Platelet CLEC-2 in Arterial Thrombosis

项目摘要/摘要 动脉血栓性疾病，如缺血性心脏病，是导致残疾和死亡的主要原因 美国。血栓破裂部位的血小板黏附和血栓形成 动脉血流下的动脉粥样硬化斑块或受损的内皮细胞在动脉粥样硬化的发病中是必不可少的。 血栓形成。在高流速或扰动流动条件下，血小板与血管壁之间的初始相互作用是 主要由von Willebrand因子(VWF)和血小板糖蛋白Iba(GPIba)介导，随后导致 对血小板内容物的释放、聚集和凝血的激活。这些机制对于 止血和血栓形成都是目前FDA批准的抗血小板治疗的靶点。尽管他们是 有效，所有这些都有导致出血的危及生命的副作用，这大大限制了它们的临床应用。至 解决这一未得到满足的需求，关键是进一步阐明对血栓形成的基本机制的见解，但 止血是可有可无的。 最近发表的几个独立实验室的数据表明，血小板CLEC-2(C型凝集素样受体2)是 在动脉血栓形成中很重要。然而，CLEC-2如何调节动脉血栓形成尚不清楚。凝集素结构域 已知CLEC-2的结合于唾液酸化的O-多糖。我们的初步数据显示CLEC-2与GPIba相互作用 以唾液酸化依赖的方式。此外，我们的初步结果显示，CLEC-2促进GPIba- 介导整合素αIIbβ3的激活，这对体内动脉血栓的生长和稳定性至关重要。重要的是 阻断CLEC-2功能不会延长体内出血时间。因此，我们假设CLEC-2是 对于GPIba介导的血小板激活至关重要，这是动脉血栓生长和稳定所必需的。为了测试这一点， 我们将1)检验CLEC-2通过相互作用调节GPIba介导的血小板激活的假设 其凝集素样结构域和唾液酸化的O-葡聚糖作为GPIba被唾液酸化的O-葡聚糖大量修饰； 确定CLEC-2是否/如何通过促进GPIba介导的整合素aIIbb3活化来稳定动脉血栓 小鼠和人动脉血栓形成模型。 CLEC-2和GPIba在小鼠血小板上有类似的高水平表达，这两种受体在 动脉血栓形成。然而，它们在动脉血栓形成中的潜在作用机制要么是难以捉摸的 (GPIba)或未知(CLEC-2)。我们提议的研究将为这些突出的问题提供新的机械论见解 在现场的问题。这可能会导致开发一种新的有效、安全的抗血栓治疗方法。

PROJECT SUMMARY/ABSTRACT Arterial thrombotic diseases such as ischemic heart disease are the leading cause of disability and death in the United States. Platelet adhesion and formation of thrombotic platelet aggregates at the site of a ruptured atherosclerotic plaque or damaged endothelium under arterial blood flow is essential in the pathogenesis of arterial thrombosis. Under high or disturbed flow conditions, the initial interaction between platelets and the vessel wall is primarily mediated by von Willebrand factor (vWF) and platelet glycoprotein Iba (GPIba), which subsequently leads to platelet content release, aggregation, and activation of the coagulation. These mechanisms, which are critical for both hemostasis and thrombosis, are targets of current FDA-approved antiplatelet therapies. Although they are effective, all have the life-threatening side effect of causing bleeding, which significantly limits their clinical use. To address this unmet need, it is critical to further elucidate insights into mechanisms essential for thrombosis but dispensable for hemostasis. Recent published data from several independent labs show that platelet CLEC-2 (C-type lectin-like receptor 2) is important in arterial thrombosis. However, how CLEC-2 regulates arterial thrombosis is unknown. The lectin-domain of CLEC-2 is known to bind to sialylated O-glycans. Our preliminary data showed that CLEC-2 interacts with GPIba in a sialylation-dependent manner. Furthermore, our preliminary results reveal that CLEC-2 promotes GPIba- mediated activation of integrin αIIbβ3, which is critical for arterial thrombus growth and stability in vivo. Importantly, blocking CLEC-2 function does not prolong the bleeding time in vivo. Therefore, we hypothesize that CLEC-2 is critical for GPIba-mediated platelet activation that is required for arterial thrombus growth and stability. To test this, we will 1) test the hypothesis that CLEC-2 regulates GPIba-mediated platelet activation through interaction between its lectin-like domain and sialylated O-glycans of GPIba as GPIba is heavily modified by sialylated O-glycans; 2) determine if/how CLEC-2 stabilizes the arterial thrombus by facilitating GPIba-mediated integrin aIIbb3 activation using mouse and human arterial thrombosis models. CLEC-2 and GPIba are expressed at similar high levels on murine platelets, and both receptors are essential in arterial thrombosis. However, the mechanisms underlying their role in arterial thrombosis have been either elusive (GPIba) or unknown (CLEC-2). Our proposed study will provide new mechanistic insights into these outstanding questions in the field. It may lead to the development of a new effective and safe anti-thrombosis therapy.