INVITRO STUDIES ON THROMBIN GENERATION IN CITRATED, R-HIRUDINIZED AND HEPARINIZED WHOLE-BLOOD

The generation of thrombin in citrated, r-hirudinized and heparinized whole blood (final concentrations 10, 25, 50-mu-g/ml) was studied in the presence or absence of various activators. Whole blood from healthy volunteers was activated either by glass or dextran sulfate (contact activation; CA) as well as by thromboplastin/calcium chloride (extrinsic activation; EA) and by ellagic acid/cephaloplastin/calcium chloride (intrinsic activation; IA) and incubated 10 (CA, EA, IA) and 30 (CA) min at 37-degrees-C. After the incubation period plasma levels of prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin III complex (TAT) were measured utilizing ELISA methodology. After EA or IA no blood clotting occurred in all r-hirudinized samples and in heparinized blood at the highest concentration used. Despite inhibition of clotting F1 + 2 levels were increased both in r-hirudinized and in heparinized blood. However, in blood anticoagulated with heparin F1 + 2 levels after EA, IA and especially after CA were markedly lower than in r-hirudinized blood. Furthermore, in r-hirudinized blood an increase of TAT levels was found. The enhancement of F1 + 2 and TAT levels by r-hirudin was concentration-dependent and was lower in the higher r-hirudin concentration. The results indicate that in r-hirudinized whole blood significant amounts of thrombin can be generated which may elicit thrombin-mediated feedback mechanisms.