[51] Upon platelet activation, this asymmetric orientation of membrane phospholipids is disrupted resulting in calcium-dependent exposure PS on the platelet surface. It is well known that PS surface exposure is a major component of normal hemostasis because it supports platelet procoagulant functions. Formation of the prothrombonase complex on platelet membrane surface further enhances platelet activation by facilitating conversion of prothrombin to thrombin.[43, 54]

Upon cellular activation, cytoplasmic phospholipase A2 (cPLA2) hydrolyzes PUFAs from the lipid membrane generating free PUFAs. Liberation of PUFAs from the platelet membrane is achieved in an agonist dependent manner. Platelet stimulation by agonists including thrombin, ADP, and collagen cause the translocation of cPLA2α to the plasma membrane where it cleaves PUFAs. A study (reference) shows that the most abundant phospholipid in the platelet is phosphatidylcholine, containing a number of PUFAs including arachidonic acid (AA) (13.5%), linoleic acid (LA) (7.9%), dihomo-γ-linolenic acid (DGLA) (2.1%), Eicosadienoic acid (EDA) (0.6%), docosahexaenoic acid (DHA) (0.6%), eicospentaenoic acid (EPA) (0.2%), Alpha-linolenic acid (ALA) (0.1%), and γ -linolenic acid (GLA) (0.07%).[55] This study also shows that supplementation with ω-6 PUFAs affects the phospholipid composition of the platelet plasma membrane[55]. Additionally, studies comparing individuals from inland and coastal communities in Europe show that differences in diet (i.e. consumption of fish products) affect both the phospholipid composition of the platelet plasma membrane and platelet function.[56–58] AA is a major component of membrane phospholipids which is required for prostaglandin synthesis and is also responsible for accelerating some phases of the coagulation process.[59] The composition of the phospholipids in the platelet membrane is dynamic in nature, and because many of the fatty acids that make up the phospholipid bilayer are not produced in the body, their content is primarily regulated by dietary intake.[60] Therefore, polyunsaturated fatty acid content in the platelet membranes varies, depending on diet.

The effects of dietary intake on primary prevention of coronary heart disease have been a long-standing interest. Early studies have shown dietary supplementation of fatty acids increase fatty acid incorporation into the platelet lipid membrane.[55] Recent research work has revealed a number of lipid products, derived from ω-3 or ω-6 PUFAs, regulate and alter platelet function (Figure 3).[6, 40, 61, 62] Understanding how these newly identified lipids regulate platelet function will aid in our understanding of how diet alteration or fatty acid supplementation affect platelet function and modulate hemostasis and thrombosis in the vessel.

Figure 3. Regulation of platelet function and thrombosis by ω-3 and ω-6 PUFAs.

PUFAs are an essential component of the platelet phospholipid membrane with the main ω-3 and ω-6 PUFAs being oxygenated by two important oxygenases (COX-1 and 12-LOX) to produce oxylipins in platelets. ω-3 and ω-6 PUFA oxylipin metabolites will further contribute to the regulation of platelet function in hemostasis and thrombosis.

4.1. Omega-3 fatty acids and platelet function

ω-3 PUFAs are a class of essential fatty acids that are an integral part of cell membranes throughout the body and affect the function of the cell receptors in these membranes. Epidemiological evidence suggests that a ω-3 fatty acid-rich diet promotes beneficial cardiovascular, neurological, and anti-inflammatory health effects.[63–67] However, the underlying biochemical mechanisms facilitating these beneficial effects are yet to be fully elucidated. Unlike most types of fats that can be made from other fats or raw materials in the human body, ω-3 PUFAs must be obtained from diet.[68] There are three main ω-3 PUFAs obtained from food: eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) come mainly from fish while alpha-linolenic acid (ALA), the most common omega-3 fatty acid in most human diets, is found in vegetable oils and nuts (especially walnuts), flax seeds and flaxseed oil, leafy vegetables, and some animal fat, especially in grass-fed animals.[69] Despite the sources of these PUFAs being known, it can be challenging to get the appropriate amount of EPA and DHA through diet alone. Dietary Guidelines for Americans states that an average daily consumption of 250 mg of EPA and DHA is associated with reduced cardiac deaths among individuals with and without preexisting CVD.[70] Studies suggest that ALA is mainly used for energy and its conversion into EPA and DHA is very limited.[71] We and others report that ω-3 fatty acids act on the platelet membrane to reduce platelet aggregation and thromboxane release by acting on COX-1 and 12-LOX, the two important oxygenases involved in metabolizing fatty acids into oxylipins in platelets.(Figure 3)[61] Our earlier studies comparing the reactivity of six essential fatty acids with 12-LOX show that EPA, AA, and DGLA react comparably, ALA reacts more slowly, and EDA and LA do not react at all.[43] It has been reported that exogenous addition of DHA and EPA inhibits human platelet aggregation in vitro but DHA seems to have a much more potent effect on human platelet function.[72] Even with the known effect, the mechanism by which supplementation with ω-3 PUFAs decreases platelet aggregation is remains unknown. It has been reported that ω-3 PUFAs incorporate into platelet membrane phospholipids, leading to a concomitant reduction of ω-6 PUFAs along with an increase in EPA.[73] EPA can then compete with AA and inhibit the cyclooxygenase-1 pathway. The decrease of platelet aggregation by ω-3 PUFAs has also been attributed to a decrease in thromboxane A2 and an increase in prostaglandins, thromboxanes[74], and the synthesis of nitric oxide in endothelial cells.[75] A recent study also showed ω-3 PUFA–derived lipid metabolites can originate from the crosstalk between the endocannabinoid and cytochrome P450 (CYP) epoxygenase metabolic pathways. ω-3 endocannabinoid epoxides epoxyeicosatetraenoic acid-ethanolamide (EEQ-EA) and epoxydocosapentaenoic acid-ethanolamide (EDP-EA) derived from DHA and EPA, respectively, exert anti-inflammatory, vasodilatory, and reciprocally modulate platelet aggregation.[76]

The evidence of cardioprotection in published studies is derived from many sources, including epidemiological studies analyzing populations with high dietary ω-3 PUFAs intake.[77] The strongest evidence for a beneficial effect of ω-3 fats is mostly related to the heart.[78] Supplementation with ω-3 PUFAs has been reported to have several beneficial effects including reducing cardiovascular mortality[79, 80], improved lipid profile[81, 82], anti-inflammatory effects[83], reducing cardiac arrhythmias [82], vasodilatory mechanisms[84] and anti-platelet effects.[85] However, pinpointing the cardioprotective therapeutic effects of ω-3 PUFAs remains challenging, as large clinical trials predominantly demonstrated neutral effects of ω-3 PUFAs in placebo-controlled studies. The OPERA trial showed no alterations in the number or severity of atrial fibrillation events or incidents of myocardial infarction or stroke.[86] Furthermore, the Risk and Prevention Study[65] and the Alpha Omega trials[87] showed no alterations in cardiovascular-derived incidents of mortality, and the OMEGA-PAD I trial[88] showed no alterations in vascular endothelial cell functions. Several interventional studies were reviewed by Begg et al. and largely concluded that ω-3 PUFAs may provide a clinical benefit, but the degree and nature of ω-3 PUFA cardioprotection remains unclear.[89] Thrombosis, like many of the other endpoints examined in ω-3 PUFA-oriented clinical trials, also shows mixed results[86, 90–93]. Although supplementation of ω-3 PUFAs has been shown to reduce platelet aggregation and activation in healthy subjects, a higher than recommended dose of ω-3 PUFAs may be needed due to platelet hyperactivite prothrombotic conditions such as in CVD.

It is worthwhile to mention that the prevalence of CVD is closely associated with diet.[94] Anthropological and epidemiological studies as well as studies at the molecular level indicate that human beings evolved on a diet with a ratio of omega-6 to omega-3 essential fatty acids (EFA) of ~1, whereas in Western diets the ratio is anywhere between 15/1 and 16.7/1.[95] Some studies suggest that this alteration of the ratio of ω-6 to ω-3 essential fatty acids in Western diets promotes the pathogenesis of many diseases including cardiovascular disease, cancer, and inflammatory and autoimmune diseases, whereas increased levels of ω −3 PUFA (a lower ω-6/ω-3 ratio), exert suppressive effects.[49, 50] Studies also compared the effects of ω-3 PUFAs on platelet activity in men and women. Study results suggested that men are more likely to benefit from supplementation with EPA, whereas women are more responsive to DHA.[96] While these observations are interesting, the effects of EPA and DHA supplementation on individuals of different ages and sexes should be investigated in future studies.

4.2. Omega-6 fatty acids and platelet function

ω-6 PUFAs are another class of essential fatty acids that are abundant components of cell membranes and serve as precursors to bioactive lipid mediators. ω-6 PUFAs need to be obtained from food because humans are not able to synthesize them. ω-6 PUFAs are a major component of the Western diet, derived from poultry, nuts, seeds and vegetable oils.[97] Linoleic acid (LA) is a precursor to the variety of ω-6 series of fatty acids including AA, gamma-linolenic acid (GLA), and dihomo-γ-linolenic acid (DGLA).[97, 98] LA is by far the most common fatty acid found in the human food supply. It is a primary fatty acid found in cooking oils, including vegetable, corn, canola, peanut, soybean, safflower, and sunflower oils. GLA is present in less commonly known oils—mainly borage, black currant seed, and evening primrose. AA is found in red meat, eggs, and poultry products. AA is one of the most abundant fatty acids in platelet membranes, granules, and soluble fractions. [99] AA is the precursor of thromboxane and prostacyclin, two of the most active compounds related to platelet function. The role of AA in regulation of platelet function has been extensively studied for decades. It is well demonstrated that COX-1 oxidizes AA to generate prostanoids (prostaglandins (PGs) and thromboxanes (TXs)) series to regulate platelet function.[6, 61, 62] Released TXA2 acts as a soluble agonist like adenosine diphosphate (ADP) to amplify platelet activation through its thromboxane receptor (TPα) on platelets and exerts prothrombotic properties.[61] In contrast, PGI2 (prostacyclin), a well-characterized vasodilator[100], has been shown to activate adenylate cyclase in the platelet via the prostacyclin (IP) receptor and in turn antagonizes platelet aggregation.[101]

ω-6 PUFA, DGLA, has been shown to play a role in inhibiting platelet aggregation ex vivo, although the exact oxylipin products by cyclooxygenase-1 (COX-1) or platelet 12-lipoxygenase responsible for the inhibitory effects of DGLA on platelet function remain unclear (Figure 3).[61, 102] [62] For a long time, the antiplatelet effects of DGLA have been primarily attributed to the COX-1–derived prostanoid metabolites (TXA1 and prostaglandin E1), although the DGLA-derived products of COX-1 are produced in low amounts in platelets.[61, 62, 102, 103]. In our earlier study, we show that the bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, αIIbβ3 activation, Rap1 activation, and clot retraction in ex vivo.[43]. This observation was further confirmed by our recently published study showing that ω-6 PUFA, DGLA, exhibits cardioprotective properties through its reduced oxidized lipid form 12(S)-HETrE by inhibiting platelet activation and thrombosis in vivo via a Gαs-linked GPCR-dependent manner.[62] These results strongly indicate that as DGLA may be competitively oxidized by both COX-1 or 12-LOX pathways, its inhibitory effect on platelet function may be dictated by the downstream oxylipin products from these two pathways.

5. In vivo effect of omega 3 and 6 PUFAs on arterial thrombosis in preclinical animal models

Despite the lack of mechanistic and in vivo evidence of anti-thrombotic benefits of ω-3 and ω-6 PUFAs, dietary supplementation with PUFAs is commonly used for their potential cardioprotective effects, including their antiplatelet effects. It is difficult to reconcile the observed antiplatelet effects of ω-3 and ω-6 PUFAs or oxylipin products in vitro with clinically relevant study results.[98, 104] Early studies using animal models of arterial or venous thrombosis indicate PUFA supplementation in diet may alter thrombosis.[105–107] However, results from those studies are not conclusive. In the last decade there have been significant advances in preclinical in vivo animal models of thrombosis using real-time intravital microscopy.[30, 31, 46] Recently, we reported the effects of ω-3 PUFA supplementation on platelet function in vivo in mice after 6 weeks of DHA/EPA-enriched diet using two well-established intravital microscopy murine models of thrombosis (Figure 4).[108] In a laser-induced cremaster arteriole thrombosis model, we found that platelet accumulation and fibrin formation in thrombi were attenuated in the DHA/EPA-fed mice compared to control diet. Intriguingly, the level of P-selectin positive platelets that accumulated at the thrombus core was similar between the two groups of mice as observed under confocal intravital microscopy. Vessel occlusion was significantly delayed in mice on a DHA/EPA diet in a FeCI₃-induced carotid artery thrombosis model. Despite this, DHA/EPA had relatively minimal effects on agonist-induced secretion, aggregation, and adhesion. DHA/EPA was observed to modulate platelet-mediated thrombin generation in vitro. Taken together, these study results support a role for ω-3 PUFA supplementation as a means to achieve cardioprotective effects by attenuating platelet function resulting in modulation of thrombus formation and vessel occlusion.

Figure 4. Attenuation of thrombosis following the DHA/EPA rich-diet.

Represented figure shows the thrombus formation in response to vessel injury in individual on a control diet or EPA/DPA-rich diet. Compared to the thrombi in the control diet (upper panel), thrombus formation is attenuated in the DHA/EPA-rich diet. DHA/EPA-rich diet resulted in a decrease of platelet accumulation on outer layers (shell region) of thrombi without affecting the overall platelet accumulation at the center (core region) of thrombi.

More intriguing in vivo evidence and mechanistic insights were obtained recent study on the 12-LOX-derived oxylipin of omega-6 PUFA DGLA, 12(S)-HETrE.[62] 12-HETrE, a DGLA-derived metabolite of 12-LOX, reduces thrombus growth in a laser-induced injury model of thrombosis, but the inhibitory effects of DGLA on platelet-mediated thrombus formation are 12-LOX dependent. Despite the strong antithrombotic effects, DGLA did not impair hemostasis. This study provided the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit–linked G-protein–coupled receptor–dependent manner. Furthermore, the antiplatelet effects of 12-HETrE are partially dependent on IP signaling providing further insight into the mechanism by which DGLA supplementation inhibits platelets function.

Taken together, results from these preclinical studies provided important mechanistic insights on how ω-3 and ω-6 PUFAs regulate platelet function and modulate thrombosis in vivo.

6. Conclusions

Adequate platelet reactivity is required for platelet adhesion and aggregation at the site of vascular injury to maintain hemostasis. However, excessive platelet reactivity can lead to the formation of occlusive thrombi, the predominate underlying cause of myocardial infarction and stroke. ω-3 and ω-6 polyunsaturated fatty acids are an essential component of the platelet phospholipid membrane and play a major role in regulation of platelet function. Dietary supplementation with ω-3 or ω-6 PUFAs may alter platelet lipid membrane phospholipid composition and affect platelet function, which, in turn, may alter the progression and thrombotic complications of cardiovascular disease. Therefore, mechanistic insights into how PUFAs and metabolites affect platelet function could have therapeutic potential.

While current anti-platelet therapies are successful in reducing the mortality rate associated with CVD, thrombotic complications of CVD remain a challenge and there is a great need to develop new therapies. This review has discussed the role of platelets in hemostasis and in the pathophysiology of CVD. We overviewed the effect of ω-3 and ω-6 PUFAs on platelet function with special emphasis on the effects on thrombus formation in vivo, including recent mechanistic insights and evidence for their cardioprotective effects. With an overwhelming amount of controversial data on the potential therapeutic effects of ω-3 and ω-6 PUFAs on platelet function, it is important to elucidate the specific factors contributing to the overall inhibition of unwarranted platelet activation leading to thrombosis. Very few studies to date have investigated the individual efficacy of the major ω-3 and ω-6 PUFAs and their oxylipin metabolites on platelet thrombosis.

In the future it would be beneficial to evaluate the changes of the levels of PUFAs in the platelet membrane following supplementation or diet change and further elucidate the specific contributions of each individual fatty acid in the regulation of platelet function. It is imperative to uncover the underlining mechanisms of PUFAs in the regulation of platelets to reduce morbidity and mortality associated with cardiovascular disease.

Highlights.

• Dietary supplementation of ω-3 and ω-6 PUFAs has long been used to slow the progression of CVD and to prevent acute cardiovascular events. In this review, we will outline the role of platelet physiology in hemostasis and the effect of ω-3 and ω-6 PUFAs on platelet function, with special emphasis on in vivo effects on hemostasis and thrombosis due to the role of PUFAs and their bioactive lipids in circulation. Further, recent mechanistic insights and evidence for cardio-protective effects of PUFAs and their bioactive lipids will be discussed.