Regulation of Platelet Reactivity by S1P Signaling

S1P 信号传导调节血小板反应性

• 批准号: 10183303
• 负责人: ULHAS P NAIK
• 金额: $ 52.31万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10183303
• 关键词: Ablation; Affect; Attenuated; Binding; Binding Proteins; Blood Platelets; CRISPR/Cas technology; Calcium Binding; Cardiovascular Diseases; Cells; Choline; Data; Defect; Enzymes; Generations; Genetic; Genotype; Growth; Heritability; Human; Hydrolysis; Impairment; In Vitro; Incidence; Integrin Binding; Ischemic Stroke; Link; Lipid Binding; Membrane; Membrane Microdomains; Middle Cerebral Artery Occlusion; Modeling; Morbidity - disease rate; Mus; Myocardial Infarction; Outcome; PAWR gene; Pathway interactions; Peptides; Platelet Activation; Platelet aggregation; Play; Positioning Attribute; Production; Proteins; Race; Regulation; Research Personnel; Rest; Role; SPHK1 enzyme; Signal Transduction; Site; Sphingomyelinase; Sphingomyelins; Sphingosine; Sphingosine-1-Phosphate Receptor; Stroke; Surface; Testing; Therapeutic Intervention; Thrombin; Thrombosis; Thrombotic Stroke; Thrombus; Variant; atherosclerotic plaque rupture; combat; disparity reduction; ethnic difference; in vivo; mortality; new therapeutic target; phosphatidylcholine transfer protein; platelet function; protease-activated receptor 4; racial difference; racial disparity; receptor; recruit; small molecule inhibitor; sphingosine 1-phosphate; stroke outcome; synthetic enzyme

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.

人类发病和死亡的主要原因是心血管疾病（CVD）。种族被认为 是心血管疾病结果的重要决定因素，因为与白人相比， 即使考虑到其他混杂因素，也不能降低CVD的发病率。心肌梗死和卒中结果 在动脉粥样硬化斑块破裂部位形成闭塞性血小板血栓。最近，埃德尔斯坦医生 和同事们已经表明，血小板对蛋白酶激活受体4-激活的反应性的差异， 肽（PAR 4-AP）是可遗传的，并且与种族差异有关。他们已经确定了两个可能的 候选人，PAR 4蛋白的变体（120 A-T）和磷脂酰胆碱转移的表达水平增加 蛋白质（PCTP）有助于观察到的血小板对PAR 4-AP的反应性增加， 看到的差异。然而，这两种独立的蛋白质如何调节血小板对PAR 4-AP的反应性尚不清楚 没有很好地理解。我们假设高反应性PAR 4变异体与中性粒细胞相关， 鞘磷脂酶（n-SMase）是参与S1 P合成的关键酶，S1 P是一种有效的生物活性脂质， 其受体在血小板表面上以增强血小板反应性。提高PCTP水平， S1 P合成所需的鞘磷脂（SM）量。总之，它们增强了血小板的募集， 血栓的生长从而影响CVD的结果。为了验证这一假设，我们提出以下建议： 三个具体目标。具体目的1：评估S1 P合成途径在影响PAR 4诱导的细胞凋亡中的作用。 血小板中的信号。我们将评估凝血酶/PAR 4-AP是否会差异诱导n-SM酶活性以及 以PAR 4基因型特异性方式的S1 P水平。接下来，我们将确定PAR 4与n-SMase的结合是否是 基因型特异性以及这种关联是否会影响PAR 4与Gαq的关联。而且我们 将测试S1 P合成途径的酶和血小板上S1 P的S1 PR 1/2受体的阻断是否会 以PAR 4基因型依赖性方式减弱PAR 4-AP诱导的血小板功能。我们还将确定， 使用CRISPR/Cas9技术在MEG-01细胞中基因切除S1 P合成的关键酶 PAR 4高反应性变体将挽救血小板高反应性。在具体目标2中，我们将评估监管 血小板中S1 P的合成。研究表明，CIB 1与Sphk 1/2结合，并调节它们的活性。 细胞我们将首先使用Cib 1-/-小鼠血小板确定Cib 1是否调节凝血酶诱导的S1 P合成。在 具体目标3，我们将评估血小板源性S1 P在血栓形成和短暂性缺血中的调节作用。 中风（tMCAO）。我们将评估小鼠中血小板特异性Cib 1缺失或S1 P信号传导抑制是否 表达人PAR 4变体将差异性地改变体内血栓形成和缺血性中风结果。以来 血栓形成和缺血性中风的种族差异已经有很好的记录，我们的研究可能会确定因素 负责这种差异，并提供治疗干预的途径。