Thrombotic vascular disease is a significant cause of morbidity and mortality in developed nations. Current treatments for thrombosis utilize either thrombolytic therapy with plasminogen activators (PAs) or direct vascular reperfusion with angioplasty or other surgical techniques. Though these treatments have a high rate of success, approximately 20% of patients fail to respond to thrombolytics and many patients undergoing angioplasty suffer from subsequent restenosis. Plasminogen activator inhibitor-1 (PAI-1) is a central regulatory protein of the fibrinolytic system and a variety of other biological systems involving plasminogen activation. While the role of PAI-1 in human disease is currently poorly defined, several observations suggest that PAI-1 is critical for the regulation of normal hemostasis. Deficiency of PAI-1 results in a mild to moderate bleeding disorder. Overexpression of PAI-1 appears to confer increased risk for thromboembolic disease and elevated PAI-1 levels have been associated with premature myocardial infarction. PAI-1 may also play a major role in the clinical response to thrombolytic therapy with tissue plasminogen activator (tPA). Several studies have demonstrated that PAI-1 is the major factor responsible for the resistance of platelet rich thrombi to lysis, suggesting that inhibition of PAI-1 activity might be a useful strategy for increasing the efficacy of thrombolytic therapy. This proposal will investigate potential methods to disrupt PAI-1 function using a combination of biochemical, physicochemical and molecular approaches. Synthetic peptides will be developed that disrupt PAI-1 functional activity. Preliminary results indicate that platelet PAI-1 activity can be completely blocked in an in vitro clot lysis assay with a 14 residue peptide. The first part of the proposal will focus on characterizing the structural and functional basis for this specific and rapid inactivation of PAI-1 by this inactivating peptide together with three other peptides that also inactivate PAI-1 in vitro. Additional peptides will then be designed and synthesized using a rational approach to optimize the present sequence for maximum efficacy. In the second phase, synthetic combinatorial peptide libraries and two phage display libraries of random 6 mer and 15 mer peptides will be screened for their ability to block PAI-1 function. Identified peptide sequences will be synthesized as free peptides and examined for their potential to inactivate PAI-1 in vitro. In addition, these libraries will be screened to identify sequences that compete for PAI-1 binding to vitronectin. Sequences identified in this latter screen may be useful in preventing restenosis by targeting the disruption of PAI-1 function to the subendothelial matrix compartment. In the last part of the proposal, reagents will be developed to examine the efficacy of identified peptides for enhancing thrombolysis in vivo.
Showing the most recent 10 out of 70 publications
