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Thrombin generation (TG) assessed by a calibrated automated thrombogram (CAT) could facilitate the evaluation of the inhibitory capacity of anticoagulant drugs and could be used, if required, for monitoring their effects on coagulation [1,2]. In this context, several antithrombotic agents have recently been studied by our group and others to determine their effect on TG [3,4].

We compared the influence of r-Hirudin, Melagatran, Dabigatran, Argatroban, Fondaparinux and Danaparoid on TG in order to elucidate some information about their mechanism of action.

Increasing concentrations of r-Hirudin (Pharmion Laboratory, Paris, France), Argatroban, Melagatran, Dabigatran (kindly provided by Mitsubishi Pharma Corporation [Tokyo, Japan], Astra-Zeneca [Mölndal, Sweden] and Boehringer Ingelheim [Dortmund, Germany], respectively), Fondaparinux (GlaxoSmithKline, Brentford, UK) and Danaparoid (Organon Laboratory, Puteaux, France) were spiked into a pool of citrated platelet-poor normal human plasma (PPP). The usual CAT parameters [1] were determined with a thrombinoscope version 3.0.029 [5].

Coagulation was triggered with PPP reagent (Biodis/Stago Laboratory, Asnières France; 5 pm tissue factor and 4 μm phospholipids final plasma concentrations).

For each drug, using Microsoft®excel software, we determined the concentrations that double lag time (LT) and time-to-peak (TTP) and those required for a 50% reduction (IC50) of peak and endogenous thrombin potential (ETP). The results are expressed in μmol L−1 and μg mL−1, except for Danaparoid where anti-factor Xa units mL−1 are used (Fig. 1).

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Figure 1.  Calibrated automated thrombograms (CATs) obtained with increasing concentrations of (A) Hirudin, (B) Dabigatran and (C) Danaparoid. The concentrations required for lag time and time-to-peak doubling time and 50% reduction (IC50) of peak and endogenous thrombin potential are indicated. The square brackets indicate the therapeutic ranges of the concentrations used for each anticoagulant.

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Each numerical value is the mean of measurements in triplicate and every experiment has been repeated at least three times with different pools of citrated normal human plasma. The intra- and inter-assay coefficients of variation range from 3% to 23% for these measurements without a significant modification of the patterns of the CATs (results not shown).

The results demonstrate a significantly greater prolongation of LT and TTP by r-Hirudin as compared to the five other anticoagulant agents. Moreover, at therapeutic ranges, r-Hirudin, in contrast to the other anticoagulants, does not reduce peak and ETP. However, at higher concentrations of r-Hirudin over the therapeutic range at about 3 μg mL−1, a moderate decrease of the peak and ETP values is observed. At even higher concentrations, at about 5 μg mL−1, TG is completely suppressed.

The CAT patterns of Melagatran, Dabigatran and Argatroban are different from that of r-Hirudin as the prolongation of the LT is much less pronounced, while an important reduction of peak and ETP is clearly evidenced (Fig. 1). Interestingly, Melagatran and Dabigatran have very similar inhibitory activities, while that of Argatroban is moderately weaker.

Danaparoid and Fondaparinux induce almost no prolongation of LT in contrast to a pronounced reduction of peak and ETP. Interestingly, in the work of Warkentin et al. [6], the concentrations doubling activated partial thromboplastin time with Melagatran, Argatroban and r-Hirudin were similar to those found in this experimental study. Thus, on a molar basis, direct thrombin inhibitors are more active in delaying the initiation of TG than factor (F) Xa indirect inhibitors, which in contrast exert a greater influence on peak and ETP (propagation phase) than on LT.

A paradoxical increase of the peak height was observed at r-Hirudin concentrations from 0 to 0.30 μmol L−1 (Fig. 1). The mechanism of the unexpected result with r-Hirudin could be related to an inaccurate estimation of the mathematical algorithm, which assumes the likely contribution of α2-macroglobulin to ETP. A similar finding with Melagatran has been attributed to an error in the mathematical algorithm to substract end-level α2-macroglobulin–thrombin activity in the presence of small molecules of direct thrombin inhibitor [7]. A similar explanation might be suspected for r-Hirudin although it has not been investigated.

The IC50 for ETP and peak were comparable with Melagatran, Dabigatran, Argatroban and Danaparoid, while they were different with Fondaparinux.

Briefly, the LT and TPP are more responsive to direct thrombin inhibitors Melagatran, Dabigatran and Argatroban than peak and ETP; however, Fondaparinux exhibits an opposite result (Fig. 1).

The results show that ETP is not the most informative parameter with which evaluate the anticoagulant activity of these agents, as it is not significantly decreased by therapeutic doses of Hirudin. The other parameters (LT, TTP, peak) could be more informative than ETP for some anticoagulant drugs.

Thus, three different patterns of CAT have been demonstrated: that of r-Hirudin, that of Melagatran, Dabigatran, and Argatroban, and that of Danaparoid and Fondaparinux (Fig. 1). It should be noted that the results obtained with Melagatran and Dabigatran are very similar; this observation is important as the results of clinical trials with Melagatran have clearly shown its antithrombotic activity.

The patterns of TG inhibition by Danaparoid and Fondaparinux are different from those determined for the four other anticoagulants. This is probably because of the different mechanisms of action. Inhibition of FXa has a greater effect on the rate of the propagation phase of TG whereas inhibition of thrombin affects the chronometric parameters more.

In this work, we show that antithrombotic activity can be associated with different alterations of TG. Three different patterns of CAT have been obtained with six different anticoagulants, which are active in vivo. The results could help to determine the blood concentrations required for effective anticoagulation or, reciprocally, alterations to the thrombogram that are observed at therapeutic concentrations. Moreover, they could be useful for laboratory monitoring of the treatment with these different drugs.

Disclosure of Conflict of Interests

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  2. Disclosure of Conflict of Interests
  3. References

The authors state that they have no conflict of interest.

References

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  2. Disclosure of Conflict of Interests
  3. References
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