PLATELET CELL ADHESION MOLECULES

P-选择素是存在于血小板和内皮细胞的储存颗粒中的细胞粘附分子。在细胞刺激后，蛋白质被转移到质膜，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。本申请代表了P-选择素和PSGL-1的生物学研究的继续。 由于PSGL-1已显示与所有选择素结合，因此将通过荧光光谱法分析可溶性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。在过去的资助期内，我们通过同源重组制备了PSGL-1缺陷小鼠，并完成了初步表征。来自该小鼠的细胞将用于通过在使用嗜中性粒细胞和T淋巴细胞的平行板离体测定中在滚动条件下比较PSGL-1（-/-）、（+/-）和（+/+）白细胞与P-选择素、E-选择素和L-选择素的相互作用来确定PSGL-1在选择素功能中的生理作用。PSGL-1缺陷小鼠和双敲除小鼠包括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-选择素缺失小鼠将用于模型系统中以确定PSGL-1的生理功能。 待研究的病理过程包括非免疫介导和T细胞介导的皮肤炎症、创伤和TNF后白细胞滚动、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的模型。为了理解血小板活化或P-选择素与白细胞上的P-选择素配体结合诱导的信号转导和效应子功能的分子基础，将分析PSGL-1通过P-选择素诱导血小板中的Ca 2+通量。此外，将使用胞质尾的二聚体构建体检查P-选择素和PSGL-1的胞质尾分别与血小板和单核细胞中的胞质信号传导蛋白的结合。如果这些研究表明PSGL-1和ESL-1不是E-选择素的生理学上关键的反受体，或者存在另一种P-选择素配体的证据，我们建议从WEHI细胞制备的白细胞文库表达克隆新的E-选择素配体，并从分离自PSGL-1缺失小鼠的嗜中性粒细胞制备的文库表达克隆新的P-选择素配体。 将对推定的配体进行全长cDNA的表征，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了解生理相关的受体和反受体，定义炎症过程中的细胞-细胞相互作用。

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.