Shear Stress Directly Regulates Platelet αIIbβ3 Signaling.

Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced by an integrin’s ligand-bound extracellular domain through its β subunit’s cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin αIIbβ3 is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on αIIbβ3, we examined αIIbβ3/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that α-actinin, myosin heavy chain and Syk co-immunoprecipitate with αIIbβ3 in resting platelets, and that 120 dynes/cm² shear stress leads to their disassociation from αIIbβ3. Shear-induced disassociation of α-actinin and myosin heavy chain from the β3 tail is unaffected by blocking VWF binding to GpIb-IX-V but abolished by blocking VWF binding to αIIbβ3. Syk’s disassociation from β3 is inhibited when VWF binding to either GpIb-IX-V or αIIbβ3 is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine phosphorylated SLAP-130 are inhibited by blocking ligand binding to αIIbβ3 but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary (CHO) cells expressing human αIIbβ3 demonstrate calcium and shear-dependent (1 and 10 dynes/cm²) attachment to glass cover slips coated with 20 μg/ml VWF. CHO cells expressing αIIbβ3 with β3 truncated of its cytoskeletal binding domains at C-terminal residue 716 demonstrate decreased VWF-dependent adhesion and cohesion in response to 1 dyne/cm² shear stress. These results support the hypothesis that shear stress directly modulates αIIbβ3 function, and suggest that shear-induced αIIbβ3-mediated signaling regulates platelet aggregation by directing the release of α-actinin and myosin heavy chain from the β3 tail.