von Willebrand factor binding to platelet glycoprotein Ib-IX-V stimulates the assembly of an α-actinin-based signaling complex

Summary. Background: Pathological shear stress induces platelet aggregation that is dependent on von Willebrand factor (VWF) binding to glycoprotein (Gp)Ib‐IX‐V and phosphatidylinositol 3‐kinase activation. We tested the hypothesis that pathological shear stress stimulates phosphatidylinositol 3,4,5‐trisphosphate (PIP₃) synthesis by directing the assembly of a molecular signaling complex that includes class IA phosphatidylinositol 3‐kinase (PI 3‐KIA). Methods: Platelets were subjected to 120 dynes cm^?2 shear stress in a cone‐plate viscometer. Resting and sheared platelets were lyzed, immunoprecipitations of PI 3‐KIA performed, or lipids extracted for PIP₃ measurements. α‐Actinin was incubated with phosphatidylinositol 4,5‐bisphosphate (PIP₂), immunoprecipitated, and used as a substrate for in vitro PI 3‐KIA activity. Results: Pathological shear stress induces biphasic PIP₃ production. In resting platelets, PI 3‐KIA associates with α‐actinin and PIP₂. After exposure to shear stress, α‐actinin and PIP₂ rapidly disassociate from PI 3‐KIA. PI 3‐KIA then gradually re‐associates with PIP₂ and α‐actinin, and this complex becomes linked to GpIbα through the cytoskeleton. PIP₃ production and the observed changes in the association between α‐actinin, PIP₂, and PI 3‐KIA are inhibited when VWF binding to GpIbα is blocked. In a cell‐free system, α‐actinin binds PIP₂ and when the α‐actinin–PIP₂ complex is added to platelet PI 3‐KIA, PIP₃ production is stimulated. Conclusions: These results suggest that pathological shear‐induced VWF binding to GpIb‐IX‐V stimulates PIP₃ production through the assembly of an α‐actinin‐based complex that colocalizes PI 3‐KIA with substrate PIP₂.