Investigation into the mechanism of the thrombin receptor function of GPIB

描述(申请人提供)：在美国，超过8300万成年人患有心血管疾病，这使他们非常容易心脏病发作和中风。在美国，33%的死亡是由心脏病和中风引起的。在许多这样的病例中，潜在的原因是动脉血栓形成，其中血小板起着关键作用。血小板对于血栓形成和止血是必不可少的。当血管受损时，循环中的血小板迅速附着在损伤部位，被激活，并聚集形成原发血栓。最初的血小板与内皮下基质蛋白的黏附，特别是在高剪切率条件下，是由von Willebrand因子(VWF)及其受体--血小板膜糖蛋白(GP)Ib-IX-V复合体(GPIb)之间的相互作用所介导的。VWF和GPIB的相互作用不仅在启动血小板黏附中起重要作用，而且在传递对充分激活血小板至关重要的信号方面也是重要的。大量报道表明，GPIB可促进凝血酶诱导的血小板活化。然而，凝血酶与GPIB的结合是如何调节的，凝血酶与GPIB的结合如何促进血小板的激活仍不清楚。利用基因工程细胞表达截短形式的GPIB和GPIB基因敲除小鼠，我们证明了GPIB与细胞内信号蛋白14-3-3的相互作用是刺激所必需的 GPIB在凝血酶诱导的血小板活化中的作用我们认为，14-3-3与GPIB的结合是低剂量凝血酶激活血小板所必需的。利用分子生物学和骨髓移植相结合的方法，我们将测试这种相互作用的缺失是否可以阻止凝血酶诱导的小鼠的血小板反应，这些小鼠表达的GPIB在关键的14-3-3结合位点发生突变。我们推测14-3-3与GPIB的结合在凝血酶诱导的血小板活化中起着重要的刺激作用。这项提案的具体目标是：1)确定14-3的职能作用 3-GPIB在凝血酶诱导的血小板活化中的相互作用2)研究GPIB促进血小板对凝血酶反应的机制。最终，了解凝血酶信号是如何由14-3-3-GPIB相互作用调节的，将导致开发治疗心脏病发作和中风患者的新药。

DESCRIPTION (provided by applicant): In the United States, more than 83 million adults live with cardiovascular diseases making them highly susceptible to heart attack and stroke. 33 percent of all deaths in the U.S. are caused by heart diseases and stroke. In many of these cases the underlying cause is arterial thrombosis, in which platelets play a critical role. Plateles are essential to thrombosis and hemostasis. Upon damage of a blood vessel, circulating platelets rapidly adhere to the site of injury, become activated, and aggregate to form a primary thrombus. The initial platelet adhesion to subendothelial matrix proteins, particularly at high shear rate conditions, is mediated by the interaction between von Willebrand factor (VWF) and its receptor, the platelet glycoprotein (GP) Ib-IX-V complex (GPIb). The interaction of VWF and GPIb is not only important in initiating platelet adhesion, but also for transmitting signals important for full platelet activation. Numerous reports have implicated GPIb in promoting thrombin induced platelet activation. However, it is still unclear how thrombin binding to GPIb is regulated and how thrombin binding to GPIb promotes platelet activation. Using cells genetically engineered to express a truncated form of GPIb¿ and GPIb¿ knockout mice, we show that the interaction of GPIb with the intracellular signaling protein 14-3-3 is required for the stimulatory role of GPIb in thrombin-induced platelet activation. We believe that the binding of 14-3-3 to GPIb is required for platelets to become activated in response to low doses of thrombin. Using molecular biology in combination with bone marrow transplantation methods we will test whether deletion of this interaction prevents thrombin-induced platelet responses in mice expressing GPIb with a mutation in a critical 14-3-3 binding site. We hypothesize that the binding of 14-3-3 to GPIb plays an important stimulatory role in thrombin-induced platelet activation. The specific aims of this proposal are: 1) to determine the functional role of the 14-3 3-GPIb interaction in thrombin-induced platelet activation using platelets expressing 14-3-3 binding deficient GPIb and 2) to investigate the mechanism by which GPIb promotes the platelet response to thrombin. Ultimately understanding how thrombin signaling is regulated by the 14-3-3-GPIb interaction will lead to the development of novel drugs for the treatment of patients suffering from heart attack and stroke.