PLATELET CELL ADHESION MOLECULES

p -选择素是一种细胞粘附分子，存在于血小板和内皮细胞的储存颗粒中。在细胞受到刺激后，该蛋白被转移到质膜，在那里它作为中性粒细胞和单核细胞上的PSGL-1的白细胞受体发挥作用。目前的应用代表了p -选择素和PSGL-1生物学研究的延续。由于PSGL-1已被证明与所有选择素结合，因此将利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。通过比较PSGL-1（-/-）、（+/-）和（+/+）白细胞与p -选择素、E-选择素和l -选择素在滚动条件下的相互作用，利用中性粒细胞和T淋巴细胞进行平行板离体实验，利用该小鼠的细胞来确定PSGL-1在选择素功能中的生理作用。我们将利用PSGL-1缺失小鼠和双敲除小鼠，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，在模型系统中确定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模型、创伤和TNF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 -选择素结合白细胞上p -选择素配体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Ca2+通量。此外，p -选择素和PSGL-1的细胞质尾部分别与血小板和单核细胞的细胞质信号蛋白的结合将使用细胞质尾部的二聚体结构进行检测。如果这些研究表明PSGL-1和ESL-1在生理上不是e -选择素的关键反受体，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克隆一种新的e -选择素配体，从PSGL-1缺失小鼠分离的中性粒细胞文库中制备一种新的P-选择素配体。将对推测的配体进行全长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炎症过程中定义细胞-细胞相互作用的生理相关受体和反受体。

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.