Thrombin Generation Assay

Blood samples were drawn into citrate-theophylline-adenine-dipyridamol (CTAD) test tubes (Vacuette® Greiner, Kremsmünster, Austria; 9:1 v/v) to minimize the effect of circulating microparticles, and immediately centrifuged at 4,500 g for 15 min. Platelet-poor-plasma (PPP) was then stored at −80°C for subsequent testing.

We performed TGA using the fully automated Ceveron® alpha TGA analyzer (Technoclone, Vienna, Austria; Software Release V 2.1.2.2). For the conventional test, 15 μl reagent RC high (Ceveron® TGA RC high; Technoclone, Vienna, Austria), 35 μl CaCl₂, and 20 μl reaction buffer are added to 40 μl PPP (thawed to 37°C) to initiate the coagulation process. Thrombin generated during the clotting process cleaves 40 μl of Z-Gly-Gly-Arg-AMC, a fluorogenic substrate (Ceveron® TGA substrate, Technoclone, Vienna, Austria). The concentration of thrombin is detected and plotted against time, resulting in a thrombin generation curve characterized by the following parameters: lag time (tLag, min), time to peak thrombin level (tPeak, min), and peak thrombin level (Peak, nM), after which the concentration of thrombin decreases (Figure 1). The velocity index (VI, nM min⁻¹) is defined as Peak/(tPeak–tLag), the area under the curve depicts the endogenous thrombin potential (ETP, nM) (11). As PPP lacks endothelial cells containing thrombomodulin (TM), we performed a modified test in which recombinant human TM (Sekisui Diagnostics, LLC, Stamford, USA) is added at a concentration of 2 nmol L⁻¹ to activate the protein C pathway and detect both pro- and anticoagulant determinants of hemostasis. In each blood sample ETP was determined using the conventional test assay (without thrombomodulin) and the TM-modified test assay in duplicate.

Parameters of the thrombin generation assay (TGA) (19).

To better understand the pro- and anticoagulant forces in our patient cohort, we related ETP levels determined in the conventional assay (ETP_−TM) to those obtained from the TM-modified assay (ETP_+TM). ETP was then expressed as “ETP ratio” and calculated both for patients (ETP ratio patient) and standardized plasma of healthy donors (ETP ratio standardized plasma) (Technofrozen Control N, REF 5021100, Technoclone, Vienna, Austria). Standardized plasma of healthy donors was obtained from pooled plasma after plasmapheresis and fulfilled the following criteria: PT 75–150%, FVIII 75–150%, fibrinogen 200–450 mg dl⁻¹. Higher ratios of ETP_+TM /ETP_−TM – mirroring a certain resistance to the anticoagulant activity of TM – were interpreted as increased procoagulant imbalance (12).

To enhance the sensitivity and reproducibility of TM-modified measurements, we followed the recommendation of Tripodi (12) and used the following formula to calculate a “normalized ETP ratio,” comparing ETP ratios of patients to those of standardized plasma:

ETP-PT_TM, endogenous thrombin potential determined in the individual patient in the presence of thrombomodulin; ETP-PT_−TM, endogenous thrombin potential determined in the individual patient in the absence of thrombomodulin (= conventional TGA); ETP-NM_TM, endogenous thrombin potential determined from pooled normalized plasma samples in the presence of thrombomodulin; ETP-NM_−TM, endogenous thrombin potential determined from pooled normalized plasma samples in the absence of thrombomodulin (= conventional TGA).