Pathological shear stress stimulates the tyrosine phosphorylation of α-actinin associated with the glycoprotein Ib-IX complex

Shear-induced platelet responses are triggered by VWF binding to the platelet GpIb-IX complex, and there is evidence that this ligand-receptor coupling stimulates transmembranous signaling through the cytoplasmic tail of glycoprotein (Gp) Ibα. To investigate the mechanism by which signaling is effected, new molecular interactions involving GpIb-IX that develop in response to pathological shearing stress were examined in intact human platelets. Exposure to shear, but not α-thrombin, results in the co-immunoprecipitation of the actin cross-linking protein α-actinin with the GpIb-IX complex. Blockers of VWF binding to Gplbα or actin·polymerization inhibit the association of α-actinin with the GpIb-IX complex, but the association of α-actinin with the GpIb-IX complex is not affected by inhibiting VWF binding to platelet integrin αIIbβ3 (GpIIb-IIIa). α-Actinin becomes tyrosine phosphorylated in response to pathological shear stress, and phosphorylated α-actinin associates with Gplb-IX. In resting platelets, class IA heterodimeric phosphatidylinositol 3-kinase (PI 3-K) and protein kinase N (PKN) associate with nonphosphorylated α-actinin. Shear stress causes PI 3-K to disassociate from α-actinin, while it stimulates PKN binding to α-actinin. These results demonstrate that shear-induced VWF binding to GpIbα causes enhanced binding of cytoskeletal α-actinin to GpIb-IX and suggest that α-actinin, perhaps through tyrosine phosphorylation, serves as an adapter for a signaling complex that could regulate VWF-induced platelet aggregation.