Platelet Junctional Adhesion Molecule-A Regulates Thrombosis by Negatively Regulating Outside-in Signaling through Integrin αIIbβ3.
Authors: Naik Meghna Ulhas , Stalker Timothy J. , Brass Lawrence F , Naik Ulhas P 
Published: October 2019

Abstract 155

Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of signaling events called inside-out signaling that leads to the activation of integrin αIIbβ3. Upon ligand binding to integrin αIIbβ3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. How these signaling events are regulated is not well understood. We have previously identified JAM-A, a junctional adhesion molecule, on the platelet surface. Genetic ablation of Jam-A significantly (P<0.00001) shortened tail bleeding times (mean 54 seconds) compared to wild-type littermates (mean 98 seconds), suggesting an enhanced clot formation. FeCl3-induced carotid artery thrombosis and laser-induced cremaster arteriole thrombosis, two well recognized in vivo thrombosis model, both showed greatly shortened time of vessel occlusion and increased thrombus formation compared to wild-type (WT). Since JAM-A is expressed both on the endothelium and on platelets, the observed defect could arise from the lack of JAM-A in either cell type. To identify the involvement of platelet derived JAM-A, we performed a collagen-epinephrine-induced pulmonary thromboembolism assay. In this assay, pulmonary vessel occlusion occurs primarily through platelet thrombus without injury to the endothelium. We found that a significantly increased number of Jam-A−/− mice died within two minutes compared to WT mice. When analyzed for the extent of pulmonary vascular occlusion by Evans blue exclusion as well as histochemical analysis, we found a significantly greater extent of thromoembolism in Jam-A−/− mice compared to WT mice. Consistent with this finding, agonist-induced platelet aggregation, but not secretion, was significantly enhanced in Jam-A−/− platelets. Interestingly, however, the expression or activation of integrin αIIbβ;3 was not affected by Jam-A deficiency. When we analyzed the rate of clot retraction, we found that Jam-A−/− platelets showed 85% clot retraction within 90 minutes compared to only a 20% clot retraction in WT platelets, indicating that the absence of Jam-A significantly increases the rate of clot retraction (P<0.0001). JAM-A was found to associate with integrin αIIbβ3 in unactivated human platelets, but this association was disrupted during outside-in signaling as determined by co-immunoprecipitation assay suggesting that JAM-A may suppress signaling through the integrin. To delineate the molecular mechanism influenced by JAM-A, we analyzed the known signaling cascade involved in clot retraction. We found that outside-in signaling-induced activation of ERK1 and p38 MAP kinase was significantly enhanced in Jam-A−/− platelets. However, activation of focal adhesion kinase was unaffected in Jam-A−/− platelets compared to WT. Furthermore, outside-in signaling-induced phosphorylation of the myosin light chain was increased in Jam-A−/− platelets. These in vivo and in vitro results clearly show that JAM-A negatively regulates outside-in signaling through integrin αIIbβ;3, thus protecting from thrombosis.

Disclosures:

No relevant conflicts of interest to declare.