Calcium Independent Events Trigger VWF-Dependent Shear-Induced Procoagulant Platelet Formation

Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracellular mechanisms mediating the initiation of PSer externalization in apoptotic (Bax/Bak) and agonist-stimulated (sustained cytosolic calcium and cyclophilin D-mediated mPTP formation) platelets have been identified. In contrast, little is known about the intracellular processes mediating shear-induced procoagulant platelet (SiPP) formation.

To investigate the intracellular mechanisms regulating SiPP formation, either human or murine platelets were sheared on a plate-cone viscometer for 3 min at 10,000 s^-1 in various conditions as indicated. Platelets were differentially labeled and evaluated by flow cytometry, and platelets with PSer externalization were defined as SiPPs.

In shear conditions engagement of VWF with GPIb-a induces unfolding of the membrane proximal GPIb-a mechanosensing domain. That this triggering mechanism is essential for SiPP formation was supported by the marked inhibition of SiPP formation in the absence of VWF, in VWF with deletion of the GPIbα-binding A1 domain, and in the presence of antibodies blocking the interaction of GPIb-IX and VWF. Calcium-dependent processes play an essential role in agonist-initiated PSer externalization. However, extracellular Ca²⁺ is not required for SiPP formation. Inhibition of Ca²⁺ entry, removal of extracellular Ca²⁺ and chelation of intracellular Ca²⁺, all had no effect on SiPP formation. Caspase activation mediates PSer externalization in the apoptotic platelet, but neither pharmacologic inhibition of caspases nor calpain inhibitors prevented SiPP formation. In contrast to the negligible roles of calcium- and caspase mediated events, SiPP formation is abrogated both in the absence of the mPTP potentiator cyclophilin D and in cyclosporine A-treated platelets. Furthermore, SiPP formation was closely correlated with mPTP formation as measured by loss of mitochondrial transmembrane potential. Together, these results identify a new mechanistic pathway of SiPP formation distinct from previously described initiating mechanisms for PSer externalization. Targeting of this distinct pathway may provide a novel pharmacologic strategy for the treatment of high shear hematopathologies, like arterial thrombosis.