Julie Oestreich Fellowship: The Effects of P2Y12 Polymorphism on Platelet Receptor Density and Activation

The goal of this research proposal is to determine if polymorph isms in the P2Y12 receptor gene contribute to increased platelet expression of the P2Y12 receptor and subsequent platelet activation events. Inhibiting the P2Y12 receptor with clopidogrel is crucial for preventing major adverse cardiovascular events. Patient responses to clopidogrel, however, are extremely variable, and negative outcomes have been attributed to differences in drug effectiveness. Although the mechanisms of clopidogrel resistance are controversial. recent evidence suggests genetic variation of the P2Y12 receptor is one important factor. The H2 haplotype of the P2Y12 gene has been associated with increased ADP-induced platelet aggregation compared to the wild-type allele, H1. Furthermore, the H2 haplotype is common as one-third of the population is a carrier. Despite these observations, the mechanisms of hyper-responsiveness associated with the H2 allele are not well characterized. We hypothesize that human carriers of the H2 allele have increased P2Y12 receptor density and/or enhanced P-selectin and fibrinogen receptor expression compared to matched H1/H1 controls. We also hypothesize that the H2 haplotype is associated with increased intracellular signaling, demonstrated by changes in platelet VASP phosphorylation and Akt activation. Three specific aims are proposed to test these hypotheses. First, we will determine the effect of the H2 haplotype on platelet P2Y12 receptor protein expression by immunoblotting washed human platelets from H1/H1, H1/H2, and H2/H2 healthy volunteers. Secondly, we will evaluate the effect of the H2 haplotype on cytosolic platelet events mediated by the Gicoupled P2Y12 receptor. Phosphorylated VASP will be examined by flow cytometry in platelets incubated with prostaglandins and ADP, and immunoblotting will quantify platelet levels of the effector protein Akt. Finally, we will elucidate the effect of the H2 haplotype on platelet surface markers of activation by stimulating whole blood from healthy volunteers with ADP and analyzing by flow cytometry to determine platelet expression of Pselectin and the fibrinogen receptor. Platelet activation will also be analyzed by ADP-stimulated aggregometry of platelet-rich plasma. These experiments will provide novel information regarding the mechanism of increased aggregation associated with the H2 haplotype and contribute to the development of haplotypespecific treatment strategies.