Human platelet PAR4: novel activation, interindividual variation, and neutrophil interactions in vivo and in vitro

人血小板 PAR4：体内和体外的新激活、个体差异和中性粒细胞相互作用

基本信息

批准号：
10569045
负责人：
PAUL F. BRAY
金额：
$ 53.67万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-08 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10569045
关键词：
Adherence Adhesions Agonist Alleles Binding Biology Black Populations Blood Platelets Brain Ischemia Brain hemorrhage Cathepsin G Cell Line Clinical Clinical Data Co-Immunoprecipitations Cyclic AMP Cytoplasmic Granules Data Disease Drug Targeting Drug or chemical Tissue Distribution Epoprostenol F2R gene Funding G-Protein-Coupled Receptors Gases Gene Frequency Generations Genetic Genotype Goals Heart Hemorrhage Heterodimerization Human Hyperactivity In Vitro Incidence Individual Infarction Ischemia Ischemic Stroke Kinetics Leukocytes Ligands Liver Lung Mediating Megakaryocytes Molecular Molecular Genetics Mus Neutrophil Infiltration Outcome Patients Peptide Hydrolases Peptides Pharmacogenetics Phosphatidylserines Physiology Platelet Activation Platelet aggregation Property Prostaglandins I Proteins RNA Race Reperfusion Injury Research Resistance Risk Role Serine Protease Signal Pathway Signal Transduction Site Stroke Thrombin Thrombin Receptor Thrombus Time Tissues Variant Vascular Diseases Work artery occlusion desensitization drug development genetic manipulation in vivo in vivo Model inter-individual variation mouse model mouse protease-activated receptor 4 neutrophil novel platelet function protease-activated receptor 3 protease-activated receptor 4 receptor recruit response shear stress stroke model stroke outcome stroke risk transcriptome user-friendly web-based tool

项目摘要

This application is a new submission extending prior research funded by HL102482, initially funded in 2009 to identify the genetic basis for inter-individual variation in platelet function that contributes to ischemic occlusion of arteries. We generated a human reference platelet transcriptome, developed a public, user-friendly interactive web tool to query platelet function and RNA-protein associations, and discovered platelet aggregation through the protease activated receptor-4 (PAR4) thrombin receptor was greater in blacks than whites. We showed this difference was due to a PAR4 Ala120Thr substitution with racially divergent allele frequencies (Thr120: .63 blacks; .19 whites). The Thr120 variant had increased sensitivity to thrombin and demonstrated ex vivo thrombus formation under arterial, but not venous, shear stress. Genotyping >12,000 patients demonstrated the Thr120 allele was associated with an increased risk of ischemic stroke and less bleeding. More recent work has led to new data relevant to basic and clinical aspects of platelet PAR biology, and the overall goals of the current application are to study (1) PAR4 interactions with other GPCRs and proteases and (2) the effect of human PAR4 (hPAR4) and the Ala120Thr variant in an in vivo model of brain ischemia/reperfusion (I/R) injury. PAR4 has slower signaling kinetics than PAR1, and our new data shows that PAR4 co-immunoprecipitates with platelet PGI2 receptor (IP). Compared to PAR4 Ala120, Thr120 is relatively resistant to desensitization in the presence of prostacyclin (PGI2), and generates less cAMP after activation in human platelets and primary megakaryocytes (MKs). We hypothesize IP cross-talks with PAR4, and Aim 1 will assess the role of IP/Gas in PAR signaling and desensitization in genetically altered human MKs and platelets from our novel humanized PAR4 mouse lines. The neutrophil serine protease, cathepsin G (CatG), is a potent platelet agonist that activates platelet PAR4, but not PAR1, and we show CatG induces more platelet activation of PAR4 Thr120 than Ala120. For the first time, we identified CatG enzymatic cleavage sites in PAR4, one of which generates a novel tethered ligand, SRALLLGWVPTR, which induces human platelet aIIbb3 activation, granule release and aggregation. Aim 2 will study CatG-platelet PAR4 interactions. Platelet PAR4, not PAR1, is critical for leukocyte recruitment, rolling and adhesion, and our preliminary data show that murine brain I/R injury in our humanized PAR4 mouse line is hPAR4- and neutrophil-dependent. The role of hPAR4 in stroke and brain hemorrhage after I/R injury has been poorly studied, and the goal of Aim 3 is to utilize our hPAR4 mouse lines to determine how hPAR4 mediates platelet and neutrophil activities in a murine stroke model, and to assess the pharmacogenetic effect of the Ala120Thr variant on hPAR4-mediated infarct, hemorrhage and platelet signaling pathways. Successful completion of the proposed studies is expected to enhance the understanding of the molecular basis of inter-individual variation in human platelet biology and PAR4-expressing tissues, and provide groundwork for individualized anti-platelet therapies in disorders with racial predilections.

本申请是一项新提交的申请，扩展了 HL102482 资助的先前研究，最初于 2009 年资助确定导致血小板功能缺血性闭塞的个体间变异的遗传基础动脉。我们生成了人类参考血小板转录组，开发了一个公共的、用户友好的交互式查询血小板功能和 RNA-蛋白质关联的网络工具，并通过以下方式发现血小板聚集黑人的蛋白酶激活受体 4 (PAR4) 凝血酶受体比白人更强。我们展示了这种差异是由于 PAR4 Ala120Thr 替换具有种族不同的等位基因频率（Thr120： .63 黑人； .19白人）。 Thr120 变体对凝血酶的敏感性增加，并在离体实验中得到证明在动脉剪切应力而非静脉剪切应力下形成血栓。超过 12,000 名患者的基因分型证明 Thr120 等位基因与缺血性中风风险增加和出血减少相关。最近的工作有产生了与血小板 PAR 生物学的基础和临床方面相关的新数据，以及当前的总体目标应用程序是研究 (1) PAR4 与其他 GPCR 和蛋白酶的相互作用以及 (2) 人类脑缺血/再灌注 (I/R) 损伤体内模型中的 PAR4 (hPAR4) 和 Ala120Thr 变体。 PAR4 信号传导动力学比 PAR1 慢，我们的新数据显示 PAR4 与血小板发生免疫共沉淀 PGI2 受体 (IP)。与 PAR4 Ala120 相比，Thr120 在存在下相对抗脱敏前列环素 (PGI2)，在人血小板和原代巨核细胞中激活后产生较少的 cAMP （MK）。我们假设 IP 与 PAR4 发生串扰，目标 1 将评估 IP/Gas 在 PAR 信号传导中的作用，来自我们的新型人源化 PAR4 小鼠系的基因改造人类 MK 和血小板脱敏。中性粒细胞丝氨酸蛋白酶组织蛋白酶 G (CatG) 是一种有效的血小板激动剂，可激活血小板 PAR4，但 PAR1 则不然，而且我们发现 CatG 比 Ala120 更能诱导 PAR4 Thr120 的血小板活化。对于第一个当时，我们在 PAR4 中发现了 CatG 酶切位点，其中一个位点生成了一种新型的束缚配体， SRALLLGWVPTR，诱导人血小板 aIIbb3 激活、颗粒释放和聚集。目标2将研究 CatG-血小板 PAR4 相互作用。血小板 PAR4，而不是 PAR1，对于白细胞的募集、滚动和迁移至关重要。粘附，我们的初步数据表明，我们的人源化 PAR4 小鼠系中的鼠脑 I/R 损伤是 hPAR4- 和中性粒细胞依赖性。 hPAR4 在 I/R 损伤后中风和脑出血中的作用尚不清楚研究，目标 3 的目标是利用我们的 hPAR4 小鼠系来确定 hPAR4 如何介导血小板和小鼠中风模型中的中性粒细胞活性，并评估 Ala120Thr 的药物遗传学效应 hPAR4 介导的梗塞、出血和血小板信号通路的变异。圆满完成了拟议的研究有望增强对个体间变异的分子基础的理解人类血小板生物学和 PAR4 表达组织中的研究，并为个体化抗血小板提供基础具有种族偏好的疾病的治疗。