Thrombin generation: phenotypic quantitation

An individual's ability to generate thrombin following tissue factor stimulus was evaluated in 13 healthy male donors in a 6-month study. Thrombin generation in whole blood collected by phlebotomy, contact pathway suppressed by the presence of 100 mug mL(-1) corn trypsin inhibitor, was initiated by the addition of 5 pM tissue factor/10 nM phospholipid. Reactions were quenched at 20 min by the addition of an ethylenediaminetetraacetic acid (EDTA), benzamidine, FPRck cocktail. Thrombin generation was determined by an ELISA for thrombin-antithrombin III (TAT) complex formation. Results showed that the levels of TAT observed varied from 245 to 775 nM. Thrombin production was consistent within each individual, CVi = 11.6%, but varied significantly within the group, CVg = 25.2%, and correlated inversely with an individual's clotting time (r = -0.54, P = 0.07). No correlations were individually observed between TAT and C-reactive protein, antithrombin III, factors II, V, VII, VIII, IX and X, fibrinogen and prothrombin time. However, computer simulations, which integrated each individual's coagulation factor levels using the Speed Rx method (Hockin et al., J Biol Chem 2002; 277: 18322), predicted maximum active thrombin levels (ranging from calculated values of 220-500 nM) consistent with the empirically determined values. Overall, these data suggest that thrombin generated in whole blood exclusively by tissue factor stimulation can be used as an integrative phenotypic marker to determine an individual's response to a tissue factor challenge.