Clathrin-mediated integrin αIIbβ3 trafficking controls platelet spreading

Dynamic endocytic and exocytic trafficking of integrins is an important mechanism for cell migration, invasion, and cytokinesis. Endocytosis of integrin can be classified as clathrin dependent and clathrin independent manners. And rapid delivery of endocytic integrins back to the plasma membrane is key intracellular signals and is indispensable for cell movement. Integrin alpha IIb beta 3 plays a critical role in thrombosis and hemostasis. Although previous studies have demonstrated that internalization of fibrinogen-bound alpha IIb beta 3 may regulate platelet activation, the roles of endocytic and exocytic trafficking of integrin alpha IIb beta 3 in platelet activation are unclear. In this study, we found that a selective inhibitor of clathrin-mediated endocytosis pitstop 2 inhibited human platelet spreading on immobilized fibrinogen (Fg). Mechanism studies revealed that pitstop 2 did not block the endocytosis of alpha IIb beta 3 and Fg uptake, but inhibit the recycling of alpha IIb beta 3 to plasma membrane during platelet or CHO cells bearing alpha IIb beta 3 spreading on immobilized Fg. And pitstop 2 enhanced the association of alpha IIb beta 3 with clathrin, and AP2 indicated that pitstop 2 inhibit platelet activation is probably due to disturbance of the dynamic dissociation of alpha IIb beta 3 from clathrin and AP2. Further study demonstrated that Src/PLC/PKC was the key pathway to trigger the endocytosis of alpha IIb beta 3 during platelet activation. Pitstop 2 also inhibited platelet aggregation and secretion. Our findings suggest integrin alpha IIb beta 3 trafficking is clathrin dependent and plays a critical role in platelet spreading, and pitstop 2 may serve as an effective tool to address clathrin-mediated trafficking in platelets.