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3247 A Tyrosine-Serine Phosphorylation Switch in the JAM-A Cytoplasmic Domain Regulates In Vivo Thrombus Growth and Stability

Program: Hemostasis and Thrombosis
Session: Platelet Activation and Biochemistry
Tuesday, December 13, 2011, 12:00 PM-1:30 PM
Room 24 (San Diego Convention Center)

Meghna Ulhas Naik, MS and Ulhas P Naik, Ph.D. FAHA

Biological Sciences, University of Delaware, Newark, DE

Dysregulation of the endogenous negative regulators present within the platelets may aid the thrombotic complications seen in various diseases. How endogenous negative regulators suppress platelet functions is not well understood. We have shown that junctional adhesion molecule A (JAM-A), a member of CTX (Cortical Thymocyte marker of the Xenopus) family is expressed on platelets. To understand the role of JAM-A in thrombosis, we used the Jam-A knockout mouse. 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, (P<0.002) and significantly increased susceptibility to pulmonary thromboembolism (P<0.008). Platelet functional studies revealed that Jam-A null platelets were hyperaggregate to physiological agonists. Surprisingly, inside-out signaling events were not affected, whereas outside-in signaling such as platelet spreading and clot retraction were significantly enhanced (P<0.0001) in Jam-A null platelets compared to wild type (WT). When we analyzed outside-in signaling events such as b3 tyrosine phosphorylation and c-Src phosphorylation, we found both to be significantly enhanced in Jam-A null platelets. Interestingly, we found an absence of inhibitory Y529 phosphorylation of c-Src in resting Jam-A null platelets compared to WT. Furthermore, we found that in WT platelets, Csk, a C-terminal Src kinase which is responsible for Y529 phosphorylation, was abundantly present in the integrin aIIbb3 immunoprecipitate, but was completely absent in the integrin immunoprecipitate of the Jam-A null platelet lysates, suggesting that JAM-A may be responsible for the recruitment of Csk to the integrin complex in resting platelets. We found that JAM-A associates with integrin aIIbb3 as well as Csk on unactivated platelets and dissociates upon platelet activation. We also found that JAM-A is tyrosine phosphorylated in resting platelets and is rapidly dephosphorylated upon platelet activation. This tyrosine phosphorylation was necessary for the association of Csk. On the other hand, in resting platelets, a minimally phosphorylated S284 residue of JAM-A is rapidly phosphorylated upon agonist stimulation. This phosphorylation was inhibited by PKC inhibitors GF109203X and Ro32-0432. Interestingly, when platelets are allowed to spread on immobilized fibrinogen, JAM-A does not localize to the inter-platelet contact. However, addition of agonist promptly induces accumulation of JAM-A at these contacts. This accumulation can be completely inhibited by PKC inhibitors. These results strongly suggest that tyrosine phosphorylated JAM-A is an endogenous negative inhibitor of platelet activation and thus thrombosis. It does so by recruiting Csk to the integrin, which negatively regulates c-Src activation and thus suppresses the initiation of thrombus growth by attenuating outside-in signaling. Upon agonist stimulation, JAM-A is dephosphorylated on the tyrosine and phosphorylated on the serine residue by PKC, allowing the dissociation of Csk and accumulation of JAM-A at the inter-platelet contacts, which likely provides stability to the clot.

Disclosures: No relevant conflicts of interest to declare.

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