Thrombolytic treatment with plasminogen activators (PA) such as tissue plasminogen activator (tPA) has been used not only for treatment of acute myocardial infarction, but also peripheral arterial occlusion (PAO). However, the risk of life-threatening intracranial hemorrhage is measurably high (0.4-2.9%), particularly for PAO, where treatment can last for 1-2 days. Additionally, problems with long, retracted clots and poor circulation results in depletion of available plasmin, the PA substrate, near the PAO thrombus, rendering the PA only partially effective. Plasmin is the enzyme which actually dissolves the clot. Consequently, thrombolytic therapy of PAO with PA is not officially sanctioned by the FDA; but it still constitutes approximately 25% of all procedures for treating this condition in the US. Recent published animal studies have indicated that catheter-administered plasmin (Plm) or its des-kringle derivatives might be more appropriate alternatives to PA, since it has a very short half-life when circulating systemically, and therefore does not result in hemorrhaging. Additionally, there is no problem with plasmin depletion at long, retracted clots. Plasmin is notoriously unstable at every level, from isolation, purification, formulation, to administration. It is usually isolated from human plasma, so its use carries the risk of HIV, HBV, or HCV infection.
Aim 1 In this proposal, Principal Investigator intends to produce 2 des-kringle derivatives of plasmin in their zymogenic form, mini-plasminogen (mini-Plg) and microplasminogen (mu Plg) recombinantly as inclusion bodies in an E. coli expression system, refold them at high yield and functionality using Proteomtech's patented Pt-Fold expression system, activate them to their active forms-miniplasmin (mini-Plm) and microplasmin (mu Plm) in vitro, and characterize their structural and functional properties in vitro.
Aim 2 The recombinant products will be tested as a potential PAO therapeutic in vivo using a rabbit extracorporeal loop thrombolysis model and also evaluated for bleeding at distal sites using a rabbit ear-puncture re-bleed model. Mini-Plm and mu Plm display different properties in clot binding, kinetics of fibrinolysis, and in-vivo half-life, so it is necessary to evaluate both of them to identify the superior molecule for product development. ? ? ?
