STUDIES OF PLATELET ADHERENCE TO OSTEOPONTIN

细胞黏附在骨肉瘤的发生和发展中起着重要作用 动脉硬化。例如，血小板基质和血小板-血小板 粘附性是动脉粥样硬化最终事件的原因 病程、急性冠脉闭塞和中风。我们已经证明了 激活的，但不是静止的，血小板黏附于骨桥蛋白(OPN)，一种 钙化动脉粥样硬化的细胞外基质成分 斑块，但不是正常动脉壁的。因此，血小板粘附性 TO OPN可能是血小板形成血栓的过程的一部分 破裂的动脉粥样硬化斑块。血小板对骨桥蛋白的粘附性 主要由整合素αvbeta3介导。因此，αvbeta3， 与AlphaIIbbeta3一样，存在于静息状态下的血小板表面 处于非活动状态，并被血小板激动剂激活。第二种潜力 血小板上的OPN受体为CD44。CD44是透明质酸的受体 酸在许多细胞上，已被报道与OPN在 阳离子和RGD不依赖的方式。这个项目的目标是 了解OPN与的交互的结构基础 血小板的各种受体。在特定的目标1中，我们将在体外使用 突变以交换Alphav和AlphaIIb的选定片段和 测定结构性和激动剂刺激的整合素与纯化的整合素的结合 OPN。研究将集中在细胞质结构域序列和 RGD-多肽交联点。此外，对生物多样性的作用的研究 CD44在血小板与OPN的黏附中将被执行。在具体目标2中， 我们将确定激活αvbeta3的信号通路。建议 实验将重点放在蛋白质和脂肪激酶的作用上， 肌动蛋白细胞骨架和小GTP结合的Rho家族成员 调节Alphavbeta3配体结合活性的蛋白质。具体而言 目的3.研究矩阵识别的结构基础 Alphavbeta3的蛋白质。ITS所需的OPN的特性 通过含有αv整合素的识别以及结合 将确定各种OPN片段与Alphavbeta3的亲和力。 将使用简单和复杂的流动系统来研究OPN的能力 为了支持激活的和未激活的血小板在 剪切应力的存在。最后，我们确定了结构和 用核磁共振方法研究不同OPN片段的动态特性 为这些研究得出的信息将被用来合成 限制性Alphavbeta3多肽和多肽模拟学。

Cell adhesion plays a prominent role in the initiation and evolution of atherosclerosis. For example, platelet-matrix and platelet-platelet adhesion are responsible for the ultimate events in the atherosclerotic process, acute coronary occlusion and stroke. We have shown that activated, but not resting, platelets adhere to osteopontin (OPN), a component of the extracellular matrix of calcified atherosclerotic plaques, but not of the normal arterial wall. Thus, platelet adherence to OPN could be part of the process whereby platelets form thrombi on disrupted atherosclerotic plaques. Platelet adherence to OPN is predominantly mediated by the integrin alphavbeta3. Thus, alphavbeta3, like alphaIIbbeta3, is present on the surface of resting platelets in an inactive state and is activated by platelet agonists. A second potential receptor for OPN on platelets is CD44. CD44 is a receptor for hyaluronic acid on many cells and has been reported to interact with OPN in a cation and RGD-independent manner. The goal of this project is to understand the structural basis for the interaction of OPN with platelet various receptors. In Specific Aim 1, we will use in vitro mutagenesis to exchange selected segments of alphav and alphaIIb and measure constitutive and agonist-stimulated integrin binding to purified OPN. Studies will focus on the role of cytoplasmic domain sequences and RGD-peptide cross-linking sites. In addition, studies of the role of CD44 in platelet adhesion to OPN will be performed. In Specific Aim 2, we will identify signaling pathways that activate alphavbeta3. Proposed experiments will focus on the role of protein and lipid kinases, the actin cytoskeleton, and members of the Rho family of small GTP-binding proteins in regulating alphavbeta3 ligand binding activity. In Specific Aim 3, we will study the structural basis for the recognition of matrix proteins by alphavbeta3. The features of OPN required for its recognition by alphav-containing integrins, as well as the binding affinities of various OPN fragments for alphavbeta3 will be determined. Simple and complex flow systems will be used to study the ability of OPN to support the adherence of both activated and unactivated platelets in the presence of shear stress. Lastly, we determine the structural and dynamic properties of various OPN fragment by NMR methods and the information derived for these studies will be used to synthesize constrained alphavbeta3 peptides and peptidomimetics.