Thrombin generation capacity is enhanced by low antithrombin activity and depends on the activity of the related coagulation factors

In this study, the following results were obtained: 1) As AT activities were decreased, the amount of thrombin generation was increased and changed significantly at 10–50% of the AT activities, and it was considered that the threshold was 30%, 2) The type of AT concentrate supplementations did not affect thrombin generation, 3) The time to the peak of thrombin generation was approximately constant regardless of the AT activities, and 4) Total thrombin generation was increased with increasing coagulation factor activities.

The molecular weight of prothrombin is 72 kDa, and its concentration in plasma is 100 µg/mL [22]; thus, its molar concentration is 1.39 mmol/L. Since the molar concentration of AT is 2.57 mmol/L [12], it indicates that the AT molar concentration is approximately two-fold that of prothrombin. The AT forms a complex with thrombin in a 1:1 proportion. When the AT concentration is in excess of that required for thrombin inhibition under physiological conditions, then it is considered that the AT would be able to suppress the thrombin activity quickly due to the higher than sufficient molar concentrations. When the AT activities are decreased to below 50%, the thrombin generation capacity may overcome the suppression activity by the AT because the AT molecule concentrations would be insufficient to inhibit the generated thrombin. In the present results, a significant increase in thrombin generation was observed below 50% of the AT activities, which was consistent with the theoretical values calculated from the molar concentrations. In particular, thrombin generation was increased remarkably when the AT activities were < 30% (Fig. 1), and it was considered that the threshold line of low AT activities was 30%.

An AT activity threshold line was also investigated in several studies, and the decreased levels of AT activities have been shown to be associated significantly with an increased mortality in patients with sepsis, with < 50% of AT activities showing a sharp increase in mortality due to organ failures from thrombotic tendencies [23]. In terms of finding a target area during clinical AT replacement therapy, it has been reported that it was effective in septic DIC in cases where the AT activities were < 43%, instead of 70% [20]. The threshold line established in the clinical studies was similar to the one in our study; it was considered that our study performed in vitro reflected the physiological conditions and the thrombotic tendencies caused by high thrombin generation derived at low AT activities. Moreover, monitoring of the AT activities during AT supplementation has been reported to be useful in predicting patient outcomes [24]. In our study, the results showed that low AT activities resulted in increased thrombin generation, and it was considered that these data reflected the thrombin generation reaction in vivo. Therefore, a thrombin generation assay may find the potential thrombosis risk in low AT activity patients and be useful in predicting the prognosis of patients receiving AT supplementation. Recently, during the coronavirus disease 2019 (COVID-19) pandemic, many studies revealed that the thrombosis tendency was often seen in COVID-19 patients and that the incidence was higher in severe cases [25]. These severely ill patients presented with coagulopathies, and Tang et al. reported that 71.4% of the non-surviving COVID-19 patients fulfilled the criteria of DIC, whereas only 0.6% of the survivors met the criteria [26]. It was also reported that since the AT activities were maintained to approximately 80%, supplementation of the AT was not necessary in most of the cases [27]. However, a low AT activity is one of the risks of thrombosis. To prevent coagulopathy in COVID-19 patients, AT activity measurement is necessary, and a thrombin generation assay might be useful in predicting thrombosis tendencies.

Under conditions in which AT was fixed at 30% (Figs. 5A and 6A), thrombin generation was increased proportionally, with an increase in the concentration of coagulation factors, especially for prothrombin. This is because thrombin production increased in proportion to each prothrombin concentration, since the conditions in which PT was present were more than that of AT (PT% ranged from 33 to 100%). In contrast, under conditions in which AT was fixed at 50% (Figs. 5B and 6B), thrombin production increased only when PT% exceeded 50%. At 33% and 40% PT, thrombin production was low because AT activity sufficiently exceeded prothrombin concentration; when PT% exceeded 50%, thrombin production increased with prothrombin concentration (PT%) because of the dominance of prothrombin. It can be suggested that using global assays, the thrombin generation reflected the balance between the amount of coagulation factors, including prothrombin and AT activities. Although the thrombin generation and cumulative production increased in normal PT%, the values of time to peak were at the same levels in five different PT% activity samples. It means that the time from coagulation cascade activation to thrombin generation was not so different in these samples, and the time to peak values were independent of the activity of coagulation factors. Theoretically, AT inhibits not only thrombin but also other serine proteases, such as activated factors XI, X, and IX. The similar values of the times to the peaks indicated that the inhibition proportion of AT to these serine proteases did not change in the five different PT% activity samples because the times from activation to thrombin generation could be prolonged if AT inhibited these serine proteases in the earlier phase of coagulation cascade. The thrombin generation assay system may evaluate the effects of coagulation factors in addition to AT.

Conventionally, plasma-derived AT concentrates derived from human plasma have been used for the AT concentrates; however, in recent years, recombinant AT concentrates, which suppress the risk of transmission of infectious diseases, have been employed. It has been previously reported that there were no differences shown in the administration of recombinant AT concentrates or plasma-derived AT concentrates in healthy volunteers or DIC patients in clinical studies [28, 29], and the present results also showed no differences between the different types of AT concentrates. Thus, it was considered that the function of the recombinant AT concentrates was the same as that of the plasma-derived concentrates, and the same threshold line could be used for predicting patient outcomes and the monitoring of AT activities.

Limitations

The number of samples used in this study was small. Furthermore, the results may be biased due to the use of only specific plasma samples. We also showed that the threshold established from the thrombin generation assay was similar to that shown in clinical studies. Although it was considered that the assay reflected the physiological conditions, there were many cases with low AT activities. In this study, the in vitro and in vivo data were insufficient to draw comparisons.

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