Protamine-induced reductions of endothelial cell ATP

Protamine, a polycationic protein used to reverse heparin anticoagulation, is frequently associated with decreased oxygen consumption, systemic hypotension, pulmonary artery hypertension, and bradycardia. This investigation examines the hypothesis that these events reflect toxic effects of protamine on endothelial cells. Cultured bovine pulmonary artery endothelium was exposed to protamine (12.5 to 500 micrograms/ml, corresponding to clinical doses 0.75 to 30 mg/kg), either alone (n = 6) or 3 minutes after exposure to heparin, 0.1 IU/microgram protamine (n = 6). ATP was measured 1 to 180 minutes after protamine by a luciferase-luciferin assay and cell viability determined by trypan blue exclusion. Ultrastructure was assessed by transmission electron microscopy. Polylysine, 25 micrograms/ml, a cytotoxic polycationic agent, was also studied. Dose-dependent reductions in ATP (range, -11% to -51%) and ATP per viable cell (up to -41%) occurred. Decreases in ATP did not occur until after 30 minutes with protamine alone, compared with differences as early as 1 minute after protamine with prior heparin. Progressive mitochondrial injury was noted evident by swollen cristae, vacuolization, and eventual disruption. Polylysine caused similar changes. Protamine decreases endothelial cell ATP and prior heparin exposure accelerates this effect. The toxicity may reside in the positive charges on these molecules and mitochondrial damage may account for reductions in cellular ATP and systemic oxygen consumption.