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.
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