Dok-2 Adaptor Protein Regulates the Shear- dependent Adhesive Function of Platelet Integrin αIIβ3 in Mice*

Background: Dok proteins are negative regulators of immunoreceptor signaling and, potentially, integrin adhesion receptors. Results: Deficiency of Dok-2 results in enhanced shear-dependent integrin adhesion in platelets, leading to accelerated platelet thrombus growth. Conclusion: Dok-2 is a shear-specific negative regulator of blood clot formation. Significance: Dok-2 regulates biomechanical platelet adhesion, and targeting this molecule may provide new avenues to regulate thrombosis. The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin (IIb3), raising the possibility that it participates in integrin (IIb3) outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin (IIb3)-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P-2 accumulation. Although agonist-induced integrin (IIb3) affinity regulation was unaltered in Dok-2(-/-) platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin (IIb3) adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.