PLATELET CELL ADHESION MOLECULES

P-选择素是一种存在于血小板和血管内皮细胞中的蛋白质，在 血管损伤后的炎症和血栓形成。P-选择素是 一种细胞黏附分子，驻留在静息的α颗粒中 血小板和内皮细胞的韦贝尔-帕莱德小体。vt.在.的基础上 在这些细胞的刺激下，蛋白质被转移到血浆中 作为中性粒细胞的白细胞受体的膜 单核细胞。自从我们12年前发现这种蛋白质以来，我们的 实验室与其他实验室一起，对这种蛋白质进行了表征， 确定了它的功能并确定了反受体的特征 它在白细胞上识别的物质。当前应用程序表示一个 继续研究P-选择素的结构， 白细胞和细胞表面P-选择素配体的结构和生物学特性 相互作用刺激的效应器功能和信号转导 P-选择素与P-选择素配基的结合。确定三个 凝集素的空间结构及P-EGF凝集素-EGF结构域 选择素，这些结构域将在细菌中表达 系统以获得适量的生物活性多肽。 这些域将按照唾液酸化的方式进行功能表征 Lewis x结合、钙离子结合和细胞黏附抑制。 定义这些结合功能的P-选择素上的氨基酸残基 将通过定点突变进行鉴定。结晶和 凝集素结构域三维结构的测定 唾液酸化Lewis x存在和不存在时凝集素-EGF结构域 将与威廉·韦斯博士合作进行。讯号 P-半胱氨酸的细胞激活诱导的信号转导和效应功能 将在血小板中研究选择素与P-选择素配体的结合 和内皮细胞，特别注意P-蛋白的磷酸化。 血小板活化和内皮细胞刺激过程中的选择素。这个 作用于P-选择素的激酶和磷酸酶将被识别。 P-选择素配体表达诱导的效应功能 P-选择素结合过程中的细胞，包括中性粒细胞和单核细胞 将进行分析，如P-选择素配体的磷酸化。这个 P-选择素配体PSGL-1的生物学特性将通过以下位点进行评估： 特异性突变以确定推定基因的功能 前肽、14个十二聚体重复序列和胞质尾部。这个 完整的P-选择素配体将通过识别以下任一项来定义 与PSGL-1或酶形成复合体的其他蛋白质 翻译后修饰PSGL-1以产生全功能的P- 选择素配体。此外，我们还建议克隆E-选择素配体 可以直接与P-选择素配体和L-选择素进行比较 莱兰德。这些研究应该有助于我们对血小板的理解 和血管生物学，特别强调选择素在 血栓形成、止血和炎症反应。拨款建议 是一项新的拨款，但实际上是对 项目V资助项目HL42443(血液中的膜蛋白 凝聚)。

P-selectin, a protein of platelets and endothelials cells, plays a role in inflammation and thrombosis following vascular injury. P-selectin is a cell adhesion molecule that resides in the alpha granules of resting platelets and the Weibel-Palade bodies of endothelial cells. Upon stimulation of these cells, the protein is translocated to the plasma membrane where it functions as a leukocyte receptor for neutrophils and monocytes. Since our discovery of this protein 12 years ago, our laboratory, in tandem with others, has characterized this protein, defined its function and determined the features of the counterreceptor that it recognizes on leukocytes. The current application represents a continuation of studies that address the structure of P-selectin, the structure and biology of the P-selectin ligand on leukocytes, and cell effector function and signal transduction stimulated by the interaction of P-selectin with the P-selectin ligand. To determine the three dimensional structure of the lectin and the lectin-EGF domains of P- selectin, these domains will be expressed in a bacterial expression system to obtain suitable quantities of biologically active peptide. These domains will be functionally characterized in terms of sialylated Lewis x binding, calcium ion binding and inhibitor of cell adhesion. Amino acid residues on P-selectin that define these binding functions will be identified by site-specific mutagenesis. Crystallization and determination of the three dimensional structure of the lectin domain and the lectin-EGF domain in the presence and absence of sialylated Lewis x will be performed in collaboration with Dr. William Weis. Signal transduction and effector function induced by cell activation of P- selectin binding to the P-selectin ligand will be studied in platelets and endothelial cells, with special attention to phosphorylation of P- selectin during platelet activation and endothelial cell stimulation. The kinases and phosphatases that act on P-selectin will be identified. Finally, effector functions induced in P-selectin ligand-expressing cells, including neutrophils and monocytes, during P-selectin binding will be analyzed, such as phosphorylation of the P-selectin ligand. The biology of PSGL-1, the P-selectin ligand, will be evaluated by site- specific mutagenesis to determine the function of the putative propeptide, the 14 dodecameric repeats and the cytoplasmic tail. The complete P-selectin ligand will be defined by identifying either additional proteins that form a complex with PSGL-1 or enzymes that posttranslationally modify PSGL-1 to yield the fully functional P- selectin ligand. Furthermore, we propose to clone the E-selectin ligand to allow direct comparison to the P-selectin ligand and the L-selectin ligand. These studies should contribute to our understanding of platelet and vascular biology, with specific emphasis on the role of selectins in thrombosis, hemostasis and the inflammatory response. The grant proposal is officially a new grant, but it is actually the competitive renewal of Project V of Program Project Grant HL42443 (Membrane Proteins in Blood Coagulation).