Junctional Adhesion Molecule-A Is a Novel Csk-Binding Protein Which Recruits Csk to the Integrin a_IIbb₃ and Suppresses Outside-In Signaling In Platelets

Abstract 2120

Platelet activation is regulated by both positive and negative regulators present within the platelets so that unwanted activation is suppressed, but, when needed, occurs rapidly. A significant amount of effort has been devoted towards understanding the positive regulators of platelet function. However, very little is known about the negative regulators. Dysregulation of the endogenous negative regulators may aid the thrombotic complications seen in various diseases. Upon ligand binding to integrin a_IIbb_3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. How endogenous negative regulators suppress these events is not well understood. We have previously identified junctional adhesion molecule A (JAM-A), on the platelet surface. We found that Jam-a knockout mice show a prothrombotic phenotype as assessed by significantly (P<0.00001) shortened tail bleeding time, decreased carotid vessel occlusion time and increased pulmonary thromboembolism. Platelet functional studies revealed that Jam-a null platelets were hyperactive to physiological agonists. Surprisingly, inside-out signaling events were not affected in Jam-a null platelets. It is therefore possible that observed hyper-reactivity of Jam-a null platelets could be due to enhanced outside-in signaling. To test this, we performed a clot retraction assay. The clot retraction in wild type (Wt) occurred normally, which began after 1 h and about 50% was completed by 2 h. On the contrary, in Jam-a null platelets, the process of clot retraction was significantly enhanced (P<0.0001). It was initiated before 1h and was completed within 2 h. When analyzed for outside-in signaling events such as b₃ tyrosine phosphorylation and c-Src phosphorylation, we found both significantly enhanced in Jam-a null platelets. To assess the mechanism by which JAM-A suppresses outside-in signaling, we analyzed the phosphorylation status of JAM-A. Interestingly, JAM-A was found to be phosphorylated on Y280 in unactivated platelets and rapidly dephosphorylated upon initiation of outside-in signaling. On the other hand, in resting platelets, a minimally phosphorylated S284 residue of JAM-A is rapidly phosphorylated, suggesting that there is a dephosphorylation/phosphorylation switch that may be involved in regulating outside-in signaling. Furthermore, we found that JAM-A associates with integrin a_IIbb₃ in unactivated human platelets, but this association was disrupted during outside-in signaling as determined by co-immunoprecipitation. We also found Csk, a C-terminal Src kinase, coimmunoprecipitating (IP) with JAM-A from resting, but not activated, platelet lysates, suggesting that JAM-A may be recruiting Csk to unactivated integrins and thus suppressing signaling. To test this, we analyzed association of Csk with integrin in Jam-a null platelets and found that in Wt platelets Csk was abundantly present in the integrin IP, but was completely absent in the integrin IP of the Jam-a null platelet lysates. These results clearly suggest that JAM-A recruits Csk to the integrin and thus suppresses outside-in signaling.