Caution in the interpretation of continuous thrombin generation assays

During the last several years, an increasing number of studies have been published in which continuous thrombin generation assays are used for the evaluation of processes related to tissue factor(TF-) initiated thrombin generation in citrated plasma [1–3]. In all of these assays, a low-molecular-weight thrombin substrate is present at a concentration usually exceeding that of its Km [4]. Thrombin plays a crucial role in its own generation during the initiation (lag) phase of TF-triggered coagulation. During the initiation phase, picomolar amounts of factor (F) X and FIX are activated and small amounts of thrombin produced, which activates the procofactors V and VIII to their corresponding active forms and FVII and FXI to their corresponding enzymes [5]. The resulting products lead to most thrombin generation during the subsequent propagation phase. An exogenous thrombin substrate present during the initiation phase will compete with critical endogenous thrombin substrates present in plasma. As a consequence, the activation of the natural thrombin substrates will be delayed, the initiation phase prolonged and the onset of the propagation phase delayed. During the propagation phase of thrombin generation, when large amounts of thrombin are produced at a high rate, an exogenous thrombin substrate will interfere with thrombin inhibition by the natural inhibitors present in plasma, leading to elevated values for total thrombin as well as to higher peak thrombin concentrations. Figure 1 represents TF-initiated thrombin generation in a synthetic coagulation proteome in the absence and in the presence of 416 lM synthetic thrombin substrate Z-Gly-GlyArg-aminomethylcoumarine [1]. In a reaction initiated with 5 pM TF in the presence of 4 lM phospholipid, the addition of substrate prolongs the initiation phase from 120 s to 340 s and the time to thrombin peak from 240 to 480 s. The peak thrombin concentration increases from 330 nM in the absence of substrate to 430 nM in its presence and total thrombin generated is doubled (1210 and 2370 nM min, respectively). Similarly, the addition of 416 lM substrate to contact pathwayinhibited nine-donor pooled plasma triggered by 5 pM TF prolongs the clotting time from 269 to 435 s. Thus, the presence of a synthetic substrate in a continuous thrombin generation assay compromises the majority of the events occurring during the TF pathway to thrombin and all parameters measured during this process, i.e. the initiation phase duration (approximately equal to the clotting time), time to peak thrombin and peak thrombin value, and total thrombin produced in the reaction. Of course, the inhibitory effect of a synthetic substrate could be decreased by lowering the concentration of the substrate and/or its affinity for thrombin. However, in the assay, where the key event is substrate hydrolysis by thrombin, an interference of such a substrate with processes leading to TF-initiated thrombin generation is inevitable, especially in experiments designed to evaluate the influence of coagulation factor deficiencies, such as hemophilia A, B and C, on thrombin generation. These experiments have to be carried out at (sub)picomolar TF concentrations [6–8], which leads to aprolonged initiationphase andan increased sensitivity to an exogenous substrate.