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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Modified Research Career Development Award (K04)
Project #
5K04HL002113-02
Application #
3074280
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1988-04-01
Project End
1993-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
American National Red Cross
Department
Type
DUNS #
003255213
City
Washington
State
DC
Country
United States
Zip Code
20037
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Kounnas, M Z; Argraves, W S; Strickland, D K (1992) The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330. J Biol Chem 267:21162-6
Strickland, D K; Larsson, L J; Neuenschwander, D E et al. (1991) Reaction of proteinases with alpha 2-macroglobulin: rapid-kinetic evidence for a conformational rearrangement of the initial alpha 2-macroglobulin-trypsin complex. Biochemistry 30:2797-803
Striekland, D K; Ashcom, J D; Williams, S et al. (1991) Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen. J Biol Chem 266:13364-9
Zucker, S; Lysik, R M; Zarrabi, M H et al. (1991) Proteinase-alpha 2 macroglobulin complexes are not increased in plasma of patients with cancer. Int J Cancer 48:399-403
Strickland, D K; Ashcom, J D; Williams, S et al. (1990) Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor. J Biol Chem 265:17401-4
Ashcom, J D; Tiller, S E; Dickerson, K et al. (1990) The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin. J Cell Biol 110:1041-8
Orthner, C L; Madurawe, R D; Velander, W H et al. (1989) Conformational changes in an epitope localized to the NH2-terminal region of protein C. Evidence for interaction of protein C domains. J Biol Chem 264:18781-8
Stoops, J K; Bretaudiere, J P; Strickland, D K (1989) Electron microscope studies of human alpha 2-macroglobulin-chymotrypsin complex: demonstration that the two structures assigned to native and proteolyzed alpha 2-macroglobulin represent two views of the proteolyzed molecule. Biochem Biophys Res Commun 161:216-20
Larsson, L J; Neuenschwander, D E; Strickland, D K (1989) Reaction of proteinases with alpha 2-macroglobulin: evidence for alternate reaction pathways in the inhibition of trypsin. Biochemistry 28:7636-43