Lipopolysaccharide-Induced Hemolysis Is Abolished by Inhibition of Thrombin Generation but Not Inhibition of Platelet Aggregation

Abstract

In human sepsis, hemolysis is an independent predictor of mortality, but the mechanisms evoking hemolysis have not been fully elucidated. Therefore, we tested the hypotheses that (1) lipopolysaccharide (LPS)-induced hemolysis is dependent on thrombin generation or platelet aggregation and (2) red cell membranes are weakened by LPS. Anesthetized male Wistar rats were subjected to LPS or vehicle for 240 min. The effects of hemostasis inhibition on LPS-induced hemolysis were investigated by use of the thrombin inhibitor argatroban or the platelet function inhibitor eptifibatide. Free hemoglobin concentration, red cell membrane stiffness and red cell morphological changes were determined by spectrophotometry, atomic force microscopy, and light microscopy. Efficacy of argatroban and eptifibatide was assessed by rotational thrombelastometry and impedance aggregometry, respectively. LPS markedly increased free hemoglobin concentration (20.8 μmol/l ± 3.6 vs. 3.5 ± 0.3, n = 6, p < 0.0001) and schistocytes, reduced red cell membrane stiffness, and induced disseminated intravascular coagulation. Inhibition of thrombin formation with argatroban abolished the increase in free hemoglobin concentration, schistocyte formation, and disseminated intravascular coagulation in LPS-treated animals. Eptifibatide had no inhibitory effect. The LPS evoked decrease of red cell stiffness that was not affected by argatroban or eptifibatide. LPS causes hemolysis, schistocyte formation, and red cell membrane weakening in rats. The thrombin inhibitor argatroban but not the platelet inhibitor eptifibatide abolished hemolysis and schistocyte formation. Thus, LPS-induced hemolysis depends on disseminated intravascular coagulation, possibly enhanced by red cell membrane weakening. Clinical studies are necessary to investigate whether thrombin antagonists can decrease hemolysis and mortality in sepsis.

Keywords: hemolysis; hemostasis; lipopolysaccharide; red cell membrane stiffness.