Race differences in platelet reactivity: is protease activated receptor 4 a predictor of response to therapy?

A number of factors influence the incidence and severity of cardiovascular disease such as environmental exposures, medical adherence, and genetic polymorphisms.1 One of the genetic components is a heritable inter-individual variation in platelet reactivity, which is greater in black individuals than whites. This correlates strongly with the historical observations that black individuals develop cardiovascular disease at a younger age and have a higher incidence of mortality from the disease.2-4The Platelet RNA and eXpression (PRAX1) study was designed to identify the specific genetic components that correlate withvariations in platelet reactivity.5 This study recently identified an expression profile of mRNAs and microRNAs that are associated with race and PAR4 reactivity. Thrombin is a potent platelet activator that initiates signaling in platelets and other cells via the GPCRs called protease activated receptors (PARs); human platelets express PAR1 and PAR4.6 PARs are activated by proteolytic cleavage of the N-terminus to expose the tethered ligand, which interacts with the extracellular loops of the receptor. The primary determinant of PAR activation is the nature of the enzyme-substrate interaction, which determines the rate of proteolysis of the N-terminus. PAR1 is more efficiently cleaved by thrombin than PAR4. As a result, PAR4 is often thought of as a low affinity backup receptor with redundant function since PAR1 and PAR4 initiate overlapping signaling cascades. Like many GPCRs, PARs form homo- and hetero-oligomers (see Figure). 7 These lateral associations of PAR4 directly influence its activation and signaling.8-11For example, interactions between PAR1 and PAR4 result in a 6-10 fold increase in the rate of PAR4 cleavage.8, 9PAR4 also forms hetero-oligomers with P2Y12, which regulates arrestin-2 recruitment and AKT signaling in platelets.The interactions with P2Y12 and PAR1 put PAR4 at the center of two important pathways for platelet activation. Therefore, any alterations in PAR4 reactivity have the potential to dramatically influence platelet signaling. Figure The activation of P2Y12, PAR1 and PAR4 are key events in platelet activation, which are regulated by homo- and hetero-oligomers(top panel). In the presence of clopidogrel and vorapaxar, PAR4 becomes a primary signaling receptor on the platelet surface . The numerous studies on PAR1 have lead to the successful development of the antagonist vorapaxar (Zontivity), which was approved for clinical use in 2014.12 Vorapaxar is a first-in-class antiplatelet agent directed to PAR1. Vorapaxar directly competes for the ligand-binding site on PAR1 to prevent activation. Recently, a high-resolution structure of PAR1 bound to vorapaxar was solved and provides exquisite detail regarding its specific contacts with PAR1.13In 2012, the results of two simultaneous Phase 3 clinical trials with vorapaxar (TRACER and TRA 2°P-TIMI 50) and a subsequent subgroup analysis were reported.14-16The TRACER trial, whichwas designed to determine the effectiveness of vorapaxar in patients with high-risk acute coronary syndrome (ACS), did not reach its primary endpoint and was terminated early due to a >2-fold increase in the rate of intracranial hemorrhage. In contrast, the TRA 2°P-TIMI 50 trialwas designed to determine the effectiveness of vorapaxar at reducing the secondary major cardiovascular events in patients with a history of myocardial infarction, peripheral artery disease, or stroke. The TRA 2°P-TIMI 50 trial showed a 12% reduction in cardiovascular death and ischemic complications in the overall study group. However, a secondary analysis that excluded individuals with a history of stroke showed that the reduction in primary the endpointsincreased to 18%. The combination of these studies ultimately lead to FDA approval with a black box warning about the increased bleeding risk in individuals with a history of stroke, transient ischemic attack, intracranial hemorrhage, or active bleeding.12Finally, it is of note that vorapaxar has not been tested as a single agent and is recommended to be used in conjunction with a P2Y12 antagonist. This combination of drugs further drives platelet activation through PAR4 (see Figure). Previously, Edelstein and colleagues examined 154 healthy individuals who self identified as black or white and determined that differences in platelet reactivity were dependent upon PAR4 activation.5The degree of aggregation in response to PAR4 agonist peptide (AYPGKF) was higher in platelets from black individuals versus white individuals. The dependence on PAR4 was confirmed by activating platelets with thrombin in the presence of a PAR1 antagonist. From there the authors found that blacks had 4-fold higher expression of phosphatidylcholine transfer protein (PCTP) mRNA and this correlated with increased PAR4 dependent aggregation and Ca2+ mobilization. In contrast, alterations in the DLK1-DIO3miRNA cluster were associated with white individuals and decreased PAR4 reactivity. This was the first report to demonstrate that PAR4 correlates with differences in platelet activity between black and white populations. The article by Tourdotand colleagues appearing in this issue of ATVB expands upon the previous work done by Edelstein et al.5, 17This new studyexamined platelet reactivity in a diverse population of white and black individuals from the Philadelphia area. This report confirmed that platelets from black individuals are more responsive to low concentrations (25-100 μM) of PAR4 agonist peptide and that this effect is due to an intrinsic property of platelets and not plasma components. In order to mimic the current standard of care, the authors measured PAR4 activation in the presence of a P2Y12 antagonists and aspirin. These experiments determined that the differences in platelet reactivity were primarily through PAR4 and not thromboxane or ADP signaling. The authors measured PAR4 surface expression on platelets from white and black individuals and found no differenceverifying that the observed signaling differences were not simply due to variable PAR4 expression. Similar to the previous report, Tourdot and colleagues linked the altered PAR4 signaling to the Gq axis.5 Black individuals had an enhanced Ca2+ response and this correlated with increased Rap1 activation, pleckstrin phosphorylation, and ultimately αIIbβ3 activation. The precise mechanism of the heightened Gq signaling through PAR4 is yet to be identified. The authors did not observe obvious differences in expression levels of key platelet proteins, which suggests there are alterations in the regulation of the signaling cascade. Since the differences are specific to PAR4 and not Gq signaling in general, future studies will need to examine the initiation of the PAR4 pathway to determine the genetic causes. The study by Tourdotand colleagues pinpoints a specific mechanism through enhanced PAR4 activation and Gq signaling that may contribute to the higher risk of stroke and myocardial infraction that is observed in black populations.17This study and the recent report by Edelstein and colleagues are timely and have important clinical implications given the recent approval of the PAR1 antagonist vorapaxar. For patients on vorapaxar, thrombin signaling will be exclusively funneled through PAR4 (see Figure). Therefore, individuals with increased PAR4 reactivity may be less protected from adverse cardiovascular events by vorapaxar. In contrast, heightened PAR4 signaling may protect from the bleeding complications that are observed in specific patient populations on vorapaxar. The answer to these questions will require carefully planned clinical trials. Finally, these studies raise the potential of PAR4 as a therapeutic target. This intriguing possibility will require substantial time and resources to fully develop and validate. The more immediate clinical application of these exciting new findingsmay lie in the ability to predict patient response to specific therapies. Time will tell how widespread vorapaxar will be used clinically. Monitoring PAR4 activity may aid in these clinical decisions.