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

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 reassociates 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.