Adaptor Protein CIN85 Supports Platelet Integrin αIIbβ3 outside-in Signaling and Thrombus Formation

Abstract

Activation of platelets in the setting of an atherosclerotic rupture contributes to thrombosis. Platelet responses at these sites are dependent on agonist induced inside-out and integrin αIIbβ3 mediated outside-in signaling processes, which in turn are regulated by protein kinases and phosphatases. Reversible tyrosine and/or serine/threonine phosphorylation dependent assembly of effector proteins propagate signaling downstream of platelet receptors. Adaptor proteins are key effectors in signal transmission because they are endowed with multiple structural domains, which enable them to engage with a wide variety of proteins in a spatial and temporal fashion to fine tune signaling. Emerging studies in the field suggest a potential for targeting integrin-induced outside-in signaling processes to attenuate thrombotic responses. During the course of our studies to understand how the catalytic subunit of protein phosphatase 2A (PP2Ac) regulates platelet integrin outside-in signaling, we previously identified a new complex of PP2Ac with an adaptor protein CIN85 (Cbl-interacting protein of 85kDa) in human platelets. Disruption of an endogenous PP2Ac-CIN85 complex with a cell permeable myristylated P3 synthetic peptide decreased platelet integrin αIIbβ3-dependent signaling functions. However, since the adaptor protein CIN85 has not been characterized in platelets, the contribution of CIN85 in integrin signaling is unknown. To explore the potential role of CIN85 in integrin function, we generated a platelet specific CIN85-/- mice by crossing the CIN85 flox/flox mice with a PF4Cre mice. Immunoblotting studies confirmed that platelets but not the non-megakaryocytic tissues from CIN85-/- mice lost the ~85kDa CIN85 protein. Loss of CIN85 did not significantly alter agonist-induced primary aggregation response, suggesting comparable inside-out signaling response. In contrast, integrin αIIbβ3 outside-in signaling responses such as spreading on immobilized fibrinogen and fibrin-mediated clot retraction was decreased in CIN85-/- platelets. Activation of Src that promotes integrin outside-in signaling was decreased in fibrinogen engaged CIN85-/- platelets. Perfusion of whole blood from the CIN85-/- mice on collagen at a shear rate of 1000 s-1 revealed significantly decreased platelet adhesion and thrombus formation. PLCγ2 is activated downstream of platelet collagen receptor engagement, and CIN85-/- platelets showed decreased PLCγ2 Tyr 529 phosphorylation. These studies indicate that the adaptor protein CIN85 supports platelet integrin outside in signaling functions. To extend these findings in human platelets, we disrupted CIN85-PP2A complex with the myristylated P3 peptide. P3 peptide but not the scrambled peptide significantly decreased human platelet adhesion and thrombus formation on collagen at a shear rate of 1000 s-1. Thus, loss of CIN85 in murine platelets or disruption of CIN85-PP2A complex in human platelets attenuates integrin outside-in signaling and thrombus formation. These studies suggest that CIN85 may represent a new potential anti-thrombotic target. DisclosuresNo relevant conflicts of interest to declare.