Pre-administration of a carboxypeptidase inhibitor enhances tPA-induced thrombolysis in mouse microthrombi: Evidence from intravital imaging analysis

Abstract

Introduction: Thrombolysis using recombinant tissue-type plasminogen activator (rt-PA) is the pharmacological treatment of choice in acute thrombotic events. However, a narrow therapeutic window and bleeding complications limit its use. We describe the role of carboxypeptidase inhibitor from potato tuber (PTCI), an inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa), on Glu-plasminogen accumulation and microthrombus dynamics in vivo and demonstrate its influence on rt-PA-mediated thrombolysis. Materials and methods: In conjunction with real-time intravital two-photon excitation fluorescence microscopy, we produced and imaged laser-induced microthrombi in the mesenteric venules of Green Fluorescent Protein (GFP)-expressing mice. We examined microthrombus dynamics and thrombolysis patterns in vivo by measuring the changes in the fluorescence intensity of labeled Glu-plasminogen following administration of epsilon aminocaproic acid (EACA), PTCI, and rt-PA. Results: PTCI enhanced Glu-plasminogen accumulation at the core of the thrombus by inhibiting TAFIa, while EACA inhibited this process. Exogenous rt-PA effectively triggered Glu-plasminogen activation within the thrombus and promoted thrombolysis. Administration of PTCI and rt-PA together showed no significant benefit on thrombolysis compared to rt-PA administration alone. However, early-phase systemic administration of PTCI before thrombolytic therapy by rt-PA expedited 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). Conclusions: PTCI potentiates rt-PA-mediated thrombolysis when administered early in acute thrombotic events. Further studies are warranted to explore the potential of TAFI inhibitors as adjunct agents in thrombolysis or thromboprophylaxis.

Visualization of Domain- and Concentration-Dependent Impact of Thrombomodulin on Differential Regulation of Coagulation and Fibrinolysis

“…40 The border of the vascular injury site surrounded by normal vascular endothelial cells could be the candidate place where both thrombin activity and TM are expected to exist at sufficient amounts to efficiently activate TAFI. Further analysis to determine the spatiotemporal regulation of TAFI activation on TM-expressing vascular endothelial cells, as well as in vivo real-time imaging techniques performed by our group [43][44][45] and others, [46][47][48] seems to contribute to the understanding of when and where to activate TAFI and protein C.…”

Thrombus composition and thrombolysis resistance in stroke

“…Finally, Mathews et al. [ 159 ] showed that CPI, a natural carboxypeptidase inhibitor, accelerated rt-PA–mediated thrombolysis in mice, when given prior to thrombolytic therapy initiation.…”