Abstract 450: Phosphorylation Of Platelet αIIbβ3 Is Crucial For Arterial Thrombosis In Vivo And Microparticle Generation

Abstract

Functional activity of platelet fibrinogen receptor αIIbβ3 is crucial for hemostasis and thrombosis. The process of αIIbβ3 activation in platelet aggregation is tightly regulated. It has been previously shown that β3 subunit of the complex undergo tyrosine phosphorylation, which, in turn, is believed to control recruitment of several intracellular adaptors. Mutations of Tyr747/ Tyr759 within the cytoplasmic domain of αIIbβ3 (DiYF substitution) were found to result in reversible platelet aggregation. To assess whether αIIbβ3 tyrosine phosphorylation is critical for arterial thrombosis, we utilized intravital microscopy to monitor thrombus formation in vivo in WT and DiYF mice. Compared to WT, DiYF mice exhibited delayed platelet adhesion and reduced rate of thrombus formation at the initial stages of thrombosis. Likewise, isolated DiYF platelets exhibited reduced adhesion to collagen under in vitro sheer conditions compared to WT. The progression phase of thrombosis in vivo was similar in WT and DiYF mice. The most dramatic difference was observed at the final phase of thrombus formation since it took 3-times longer for blood vessels in DiYF mice to occlude compared to WT. To specifically address the role if β3 phosphorylation in platelet αIIbβ3 vs αvβ3 on leukocytes and vascular cells, we transfused labeled WT and DiYF platelets into irradiated WT mice with low blood cells and platelet counts. It was found that transfusion of DiYF but not WT platelets resulted in reversal of the thrombotic phenotype and significantly prolonged blood vessel occlusion time in vivo. Similar differences were observed in tail bleeding test. Importantly, we have found that the lack of β3 phosphorylation impaired an ability of platelets to generate microparticles in response to activation, an event believed to be critical for the final stages of thrombosis. When stimulated with thrombin and PMA, DiYF platelets shed ~50% less Annexin V-positive microparticles as compared to WT platelets. Thus, β3 tyrosine phosphorylation of αIIbβ3 in platelets is crucial for the microparticles generation by activated platelets and the overall process of arterial thrombosis in vivo.