Chemical Genetic Analysis of Platelet Granule Secretion

描述(申请人提供)：血小板是动脉血栓形成的重要组成部分。干预血小板信号转导可以显著降低血小板介导的血栓性疾病的发病率和死亡率，如心脏病发作、中风和外周血管疾病。然而，抑制血小板活化的最佳治疗靶点的确定已经放缓，因为血小板是无核的，不受经常用于研究有核细胞中的信号转导机制的遗传操作的影响。相比之下，血小板的现成可获得性及其暴露于激动剂后发生深刻表型变化的能力使其成为使用小分子进行研究的流行细胞模型。我们已经开发了一种高通量的检测方法来鉴定新型的抗血小板药物，这些药物通过密集的颗粒分泌来抑制血小板的激活。这项工作的目标是发现控制血小板颗粒分泌的新分子靶点。我们将与分子图书馆筛选中心网络(MLSCN)内的一个机构合作，扩大我们筛选的化合物的数量，提高筛选的速度，并优化具有抗血小板活性的化合物。我们将在两个相关的检测中评估颗粒分泌。第一种方法将确定抑制SFLLRN诱导的致密颗粒分泌的化合物，SFLLRN通过蛋白酶激活受体1刺激血小板。第二种方法将识别抑制I型胶原诱导的致密颗粒分泌的化合物，I型胶原通过糖蛋白VI激活血小板。这一策略将有助于鉴定通过不同机制抑制血小板功能的化合物。一旦确定，化合物将在先前建立的一系列血小板功能测试中进行测试，并在体内血栓形成的小鼠模型中评估抗血栓潜力。

DESCRIPTION (provided by applicant): Platelets are essential components of arterial thrombosis. Interference with platelet signal transduction can dramatically reduce morbidity and mortality of platelet-mediated thrombotic disease such as heart attack, stroke, and peripheral vascular disease. Identification of optimal therapeutic targets for inhibition of platelet activation has been slowed, however, because platelets are anucleate and not amenable to genetic manipulations frequently used to study signal transduction mechanisms in nucleated cells. In contrast, the ready availability of platelets and their ability to undergo profound phenotype change upon exposure to agonists has made them a popular cellular model for studies using small molecules. We have developed a high throughput assay to identify novel anti-platelet agents that inhibit platelet activation as monitored by dense granule secretion. The goal of this undertaking is to discover new molecular targets that control platelet granule secretion. In collaboration with a facility within the Molecular Library Screening Center Network (MLSCN), we will expand the number of compounds that we screen, improve the rate at which screening is performed, and optimize compounds with antiplatelet activity. We will evaluate granule secretion in two related assays. One will identify compounds that inhibit dense granule secretion induced by SFLLRN, which stimulates platelets via protease-activated receptor 1. The second assay will identify compounds that inhibit dense granule secretion induced by collagen I, which activates platelets via glycoprotein VI. This strategy will facilitate identification of compounds that inhibit platelet function by diverse mechanisms. Once identified, compounds will be tested in a previously established battery of platelet function tests and evaluated for antithrombotic potential in an in vivo murine model of thrombus formation.