EFFECT OF PF4 ON LIPOPROTEIN METABOLISM/ATHEROSCLEROSIS

PF4 对脂蛋白代谢/动脉粥样硬化的影响

• 批准号: 6490294
• 负责人: Bruce S Sachais
• 金额: $ 13.12万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-15 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6490294
• 关键词: apolipoproteins; atherosclerosis; atherosclerotic plaque; blood lipoprotein metabolism; cell line; genetically modified animals; human subject; laboratory mouse; low density lipoprotein; low density lipoprotein receptor; platelet activation; platelet factor 4; receptor binding; receptor sensitivity; surface plasmon resonance; transfection

(Adapted from applicant's abstract) Atherosclerosis is the most common cause of death in the United States. Elevated plasma levels of LDL are a major risk factor for the development of this disease. The major pathway by which LDL is catabolized is via the LDL-receptor (LDL-R). Therefore, factors that impede LDL-LDL-R interactions promote atherosclerosis. An unexplored question is whether platelet activation modulates LDL-R function. Whereas the role of platelets in the terminal thrombotic phase of this disease is well- established, it is less certain whether persistent platelet activation accelerates the pathogenesis of the atherosclerotic plaque. In view of the fact that certain clusters of positively charged residues on apolipoprotein Beta-100 are required for optimal binding of LDL to the LDL-R, the investigators tested the hypothesis that platelet factor 4 (PF4], an abundant lysine rich protein released upon platelet activation, can complete for receptor binding and thereby impede lipoprotein clearance and catabolism. Pilot data provided support for this hypothesis by demonstrating that PF4 binds to the LDL-R with nM affinity, inhibits the binding and degradation of LDL in vitro, and prolongs the plasma clearance of LDL in vivo. It is now proposed to study the biochemicals basis of the PF4-LDL-R interaction in greater detail and to develop models to elucidate the role of this platelet protein in the development of atherosclerosis through the following specific aims; 1) Specific Aim 1: Characterization of PF4 binding to the LDL-R and it's consequences in vitro. The binding kinetics of PF4 to cell lines that overexpress LDL-R as well as to recombinant soluble receptor will be measured using surface plasmon resonance. The effect of PF4 on the binding and cellular metabolism of LDL and apoE will be studied using cells that are genetically lacking or overexpress LDL-R and in which the level of proteoglycan expression has been controlled. Specific Aim 2: Effect of PF4 on lipoprotein metabolism and atherosclerosis in vivo. Adenoviral-mediated gene transfer of PF4 will be used to analyze changes in LDL clearance and endogenous lipoprotein levels in vivo. The propensity to develop hyperlipidemia and atherosclerosis will be examined in transgenic mice that overexpress human PF4. These studies are designed to gain insight into a novel mechanism by which persistent platelet activation may promote the development of atherothrombotic disease. An understanding of the structural basis of the PF4-LDL-R interaction may identify a potential locus for therapeutic intervention. This research proposal is part of comprehensive training program designed to prepare the applicant for a career as an independent investigator in the field of vascular biology.

(摘自申请人的摘要)动脉粥样硬化是最常见的原因 在美国的死亡。血浆低密度脂蛋白水平升高是一个主要风险 导致这种疾病发展的因素。低密度脂蛋白的主要途径是 分解是通过低密度脂蛋白受体(低密度脂蛋白受体)。因此，阻碍因素 低密度脂蛋白-低密度脂蛋白-受体相互作用促进动脉粥样硬化。一个未被探索的问题是 血小板活化是否调节低密度脂蛋白受体功能。鉴于这一角色 这种疾病的终末血栓形成阶段的血小板很好- 目前还不太确定持续的血小板激活 加速动脉粥样硬化斑块的发病。鉴于 载脂蛋白上某些带正电的残基簇 β-100是低密度脂蛋白与低密度脂蛋白受体最佳结合所必需的， 研究人员测试了一种假设，即血小板因子4(PF4)，一种丰富的 富含赖氨酸的蛋白质在血小板激活时释放，可完成 受体结合，从而阻碍脂蛋白清除和分解代谢。 试点数据通过证明PF4提供了支持这一假设 以NM亲和力与低密度脂蛋白受体结合，抑制低密度脂蛋白的结合和降解 并延长体内低密度脂蛋白的血浆清除量。现在是时候了 建议研究PF4-LDL-R相互作用的生物化学基础 更多的细节，并开发模型来阐明这种血小板的作用 蛋白质通过以下特异性途径参与动脉粥样硬化的发展 AIMS；1)特定目标1：PF4与低密度脂蛋白受体结合的特性及其 在体外产生的后果。PF4与细胞系结合动力学的研究 将检测过表达的低密度脂蛋白受体和重组的可溶性受体 使用表面等离子激元共振。PF4对结合力和亲和力的影响 低密度脂蛋白和载脂蛋白E的细胞代谢将使用以下细胞进行研究 遗传缺乏或过度表达低密度脂蛋白受体，在这种情况下， 蛋白多糖的表达已经得到了控制。具体目标2：PF4对 脂蛋白代谢与体内动脉粥样硬化。腺病毒介导的基因 转移的PF4将用于分析低密度脂蛋白清除和 体内内源性脂蛋白水平。发展的倾向 高脂血症和动脉粥样硬化将在转基因小鼠中进行检测 过表达人PF4。 这些研究旨在深入了解一种新的机制，通过这种机制 血小板持续活化可能促进动脉粥样硬化血栓形成 疾病。对PF4-LDL-R结构基础的理解 相互作用可能为治疗干预确定一个潜在的基因座。这 研究计划是全面培训计划的一部分，旨在 为申请人在该领域成为一名独立调查员做好准备 血管生物学的研究。