PLATELET CELL ADHESION MOLECULES

P-选择素是一种细胞黏附分子，存在于血小板和内皮细胞的储存颗粒中。在细胞刺激下，蛋白质被转移到质膜上，在那里它作为中性粒细胞和单核细胞上PSGL-1的白细胞受体发挥作用。目前的应用代表了对P-选择素和PSGL-1生物学研究的继续。由于PSGL-1已被证明可与所有选择素结合，因此将用荧光光谱分析可溶性PSGL-1与可溶性P-选择素、E-选择素和L-选择素的动力学和平衡结合。在过去的资助期间，我们通过同源重组的方法制备了PSGL-1缺陷小鼠，并完成了初步的鉴定。该小鼠的细胞将用于比较PSGL-1(-/-)、(+/-)和(+/+)白细胞在滚动条件下与P-选择素、E-选择素和L-选择素的相互作用，以确定PSGL-1在选择素功能中的生理作用。将PSGL-1基因缺陷小鼠和包括PSGL-1缺失/P-选择素缺失小鼠、PSGL-1缺失/E-选择素缺失小鼠和PSGL-1缺失/L-选择素缺失小鼠在内的双基因敲除小鼠用于模型系统，以确定PSGL-1的生理功能。有待研究的病理过程包括非免疫和T细胞介导的皮肤炎症模型、创伤和肿瘤坏死因子后的白细胞滚动、实验性肾小球肾炎、化学性腹膜炎、细菌性肺炎、血栓形成、动脉粥样硬化、伤口愈合和血小板滚动。为了了解血小板活化或P-选择素与白细胞P-选择素配体结合所诱导的信号转导和效应功能的分子基础，我们将分析PSGL-1通过P-选择素诱导血小板内钙离子流动的机制。此外，P-选择素和PSGL-1的胞浆尾巴分别与血小板和单核细胞的胞浆信号蛋白的结合将使用胞浆尾巴的二聚体结构来检测。如果这些研究表明PSGL-1和ESL-1不是E-选择素的生理关键拮抗剂，或者有证据表明存在另一种P-选择素配体，我们建议从WEHI细胞制备的白细胞文库中表达克隆一种新的E-选择素配体，并从PSGL-1缺失小鼠的中性粒细胞中分离出一种新的P-选择素配体。这些假定的配体将接受其全长cDNA的鉴定，并将其预测的氨基酸序列与PSGL-1、ESL-1和GlyCAM-1的氨基酸序列进行比较。这些研究将有助于我们理解在炎症过程中定义细胞-细胞相互作用的生理相关受体和对抗受体。

P-selectin is a cell adhesion molecule that resides in the storage granules of platelets and endothelial cells. Upon cell stimulation, the protein is translocated to the plasma membrane where it functions as a leukocyte receptor for PSGL-1 on neutrophils and monocytes. The current application represents a continuation of studies of the biology of P-selectin and PSGL-1. Since PSGL-1 has been shown to bind to all selectins, the kinetic and equilibrium binding of soluble PSGL-1 to soluble P-selectin, E-selectin and L-selectin will be analyzed by fluorescence spectroscopy. During the past grant period, we have prepared a PSGL-1 deficient mouse by homologous recombination and have completed the initial characterization. Cells from this mouse will be used to establish the physiologic role of PSGL-1 in selectin function by comparing the interaction of PSGL-1 (-/-), (+/-) and (+/+) leukocytes with P-selectin, E- selectin and L-selectin under rolling conditions in a parallel plate ex vivo assay using neutrophils and T lymphocytes. The PSGL-1 deficient mouse and double knockout mice including PSGL-1 null/P-selectin null mice, PSGL-1 null/E-selectin null mice and PSGL-1 null/L-selectin null mice will be employed in model systems to determine the physiologic function of PSGL-1. Pathologic processes to be studied include models of non- immune mediated and T-cell mediated skin inflammation, leukocyte rolling following trauma and TNF, experimental glomerulonephritis, chemical peritonitis, bacterial pneumonitis, thrombosis, atherosclerosis, wound healing and platelet rolling. To understand the molecular basis of signal transduction and effector function induced by platelet activation or P-selectin binding to the P-selectin ligand on leukocytes, the induction of Ca2+ flux in platelets by PSGL-1 via P-selectin will be analyzed. Furthermore, binding of cytoplasmic tails of P-selectin and PSGL-1 to cytoplasmic signalling proteins in platelets and monocytes respectively will be examined using dimer constructs of cytoplasmic tails. If these studies indicate that PSGL-1 and ESL-1 are not physiologically critical counterreceptors for E-selectin or that there is evidence for another P- selectin ligand, we propose to expression clone a novel E-selectin ligand from a leukocyte library prepared from WEHI cells and a novel P-selectin ligand from a library prepared from neutrophils isolated from the PSGL-1 null mouse. The putative ligands will undergo characterization of their full length cDNAs and comparison of their predicted amino acid sequences with that of the PSGL-1, ESL-1 and GlyCAM-1. These studies will contribute to our understanding of the physiologically relevant receptors and counterreceptors that define cell-cell interaction during inflammation.