When platelets are simultaneously stimulated by multiple agonists, such as thrombin and collagen, a subpopulation of procoagulant platelets is formed that is characterized by phosphatidylserine (PS) exposure, integrin αIIbβ3 inactivation, and a rounded morphology in a process that is dependent on the mitochondrial permeability transition pore (mPTP) regulatory protein cyclophilin D (CypD). Recently, we have found that, in the absence of platelet CypD, platelet accumulation is markedly accentuated both in vitro and in vivo indicating that CypD-dependent procoagulant platelet formation may limit thrombus growth. Interestingly, the importance of CypD in limiting platelet accumulation was most evident in shear stress conditions consistent with arterial flow. In low (300 s−1) and high (1500 s−1) shear conditions, CypD null platelet accumulation was increased 1.5- and 4-fold compared to wild type platelets, respectively.
Platelet activation and procoagulant platelet formation were examined in various shear conditions. In platelets subjected to increasing shear, PS exposure, but not P-selectin expression or integrin αIIbβ3 activation, was observed in a platelet subpopulation. When subjected to high shear in vitro as many as 70 % of platelets expressed high levels of PS on their surface within 5 min, and shear-dependent PS exposure was observed in as little as 30 seconds in high shear stress conditions. Previously we demonstrated that agonist-initiated PS exposure is closely associated with integrin αIIbβ3 cleavage and inactivation. In shear-treated, as in strongly-stimulated platelets, integrin αIIbβ3 cleavage was closely associated with PS exposure. PS exposure and integrin αIIbβ3 cleavage were not observed in the absence of VWF. In contrast to agonist-initiated PS exposure, shear stress-induced PS exposure was observed even in the absence of extracellular calcium.
In a previous study, platelet PS exposure and apoptosis induced by ristocetin were found to be closely associated with an increase in the expression of the BH3-proteins, Bax and Bak. This finding, along with the observation that shear-stress dependent PS exposure occurred even in the absence of extracellular calcium led us to examine the role of Bax and Bak. However, shear stress-induced PS exposure was unaffected in Bax/Bak null platelets. In contrast, both shear stress-induced PS exposure and integrin αIIbβ3 cleavage were markedly blunted in CypD null platelets. Furthermore, shear stress induced-PS exposure was closely associated with loss of mitochondrial transmembrane potential, together indicating the importance of mPTP formation in the regulation of PS exposure in high shear stress conditions. These data identify a novel VWF- and CypD-dependent pathway that results in rapid PS exposure and integrin αIIbβ3 cleavage in high-shear stress conditions. This shear stress-initiated pathway of PS exposure, distinct from thrombin-collagen initiated PS exposure, does not require extracellular calcium and is not associated with P-selectin expression.
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