Thrombin generation: phenotypic quantitation

Abstract

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 micro g 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.

The plasma hemostatic proteome: thrombin generation in healthy individuals

“…The extremes were £ 311nM (P5) and ‡ 650nM (P95). This range of thrombin maximum levels in the LETS control population (n ¼ 473), overlap the maximum levels determined in an empirical whole blood study of 13 healthy individuals (245-775nM, with a cumulative mean of 414nM, SD 111nM) (26). The mean of the population for total thrombin (Fig 2D) was 92lMs (SD 19lMs) with a range of 39-177lMs.…”

“…The second approach focuses on developing tests for assessing hemostatic competence, with a primary focus on the measurement of thrombin generation. These in vitro approaches include the whole blood thrombin generation test [23,24], the endogenous thrombin potential in plasma [25], or derived methods in which markers of procoagulant [26,27] or fibrinolytic activity [28][29][30] are measured.…”

Thrombin generation in different cohorts: Evaluation of the haemostatic potential

“…Both inherited (genetic) and acquired (environmental and therapeutically induced) haemostatic variations can lead to significant alterations in these profiles [23,24]. It has been shown that the coagulation factors show remarkable interindividual variation, even among healthy subjects [24].…”

“…It has been shown that the coagulation factors show remarkable interindividual variation, even among healthy subjects [24]. Several technologies have been developed to directly or indirectly measure thrombin generation profiles [23,24,25].…”

In silico thrombin generation: Plasma composition imbalance and mortality in human immunodeficiency virus

“…Thrombin generation assays can be used to define an individual's phenotype through the use of the phases of thrombin generation (initiation, propagation, termination) which are governed by the underlying kinetic reactions and can provide descriptors of the coagulation process (eg, rates, levels, and timing of thrombin formation and inhibition) 24. Studies in the healthy population have shown that thrombin generation dynamics differ largely between individuals, but are relatively stable over time in a given individual, supporting the concept that they may have utility as a phenotypic marker 25, 26, 27. The mechanism‐based bridge between thrombin generation assays and individualized risk prediction is plasma composition–based computational modeling 28, 29.…”