Endogenous suppression of integrin signaling

心血管疾病（CVD）是人类的头号杀手。大多数心血管疾病与 动脉粥样硬化和血栓形成有充分的证据表明，血小板是这两种疾病的始作俑者。 动脉粥样硬化和血栓形成。血小板在循环中保持静止状态， 内源性负调节因子，以避免无意激活。在血管损伤期间，亲- 刺激信号优先于抗刺激信号以实现快速血小板活化。很多 研究集中在促刺激信号上，而对负调节因子知之甚少。我们 已经表明JAM-A是血小板活化的内源性抑制剂。JAM-A消融 增强体内血栓形成。然而，当血小板活化时， JAM-A是未知的。我们假设JAM-A在从细胞中解离后形成二聚体， 血小板-血小板连接处的整合素复合物和支持连接组装 通过激活Rap1。R01提案的重点是界定JAM-A的作用 启动血小板-血小板连接形成，从而防止伤口失血。 因此，提出了三个具体目标。具体目标1将检验假设 血小板JAM-A通过CD9（一种四跨膜蛋白）与整联蛋白结合，形成一个多- 蛋白质复合物中的PDZ结构域依赖性的方式。我们将使用生物化学，突变和 遗传学的方法来确定这个复杂的组成部分。具体目标2将测试 血小板活化后JAM-A二聚化和组装Rap1活化的假说 复杂，以实现Rap1激活在由外向内信号。我们将使用生化， 突变和遗传学方法来描述调节JAM-A的分子机制， 依赖Rap1激活。具体目标3将检验JAM-A负责 在血小板-血小板接触处募集连接蛋白以组装功能性连接。我们 将使用转基因细胞和小鼠来评估JAM-A和其他已知的 使用体外和体内测定调节止血塞渗透性的连接蛋白。 成功完成这一提议将有助于理解交界粘连的作用 止血分子，并开发血栓形成相关的治疗干预 例如动脉粥样硬化、MI和中风的疾病。

Cardiovascular disease (CVD) is the number one killer of mankind. Most CVDs are associated with atherosclerosis and thrombosis. It is well documented that platelets are the initiators of both atherosclerosis and thrombosis. Platelets are maintained in resting state in the circulation by endogenous negative regulators to avoid unintentional activation. During vascular injury, pro- stimulatory signals override anti-stimulatory signals to achieve rapid platelet activation. Lot of research is focused on pro-stimulatory signals while little is known about negative regulators. We have shown that JAM-A is an endogenous suppressor of platelet activation. Ablation of JAM-A confers an augmentation of in vivo thrombosis. However, upon platelet activation what happens to JAM-A is not known. We hypothesize that JAM-A forms a dimer upon dissociation from the integrin complex and support junctional assembly at the platelet–platelet junctions through the activation of Rap1. This R01 proposal is focused on delineating the role of JAM-A in initiating platelet-platelet junction formation, and thus preventing blood loss through the wound. Accordingly, three Specific Aims have been proposed. Specific Aim 1 will test the hypothesis that platelet JAM-A associates with the integrin through CD9, a tetraspanin, by forming a multi- protein complex in a PDZ-domain-dependent manner. We will use biochemical, mutational and genetic approaches to identify the components of this complex. Specific Aim 2 will test the hypothesis that upon platelet activation JAM-A dimerizes and assembles Rap1-activating complex to achieve Rap1 activation during outside-in signaling. We will use biochemical, mutational and genetic approaches to delineate the molecular mechanism that regulate JAM-A- dependent Rap1 activation. Specific Aim 3 will test the hypothesis that JAM-A is responsible in recruiting junctional proteins at the platelet-platelet contacts to assemble functional junctions. We will use genetically modified cells and mice to evaluate the role of JAM-A and other known junctional proteins in regulating permeability of hemostatic plug using in vitro and in vivo assays. Successful completion of this proposal will help to understand the role of junctional adhesion molecules in hemostasis and to develop therapeutic interventions for thrombosis associated diseases such as atherosclerosis, MI, and stroke.