G-Quadruplex Aptamers to Human Thrombin Versus Other Direct Thrombin Inhibitors: The Focus on Mechanism of Action and Drug Efficiency as Anticoagulants

Abstract Three direct thrombin inhibitors (DTIs) differ in their ability to increase the International Normalized Ratio (INR) for a given prolongation of the activated partial thromboplastin time (APTT), as follows: argatroban > bivalirudin > lepirudin. This phenomenon is clinically-relevant, as it can complicate the transition to coumarin therapy (a risk factor for venous limb ischemia in patients with heparin-induced thrombocytopenia and deep-vein thrombosis, i.e., a clinical situation in which a DTI may be given). Since this observed order of INR prolongation by DTIs is the reverse of that expected based on their known relative affinities for human thrombin (see Table), we tested whether these DTIs (as well as the small-molecule thrombin inhibitor, D-Phe-Pro-ArgCH2Cl [PPACK]) also inhibit free and/or prothrombinase-bound factor Xa (FXa). Prothrombinase-bound FXa was assembled on rabbit brain phospholipids supplemented with human plasma factor X-depleted plasma, to which human FXa (0.5 nM, final) was added. We found that argatroban inhibited free human FXa, i.e., FXa in the fluid-phase (IC50 = 5 μM), whereas it inhibited bovine FXa much less effectively (IC50 = 110 μM). In contrast, only supratherapeutic concentrations of bivalirudin inhibited human and bovine FXa, whereas lepirudin had no effect (see Table). Neither therapeutic nor supratherapeutic concentrations of the three DTIs inhibited prothrombinase-bound FXa. Comparison of DTIs on INR, APTT, IIa, and Inhibition of Human and Bovine Fxa PPACK Argatroban Bivalirudin Lepirudin * published values Effect on INR N.D. Greatest Intermediate Least [DTI] doubling APTT N.D. 1 μM 0.25 μM 0.06 μM Ki for human thrombin* ~40 nM ~2 nM ~0.0001 nM IC50 human FXa 3 μM 5 μM 1,700 μM >10,000 μM IC50 bovine FXa 5 μM 110 μM 2,900 μM >10,000 μM CONCLUSION: Compared with the other two DTIs, argatroban effectively inhibits human FXa at concentrations (IC50 = 5 μM) within the order of magnitude of the usual therapeutic drug levels (about 1 to 2 μM). Inhibition of free FXa is a plausible reason why argatroban prolongs the INR more effectively than the other two DTIs.

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The Effect of Irreversible and Reversible Direct Thrombin Inhibitors and Danaparoid on Tissue Factor Mediated Thrombin Generation.

Blood ◽

10.1182/blood.v108.11.4111.4111 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4111-4111

Author(s):

Meyer M. Samama ◽

Lena Le Flem ◽

Daphne Pierre-Eugène ◽

Francois Depasse

Keyword(s):

Mechanism Of Action ◽

Thrombin Generation ◽

In Vitro Study ◽

Thrombin Inhibitors ◽

Direct Thrombin Inhibitors ◽

Blood Concentrations ◽

Antithrombotic Activity ◽

Time To Peak

Abstract Background Recently, direct thrombin inhibitors such as Hirudin, Bivalirudin, Melagatran and Argatroban have been introduced in the armamentarium of an antithrombotic drugs. Danaparoid, an anti-Xa - anti IIa agent is also used especially in Heparin induced thrombocytopenia. Objective Thrombin generation test (TGT) has been recently automated and could be useful for the evaluation of the inhibitory activity of these drugs and the measurement of their anticoagulant effects at established therapeutic concentrations. Moreover, TGT can elucidate some information about their mechanism of action. The aim was to compare Hirudin, Argatroban, Melagatran and Danaparoid while Bivalirudin was not available. Methods and results Pools of normal human plasma were spiked with increasing concentrations of the four studied drugs and TGT was determined by studying the lag time (LT) and time to peak TTP as well as the peak (P), the endogenous thrombin potential (ETP) and the mean rate of thrombin generation (velocity TG) and its inhibition once a maximal concentration has been reached (velocity TI). The mechanism of action on TGT was heterogeneous. At therapeutic concentrations, Hirudin delays dramatically thrombin generation without affecting significantly its velocity and the amount of thrombin formed. Argatroban and Melagatran induce a far less marked prolongation of LT and TTP than hirudin but they both reduce the velocity of the reaction and the amount of thrombin generated in this in vitro study. Melagatran was found more active than Argatroban but the patterns of the thrombograms were similar. Danaparoid exerts a minimal effect on LT and TTP which is associated with a very significant influence on the parameters related to the reaction of thrombin formation (velocity index, peak and ETP). In total ETP generally considered as the most informative parameter of the thrombogram was not found as relevant as predicted. Schematically, 3 different patterns for the thrombogram have been observed indicating different mechanisms of anticoagulation, all of them, clearly associated with an antithrombotic activity in vivo. Conclusion The mechanism of action of hirudin on thrombin generation is clearly different from that of Argatroban, Melagatran and Danaparoid. This work demonstrates that antithrombotic activity is associated with different alterations of TGT. The results could help to determine the blood concentrations required for an effective anticoagulation or reciprocally the alterations of the thrombogram which are associated with therapeutic efficacy. Moreover they could be useful when laboratory monitoring of the treatment with these different drugs is considered.

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The uncalibrated prothrombinase-induced clotting time test

Hämostaseologie ◽

10.1055/s-0037-1619058 ◽

2010 ◽

Vol 30 (04) ◽

pp. 212-216 ◽

Cited By ~ 9

Author(s):

R. Jovic ◽

M. Hollenstein ◽

P. Degiacomi ◽

M. Gautschi ◽

A. Ferrández ◽

...

Keyword(s):

Factor Xa ◽

Heparin Therapy ◽

Thrombin Inhibitors ◽

Coagulation Cascade ◽

Diagnostic Tools ◽

Direct Thrombin Inhibitors ◽

Functional Assay ◽

Clotting Time ◽

Coagulation Time ◽

Time Test

SummaryThe activated partial thromboplastin time test (aPTT) represents one of the most commonly used diagnostic tools in order to monitor patients undergoing heparin therapy. Expression of aPTT coagulation time in seconds represents common practice in order to evaluate the integrity of the coagulation cascade. The prolongation of the aPTT thus can indicate whether or not the heparin level is likely to be within therapeutic range. Unfortunately aPTT results are highly variable depending on patient properties, manufacturer, different reagents and instruments among others but most importantly aPTT’s dose response curve to heparin often lacks linearity. Furthermore, aPTT assays are insensitive to drugs such as, for example, low molecular weight heparin (LMWH) and direct factor Xa (FXa) inhibitors among others. On the other hand, the protrombinase-induced clotting time assay (PiCT®) has been show to be a reliable functional assay sensitive to all heparinoids as well as direct thrombin inhibitors (DTIs). So far, the commercially available PiCT assay (Pefakit®-PiCT®, DSM Nutritional Products Ltd. Branch Pentapharm, Basel, Switzerland) is designed to express results in terms of units with the help of specific calibrators, while aPTT results are most commonly expressed as coagulation time in seconds. In this report, we describe the results of a pilot study indicating that the Pefakit PiCT UC assay is superior to the aPTT for the efficient monitoring of patients undergoing UFH therapy; it is also suitable to determine and quantitate the effect of LMWH therapy. This indicates a distinct benefit when using this new approach over the use of aPPT for heparin monitoring.

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