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.
Showing the most recent 10 out of 17 publications
