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Influence of lepirudin, argatroban, and melagatran on prothrombin time and additional effect of oral anticoagulation.

Oral anticoagulation (OAC) with coumarin derivatives decreases the activities of coagulation factors II, VII, IX, and X by inhibiting their vitamin K-dependent carboxylation (1). The effectiveness and safety of therapy with oral anticoagulants for primary or secondary prophylaxis of thromboembolism are usually monitored by the prothrombin time (PT), expressed as international normalized ratio (INR), prothrombin ratio, or percentage of normal (2,3). INR was standardized through large international reference studies (4-7).

The direct thrombin inhibitors (DTIs) lepirudin and argatroban are used to achieve effective anticoagulation in patients with heparin-induced thrombocytopenia with or without thrombosis (type II) (8-10). Melagatran is currently under investigation in clinical trials (11-14). DTIs prolong clotting times in PT assays and therefore interfere with oral anticoagulants (15-18). During treatment of deep venous thrombosis, heparins or DTIs are switched to oral anticoagulants. During treatment for invasive diagnostics or surgery, patients on oral anticoagulant therapy may temporarily be switched to a DTI. Decreased thrombin activity in the plasma of these patients leads to prolongations of the PT (15,16,18). These additive effects make it difficult to adjust dosage of either of the drugs during concomitant use. In the case of heparins, additive effects are antagonized by addition of protamine or heparinase to PT reagents (19). For DTIs, such antagonists are missing. Antibodies against hirudin have been unsuitable for neutralizing the drug's anti-factor IIa effects because of polyclonality, producing neutralizing or enhancing antibodies, depending on the individual (20,21). Argatroban and melagatran are small molecules and have not been reported to be antigenic. Without the ability to eliminate the additive effects of DTIs and oral anticoagulants on PT, it is important to address the issue of handling these interactions in clinical practice and to individually investigate them for each drug in vitro and ex vivo.

Here we describe the additive and synergistic actions of the DTIs lepirudin, argatroban, and melagatran on the effects of the oral anticoagulant phenprocoumon on PT. These synergisms interfere with the analysis and dose adjustment of oral anticoagulants during concomitant therapy periods. Data were derived from plasma from healthy volunteers and from patients treated with the vitamin K antagonist phenprocoumon.

Blood from 6 healthy volunteers and 10 patients undergoing treatment with the vitamin K antagonist phenprocoumon (Hoffmann-La Roche) was collected by clean cubital vein puncture into plastic vials containing 38 mL/L sodium citrate (9 mL of plasma in 1 mL of citrate). All donors (volunteers and patients) gave informed consent in accordance with the current revision of the Helsinki Declaration. After centrifugation (1800g for 10 min), plasma samples were shock frozen in liquid nitrogen, stored at -80 [degrees]C, and analyzed within 4 weeks. After thawing, plasma samples were supplemented with lepirudin (molecular mass ~6500 Da; obtained from Aventis) and argatroban (molecular mass 526.7 Da; kindly provided by Mitsubishi Chemical Corp., Tokyo, Japan) in concentrations ranging from 300 to 3000 [micro]g/L. Melagatran (molecular mass 473.6 Da; courtesy of Astra Zeneca, Melndal, Sweden) was added at lower concentrations, between 30 and 1000 [micro]g/L, because of its higher gravimetric potency observed in preliminary experiments.

Clotting time measurements were carried out in a KC 10a microdevice (22) from Amelung Co. PT was determined with a recombinant thromboplastin reagent (Aventis Behring; lot number 526935; international sensitivity index = 1.09). With this thromboplastin reagent, clotting times can be determined up to ~600 s with this device. Interassay CVs were 10%, 8.7%, and 9.9% at clotting times of 10, 50, and 300 s, respectively (n = 12). To start the clotting time assay, 50 [micro]L of plasma was incubated at 37 [degrees]C for 3 min (micromethod). To initiate clot formation, 100 [micro]L of PT reagent (dissolved according to the manufacturer's instructions) was added. The clotting times (PT) were transformed into INRs with an equation appropriate for the KC 10a device, obtained from the manufacturer of the thromboplastin reagent: PT (in seconds) x 0.09 + 0.2 = INR. The mean value obtained for the healthy volunteers was 10.2 [+ or -] 0.3 s, corresponding to an INR range of 1.1 [+ or -] 0.03.

All data are presented as the mean [+ or -] SD. Calculation factors (ng x factor = nmol/L) for transformation of the data from gravimetric scaling to an equimolar scale were 0.154 for lepirudin, 1.901 for argatroban, and 2.11 for melagatran, respectively. For reverse transformation, the factors were 6.5 for lepirudin, 0.526 for argatroban, and 0.474 for melagatran.

The PT was prolonged in OAC plasma (OACP; samples from patients undergoing a stable phase of OAC therapy) to 26.0 [+ or -] 5.0 s. All DTIs prolonged PT values in a concentration-dependent manner in plasma from the controls and patients receiving OAC therapy. The results are displayed in Fig. 1 in double scaling (nmol/L and [micro]g/L). In Fig. 1, the upper therapeutic gravimetric concentrations (11, 23,24) are compared in equimolar scaling. Lepirudin at 308 nmol/L (2000 [micro]g/L) prolonged PTs to 12.4 [+ or -] 0.5 s and 70.3 [+ or -] 36.8 s in plasma samples from healthy volunteers (normal plasma) and OACP samples. Argatroban at 1901 nmol/L (1000 [micro]g/L) yielded PTs of 25.7 [+ or -] 3.2 s in normal plasma and 94.5 [+ or -] 26.4 s in OACP, respectively. Melagatran at 633 nmol/L (300 [micro]g/L) prolonged PTs to 16.8 [+ or -] 1.0 s in normal plasma and 117.8 [+ or -] 47.2 s in OACP. The INRs can be calculated with the equation given above.

[FIGURE 1 OMITTED]

OAC strongly influenced all concentration-PT relationships. The influence of PT reagents on the PT coagulation assay has been reported (16,25). Here we describe the actions of various DTIs on the prothrombin coagulation assay with the use of a single thromboplastin reagent. The effects of argatroban and melagatran were influenced by OAC throughout the entire concentration range tested. In contrast, the influence of lepirudin on the PT was more strongly affected by OAC at higher concentrations [308 nmol/L (2000 [micro]g/L)]. Up to this concentration, only a slight effect occurred. Lepirudin and hirulogs are directed against the active catalytic site as well as against the anion-binding exosite of thrombin (26,27). Argatroban and melagatran are monovalent inhibitors of the catalytic active site of thrombin (28). The anion-binding exosite is responsible for the recognition of fibrinogen and may play an important role in mediating the different inhibition patterns of lepirudin compared with argatroban or melagatran. Recently, a novel oligopeptidic compound was described that specifically inhibits the anion-binding exosite. In a rat venous thrombosis model, its median effective dose was lower than that of recombinant hirudin or of argatroban (29).

In addition to differences regarding target sites and affinities of DTIs, the data presented here may also indicate some other interactions. These interferences could be mediated by other clotting factors affected by vitamin K antagonism. During OAC, in addition to decreased thrombin activity, concentrations of factors VII, IX, and X are decreased. Changes in the relationships of factor concentrations as a result of vitamin K antagonism could in turn alter feedback mechanisms between different factors of the coagulation cascade. Feedback mechanisms between thrombin and coagulation factors V, VII, and X are described in the literature (30). Finally, certain DTIs lack specificity for factor II and additionally inhibit factor X (31). Such a mechanism could partly explain the lack of uniformity of interactions between OACs and direct thrombin antagonists. Carboxylation of prothrombin may affect the conformation of the anionic binding site. Decreased carboxylation of factor II with OACs could in turn alter the accessibility of the binding site. Furthermore, the anion-binding exosite could be activated by a higher relative inhibitor concentration in OACP samples. Slow-binding inhibitor-receptor interactions can be potentiated by higher relative inhibitor concentrations with OACs (32). The precise mechanisms of the interactions remain, however, to be further investigated. A pharmacokinetic interference seems to be unlikely. OAC with warfarin does not alter the pharmacokinetics of napsagatran, but strongly affects its pharmacodynamics (33).

On the basis of our results and the interpretation described, it seems difficult to predict to what extent PTs or INRs are affected during concomitant therapy. Models described in the literature deal with a single DTI (argatroban or lepirudin) combined with either warfarin or phenprocoumon (15,16,18, 34). When the same DTI is used, results are similar with both vitamin K antagonists because the actions of phenprocoumon and warfarin on clotting times are mediated by comparable lowering of clotting factor concentrations (unpublished results). In contrast, each competitive thrombin inhibitor has its own kinetics at its binding site(s). Upper therapeutic limits are ~308 nmol/L (2000 [micro]g/L) for lepirudin, 633 nmol/L (300 [micro]g/L) for melagatran, and 1901 nmol/L (1000 [micro]g/L) for argatroban. Our results demonstrate considerable differences between the effects of DTIs on PT values. These differences are even more pronounced in plasma from patients during stable OAC therapy.

Considering these differences, it might be difficult to establish a single model for all DTIs to predict PT (INR) prolongations during concomitant application periods. Simultaneous measurements of PT and of the concentrations of the DTIs by specific coagulation testing methods (e.g., ecarin clotting time), chromogenic (S-2238), ELISA, or chromatographic assays may improve judgment about the correct dosing during periods of concomitant therapy with vitamin K antagonists and DTIs. At present, when patients are switched from heparins and low-molecular-weight heparins to OAC, the current American College of Chest Physicians guidelines recommend discontinuation of heparins when the INR is in the therapeutic range (2,3) for 2 consecutive days (35). However, the effects of heparins can be antagonized by adding protamine or heparinase to PT reagents, as mentioned above, although no antagonists are yet available for DTIs. On the basis of the data presented, more accurately detailed dose adjustment regimens may be required for concomitant treatment with vitamin K antagonists and each DTI.

We thank Christina Giese, Antje Hagedorn, and Inge Trdger for excellent laboratory work and patient care. This study was supported by a grant from the Faculty of Clinical Medicine Mannheim, University of Heidelberg.

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Tivadar Fenyvesi, Ingrid Joerg, and Job Harenberg *

Fourth Department of Medicine, University Hospital Mannheim, Ruprecht-Karls-University Heidelberg, Theodor-Kutzer-Ufer 1, 68167 Mannheim, Germany;

* author for correspondence: fax 49-621-383-3308, e-mail j-harenberg@t-online.de
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Title Annotation:Technical Briefs
Author:Fenyvesi, Tivadar; Joerg, Ingrid; Harenberg, Job
Publication:Clinical Chemistry
Date:Oct 1, 2002
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