The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3)-binder Rasa3 Regulates Phosphoinositide 3-kinase (PI 3-kinase)-dependent Integrin αIIbβ3 Outside-in Signaling

The class I phosphoinositide 3-kinase (PI 3-kinase) family of lipid kinases plays an important role in integrin αIIbβ3 function, thereby supporting thrombus growth and consolidation. Here, we identify the Ras/Rap1GAP Rasa3 (GAP1IP4BP) as a major phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3)-binding protein in human platelets and a key regulator of integrin αIIbβ3 outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI 3kinase-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin αIIbβ3-expressing CHO cells blocked Rap1 activity and integrin αIIbβ3-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 effect integrin αIIbβ3-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 PI 3-kinase inhibitors. Together, these results support an important role for Rasa3 in PI 3-kinase-dependent integrin αIIbβ3-mediated outside-in signaling and cell spreading.