Platelet CLEC-2 in Arterial Thrombosis

项目总结/摘要 动脉血栓性疾病，如缺血性心脏病，是老年人残疾和死亡的主要原因。 美国的破裂部位血小板粘附和血栓性血小板聚集体的形成 动脉粥样硬化斑块或动脉血流下受损的内皮在动脉粥样硬化的发病机制中是必不可少的。 血栓形成在高流量或扰动流量条件下，血小板与血管壁之间的初始相互作用是 主要由血管性血友病因子（vWF）和血小板糖蛋白Iba（GPiba）介导，随后导致 以血小板内容物的释放、聚集和激活凝血。这些机制对于 止血和血栓形成都是目前FDA批准的抗血小板疗法的目标。虽然他们是 有效，但都有引起出血的危及生命的副作用，这大大限制了它们的临床使用。到 为了解决这一未满足的需求，进一步阐明血栓形成的基本机制至关重要， 止血药。 最近发表的来自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结合唾液酸化的0-聚糖。我们的初步数据表明CLEC-2与GPIba相互作用 以唾液酸化依赖的方式。此外，我们的初步研究结果表明，CLEC-2促进GPiba- 介导的整合素αIIbβ3活化，这对体内动脉血栓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介导的血小板活化至关重要，这是动脉血栓生长和稳定所需的。为了验证这个， 我们将1）检验CLEC-2通过以下相互作用调节GPIba介导的血小板活化的假设： 其凝集素样结构域和GPIba的唾液酸化O-聚糖，因为GPIba被唾液酸化O-聚糖严重修饰; 2） 确定CLEC-2是否/如何通过促进GPIba介导的整合素aIIbb 3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在小鼠血小板上以相似的高水平表达，并且这两种受体在血小板活化中是必需的。 动脉血栓形成然而，它们在动脉血栓形成中的作用机制要么是难以捉摸的， （GPiba）或未知（CLEC-2）。我们提出的研究将提供新的机制的见解，这些突出的 现场提问。这可能会导致一个新的有效和安全的抗血栓治疗的发展。

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.