Potent cardioprotection from ischemia–reperfusion injury by a two‐domain fusion protein comprising annexin V and Kunitz protease inhibitor

Abstract

Considerable evidence suggests that coagulation proteases (tissue factor [TF]/activated factor VII [FVIIa]/FXa/thrombin) and their target protease activated receptors (PAR-1/PAR-2) play important roles in myocardial ischemia-reperfusion (I-R) injury. We hypothesized that localized inhibition of TF/FVIIa on the membrane surfaces of ischemic cells could effectively block coagulation cascade and subsequent PAR-1/PAR-2 cell signaling, thereby protecting the myocardium from I-R injury. We recently developed an annexin V-Kunitz inhibitor fusion protein (ANV-6L15) that could specifically bind to anionic phospholipids on the membrane surfaces of apoptotic cells and efficiently inhibit the membrane-anchored TF/FVIIa. In this study, we investigated the cardioprotective effect of ANV-6L15 in a rat cardiac I-R model in comparison with that of hirudin. Left coronary artery occlusion was maintained for 45min followed by 4h of reperfusion in anesthetized Sprague-Dawley rats. One minute before or 2min after coronary ligation, rats received an intravenous bolus injection of ANV-6L15 (2.5-250μgkg(-1) ), vehicle, or hirudin via bolus injection and continuous infusion. ANV-6L15 dose-dependently reduced infarct size by up to 87% and decreased plasma levels of cardiac troponin I, tumor necrosis factor-α, and soluble intercellular adhesion molecule-1, by up to 97%, 96%, and 66%, respectively, with little impact on the coagulation parameters. ANV-6L15 also ameliorated hemodynamic derangements, attenuated neutrophil infiltration and reduced Terminal deoxynucleotidyl transferase dUTP nick end labeling-positive apoptotic cardiomyocytes. Hirudin was less efficacious even at supraclinical dose. ANV-6L15 confers exceptionally potent cardioprotection and is a promising drug candidate for the prevention of myocardial I-R injury.