The applicant's primary research goal is the study of mechanisms initiating and regulating the tissue factor (TF) -dependent pathway of blood coagulation. The applicant is a member of the Hemostasis and Thrombosis Research Laboratory, Department of Medicine, University of California, San Diego, and the proposed work will be carried out in this laboratory. The laboratory director, Dr. Samuel I. Rapaport, supports the application. The candidate's immediate career goal is to establish himself and be recognized by his peers as a fully independent investigator in the field of hemostasis research. The candidate's long term goal is to direct a major laboratory at a university or research institute in which problems significant for the understanding of the mechanisms regulating TF-dependent blood coagulation in normal and pathologic states are studied and in which graduate students and post-doctoral fellows are trained. A human ovarian carcinoma cell line constitutively producing cell surface tissue factor will be used in studies addressing four major problems. The first is an analysis of reasons for a substantial discrepancy that the applicant has observed between the time required for complete factor VII binding to cell surface tissue factor and the time required to obtain maximal catalytic activity of surface-bound factor Vlla/TF. The second is to determine whether earlier results that factor VII bound to reconstituted purified TF is preferentially and rapidly activated by trace concentrations of factors Xa and IXa will also hold for factor VII bound to cell surface TF. Additionally, the effect of trace concentrations of other proteases, e.g., factor XIIA and thrombin, will be examined. A third goal is an exploration, using placental anticoagulant protein I (annexin V) as a probe, of the importance of asymmetric distribution of negatively charged phospholipids on the cell surface as a modulator of TF functional expression. A final goal is to test whether binding of factor Xa/extrinsic factor pathway (EPI) complex to cell surface factor VIIA/TF leads to internalization of the quaternary complex. In this proposed study various biochemical techniques will be employed such as radioligand binding, activation peptide release from 3H-factors X and IX, and SDS-PAGE analysis.
