Racial Differences In Thrombin-Induced Human Platelet PAR4 Reactivity

Compared to white patients, black patients have worse outcomes after acute coronary events, but there is a paucity of literature considering racial differences in platelet function. Thrombin is an especially potent in vivo platelet agonist, and no work has considered racial differences in thrombin-induced platelet aggregation. Using PAR1- and PAR4- activation peptides (APs), we recently reported that platelets from healthy black subjects (n = 70) demonstrated greater aggregation to the PAR4-AP than platelets from white subjects (n = 84) (p = 5.15 x 10^-8). There was no racial difference to PAR1-AP, ADP or CRP. The goal of the current study was to determine if this racial difference in PAR4-AP-mediated platelet reactivity was also observed with thrombin and to investigate responsible molecular and genetic mechanism(s). A detailed dose-response study (4 blacks and 3 whites) revealed that when thrombin signaling was restricted to PAR4 by inhibiting PAR1 with BMS-200261, platelets from black subjects aggregated faster than platelets from white subjects at low concentrations of thrombin. No PAR1-AP-induced aggregation occurred in the presence of BMS-200261. A subsequent replication study (an additional 5 blacks and 5 whites) again showed platelets from black subjects aggregated faster than white subjects in the absence of PAR1 signaling (p = 3.56x10^-5). DNA from all 154 subjects was genotyped for 5 million SNPs (HumanOmni5 array) and principal component analysis revealed that the genotypes segregated into two distinct groups that correlated perfectly with subject self-identified race. Gene expression profiling on leukocyte-depleted platelets from all 154 subjects revealed numerous differentially expressed (DE) RNAs associated with both race and PAR4 reactivity. The gene encoding phosphatidylcholine transfer protein (PC-TP), PCTP, showed the strongest correlation with race (p = 10^-23; q = 10^-20) and with PAR4 reactivity (p = 3.4x10^-8; q = 3.5x10^-4). PC-TP protein was higher in platelets from blacks (p = 3.8x10^-6) and levels correlated with reactivity to PAR4-AP (r = 0.249, p = 0.002). Pctp has been knocked out in mice, but we found that wild type mouse platelets express little or no Pctp protein, consistent with mouse platelet RNA data from Rowley et al (Blood 2011). However, a specific PC-TP inhibitor resulted in a reduced aggregation response to PAR4-AP, but not PAR1-AP. Transfection of a siRNA against PCTP reduced both PCTP mRNA and PC-TP protein levels, and inhibited Ca⁺ release in a megakaryocytic cell line, Meg-01 in response to PAR4-AP but not PAR1-AP. A racial difference in platelet Ca⁺ release in response to PAR4-AP treatment was also observed (p = 0.02). Platelet microRNA (miRNA) profiling from all 154 subjects also revealed numerous DE miRNAs associated with both race and PAR4 reactivity. Target prediction analysis indicated that miR-376c is a candidate for regulating PCTP expression. qRT-PCR of all 154 subjects indicated that miR-376c levels are expressed higher in platelets from whites (p = 1.47 x10^-4; q = 1.38 x10^-3), and are inversely correlated with PCTP mRNA (r = -0.214; p = 0.008), PC-TP protein (r = -0.211; p = 0.009) and PAR4 reactivity (r = -0.161; p = 0.049). Transfection of megakaryocytic cell lines or cord blood CD34⁺ derived megakaryocytes with the pre-miR-376c precursor or LNA-miR-376c inhibitor resulted in decreased (P<0.01) or increased PCTP (p = 0.0003), respectively. Co-transfection of the miRNA precursor or inhibitor with a luciferase vector containing the PCTP 3’UTR indicated the regulation was dependent on the predicted miR-376c target site. In summary, we have uncovered a racial difference in thrombin-induced PAR4 platelet activation. This finding has potential clinical significance because PAR4 is the primary means by which thrombin activates platelets in the presence of vorapaxar (a PAR1 inhibitor in clinical trials), and the risks and benefits of vorapaxar by race are unknown. This racial difference in platelet activation is mediated, in part, by PC-TP, a novel protein in platelet biology. Our data also supports racial differences in miRNA expression, one of which (miR-376c) regulates PC-TP expression. These results indicate a genomic contribution to platelet function that differs by race, emphasize a need to consider race effects when developing anti-thrombotic drugs and raise the possibility that PC-TP inhibition might be a useful anti-thrombotic strategy.