Fluorogenic Assays for Factor Vlla and Tissue Factor

DESCRIPTION (provided by applicant): Factor Vlla and Tissue Factor (TF) are essential proteins for the initiation of blood coagulation. Blood coagulation is initiated when cryptic TF becomes exposed on the surface of vascular cells where it can bind circulating factor Vlla. The factor Vlla/TF complex catalyzes the activation of certain blood zymogens that propagate the coagulation event. The amount of circulating factor Vlla has been shown to be a good indicator of hemostatic potential and for this reason is a potential risk indicator for the development of cardiovascular disease. In addition, over the past decade recombinant factor Vlla has become the drug of choice for treating hemophilia A and B patients who have developed inhibitors to factors VIII and IX respectively. Formation of the factor Vlla/TF complex is also the basis of specific coagulation assays. The Prothrombin time (PT assay) utilizes either natural or synthetic thromboplastin reagents to initiate coagulation in-vitro. Thus in addition to its in-vivo role, TF also has in-vitro applications. Furthermore, TF is not only present in the vasculature, but also in a numerous other tissues and cells including brain, lung, placenta, platelets, monocytes and tumor cells. In addition to its "normal" role in hemostasis, it is also known to be involved in the metastasis of tumor cells. The fact that TF and factor Vlla play such important roles both in-vivo and in-vitro, indicates that rapid and direct assays for these proteins could be of great utility. At the present time, reliable assays for factor Vlla and tissue factor that can be applied to simple as well as complex biological systems do not exist. The goal of this phase I proposal is to demonstrate the feasibility of developing fluorogenic-based assays that may be used to directly measure factor Vlla and TF in simple and complex biological systems. To accomplish this goal, the following specific aims are proposed: 1) To utilize aminonapthalene-based fluorogenic substrates for factor Vlla to develop basic assays that may be used to quantitate factor Vlla and TF in simple and well defined systems; b) to further develop the assay for tissue factor and demonstrate the ability to measure TF in more complex biological systems; and c) to further develop the assay for factor Vlla and demonstrate the ability to measure factor Vlla in more complex biological systems.