Summary
To investigate the relative importance of direct inhibition of thrombin by complex
formation and of inhibition of thrombin generation to the mechanisms by that unfractionated
heparin (UH) and recombinant hirudin (rH) exert their anticoagulant effects, thrombin-antithrombin
III complex (TAT) and thrombin-hirudin complex (THC) formation was compared with the
generation of thrombin and prothrombin fragments 1 + 2 (F 1 + 2). Clotting was initiated
by activation of citrated plasma in the absence or presence of UH or rH using partial
thromboplastin, ellagic acid and calcium chloride. THC was determined by means of
ELISA using specific antibodies to thrombin and rH.
Activation of citrated plasma resulted in a sudden onset of thrombin generation after
a lag phase of 2 min. Addition of 50 ng rH/ml plasma or 0.1 UH/ml plasma prolonged
the clotting time to 3 min. While the peak of thrombin was only slightly decreased
in hirudinized plasma, in heparinized plasma thrombin generation was significantly
lower than in not anticoagulated plasma. This difference was more pronounced when
the lag phase was prolonged to 5 min using 400 ng rH/ml plasma or 0.35 U UH/ml plasma.
Using 1200 ng rH/ml or 0.65 U UH/ml to obtain a clotting time of 9 min only a small
amount of thrombin could be detected in heparinized plasma, but hirudinized plasma
still showed a high peak of thrombin. F 1 + 2 showed essentially the same pattern
as thrombin. Prior to the onset of visible clot formation in all experiments using
different concentrations of UH about the same values of TAT were observed. In contrast,
when samples were anticoagulated with different amounts of rH, a dose dependent increase
of THC was detected. When calculated from TAT and THC formation, much more thrombin
was bound by rH than by AT III.
Our experiments show that after intrinsic activation of plasma UH exerts its anticoagulant
effect more by inhibiting thrombin generation, while rH suppresses thrombin generation
to a much lesser extent but inhibits clotting more by direct inhibition of thrombin
due to the formation of THC.
