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：
10340430
负责人：
PAUL F. BRAY
金额：
$ 53.95万
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/10340430
关键词：
Adherence Adhesions Agonist Alleles Amputation Binding Biology Black Populations Blood Platelets Brain Ischemia Brain hemorrhage Cathepsin G Cell Line Clinical Clinical Data 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 Internet 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 Receptor Cross-Talk 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 base 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 time use tool transcriptome user-friendly

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.

该申请是一项新提交的提交，扩展了由HL102482资助的事先研究，该研究最初于2009年资助确定血小板功能中个体间变异的遗传基础，这有助于缺血性阻塞动脉。我们产生了人类参考血小板转录组，开发了公共，用户友好的交互式查询血小板功能和RNA蛋白关联的Web工具，并通过蛋白酶激活的受体-4（PAR4）凝血酶受体在黑人中比白人大。我们展示了这种差异是由于具有种族歧视等位基因频率的Par4 Ala120ThR替代（THR120： .63黑人； .19白色）。 THR120变体对凝血酶的敏感性提高，并证明了离体血栓形成在动脉下，但不是静脉剪切应力。基因分型> 12,000名患者证明 THR120等位基因与缺血性中风的风险增加和出血相关。最近的工作有导致了与血小板生物学的基本和临床方面有关的新数据，以及当前的总体目标应用是研究（1）PAR4与其他GPCR和蛋白酶的相互作用，（2）人类的影响脑缺血/再灌注（I/R）损伤的体内模型中的PAR4（HPAR4）和ALA120TH变体。 PAR4 具有比PAR1较慢的信号动力学，我们的新数据表明，PAR4与血小板共免疫沉淀 PGI2受体（IP）。与PAR4 ALA120相比，THR120在存在下对脱敏性相对抗性 Prostacyclin（PGI2），并在人血小板和原发性巨核细胞中激活后产生较少的营地（MK）。我们假设使用PAR4的IP串扰，AIM 1将评估IP/气体在PAR信号传导中的作用我们新型的人源性PAR4小鼠系的遗传变化的人类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，但不是PAR1，我们显示CATG诱导PAR4 THR120的血小板激活比ALA120更多。第一个时间，我们确定了PAR4中的CATG酶促切割位点，其中一个产生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。 AIM 2意志研究CATG-Al-PAR4相互作用。血小板PAR4而不是PAR1对于白细胞招募，滚动和粘附，我们的初步数据表明，人源化PAR4小鼠系中的鼠大脑I/R损伤是HPAR4- 和中性粒细胞依赖性。 I/R损伤后HPAR4在中风和脑出血中的作用很差研究了，目标3的目标是利用我们的HPAR4鼠标线来确定HPAR4如何介导血小板和鼠卒中模型中的中性粒细胞活性，并评估ALA120TH的药物遗传学作用 HPAR4介导的梗塞，出血和血小板信号通路的变体。成功完成预计拟议的研究将增强对个体间变异的分子基础的理解在人血小板生物学和表达PAR4的组织中种族偏好的疾病疗法。