PLATELET CELL ADHESION MOLECULES

血小板细胞粘附分子

• 批准号: 2838995
• 负责人: Bruce Furie
• 金额: $ 36.38万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2838995
• 关键词: X ray crystallography; binding proteins; biological signal transduction; cell adhesion molecules; chemical binding; complementary DNA; human subject; laboratory mouse; laboratory rabbit; ligands; molecular cloning; molecular site; monocyte; neutrophil; phosphoprotein phosphatase; phosphorylation; platelet activation; platelets; posttranslational modifications; protein kinase; protein sequence; protein structure function; selectins; site directed mutagenesis; vascular endothelium

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).

P-选择素是血小板和内皮细胞的一种蛋白质， 炎症和血管损伤后血栓形成。P-选择素是 一种细胞粘附分子，存在于静息细胞的α颗粒中， 血小板和内皮细胞的韦伯-帕拉德体。后 这些细胞的刺激，蛋白质被转移到血浆中 膜，在那里它作为中性粒细胞的白细胞受体发挥作用， 单核细胞自从12年前我们发现这种蛋白质以来， 实验室与其他实验室合作，已经表征了这种蛋白质， 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。当前应用程序表示一个 继续研究，解决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 相互作用刺激的效应子功能和信号转导 与P-选择素配体结合。为了确定这三个 的凝集素和凝集素-EGF结构域的三维结构， 选择素，这些结构域将在细菌表达中表达 系统以获得适量生物活性肽。 这些结构域将在功能上表征为唾液酸化的结构域。 刘易斯x结合、钙离子结合和细胞粘附抑制剂。 P-选择素上定义这些结合功能的氨基酸残基 将通过位点特异性诱变来鉴定。结晶和 确定凝集素结构域的三维结构， 在存在和不存在唾液酸化的刘易斯x的情况下的凝集素-EGF结构域 将与William Weis博士合作进行。信号 细胞活化诱导的P- 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- 血小板活化和内皮细胞刺激过程中的选择素。的 将鉴定作用于P-选择素的激酶和磷酸酶。 最后，在P-selectin配体表达中诱导的效应子功能 细胞，包括中性粒细胞和单核细胞，在P-选择素结合期间 将被分析，如磷酸化的P-选择素配体。的 PSGL-1（P-选择素配体）的生物学将由研究中心进行评价， 特异性诱变以确定推定的 前肽、14个十二聚体重复序列和胞质尾。的 完整的P-选择素配体将通过鉴定 与PSGL-1形成复合物的其他蛋白质或 对PSGL-1进行后处理修饰，以产生全功能的P- 选择素配体此外，我们建议克隆E-选择素配体， 以允许与P-选择素配体和L-选择素配体直接比较， 配体。这些研究应有助于我们对血小板的了解 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。赠款提案 是官方的一个新的赠款，但它实际上是竞争性的更新， HL 42443计划资助项目五（血液膜蛋白 凝血）。