The objectives of this research proposal are to define the biochemical role of activated protein C (APC) in regulating the expression of fibrinolytic activity. Our approaches will involve: (a) characterization of the plasminogen activator reported to be responsible for this effect; (b) an examination and characterization of the interaction of APC with cultured cells, and the effect upon expression of plasminogen activator activity; and (c) a characterization of structural and functional aspects of the inhibition of fibrinolysis, and the effect of APC, if any, if any, on these inhibitor molecules. In order to characterize the plasminogen activator responsible for this effect, we will rely on immunological techniques, using specific high-affinity antibodies directed against tissue activator, urokinase and kallikrein. By using the appropriate antibody coupled to Sepharose, we will isolate the plasminogen activator, and determine if a precursor form exists in plasma, capable of undergoing activation in the presence of APC and any necessary cofactor molecules. The binding of 125I-labelled APC to cultured endothelial cells, prostate adenocarcinoma cells, and human monocytes will be thoroughly examined, and the effect on expression of plasminogen activator antigen and activity measured. The binding will be characterized with regard to certain features required of a specific receptor, i.e. high affinity, limited numbers of sites per cell, specificity for APC, and the elicitation of a biological response. Certain structural features of Alpha2-antiplasmin and Alpha2-macroglubulin will also be characterized to further our understanding of the role of these molecules in the inhibition of the fibrinolytic pathway. These studies will include a characterization of the physical properties of these inhibitors and their plasmin complexes by differential scanning calorimetry, examination of the conformational alterations induced in Alpha2M as a consequence of plasmin binding, and a characteization of the structural alteration associated with the inactivation of Alpha2-antiplasmin, and the role of APC, if any, in this process. The long-term objective of this proposal is to explore several areas involved in the regulation of fibrinolysis, with the goal of improving thrombolytic therapy. It is likely that an enzyme with anticoagulant properties, such as APC, that also has been demonstrated to stimulate fibrinolysis, will be an excellent thrombolytic agent.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL030200-03
Application #
3341255
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1983-04-01
Project End
1987-11-30
Budget Start
1985-04-01
Budget End
1987-11-30
Support Year
3
Fiscal Year
1985
Total Cost
Indirect Cost
Name
American National Red Cross
Department
Type
DUNS #
003255213
City
Washington
State
DC
Country
United States
Zip Code
20037
Williams, S E; Inoue, I; Tran, H et al. (1994) The carboxyl-terminal domain of lipoprotein lipase binds to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and mediates binding of normal very low density lipoproteins to LRP. J Biol Chem 269:8653-8
Chappell, D A; Inoue, I; Fry, G L et al. (1994) Cellular catabolism of normal very low density lipoproteins via the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is induced by the C-terminal domain of lipoprotein lipase. J Biol Chem 269:18001-6
Stoops, J K; Schroeter, J P; Kolodziej, S J et al. (1994) Structure-function relationships of human alpha 2-macroglobulin. Three-dimensional structures of native alpha 2-macroglobulin and its methylamine and chymotrypsin derivatives. Ann N Y Acad Sci 737:212-28
Kounnas, M Z; Henkin, J; Argraves, W S et al. (1993) Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor mediates cellular uptake of pro-urokinase. J Biol Chem 268:21862-7
Williams, S E; Ashcom, J D; Argraves, W S et al. (1992) A novel mechanism for controlling the activity of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Multiple regulatory sites for 39-kDa receptor-associated protein. J Biol Chem 267:9035-40
Kounnas, M Z; Morris, R E; Thompson, M R et al. (1992) The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A. J Biol Chem 267:12420-3
Constantine, K L; Brew, S A; Ingham, K C et al. (1992) 1H-n.m.r. studies of the fibronectin 13 kDa collagen-binding fragment. Evidence for autonomous conserved type I and type II domain folds. Biochem J 283 ( Pt 1):247-54
Stoops, J K; Schroeter, J P; Bretaudiere, J P et al. (1991) Structural studies of human alpha 2-macroglobulin: concordance between projected views obtained by negative-stain and cryoelectron microscopy. J Struct Biol 106:172-8
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
Herz, J; Goldstein, J L; Strickland, D K et al. (1991) 39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. J Biol Chem 266:21232-8

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