Junctional Adhesion Molecule a (JAM-A) Associates with CD36 upon Platelet Activation and Cytoskeleton in a Phosphorylation-Dependent Manner to Promote Platelet-Platelet Contacts

Abstract

Background: Platelets play a fundamental role inmaintaining hemostasis. Stability of hemostatic plug is crucial for arresting blood loss during vascular breach. JAM-A is a member of the immunoglobulin superfamily that restricts α IIbβ 3 to its low affinity state by recruiting CSK to the α IIbβ 3-c-Src complex in resting platelets. Agonist-induced inside-out signaling causes JAM-A to dissociate from the α IIbβ 3-Src complex, thus facilitating activation of α IIbβ 3 and outside-in signaling. However, the function of JAM-A in the process of hemostasis is poorly understood. Aim: To evaluate the role of JAM-A in activated platelets and on hemostasis. Methods: Washed human platelets were used in these studies. Full-length cytoplasmic domain of JAM-A (cyto JAM-A) and JAM-A cytoplasmic domain lacking C-terminal PDZ-domain-binding motif (ΔPDZ cyto JAM-A) were expressed as GST-fusion proteins in bacteria using pGEX4T-1 vector. GST-fusion proteins were purified using glutathione beads. Pull-down assays were performed by incubating glutathione GST-fusion proteins beads with lysates of washed human platelets (4×10 8/mL). GST beads alone without the fusion proteins were used as a control. Immunoprecipitation studies were performed using washed human platelets (4x10 8/mL) lysed with CHAPS lysis buffer and precleared by using Protein G Sepharose beads. Specific primary antibodies and isotype specific IgGs (control) were used for immunoprecipitation followed by western blotting. To identify protein complexes two-dimensional blue-native polyacrylamide gel electrophoresis was performed in which protein complexes were first separated based on their size (1 st dimension) and individual proteins in each complex were separated using SDS PAGE (2 nd dimension). JAM-A S 284 phosphorylation was detected using anti-Phospho-S 284. Rap1 activation assay was performed by pull-down GTP-bound Rap1 following the manufacturer's instructions. Results: CD9 and α IIbβ 3 co-immunoprecipitated with JAM-A from resting platelet lysates suggesting that CD9 is a part of the JAM-A/α IIbβ 3 complex. In a pull-down assay recombinant full-length JAM-A cytoplasmic tail fused with GST, but not JAM-A tail lacking the PDZ domain motif fused with GST pull-down CD9 and α IIbβ 3 from resting platelet lysates suggesting that PDZ-domain binding motif of JAM-A is responsible for its interaction with CD9/α IIbβ 3 complex. Interestingly, platelet activation resulted in the dissociation of JAM-A from the CD9/α IIbβ 3 complex as seen by loss of interaction between JAM-A and CD9/α IIbβ 3 in co-immunoprecipitation and blue-native PAGE assays. It is reported that platelet activation results in rapid phosphorylation of JAM-A on S 284 in a PKC-dependent manner. We found that S 284 phosphorylated JAM-A associates with CD36 and this complex moves to the Triton X-100 insoluble cytoskeletal fraction upon platelet activation suggesting that phosphorylation of JAM-A may assist its localization to cytoskeleton. Interestingly, we found that induction of conformational change in α IIbβ 3 using Mn 2+ (10μM) activates JAM-A S 284 phosphorylation. Furthermore, Mn 2+ also induced a robust activation of Rap1, a key signaling molecule involved in junctional assembly. Both JAM-A phosphorylation and Rap1 activation were significantly ( P<0.05) inhibited by a pan PKC inhibitor, bisindolylmaleimide (10μM) suggesting that S 284 phosphorylated JAM-A/CD36 complex may support junctional assembly within the platelet plug. Conclusion: JAM-A, through its cytoplasmic domain, associates with α IIbβ 3 and CD9 in resting platelets. Upon platelet activation JAM-A rapidly dissociates from this complex and is phosphorylated on S 284. Phosphorylated JAM-A then associates with CD36 and cytoskeleton leading to activation of Rap1, a protein important in junctional assembly between platelets within a hemostatic plug.