Thrombin increases cardiomyocyte acute cell death after ischemia and reperfusion

Thrombin exerts multiple actions on cardiomyocytes leading to increased intracellular Na+ and Ca2+ concentrations, and to activation of a Ca2+-independent PLA2, and has been proposed to favor the genesis of arrhythmias and ischemic injury in acute coronary syndromes. However, the influence of thrombin on cardiomyocyte cell death during ischemia–reperfusion has not been studied. A beneficial influence of low thrombin concentrations has been described in other cell types. HL-1 cardiomyocytes were subjected to simulated ischemia (SI) and reperfusion (SR) and cell death was assessed by means of LDH release to the incubation media. Thrombin dose-dependently increased cell death in normoxic cells, in cells subjected to SI, and in cells subjected to SR (by 20 ± 8%, 95 ± 32% and 35 ± 9%, respectively, at 100 U/ml). The effects of thrombin were associated to increased cytosolic Ca2+ overload, mimicked by 100 μM PAR-1 agonist peptide SFLLRNPNDKYEPF, and reversed by the direct thrombin inhibitor lepirudin (IC50 = 1.3 ± 0.2 μg/ml). The presence of thrombin during simulated ischemia–reperfusion increases cardiomyocyte cell death by a mechanism that involves activation of PAR-1 receptors and can be prevented by the direct thrombin inhibitor lepirudin.