RECEPTOR FOR FIBRIN ON ENDOTHELIAL CELLS

内皮细胞上的纤维蛋白受体

基本信息

批准号：
3082962
负责人：
JOHN K ERBAN
金额：
$ 7.51万
依托单位：
TUFTS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-12-15 至 1995-11-30
项目状态：
已结题

项目摘要

Deposition of fibrin at the site of vascular injury or inflammation is the culmination of a complex cascade of events involving soluble blood coagulation factors and cofactors. In the process fibrin assumes new biologic properties not present in the parent fibrinogen molecule. Fibrin and it degradation products induce numerous important responses including cellular retraction and migration, release of von Willebrand factor from endothelial cells, leukocyte chemotaxis, and release of growth factors. The laboratory of D. Wagner first reported that fibrin rapidly induces the release of von Willebrand factor (vWf) from human umbilical vein endothelial cells in vitro, though fibrinogen is inactive. It is the goal of this project to identify and characterize the receptor on endothelial cells which participates in signalling the release of vWF when cells are stimulated with fibrin. Studies in this laboratory already identify a potential receptor which is unrelated to the known integrin receptor for fibrinogen. Initial affinity chromatography studies with portions of the fibrin molecule which induce release of vWF demonstrate that a protein can be purified from surface labelled, solubilized endothelial cells on a matrix which contains the B-beta15-42 peptide sequence of fibrinogen. The next studies will focus on the binding to and specificity for fibrin of this polypeptide by reconstituting the labelled protein in phospholipid vesicles and assaying for vesicle binding to fibrin formed by thrombin, reptilase or contortrix enzyme. Third the major thrust of this work will concentrate on cloning the receptor for fibrin using an affinity cloning strategy which has been used successfully to identify other adhesion molecules such as ELAM-1, ICAM-1 and ICAM-2. Alternatively, sequence data derived from protein analysis will be used to clone the receptor using the polymerase chain reaction. Once cloned the cDNA will be transfected into AtT-20 cells in an attempt to demonstrate fibrin stimulated release from cells which have a regulated pathway of secretion. Site directed mutagenesis will be used in an attempt to identify the binding domain of the receptor. Hematopoietic and mesenchymal cells which participate in the hemostatic and inflammatory responses will be surveyed for evidence of the receptor by RNA hybridization studies. Antibodies to the receptor will by synthesized either by purification of sufficient antigen or by preparation of a synthetic peptide derived from cloned sequence data for use as an immunogen. We will test abnormal fibrinogens to determine whether the fibrin formed can induce release of vWF in vitro. Investigation of the interaction of fibrin with the endothelial cell is of fundamental importance and will contribute to the understanding of hemostasis, the inflammatory response and the biology of metastasis.

纤维蛋白在血管损伤或炎症部位的沉积是涉及可溶性血液的复杂级联事件的高潮凝血因子和辅因子。在此过程中，纤维蛋白假设在母纤维蛋白原分子中不存在生物学特性。纤维蛋白它降解产品引起了许多重要的回应细胞缩回和迁移，von Willebrand因子从内皮细胞，白细胞趋化性和生长因子的释放。 D. Wagner的实验室首先报道了纤维蛋白迅速诱导从人脐静脉中释放von Willebrand因子（VWF）体外内皮细胞，尽管纤维蛋白原不活跃。这是目标该项目的识别和表征内皮上的受体参与信号在细胞为时释放VWF的单元用纤维蛋白刺激。该实验室的研究已经确定潜在受体与已知的整联蛋白受体无关纤维蛋白原。最初的亲和力色谱研究与一部分诱导VWF释放的纤维蛋白分子表明蛋白质可以从标记，溶解的内皮细胞的表面纯化含有纤维蛋白原的B-Beta15-42肽序列的基质。这接下来的研究将集中于与纤维蛋白的结合和特异性通过重构磷脂中标记的蛋白质通过重构囊泡和分析囊泡结合与凝血酶形成的纤维蛋白的结合，爬行动物酶或局显酶。第三，这项工作的主要力量将使用亲和力克隆将纤维蛋白的受体克隆集中已成功用于识别其他粘附的策略 ELAM-1，ICAM-1和ICAM-2等分子。或者，序列数据源自蛋白质分析将用于使用该受体克隆聚合酶链反应。克隆后，将转染cDNA ATT-20细胞试图证明纤维蛋白刺激从具有调节途径的细胞。站点定向诱变将用于识别结合域受体。参与参与的造血和间充质细胞止血和炎症反应将进行调查，以证明通过RNA杂交研究受体。对受体的抗体将通过通过纯化足够的抗原或制备合成从克隆序列数据得出的合成肽作为用作免疫原。我们将测试异常纤维蛋白，以确定是否是否形成的纤维蛋白可以在体外诱导VWF释放。调查纤维蛋白与内皮细胞的相互作用是基本的重要性，将有助于理解止血，炎症反应和转移的生物学。