A Contractile Cytoskeletal Tether Modulates Signaling between Glycoprotein Ibα and αIIbβ3.

Abstract In resting platelets, the cytoplasmic domains of glycoprotein (Gp) Ibα and β3 integrin link the GpIb-IX-V and αIIbβ3 complex with adapter proteins, cytoskeletal elements and lipid and tyrosine kinases. Biochemical and pharmacological analyses of intact platelets and genetic analyses of recombinant GpIb-IX-V and αIIbβ3 in CHO cells point towards the hypothesis that a series of cytoskeletal proteins form a functional tether connecting the cytoplasmic domains of GpIbα and β3 integrin. To test the hypothesis that dynamic contractility of this tether modulates VWF-induced signaling between GpIb-IX-V and αIIbβ3, we examined how pathological shear stress affects connections between tethering elements and how altering the contractile function of the tether affects VWF-induced platelet adherence and aggregation. We observed in resting platelets that there is no co-immunoprecipitation of GpIbα and αIIbβ3. The large dimeric actin-binding protein filamin A co-immunoprecipitates with both GpIbα and β3 integrin, but its association with αIIbβ3 is inhibited by DNaseI, indicating that it binds indirectly to αIIbβ3 through cytoskeletal connections. These connections were investigated in resting platelets by examining a series of immunoprecipitates (IP) for co-precipitating proteins using immunoblotting (IB). We observed the following IP/IB pairs [(+) designates that the co-precipitation is DNaseI-sensitive]: β3/talin (+); talin/α-actinin (+); talin/vinculin (+); and talin/filamin A (−). We also observed that myosin heavy chain (MHC) and the tyrosine kinase Syk co-immunoprecipitate with αIIbβ3 in resting platelets. When washed platelets in buffer containing calcium (1 mM) and VWF (5 μg/ml) were sheared at 120 dynes/cm2 for 2 minutes, the cytoskeletal linkage separates: there was enhanced filamin A, actin and α-actinin binding to GpIbα, enhanced vinculin binding to α-actinin, enhanced talin binding to αIIbβ3, and both myosin and the tyrosine kinase Syk disassociated from the β3 tail. Inhibiting myosin contractility with the myosin light chain kinase (MLCK) inhibitor ML-9 (1 mM) inhibited the shear-induced association between talin and β3, as well as platelet aggregation in response to 120 dynes/cm2 in the cone plate viscometer and platelet-dependent thrombosis from whole blood onto type I collagen in a parallel plate flow chamber with a shear rate of 500 sec−1 (shear stress of ~ 20 dynes/cm2). The data presented support a model of shear-induced platelet aggregation in which a series of cytoskeletal proteins serve as a mechanotransducing scaffolding linking the cytoplasmic domains of GpIbα and β3 integrin. When GpIb-IX-V engages ligand under shearing forces, the force is transduced from GpIbα to filamin to actin/α-actinin to vinculin to talin to β3, and contractility driven by the activation of αIIbβ3-associated myosin enhances talin binding to β3, thereby effecting a conformation change that creates a ligand-receptive αIIbβ3. Such interactions may be regulated by cytosolic ionized calcium (which effects MLCK activation) and tyrosine kinases (αIIbβ3-associated Syk disassociates from β3 following shear). These results provide evidence that the structural scaffolding connecting GpIb-IX-V with αIIbβ3 localizes signaling elements to functionally important compartments that modulate αIIbβ3 activation and shear-induced platelet aggregation.