PB0630 - Early Systemic Administration of a Natural Carboxypeptidase Inhibitor Potentiates tPA-mediated Thrombolysis: Evidence from Real-time Intravital Imaging Analysis of Microthrombi in Mice

Background: Fibrinolytic therapy using recombinant tissue-type plasminogen activator (rt-PA) is widely practiced in acute thrombotic disorders including myocardial infarction and ischemic stroke. It activates Glu-plasminogen to plasmin on the surface of fibrin to promote clot lysis. However, its disadvantages include a narrow therapeutic window and bleeding complications.

Aims: To elucidate the functional role of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) and Glu-plasminogen in vivo using labeled Glu-plasminogen and a carboxypeptidase inhibitor from potato tuber (CPI), an inhibitor of TAFIa. Further, we aimed to demonstrate the effect of CPI on rt-PA-mediated thrombolysis in vivo.

Methods: Using multi-photon excitation fluorescence microscopy, laser-induced microthrombi were produced and imaged in real-time in the mesenteric venules of Green Fluorescent Protein (GFP)-expressing mice aged between 7-9 weeks. By measuring the changes in fluorescence intensity of labeled Glu-plasminogen, microthrombus dynamics and thrombolysis patterns were examined following administration of epsilon aminocaproic acid (EACA), CPI, and rt-PA.

Results: CPI promoted Glu-plasminogen accumulation at the center of the microthrombus by inhibiting TAFIa, while EACA inhibited this process. Exogenous rt-PA effectively triggered Glu-plasminogen activation within the thrombus and promoted thrombolysis. Administration of CPI alone was not effective in clot lysis and CPI given together with rt-PA did not show accelerated lysis. However, early-phase systemic administration of CPI before thrombolytic therapy by rt-PA accelerated clot lysis as evidenced by significantly faster time to reach peak Glu-plasminogen fluorescence intensity and shorter time to achieve near-complete clot lysis (P = 0.014 and P = 0.003 respectively).

Conclusion(s): Administration of the TAFIa inhibitor CPI potentiates rt-PA-mediated thrombolysis when given early in acute thrombotic events. CPI, and TAFIa inhibitors in general, show great promise in the management of thrombotic diseases. More studies are needed to assess the pharmacokinetic and pharmacodynamic profile of this group of drugs for potential use in thrombolysis or thromboprophylaxis.