The formation of a stable intravascular coronary thrombus, a critical event in the initiation of acute myocardial infarction, results from an imbalance between the competing biochemical pathways of blood coagulation and clot lysis. In this country alone 1.5 million patients suffer heart attacks each year, and approximately 25% of all deaths results from myocardial infarction. While treatment of acute myocardial infarction took a dramatic stride forward with the advent of intravenous thrombolytic therapy, there is still substantial room for improvement. A general objective of this proposal is to develop both novel therapeutic agents and new strategies for the treatment of acute myocardial infarction and other thrombotic disorders. To accomplish this task and to gain fundamental new insights into the regulation of the activity of serine proteases, our specific aims are: (1) To design, produce, and characterize zymogen-like variants of tissue type plasminogen activator (t-PA). (2) To design, produce, and characterize variant of t-PA that are resistant to inhibition by the cognate inhibitor PAI-1 and PAI-2 but are rapidly inhibited by a noncognate serpin (protein C inhibitor or antithrombin III). (3) To design, produce, and characterize variants of plasmin(ogen) that are resistant to inhibition by alpha 2 antiplasmin. To achieve these goals we utilize a broad, interdisciplinary approach. We will perform molecular modeling experiments, use knowledge-based strategies of site directed mutagenesis, complete extensive kinetic analysis of variants of t-PA and plasminogen, perform large scale protein purification, analyze the protease domain of both wild type and mutated variants of t-PA by X-ray crystallography, and conduct pilot experiments that analyze the efficacy of both novel thrombolytic agents and new strategies of thrombolytic therapy in a primate, nonhuman model of thrombosis.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
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Scripps Research Institute
La Jolla
United States
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Hervio, L S; Coombs, G S; Bergstrom, R C et al. (2000) Negative selectivity and the evolution of protease cascades: the specificity of plasmin for peptide and protein substrates. Chem Biol 7:443-53
Beck, Z Q; Hervio, L; Dawson, P E et al. (2000) Identification of efficiently cleaved substrates for HIV-1 protease using a phage display library and use in inhibitor development. Virology 274:391-401
Coombs, G S; Rao, M S; Olson, A J et al. (1999) Revisiting catalysis by chymotrypsin family serine proteases using peptide substrates and inhibitors with unnatural main chains. J Biol Chem 274:24074-9
Zhang, Y L; Hervio, L; Strandberg, L et al. (1999) Distinct contributions of residue 192 to the specificity of coagulation and fibrinolytic serine proteases. J Biol Chem 274:7153-6
Coombs, G S; Bergstrom, R C; Madison, E L et al. (1998) Directing sequence-specific proteolysis to new targets. The influence of loop size and target sequence on selective proteolysis by tissue-type plasminogen activator and urokinase-type plasminogen activator. J Biol Chem 273:4323-8
Coombs, G S; Bergstrom, R C; Pellequer, J L et al. (1998) Substrate specificity of prostate-specific antigen (PSA). Chem Biol 5:475-88
Ke, S H; Coombs, G S; Tachias, K et al. (1997) Optimal subsite occupancy and design of a selective inhibitor of urokinase. J Biol Chem 272:20456-62
Tachias, K; Madison, E L (1997) Converting tissue type plasminogen activator into a zymogen. Important role of Lys156. J Biol Chem 272:28-31
Ke, S H; Tachias, K; Lamba, D et al. (1997) Identification of a hydrophobic exosite on tissue type plasminogen activator that modulates specificity for plasminogen. J Biol Chem 272:1811-6
Ke, S H; Coombs, G S; Tachias, K et al. (1997) Distinguishing the specificities of closely related proteases. Role of P3 in substrate and inhibitor discrimination between tissue-type plasminogen activator and urokinase. J Biol Chem 272:16603-9

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