The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin α(IIb)β(3) Outside-in Signaling

The class I PI3K family of lipid kinases plays an important role in integrin α(IIb)β(3) function, thereby supporting thrombus growth and consolidation. Here, we identify Ras/Rap1GAP Rasa3 (GAP1(IP4BP)) as a major phosphatidylinositol 3,4,5-trisphosphate-binding protein in human platelets and a key regulator of integrin α(IIb)β(3) outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI3K-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin α(IIb)β(3)-expressing CHO cells blocked Rap1 activity and integrin α(IIb)β(3)-mediated spreading on fibrinogen. In contrast, Rap1GAP-deficient (P489V) and Ras/Rap1GAP-deficient (R371Q) Rasa3 had no effect. We furthermore show that two Rasa3 mutants (H794L and G125V), which are expressed in different mouse models of thrombocytopenia, lack both Ras and Rap1GAP activity and do not affect integrin α(IIb)β(3)-mediated spreading of CHO cells on fibrinogen. Platelets from thrombocytopenic mice expressing GAP-deficient Rasa3 (H794L) show increased spreading on fibrinogen, which in contrast to wild-type platelets is insensitive to PI3K inhibitors. Together, these results support an important role for Rasa3 in PI3K-dependent integrin α(IIb)β(3)-mediated outside-in signaling and cell spreading.