The proposal is focused on the mechanisms of protease control by serine protease inhibitors (SERPINS). Two different model SERPINs will be employed to investigate the mechanism underlying SERPIN activation by heparin, and the mechanism by which SERPIN: Protease complexes are bound, internalized, and processed by cells. The first long range goal is to gain insight into the molecular basis of antithrombotic therapy. The thrombin inhibitory activity of ATIII is accelerated 1,000 to 10,000 fold by heparin, but the structural and molecular basis underlying this important clinical application of heparin and related compounds, remains to be elucidated. This level of understanding is requisite to the development of alternatives to heparin therapy. This will be approached by a combination of site-directed mutagenesis within the identified heparin binding region of ATIII, followed by a quantitative biochemical assessment of the effects of these changes on heparin activation in the engineered proteins. These studies will be extended to characterize the interactions of these variants with vascular endothelial cells which represent the physiological source of heparin for ATIII-thrombin interactions. In addition, SERPIN levels are regulated by the internalization and processing of SERPIN: Protease complexes by specific cell types. This aspect of the present studies will focus on a combined biochemical and site directed-mutagenesis analysis of PN1 to probe the mechanism of SERPIN:Protease binding to cells, and the mechanism that mediates their internalization, which is requisite to processing. Based on previously obtained data, genetically engineered variants of PN1 and biologically active synthetic peptides will be used to probe the molecules responsible for the entry of Th:PN1 complexes into the cells that process them.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Pathobiochemistry Study Section (PBC)
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University of California Irvine
Anatomy/Cell Biology
Schools of Arts and Sciences
United States
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Crisp, Robert J; Knauer, Mary F; Knauer, Daniel J (2002) Protease nexin 1 is a potent urinary plasminogen activator inhibitor in the presence of collagen type IV. J Biol Chem 277:47285-91
Crisp, R J; Knauer, D J; Knauer, M F (2000) Roles of the heparin and low density lipid receptor-related protein-binding sites of protease nexin 1 (PN1) in urokinase-PN1 complex catabolism. The PN1 heparin-binding site mediates complex retention and degradation but not cell surface binding or intern J Biol Chem 275:19628-37
Knauer, D J; Majumdar, D; Fong, P C et al. (2000) SERPIN regulation of factor XIa. The novel observation that protease nexin 1 in the presence of heparin is a more potent inhibitor of factor XIa than C1 inhibitor. J Biol Chem 275:37340-6
Knauer, M F; Crisp, R J; Kridel, S J et al. (1999) Analysis of a structural determinant in thrombin-protease nexin 1 complexes that mediates clearance by the low density lipoprotein receptor-related protein. J Biol Chem 274:275-81
Mentz, S; de Lacalle, S; Baerga-Ortiz, A et al. (1999) Mechanism of thrombin clearance by human astrocytoma cells. J Neurochem 72:980-7
Kridel, S J; Knauer, D J (1997) Lysine residue 114 in human antithrombin III is required for heparin pentasaccharide-mediated activation. J Biol Chem 272:7656-60
Knauer, M F; Kridel, S J; Hawley, S B et al. (1997) The efficient catabolism of thrombin-protease nexin 1 complexes is a synergistic mechanism that requires both the LDL receptor-related protein and cell surface heparins. J Biol Chem 272:29039-45
Knauer, M F; Hawley, S B; Knauer, D J (1997) Identification of a binding site in protease nexin I (PN1) required for the receptor mediated internalization of PN1-thrombin complexes. J Biol Chem 272:12261-4
Kridel, S J; Chan, W W; Knauer, D J (1996) Requirement of lysine residues outside of the proposed pentasaccharide binding region for high affinity heparin binding and activation of human antithrombin III. J Biol Chem 271:20935-41
Smith, J W; Dey, N; Knauer, D J (1990) Heparin binding domain of antithrombin III: characterization using a synthetic peptide directed polyclonal antibody. Biochemistry 29:8950-7

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