Endogenous suppression of integrin signaling

心血管疾病是人类的头号杀手。大多数CVD都与 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起始者 动脉粥样硬化和血栓形成。血小板在循环中被维持在静息状态 内源性负性调节因子，避免无意激活。在血管损伤期间，PRO- 刺激信号优先于反刺激信号，以实现快速的血小板激活。很多 研究集中在有利于刺激的信号上，而对负面监管机构知之甚少。我们 研究表明，JAM-A是一种内源性的血小板激活抑制因子。JAM-A的消融 增强体内血栓形成。然而，当血小板被激活时，会发生什么 对Jam-A的了解是未知的。我们假设JAM-A在解离时形成二聚体 血小板-血小板连接处的整合素复合体和支持性连接组装 通过激活RAP1。这份R01提案的重点是描述JAM-A的作用 在启动血小板-血小板连接形成，从而防止通过伤口失血。 因此，提出了三个具体目标。具体目标1将检验这一假设 这种血小板粘连蛋白-A通过CD9与整合素结合，这种整合素是一种四环素，通过形成多个- 以PDZ结构域依赖的方式与蛋白质复合体结合。我们将使用生化、突变和 用遗传方法来鉴定这个复合体的成分。《特定目标2》将测试 假设当血小板激活时，JAM-A二聚化并组装RAP1激活 在自外向内信号转导过程中实现Rap1激活的复合体。我们将使用生物化学， 用突变和遗传学方法描述调控JAM-A-A的分子机制 依赖的RAP1激活。具体目标3将测试JAM-A在 在血小板-血小板接触处招募连接蛋白以组装功能连接。我们 将使用转基因细胞和小鼠来评估JAM-A和其他已知的 结合蛋白对止血栓通透性调节作用的体内外研究。 成功地完成这一建议将有助于理解连接粘连的作用 止血分子和开发血栓相关的治疗干预措施 动脉粥样硬化、心肌梗塞和中风等疾病。

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.