I. Research Plan Thrombolytic therapy remains suboptimal because of difficulties with bleeding, incomplete lysis and rethrombosis. previous attempts to improve the safety and efficacy of this therapy have all by ignored alpha2-antiplasmin (alpha2AP), the chief inhibitor of clot lysis. In this application, we propose to study the functions of alpha2AP in fibrinolysis and to use the understanding acquired to develop and test a safer, more effective thrombolytic strategy. To study alpha2AP in plasma we have developed a highly specific monoclonal antibody that inhibits soluble and fibrin-crosslinked alpha2AP. Preliminary work with this antibody has confirmed the pivotal role played by alpha2AP in retarding fibrinolysis. By inhibiting alpha2AP this antibody is synergistic with tissue plasminogen activator (t-PA) in clot lysis; at the same time, it increases the specificity of t-PA for fibrinolysis. Using this antibody and other techniques, we plan to better define the special roles played by fibrin-crosslinked, platelet-derived, and other forms of alpha2AP on fibrinolysis. This information will be exploited to obtain a more specific inhibitory antibody which can act as a clot-specific, thrombolytic agent. Our long-term goal is to design a recombinant, hybrid thrombolytic agent consisting of this inhibitory antibody coupled to a plasminogen activator. II. Candidate Training In addition to meeting scientific objectives, this application has been formulated to achieve the research training goals of the applicant. The work described in the initial """"""""Specific Aims"""""""" of the proposal is designed to further expand the candidate's training in techniques of protein chemistry and immunology. Later phases of the proposal are designed to provide the candidate's with experience in testing hypotheses in an in vivo experimental system. Finally, the last phase is design to provide training in the techniques of cloning, as well as the design, expression and characterization of new, recombinant molecules. III. Research Environment The work outlined in this application will be conducted in the Laboratory of Cellular and Molecular Research at Massachusetts General Hospital. This laboratory has had particular success in the scientific training of physicians. The laboratory currently has 19 postdoctoral fellows and 9 faculty engaged in a broad range of basic cardiac research. Regular laboratory meetings and postdoctoral seminars are a regular source for critical scientific evaluation and exposure to new techniques. There is also the broader educational interaction afforded by the Harvard Combined Seminar Series in Atherosclerosis and Vascular Biology. Consistent with the initial scientific training goals of the applicant, the sponsor will be DR. Gary R. Matsueda, a biochemist with expertise in fibrinogen and fibrinolytic research, peptide synthesis and the elicitation of highly specific monoclonal antibodies. The co-sponsor, Dr. Thomas Quertermous is a molecular biologist with unique experience in the design and expression of recombinant, hybrid fibrinolytic agents. Dr. Matsueda and Quertermous have successfully collaborated with each other in achieving scientific goals as well as the training of young scientists.
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