| Tissue plasminogen activator (t-PA) is currently the "gold" standard of thrombolytic therapy for treatment of acute myocardial infarction. The function of t-PA is to convert the inactive precursor plasminogen to plasmin, which subsequently lyses the fibrin clot and restores blood flow. However, while activating clot bound plasminogen, t-PA can also act indiscriminately on the activation of systemic plasminogen, producing excess plasmin that can degrade crucial circulating clotting factors. Therefore, bleeding is often a risk associated with thrombolytic therapy. To resolve this problem, we have designed an approach, termed ATTEMPTS (A&barbelow;ntibody T&barbelow;argeted T&barbelow;riggered E&barbelow;lectrically M&barbelow;odified P&barbelow;rodrug T&barbelow;ype S&barbelow;trategy), to deliver t-PA to the clot site in an inactive form, and then trigger its release to the active form so it may specifically activate clot bound plasminogen. This delivery system is composed of a large protein complex, consisting of two components: (i) a negatively charged fibrin targeting antibody; and (ii) a t-PA molecule modified with a positively charged species, which are bound via a tight but reversible electrostatic interaction. The antibody moiety localizes the inactive t-PA complex to the fibrin clot site. Upon administration of the triggering agent, protamine, t-PA is released and directs clot lysis on site, as opposed to non-specific activation in circulation. In vitro studies performed previously in our laboratory strongly demonstrate the feasibility of this approach in inducing clot lysis, while preserving the levels of crucial clotting factors, including fibrinogen and plasminogen.; In order to evaluate the real-time feasibility of "ATTEMPTS," in vivo studies were conducted using a rat thrombosis model, and the preliminary results are presented in this dissertation. In addition, future directions in further developing this system include using recombinant methods to produce a t-PA mutant with a specific location of incorporated positive charges; and the possibility of a "reverse" strategy of ATTEMPTS to address clinical concerns of using protamine as the triggering agent. |
