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

• 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
• 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

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.

该申请是一项新提交的申请，扩展了由HL 102482资助的先前研究，最初于2009年资助， 确定血小板功能个体间差异的遗传基础，这种差异有助于缺血性闭塞， 动脉我们生成了一个人类参考血小板转录组，开发了一个公共的，用户友好的互动 网络工具查询血小板功能和RNA-蛋白质协会，并发现血小板聚集， 蛋白酶激活受体-4（PAR 4）凝血酶受体在黑人中比白人更高。我们展示 这种差异是由于PAR 4 Ala 120 Thr置换，具有种族上不同的等位基因频率（Thr 120： 0.63黑人; 0.19白人）。Thr 120变体对凝血酶的敏感性增加，并在体外证实了Thr 120变体对凝血酶的敏感性。 在动脉而不是静脉剪切应力下血栓形成。基因分型> 12，000例患者显示， Thr 120等位基因与缺血性卒中风险增加和出血减少相关。最近的工作有 导致了与血小板PAR生物学的基础和临床方面相关的新数据，以及当前研究的总体目标。 应用是研究（1）PAR 4与其他GPCR和蛋白酶的相互作用和（2）人GPCR的作用。 PAR 4（hPAR 4）和Ala 120 Thr变体在脑缺血/再灌注（I/R）损伤的体内模型中的作用。PAR4 具有比PAR 1更慢的信号传导动力学，我们的新数据显示PAR 4与血小板共免疫沉淀， PGI 2受体（IP）。与PAR 4 Ala 120相比，Thr 120对存在的脱敏具有相对抗性。 前列环素（PGI 2），并在人血小板和原代巨核细胞活化后产生较少的cAMP （MK）.我们假设IP与PAR 4的交叉对话，目标1将评估IP/Gas在PAR信号传导中的作用， 在来自我们的新型人源化PAR 4小鼠系的遗传改变的人MK和血小板中的脱敏。 嗜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，其激活血小板PAR 4， 而不是PAR 1，我们发现CatG诱导的PAR 4 Thr 120的血小板活化比Ala 120更多。第一 同时，我们鉴定了PAR 4中的CatG酶促切割位点，其中一个产生新的栓系配体， SRALLLGWVPTR，其诱导人血小板aIIbb 3活化、颗粒释放和聚集。目标2将 研究CatG-血小板PAR 4相互作用。血小板PAR 4，而不是PAR 1，对白细胞募集、滚动和增殖至关重要。 我们的初步数据显示，在我们的人源化PAR 4小鼠系中，小鼠脑I/R损伤是hPAR 4- 和依赖性人格障碍hPAR 4在缺血再灌注损伤后卒中和脑出血中的作用一直很差， 目的3的目的是利用我们的hPAR 4小鼠系来确定hPAR 4如何介导血小板 和中性粒细胞活性，并评估Ala 120 Thr 在hPAR 4介导的梗死、出血和血小板信号传导途径上的变体。成功完成 预期拟议的研究将增强对个体间变异的分子基础的理解 在人类血小板生物学和PAR 4表达组织中的作用，并为个体化抗血小板药物提供基础 种族偏好失调症的治疗