Abnormal thrombus formation and dissolution are associated with several cardiovascular diseases including atherosclerosis and both thromboembolic and hemorrhagic conditions. One important mechanism by which fibrinolysis is regulated is through the availability and activity of plasminogen activators (PAs). The physiologically relevant vascular PA is believed to be tissue-type PA (t-PA) because of 1) its ability to bind to fibrin, 2) its dependency on fibrin for activity, 3) its production by vascular endothelial cells, and 4) its efficient and specific use as a thrombolytic agent. The long range objective of this application is to understand the control of fibrinolysis in physiological situations.
The specific aim of this proposal is to characterize the structure-function relationship of t-PA with the aid of specific immunological probes. These probes will be a library of monoclonal antibodies (MABs) to t-PA developed in this study from mice immunized with intact, native t-PA. The initial characterization of these MABs will determine whether they, 1) inhibit t-PA activity, 2) recognize native and/or denatured t-PA (conformation specific epitopes), 3) recognize the A- or B-chain of t-PA, 4) recognize t-PA bound to fibrin, 5) recognize active site 'blocked' t-PA, 6) recognize t-PA complexed to the antiactivator, and 7) are directed toward distinct epitopes (epitope mapping). This procedure should permit the development of a number of MABs along the entire t-PA molecule, some of which bind but do not block the function of t-PA, and others which bind and blocks its function. These MABs will then be analyzed for their ability to interfere with four functional activities of t-PA including its 1) active site, and its interaction with 2) fibrin, 3) plasminogen, and 4) the antiactivator. Once MABs are identified to these four functional domains, the corresponding structural domains in the t-PA molecule will be identified. These experiments will require the proteolytic and/or chemical degradation of t-PA and the identification of each of the resulting peptides with the MABs. Delineating the complex interactions that occur between t-PA and other fibrinolytic components in blood may lead to the development of new approaches for the diagnosis and treatment of various thrombotic diseases.
| Schleef, R R; Wagner, N V; Loskutoff, D J (1988) Detection of both type 1 and type 2 plasminogen activator inhibitors in human cells. J Cell Physiol 134:269-74 |
| Mimuro, J; Schleef, R R; Loskutoff, D J (1987) Extracellular matrix of cultured bovine aortic endothelial cells contains functionally active type 1 plasminogen activator inhibitor. Blood 70:721-8 |
| Schleef, R R; Wagner, N V; Sinha, M et al. (1986) A monoclonal antibody that does not recognize tissue-type plasminogen activator bound to its naturally occurring inhibitor. Thromb Haemost 56:328-32 |
