Regulation of Platelet Reactivity by S1P Signaling

心血管疾病(CVD)是人类发病和死亡的首要原因。种族被认为是 是心血管疾病结局的重要决定因素，因为与白人相比，黑人的发病率要高出两倍 即使考虑到其他混杂因素，心血管疾病的发生率也是如此。心肌梗死和卒中结果 动脉粥样硬化斑块破裂部位形成闭塞的血小板血栓。最近，埃德尔斯坦博士 和他的同事们已经证明，血小板对蛋白水解酶激活受体4激活的反应性的差异 多肽(PAR4-AP)是可遗传的，与种族差异有关。他们已经确定了两个可能的 PAR4蛋白(120A-T)变异与磷脂酰胆碱转运蛋白表达水平升高 蛋白(PCTP)有助于观察到对PAR4-AP和种族的血小板反应性增加 看到了不同之处。然而，这两个独立的蛋白质如何调节血小板对PAR4-AP的反应性是 不是很清楚。我们假设高反应性PAR4变体与中性 鞘磷脂酶(n-sMase)，参与合成S1P的关键酶，S1P是一种结合到 其受体在血小板表面，以增强血小板的反应性。增强的PCTP水平提供了更多 合成S1P所需的鞘磷脂(SM)量。他们一起促进了血小板的募集 血栓的增加会影响心血管疾病的预后。为了检验这一假设，我们提出了以下建议 三个具体目标。特异性目标1：评价S1P合成途径在影响PAR4诱导中的作用 在血小板中发出信号。我们将评估凝血酶/PAR4-AP是否会不同地诱导n-sMase活性以及 S1P水平具有PAR4基因特异性。接下来，我们将确定PAR4与n-sMase的关联是否 以及这种关联是否会影响PAR4与GαQ的关联。此外，我们 将测试阻断S1P合成途径的酶和S1P的S1PR1/2受体是否会 以PAR4基因依赖的方式减弱PAR4-AP诱导的血小板功能。我们还将确定是否 CRISPR/Cas9技术对表达S1P的MEG-01细胞S1P合成关键酶的基因消融 PAR4高反应型可挽救血小板高反应性。在具体目标2中，我们将对该规定进行评估 CIB1对血小板合成S1P的影响。已有研究表明，CIB1与Sphk1/2结合并调节其活性。 细胞。我们将首先利用CIB1-/-小鼠血小板来确定CIB1是否调节凝血酶诱导的S1P的合成。在……里面 具体目标3，我们将评估血小板衍生的S1P在调节血栓形成和短暂性脑缺血中的作用 卒中(TMCAO)。我们将评估小鼠的血小板特异性CIB1缺失或S1P信号抑制 表达人类PAR4变异体将不同地改变体内血栓形成和缺血性卒中的结果。自.以来 血栓形成和缺血性中风的种族差异已经得到了很好的记录，我们的研究可能会确定其中的因素 对这种差异负有责任，并提供治疗干预的途径。

The preeminent cause of morbidity and mortality in humans is cardiovascular disease (CVD). Race is considered to be an important determinant of the outcome of CVD since compared to whites, blacks have a twofold higher incidence of CVD even when other confounding factors are accounted for. Myocardial infarction and stroke result from formation of occlusive platelet thrombus at the site of atherosclerotic plaque rupture. Recently, Dr. Edelstein and colleagues have shown that differences in platelet reactivity to protease activated receptor 4-activating peptide (PAR4-AP) is heritable and is associated with racial disparity. They have identified two probable candidates, a variant of PAR4 protein (120A-T) and increased expression levels of phosphatidyl choline transfer protein (PCTP) to be contributing for the observed increase in platelet reactivity to PAR4-AP and ethnic differences seen. However, how these two independent proteins may regulate platelet reactivity to PAR4-AP is not well understood. We hypothesize that the hyperresponsive PAR4 variant associates with neutral sphingomyelinase (n-SMase), a key enzyme involved in synthesis of S1P, a potent bioactive lipid that binds to its receptor on the platelet surface to potentiate platelet reactivity. Enhanced levels of PCTP provide an increased amount of sphingomyelin (SM) required for S1P synthesis. Together, they enhanced recruitment of platelets in the growing thrombus thus influencing the outcome of CVD. To test this hypothesis, we propose the following three specific aims. Specific Aim 1: To evaluate the role of S1P synthetic pathway in influencing PAR4-induced signaling in platelets. We will evaluate if thrombin/PAR4-AP will differentially induce n-SMase activity as well as S1P levels in a PAR4 genotype specific manner. Next, we will determine if association of PAR4 with n-SMase is genotype specific and whether this association will influence association of PAR4 with Gαq. Furthermore, we will test if blockade of enzymes of S1P synthetic pathway and S1PR1/2 receptors for S1P on platelets will attenuate PAR4-AP-induced platelet function in PAR4 genotype dependent manner. We will also determine if genetic ablation of key enzymes of S1P synthesis using CRISPR/Cas9 technology in MEG-01 cells expressing PAR4 hyperreactive variant will rescue platelet hyperreactivity. In Specific Aim 2, we will evaluate the regulation of S1P synthesis in platelets by CIB1. It has been shown that CIB1 bind Sphk1/2 and regulate their activity in cells. We will first determine if Cib1 regulate thrombin-induced S1P synthesis using Cib1-/- mouse platelets. In Specific Aim 3, we will assess the role of platelet–derived S1P in regulation of thrombosis and transient ischemic stroke (tMCAO). We will evaluate if platelet specific deletion of Cib1 or inhibition of S1P signaling in mice expressing human PAR4 variants will differentially alter in vivo thrombotic and ischemic stroke outcomes. Since racial disparity in thrombosis and ischemic stroke has been well documented, our study may identify the factor responsible for this disparity and provide avenues for therapeutic intervention.