The objectives of this proposal are to develop an understanding of the mechanisms by which protease activity in coagulation, fibrinolysis, and inflammation is regulated. To accomplish these objectives the interaction of proteases with the plasma protease inhibitor, a2-Macroglobulin (a2M), and the interaction of the resultant complexes with cell surface receptors will be characterized. Major areas of focus will be to elucidate the mechanism by which proteinases react with this inhibitor, to access the importance and role of a2M as a regulator of protease activity in vivo, and to isolate and characterize the a2M receptor from fibroblasts. Conformational changes occurring in a2M are central to the function of the inhibitor, and not only result in inhibition of protease activity, but also generate receptor determinants on the molecule. Studies are proposed to determine the relationship between conformational changes, protease inhibition, and the generation of the receptor binding sites on the inhibitor-protease complex. Specific probes for monitoring each process have been developed, and will be extensively utilized in these studies. The specific in vivo processes in which a2M plays a key role will be addressed by isolating complexes formed under physiological conditions and identifying which specific enzymes associate with the inhibitor. Complexes will be isolated by immunoaffinity chromatography using a monoclonal antibody that is specific for a2M-protease complexes. Studies are also proposed to characterize the interaction of the inhibitor-protease complex with specific receptors. To accomplish this goal the receptor determinants on the complex will be identified by preparing anti- bodies against synthetic peptides representing regions of the molecule. The receptor will also be isolated from fibroblasts, and the properties characterized. Studies will be initiated on the amino acid sequence of regions of the receptor. Monoclonal antibodies prepared against the purified receptor will assist in characterizing subunits with the goal of identifying regions that participate in receptor function, and will be useful for comparing the structure of the receptor from various cell types and species.
