Platelet aggregation pathway

Platelet activation and coagulation normally do not occur in an intact blood vessel. After blood vessel wall injury, platelet plug formation is initiated by the adherence of the platelets to subendothelial collagen [1,2]. In high shear arterial blood, platelets are first slowed down from their blood flow velocity by interacting with the collagen-bound von Willebrand factor and are subsequently stopped by binding directly to the collagen by their glycoprotein (GP) receptor complex [2,3]. The activation of these collagen receptors on platelets after their binding to the collagen activates phospholipase C-mediated cascades (Fig. 1) [1–3]. This results in the mobilization of calcium from the dense tubular system [4,5]. An increase in intracellular calcium is associated with the activation of several kinases necessary for morphologic change, the presentation of the procoagulant surface, the secretion of platelet granular content, the activation of GPs, and the activation of phospholipase A2 (see Fig. 1) [2,5–7]. The presentation of the procoagulant surface results in the colocalization of different coagulation factors on the surface of the activated platelet, which triggers a series of zymogen conversions, resulting in the release of active thrombin from prothrombin [8]. Adenosine diphosphate (ADP), adenosine triphosphate, and serotonin are released from the dense platelet granule. Activated phospholipase A2 enzymes release arachidonic acid (AA) by the cleaving of fatty acids, especially phosphatidylcholine and phosphatidylethanolamine, at their sn-2 position [9–11]. AA is a precursor for thromboxane A2 (TBXA2) synthesis. In the first step in platelets, prostaglandin (PG)-endoperoxide synthase 1 (PTGS1; also known as cyclooxygenase 1) catalyzes the transformation of AA into cyclic endoperoxide PG G2 and H2 [9]. In platelets, PGG2 and PGH2 are then mainly converted by TBXA synthase into TBXA2 [9].

Fig. 1.

Effects of antiplatelet drugs in platelet aggregation pathway. (PA154444041; http://www.pharmgkb.org/do/serve?objId=PA154444041&objCls=Pathway). AA, arachidonic acid; cAMP, cyclic AMP; GNAS, guanine nucleotide binding protein a s; IP3, inositol 1,4,5-trisphosphate; PGD2, prostaglandin D2; PGE2, prostaglandin E2; PGG2, prostaglandin G2, PGH2, prostaglandin H2; PGI2, prostaglandin I2; PTGS1, prostaglandin (PG)-endoperoxide synthase 1; PLA2, phospholipase A2; TBXA2, thromboxane A2; TBXA2R, TBXA2 receptor; VWF, von Willebrand factor.

The mechanism of action of aspirin is the inhibition of PTGS1, thereby preventing the production of PGs and, particularly in platelets, inhibiting TBXA2 production [10–12]. In ex vivo platelet aggregation testing, aspirin affects predominantly AA-stimulated platelet aggregation through a direct pathway, and also collagen-stimulated platelet aggregation through indirect pathways. A review by Lopez Farre et al. [12] discusses further mechanisms associated with platelet response to aspirin.

The processes described above result in the local accumulation of molecules such as thrombin, TBXA2, and ADP, which are important for the further recruitment of platelets and the amplification of activation signals as described above. The secreted agonists activate their respective G protein-coupled receptors: coagulation factor II (thrombin) receptors (F2R also known as protease-activated receptor 1; F2RL3 also known as protease-activated receptor 4), TBXA2 receptor (TBXA2R), and ADP receptors (P2RY1 and P2RY12) [10,11,13–15]. The P2RY12 receptor couples to Gi, and when activated by ADP, inhibits adenylate cyclase [16]. This interaction counteracts the stimulation of cyclic AMP formation by endothelial-derived PGs, which alleviates the inhibitory effect of cyclic AMP on inositol 1,4,5-trisphosphate-mediated calcium release [14,16–20]. P2RY12 has a major role in arterial thrombosis and pharmacologic targeting of this receptor, which is an important strategy in the treatment of cardiovascular diseases [21]. Thienopyridines (ticlopidine, clopidogrel, prasugrel), a class of oral anti-platelet agents, permanently inhibit P2RY12 signaling by irreversibly binding the receptor and blocking ADP-induced platelet activation and aggregation [22].

F2R, TBXA2R, and P2RY1 couple to Gq-phospholipase C–inositol 1,4,5-trisphosphate–Ca2+ pathway, inducing shape change and platelet aggregation [14,23,24]. In addition, receptor signaling by G_12/13 (F2R; TBXA2R) contributes to morphologic changes through the activation of kinases [23,24]. Platelet adhesion, cytoskeletal reorganization, secretion, and amplification loops are all different steps toward the formation of a platelet plug. These cascades finally result in the activation of the fibrinogen receptor (GPIIb/GPIIIa) expressed on platelet cells [14,25,26]. This activation results in the exposure of the binding sites for fibrinogen, which are not available in inactive platelets. The binding of fibrinogen results in the linkage of the activated platelets through fibrinogen bridges, thereby mediating aggregation [3]. The inhibition of this receptor by GPIIb/GPIIIa inhibitors blocks platelet aggregation induced by any agonist [27,28].

The individual platelet response is variable because of polymorphisms in genes involved in the activation and aggregation of platelets, in conjunction with environmental factors, and contributes to diseases such as arterial thrombosis ([29–33], and http://www.bloodomics.org/web/). In addition to the variation in platelet physiology, platelet sensitivity to drugs targeting platelet activation and aggregation is also influenced by gene polymorphisms and clinical and environmental variables [29,31,34].

Variability in platelet response to aspirin

Not all individuals respond equally to aspirin therapy and cardiovascular events may occur during aspirin therapy, which is often referred to as ‘clinical aspirin resistance’ [12]. The term aspirin resistance has also been used in the laboratory context. Here, aspirin resistance describes persistent platelet reactivity in vitro, despite the use of aspirin, measured by various platelet function tests such as measurement of serum and urinary thromboxane metabolites, and AA-induced platelet aggregation, collagen-induced platelet aggregation, or ADP-induced platelet aggregation [35,36]. One limitation of the assessment of the various functional indexes of platelet capacity that can be measured ex vivo with in-vitro tests is the largely unknown translational relevance to the actual occurrence of platelet activation and inhibition in vivo [37]. Several studies have shown that diabetes mellitus [38–41] and obesity [35,42] are related to aspirin variability. Furthermore, polymorphisms in different genes have been associated with variability to aspirin response. The review by Feher et al. [34] found that the role of single nucleotide polymorphisms (SNPs) in PTGS1, PTGS2 (also known as cyclooxygenase 2), GPIb α (GP1BA), GPIa (ITGA2), GPIIIa (ITGB3), ADP receptor (P2RY1), and uridine 5′-diphospho-glucuronosyltransferase 1 family, polypeptide A6 (UGT1A6) in the context of aspirin resistance. The investigators concluded that the results are difficult to replicate between different research groups partially explained by the use of different platelet functions tests and that larger, population-based studies are needed to fully clarify the variability in platelet response [34]. Another recent review by Zuern et al. [29] summarized the pharmacogenomics of aspirin based on several studies investigating the effects of SNPs in PTGS1, PTGS2, ITGB3, P2RY1, and GPVI (GP6) genes with the conclusion that the reasons for insufficient response to aspirin are diverse and are still not well understood. Recent studies suggest that variants in the platelet endothelial aggregation receptor 1 (PEAR1) gene may influence agonist-stimulated platelet aggregation [43,44] and response to aspirin [43].

Variability in platelet response to clopidogrel

Clopidogrel must be metabolized into an active metabolite by liver cytochrome P-450 enzymes. Functional variants in CYP2C19 have been associated with decreased active metabolite, decreased inhibition of platelet aggregation ex vivo, and increased cardiovascular event rates in patients on clopidogrel (see also Clopidogrel PK pathway at PharmGKB PA154424674, http://www.pharmgkb.org/do/serve?objId=PA154424674&objCls=Pathway, [45]). The CYP2C19*2 loss-of-function variant was significantly associated with lower exposure to active metabolite in patients treated with clopidogrel [46,47]. Furthermore, CYP2C19*2 and other loss-of-function variants have been associated with decreased platelet responsiveness to clopidogrel ex vivo [47–49]. The decreased responsiveness and high on-clopidogrel platelet reactivity have been associated with increased cardiovascular event rates in patients on clopidogrel [47,50–54]. The large amount of evidence regarding the heterogeneity of clopidogrel response led the US Food and Drug Agency to modify the label of clopidogrel to include information regarding CYP2C19 pharmacogenetics and a boxed warning. Several recent studies discuss the value of an implementation of CYP2C19 genotyping to guide antiplatelet therapy [55–58]. Studies by Sibbing et al. [59] found that the carrier status of the gain-of-function variant CYP2C19*17 was significantly associated with enhanced response to clopidogrel and an increased risk of bleeding [60]. However, not all studies have found an independent effect of the CYP2C19*17 allele. Early candidate gene and clinical studies implicated variants in other genes involved in the metabolism and transport of clopidogrel, such as CYP3A4 [61], CYP2C9 [46,62,63], ABCB1 [51,64], and CYP2B6 [47], with clopidogrel response. However, these studies were not consistently replicated.

SNPs in the ADP receptors (P2RY1 and P2RY12), the target of clopidogrel, have also been studied in connection with platelet response variability to clopidogrel. Much of the work so far has been contradictory or has failed to identify genetic variants as significantly associated with response. No association between response to clopidogrel and P2RY1 polymorphisms was found [65,66]. The influence of P2RY12 genetic polymorphisms on clopidogrel response is not clear yet [29]. Initially, a study by Ziegler et al. [67] suggested that a genetic variation of the P2RY12 gene (34C > T, no rs number) is associated with higher numbers of cerebrovascular events in patients with peripheral artery disease receiving clopidogrel therapy. A recent study by Simon et al. [51], found no association between polymorphisms of P2RY12 and clinical outcomes in patients with acute myocardial infarction, who were treated with clopidogrel, and is in accordance with a number of other studies failing to show functional effect of P2RY12 SNPs in patients treated with clopidogrel [49,65,68–71]. Contrary to those results, a study of patients with coronary artery disease suggested a contribution of the homozygote H2 haplotype (as measured by the tagging SNP 52G > T, rs6809699) to clopidogrel resistance [72]. Another study in patients with coronary artery disease also found that common variation in the P2RY12 gene is a significant determinant of the interindividual variability in residual on-clopidogrel platelet reactivity measured by ADP-induced light transmittance aggregometry and the VerifyNow P2RY12 assay [73]. Haplotype F (tagging SNP rs2046934, i-T744C) was associated with significantly lower residual on-clopidogrel platelet reactivity compared with the reference haplotype A (tagging SNP rs6798347) [73]. The Pl^A2 polymorphism in the ITGB3 gene, which encodes the platelet membrane GPIIIa, was also found to modulate clopidogrel-mediated antiplatelet effects [74,75].

Several methods of ex vivo platelet aggregation used to define responsiveness to clopidogrel have been established, including ADP-induced light transmission aggregation, platelet function analyzer, multiple electrode aggregometry, Verify Now assay, and analysis of the degree of vasodilator-stimulated phosphoprotein phosphorylation [76]. The high assay variability and lack of standardization and definition of nonresponsiveness makes it difficult to decide which methodology reflects the true platelet activity of a patient and to evaluate the effect of genetic polymorphisms on the ex vivo platelet reactivity of antiplatelet drugs. The popular study (Do Platelet Function Assays Predict Clinical Outcomes in Clopidogrel-Pretreated Patients Undergoing Elective PCI) compared five different platelet function tests with the result that only the light transmittance aggregometry, Verify-Now, and Platelet works assays were significantly associated with the primary end point, defined as nonfatal acute myocardial infarction, stent thrombosis, and ischemic stroke, occurring more frequently in patients with high on-treatment platelet reactivity [77]. In addition to genetic factors, demographic and clinical variables, such as age [49,50,53,78], diabetes mellitus [50,78], obesity [50,53,79], smoking [80,81], acute coronary syndromes, poor left ventricular function, and renal failure [78], have been shown to influence variation in platelet function in response to clopidogrel.

Variability in platelet response to glycoprotein IIb/IIIa inhibitors

Only a small number of studies have investigated the pharmacogenomics of GPIIb/IIIa inhibitors and there are limited data available to support the relevance of genetic variants in response to these inhibitors [29]. All the studies have examined the well-known ITGB3 SNP Pl^A1/A2, also known as human platelet antigen-1 PI^A and rs5918. Michelson et al. [82] found that heterozygotes for Pl^A1/A2 were more sensitive to abciximab compared with other genotypes. Wheeler et al. [83] found reduced inhibition by abciximab in platelets in patients with the Pl^A2 SNP. A study by Sirotkina et al. [84] supports an association between this SNP and sensitivity to GPIIb/IIIa antagonists. Weber et al. [85] failed to find any association between this genotype and the inhibition of fibrinogen binding by abciximab or eptifibatide. Similarly, Schrör and Weber [86] showed that the human platelet antigen-1 genotype did not influence the inhibition of fibrinogen binding by GPIIb/IIIa receptor antagonists (abciximab, eptifibatide, and tirofiban) in healthy volunteers or patients with stable coronary heart disease.