EFFECT OF PF4 ON LIPOPROTEIN METABOLISM/ATHEROSCLEROSIS

（改编自申请人摘要）动脉粥样硬化是最常见的原因 死亡在美国。血浆LDL水平升高是一个主要风险 是导致这种疾病发展的因素。 低密度脂蛋白的主要途径是 分解代谢是通过LDL-受体（LDL-R）。 因此，阻碍 LDL-LDL-R相互作用促进动脉粥样硬化。 一个尚未探讨的问题是 血小板活化是否调节LDL-R功能。 然而， 血小板在这种疾病的终末血栓形成阶段是很好的- 建立，不太确定持续的血小板活化是否 加速动脉粥样硬化斑块的发病。 考虑到 载脂蛋白上的某些带正电荷的残基簇 β-100是LDL与LDL-R最佳结合所必需的， 研究人员测试了血小板因子4（PF 4）的假设， 在血小板活化时释放的富含赖氨酸的蛋白质，可以完成 受体结合，从而阻碍脂蛋白清除和催化。 试验数据通过证明PF 4为这一假设提供了支持。 以nM亲和力结合LDL-R，抑制LDL-R的结合和降解， 体外测定LDL的清除率，体内测定LDL的血浆清除率。 现在 建议研究PF 4-LDL-R相互作用的生化基础， 更详细的信息，并开发模型来阐明这种血小板的作用， 蛋白质在动脉粥样硬化的发展通过以下具体 1）具体目的1：PF 4与LDL-R结合的表征及其在 体外的后果 PF 4与细胞系的结合动力学， 将测量过表达LDL-R以及重组可溶性受体 使用表面等离子体共振。 PF_4对结合和增殖的影响 LDL和apoE的细胞代谢将使用以下细胞进行研究： 基因缺乏或过度表达LDL-R，并且其中 蛋白聚糖的表达受到控制。 具体目标2：PF 4对 脂蛋白代谢和动脉粥样硬化。 腺病毒介 PF 4的转移将用于分析LDL清除率的变化， 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检测， 过表达人PF 4。 这些研究旨在深入了解一种新的机制， 持续的血小板活化可能促进动脉粥样硬化性血栓形成的发展， 疾病 了解PF 4-LDL-R的结构基础 相互作用可以识别用于治疗干预的潜在位点。 这 研究计划是综合培训计划的一部分， 准备申请人的职业生涯作为一个独立的调查员在外地 血管生物学。

(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.