Detection of thrombin in human blood by ex-vivo hirudin

Hirudin, as a high-affinity, stoichiometric thrombin inhibitor (1), can serve as a sensitive probe for detection of thrombin. Thrombin-hirudin complex can be measured by ELISA (2,3) or by affinity chromatography on concanavalin-A-Sepharose using radiolabeled hirudin (4). Intravenous infusion of hirudin in pigs yielded circulating thrombin-hirudin complex reflective of potentially coagulant basal intravascular thrombin generation of 120 pM per hour. Go-infusion of lipopolysaccharide from S. equi resulted in a massive generation of thrombin-hirudin complex (approximately 2 nM per hour) as measured by ELISA (2) or by concanavalin-A-Sepharose chromatography (4). Basal thrombin generation (3 pM per hour) was also observed in humans in a hirudin dosing study (5). Retrieval of thrombin by hirudin was independent of the hirudin dose. The thrombin was inferred to have been associated with vascular endothelial thrombomodulin.