Monitoring direct FXa-inhibitors and fondaparinux by Prothrombinase-induced Clotting Time (PiCT): Relation to FXa-activity and influence of assay modifications

Pharmazentrum Frankfurt, Institute for Clinical Pharmacology, University Hospital Frankfurt am Main, Germany.
Thrombosis Research (Impact Factor: 2.45). 07/2008; 123(2):396-403. DOI: 10.1016/j.thromres.2008.05.010
Source: PubMed


FXa-activity can be measured by the Prothrombinase induced Clotting time (PiCT). The manufactured assay uses bovine FXa as component and employs a incubation period before re-calcification. Its use with new direct FXa-inhibitors is challenged by reports on decreased sensitivity.
Blood was incubated with 3 investigational, structurally related (oxazolidinones) direct FXa-inhibitors including the recently approved agent rivaroxaban (0 - 2.0 microM), with the structurally distinct direct FXa-inhibitor DX 9065a (0 - 18 microM) and with the indirect inhibitor fondaparinux (0 - 0.6 microM). We tested modifications of PiCT regarding the source of FXa (bovine or human) and the incubation step (incubation before re-calcification=2-step, no incubation =1-step), and compared results with inhibition of human or bovine FXa-activity.
The bovine 2-step PiCT showed a paradoxical decrease with all direct FXa-inhibitors, this effect is surmounted only at high concentrations and is not seen with the bovine 1-step PiCT. The decrease in PiCT is not observed in antithrombin-depleted plasma. The humanized PiCT (1 or 2 step) showed a consistent prolongation under all direct inhibitors. Fondaparinux prolonged PiCT with either assay. The correlation between PiCT and corresponding FXa-activity was significant both for humanized 2-step PiCT or bovine 1 step PiCT (r2=0.80), but the 95% prediction interval was large and covered a span of 40% FXa-activity between one agent and another.
The customary bovine PiCT should only be used to monitor direct FXa-inhibitors when modified as 1-step procedure. PiCT is not suitable to assess similarity of FXa-inhibition when different agents are interchanged.

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    • "Similarly, the APTT is not a suitable method as it is largely insensitive to these two anticoagulants. The PiCT has been evaluated by a number of groups for measurement of LMWH and fondaparinux activity (Calatzis et al, 2008; Harder et al, 2008). Although PiCT is sufficiently sensitive to LMWH, it can be influenced by other inhibitors and is therefore not specific for LMWH. "
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    • "Rivaroxaban prolongs clotting times concentration-dependently in the activated partial thromboplastin time [24], HepTest (Sekisui Diagnostics, Stamford, CT, USA [27]) [15,20] and prothrombinase-induced clotting time (PiCT) test [24,26,28]. However, for the HepTest and PiCT test, there is a paradoxical shortening of clotting time at low rivaroxaban concentrations when bovine Factor Xa is used [24]. "
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    • "The direct thrombin inhibitors lepirudin, argatroban, melagatran, as well as unfractionated and low-molecular weight heparins and fondaparinux prolong coagulation time dose dependently [31]. The effects of rivaroxaban are measured using a one-step incubation procedure in contrast to two-step incubation procedure for the other coagulation inhibitors [32]. Dabigatran also prolongs coagulation time of PiCT more using the one step-incubation procedure than with the two-step incubation procedure [33]. "
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