Influence of the surface on thrombin generation

Thrombin generation depends on the surface of the blood vessel or container. With a new ultra-sensitive and -specific thrombin assay the surface-dependent thrombin generation was quantified. Citrated blood or plasma was preincubated for 1 h (37 degrees C). Citrated blood, plasma, or plasma with 0-10 g/l hemoglobin-erythrocyte microparticles (Hb-MP) were preincubated at 23 degrees C or at 37 degrees C. Plasma samples (50 microl) were recalcified in polystyrol (PS) wells and incubated for different coagulation reaction times (CRT). Final supramolar arginine concentrations, 0.1% Triton X 100, and chromogenic thrombin substrate concentrations in the onefold km-range were added and the linear DeltaA/t was measured in the recalcified coagulation activity assay (RECA). Aprotinin or corn trypsin inhibitor were added. (i) Recalcification of plasma (in different monovettes) pre-incubated for 1 h (37 degrees C) generated the following thrombin activities after 7 min (37 degrees C): 0.74 IU/ml (polypropylene (PP)-citrate), 0.39 IU/ml (PP-EDTA), 0.06 IU/ml (PP-heparin), 1.38 IU/ml (PS), 0.63 IU/ml (1 ml volume PP), 0.13 IU/ml (15 ml volume PP), and 3.62 IU/ml (glass). (ii) Recalcification of preincubated whole blood generated up to about fivefold more thrombin. (iii) Thrombin generation is proportional to the plasmatic concentration of Hb-MP, 10 g/l Hb-MP generating about 4 IU/ml thrombin within 20 min CRT. (iv) The IC50 of aprotinin and corn typsin inhibitor on thrombin generation in RECA are about 2 KIU/ml and about 1 U/ml, respectively. The reaction wall, the preincubation temperature, and hemolysis influences thrombin generation. The RECA allows to diagnose the prothrombotic capacity of any material.