Paradoxical enhancement of the intrinsic pathway-induced thrombin generation in human plasma by melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, or heparin

2015 ◽  
Vol 136 (3) ◽  
pp. 658-662 ◽  
Author(s):  
Taketoshi Furugohri ◽  
Yoshiyuki Morishima
Keyword(s):  
Human Plasma ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Direct Thrombin Inhibitor ◽  
Thrombin Inhibitor ◽  
Factor Xa Inhibitor ◽  
Direct Factor ◽  
Intrinsic Pathway

Effects of Direct Factor Xa Inhibitor, YM150, on Clot Formation and Clot Lysis In Vitro Compared with Other Anticoagulants.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3153-3153 ◽  
Author(s):  
Seiji Kaku ◽  
Ken-ichi Suzuki ◽  
Toshiyuki Funatsu ◽  
Minori Saitoh ◽  
Hiroyuki Koshio ◽  
...  
Keyword(s):  
Human Plasma ◽  
Statistical Significance ◽  
Factor Xa ◽  
Clot Formation ◽  
Thrombin Inhibitor ◽  
Clot Lysis ◽  
Factor Xa Inhibitor ◽  
Dependent Manner ◽  
Direct Factor ◽  
Clotting Time

Abstract The objective of this study was to evaluate the effects of direct factor Xa inhibitor, YM150 and its major in vivo metabolite, YM-222714, on clot formation and clot lysis compared with other anticoagulants, such as a direct thrombin inhibitor (melagatran), a pentasaccharide (fondaparinux), low molecular weight heparin (enoxaparin) and unfractionated heparin. To assess clot lysis, the tissue plasminogen activator (tPA)-induced clot lysis assay was used with human plasma triggered by low and high levels of tissue factor (TF). Under low TF conditions, clot formation was completely prevented by melagatran at 1 μmol/L, by fondaparinux at all concentrations examined (0.1 to 1 μg/mL), by enoxaparin at 0.3 and 1 IU/mL and by heparin at 0.1 and 0.3 U/mL. Even under high TF conditions, 0.3 U/mL heparin prevented any clot formation. Although melagatran, fondaparinux, enoxaparin, and heparin potently prevented plasma clot formation under low TF conditions, under high TF conditions they were less effective at prolonging the clotting time. Under both low and high TF conditions, YM150 and YM-222714 prolonged the clotting time in a concentration dependent manner at concentrations between 0.3 and 3 μmol/L. YM150 and YM-222714 significantly accelerated clot lysis under both low and high TF conditions, but their effects were most evident under high TF conditions. Lower concentrations of melagatran (0.1 and 0.3 μmol/L) enhanced clot lysis under low TF conditions, but under high TF conditions, enhancement of clot lysis required higher melagatran concentrations (0.3 μmol/L or more). Under high TF conditions, fondaparinux enhanced clot lysis only at the highest concentration tested (1 μg/mL). Enoxaparin and heparin enhanced clot lysis under low TF conditions at the lowest test concentrations (0.1 IU/mL and 0.03 U/mL, respectively). Both also enhanced clot lysis under high TF conditions, but their effect reached statistical significance only at higher concentrations (1 IU/mL and 0.1 U/mL, respectively). These results suggested that direct factor Xa inhibitors, YM150 and YM-222714, exert stable anticoagulant effects independently of TF concentration. Both inhibitors enhanced tPA-induced fibrinolysis in human plasma clotted via the extrinsic coagulation pathway. Useful characteristics of YM150 and YM-222714, such as a linear dose response and reliable anticoagulation independent of TF concentration, may lead to the creation of an anticoagulant that is easier to use in the clinical setting than existing products. Potentially beneficial antithrombotic effects, which can be promoted by accelerating endogenous fibrinolytic pathways, may further aid in the prevention or treatment of thrombosis.

Antithrombin-independent thrombin inhibitors, but not direct factor Xa inhibitors, enhance thrombin generation in plasma through inhibition of thrombin-thrombomodulin-protein C system

2011 ◽  
Vol 106 (12) ◽  
pp. 1076-1083 ◽  
Author(s):  
Nobutoshi Sugiyama ◽  
Yoshiyuki Morishima ◽  
Toshiro Shibano ◽  
Taketoshi Furugohri
Keyword(s):  
Human Plasma ◽  
Negative Feedback ◽  
Protein C ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Direct Thrombin Inhibitors ◽  
Direct Factor

SummaryThere is increasing concern that some anticoagulants can paradoxically increase thrombogenesis under certain circumstances. Previously, we demonstrated that at certain doses a direct thrombin inhibitor, melag-atran, worsens the coagulation status induced by tissue factor (TF) in-jection in a rat model. We utilised an in vitro thrombin generation (TG) assay to determine if direct thrombin inhibitors could enhance TG in human plasma, and whether inhibition of the negative-feedback sys-tem [thrombin-thrombomodulin (TM)-protein C] contributed to the TG enhancement. TG in human plasma was assayed by means of the cali-brated automated thrombography. In this assay, direct factor Xa (FXa) inhibitors such as edoxaban and antithrombin (AT)-dependent anti-coagulants such as heparin did not increase, but simply suppressed TG. AT-independent thrombin inhibitors (melagatran, lepirudin, and active site blocked thrombin (IIai)) increased peak levels of TG (2.0, 1.6, and 2.2-fold, respectively) in the presence of 12 nM recombinant human soluble TM (rhsTM). Melagatran and lepirudin at higher concentrations began to suppress TG. In the absence of rhsTM, the enhancement of peak TG by melagatran decreased to 1.2-fold. Furthermore, in protein C-deficient plasma, AT-independent thrombin inhibitors failed to enhance TG. In addition, a human protein C neutralising antibody increased the peak height of TG in the presence of rhsTM. These results suggest that AT-independent thrombin inhibitors may activate throm-bogenesis by suppression of the thrombin-induced negative-feedback system through inhibition of protein C activation. In contrast, direct FXa inhibitors are more useful than AT-independent thrombin inhibitors in terms of lower possibility of activation of the coagulation pathway.

Comparison of the Antithrombotic Effectiveness of the Direct Thrombin Inhibitor, Dabigatran Etexilate, and Two Factor Xa Inhibitors in an A-V Shunt Model of Thrombosis in Rabbits.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3154-3154 ◽  
Author(s):  
Joanne van Ryn ◽  
Norbert Hauel ◽  
Henning Priepke ◽  
Kai Gerlach ◽  
Annette Schuler-Metz ◽  
...  
Keyword(s):  
Dabigatran Etexilate ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Direct Thrombin Inhibitor ◽  
Factor Xa Inhibitors ◽  
Thrombin Inhibitor ◽  
Factor Xa Inhibitor ◽  
Antithrombotic Activity ◽  
Antithrombotic Efficacy ◽  
Dose Dependent

Abstract Inhibition of two key serine proteases in the coagulation cascade, thrombin (IIa) and factor Xa, are currently being exploited for direct, oral antithrombotic activity in the clinic. However, it is still unclear if one form of coagulation factor inhibition is more effective than the other. Thus, the objective of this study was to test the antithrombotic efficacy of the clinically advanced compounds, the potent direct thrombin inhibitor, dabigatran etexilate and rivaroxaban, a potent direct factor Xa inhibitor in the rabbit A-V shunt model of thrombosis. In addition, another internally developed factor Xa inhibitor, BI42551, with properties similar to those in clinical development was tested. All three compounds have affinities (Ki) for their respective coagulation factor in the low nM range, i.e. human thrombin with dabigatran or human factor Xa with rivaroxaban or BI42551. In addition, each is at least >700-fold selective for its human coagulation factor, dabigatran etexilate for IIa vs Xa and the factor Xa inhibitors for Xa vs IIa. These compounds are highly selective inhibitors not only of the human enzyme, but also have similar values for rabbit thrombin and Xa, respectively. All experiments were performed according to German animal ethics guidelines. The femoral artery and vein of anesthetised rabbits were connected with polyethylene tubing containing a fixed length of suture, pre-soaked in tissue factor. Blood flow through the shunt was maintained over 40 min, after which the suture with any thrombus was removed from the shunt and weighed. The prodrug dabigatran etexilate and the factor Xa inhibitors were given in doses of 3 and 10 mg/kg orally and the rabbits were anesthetised either 90 min or at the highest dose, also 6.5 hrs after drug administration. There was a dose-dependent reduction of thrombus formation with all three compounds as compared to control. Antithrombotic efficacy at 3 and 10 mg/kg is shown as % inhibition of control measured 2 hrs after drug administration (table, columns 2&3). These effects were long-lasting, as significant antithrombotic activity was also measured 7 hrs post administration (last column). Plasma levels of all compounds were dose-dependent and clotting tests correlated well with dose. 3 mg/kg–2 hrs 10 mg/kg–2 hrs 10 mg/kg–7 hrs Dabigatran etexilate 61.7 ± 8.7 82.1 ± 5.5 59.5 ± 17.6 Rivaroxaban 43.2 ± 7.7 64.5 ± 8.1 41.0 ± 8.4 BI42551 31.1 ± 10.7 70.3 ± 3.3 39.9 ± 14.7 These results show that both thrombin and factor Xa inhibition are effective methods of inhibiting thrombosis in a rabbit AV shunt model. All drugs had potent and long-lasting effects after a single oral administration in this model, though dabigatran showed a trend to elevated antithrombotic efficacy at both 2 and 7 hrs. However, in the clinical setting differences in antithrombotic treatment may also be related to differences in pharmacokinetic profiles, drug interactions or metabolism, or the individual side effect profiles of each compound.

Antithrombin-Independent Thrombin Inhibitors, but Not Factor Xa Inhibitors, Enhance Thrombin Generation in Human Plasma Via Inhibition of Thrombin-Thrombomodulin-Protein C System.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 914-914 ◽  
Author(s):  
Yoshiyuki Morishima ◽  
Taketoshi Furugohri ◽  
Yoko Shiozaki ◽  
Nobutoshi Sugiyama ◽  
Toshiro Shibano
Keyword(s):  
Human Plasma ◽  
Tissue Factor ◽  
Negative Feedback ◽  
Protein C ◽  
Thrombin Generation ◽  
Feedback System ◽  
Direct Thrombin Inhibitor ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor

Abstract Rebound like recurrent thrombotic events are concerns about anticoagulant therapies. Withdrawal of heparins and a direct thrombin inhibitor is reported to be associated with evidence of rebound coagulation phenomenon in patients with coronary artery diseases (Ref 1). Previously we have shown that low-dose administration of a direct thrombin inhibitor, melagatran, enhances coagulation induced by tissue factor (TF) in rats (Ref 2). Objectives: To determine whether anticoagulants enhance thrombin generation in human plasma, and whether the negative-feedback system [thrombin-thrombomodulin (TM)-protein C] contributes to the enhancement. Methods: Thrombin generation in pooled human plasma was assayed by means of the calibrated automated thrombography (CAT) with the thrombinoscope software in vitro. Thrombin generation was induced by 2.5 pM tissue factor (TF) and 4 μM phospholipids. The effects of following anticoagulants were assessed: antithrombin (AT)-independent thrombin inhibitors [melagatran, recombinant hirudin (lepirudin), and active site blocked thrombin (IIai)], AT-dependent anticoagulants (heparin, dalteparin, and fondaparinux), and AT-independent FXa inhibitors (DU-176b and DX-9065a). Results: Melagatran, lepirudin, and IIai increased peak levels of thrombin generation in the presence of 8 nM recombinant human soluble TM. The effects reached maximal at 200 nM of melagatran (2.3-fold), 8.95 nM of lepirudin (1.6-fold), and 405 nM of IIai (2.2-fold). At higher concentrations, melagatran and lepirudin turned to suppress thrombin generation. Heparin, dalteparin, fondaparinux, DU-176b, and DX-9065a did not enhance thrombin generation, just exerted inhibitory effects. In the absence of TM, the enhancement by melagatran of peak thrombin generation was only 1.2-fold, suggesting the significant role of the negative-feedback system in this aggravation of thrombin generation. Since thrombin acts both the pro- and anti-coagulant, the inhibition of the negative-feedback system by these thrombin inhibitors may cause enhancement of thrombin generation. To test this hypothesis, we examined thrombin generation in protein C-deficient plasma. AT-independent thrombin inhibitors failed to enhance thrombin generation in protein C-deficient plasma. Conclusions: These results indicate that AT-independent thrombin inhibitors at low concentrations enhance thrombin generation probably due to suppression of the negative feedback system by inhibiting protein C activation. This in vitro aggravation of thrombin generation may be a possible explanation of hypercoagulation by melagatran in a rat model of TF-induced intravascular coagulation. Furthermore this phenomenon would contribute to clinical rebound like recurrent thrombotic events associated with anticoagulant therapies with these inhibitors. In contrast, AT-independent FXa inhibitors like DU-176b are less prone to induce the rebound because of lack of increase in thrombin generation.

Effects of the Oral, Direct Factor Xa Inhibitor Rivaroxaban on Platelet-Induced Thrombin Generation and Prothrombinase Activity1

2007 ◽  
Vol 47 (11) ◽  
pp. 1398-1407 ◽  
Author(s):  
Jochen Graff ◽  
Nils von Hentig ◽  
Frank Misselwitz ◽  
Dagmar Kubitza ◽  
Michael Becka ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Factor Xa ◽  
Factor Xa Inhibitor ◽  
Direct Factor

Effect of Rivaroxaban, An Oral Direct Factor Xa Inhibitor, On Whole Blood Clot Permeation and Thrombolysis: Critical Role of Red Blood Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1064-1064 ◽  
Author(s):  
Remi Varin ◽  
Shahsultan Mirshahi ◽  
Pehzman Mirshahi ◽  
Jean Chidiac ◽  
Gerald Kierzek ◽  
...  
Keyword(s):  
Blood Cells ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Permeation Rate ◽  
Factor Xa Inhibitor ◽  
Direct Factor ◽  
Fibrinolytic Enzymes ◽  
Fibrin Network ◽  
Lower Porosity ◽  
Clot Structure

Abstract Abstract 1064 Poster Board I-86 Introduction: Decreased fibrinolysis has been reported in venous thrombosis. Thrombus degradation depends on its structure: thicker fibrin fibers are permeable to blood flow and highly susceptible to fibrinolytic enzymes, while thinner fibers are poorly permeable to flow and are resistant to fibrinolysis. Thrombin concentration present at the time of gelation profoundly influences fibrin clot structure: decrease in thrombin generation leads to the formation of thick fibrin fibers and to a decrease in activation of thrombin-activated fibrinolysis inhibitor (TAFI). Rivaroxaban, an oral direct factor Xa inhibitor, is in late stage clinical development for the prevention and treatment of venous and arterial thrombosis. The objective of this study was to evaluate the effect of Rivaroxaban on whole blood (WB) clot structure and degradability by t-PA. Compared to plasma clots, WB clots might better represent the in vivo formed thrombi. Methods: 1- Clots were formed by adding to WB or to corresponding plasma, low concentration of tissue factor and calcium in the presence or absence of Rivaroxaban at therapeutic concentrations (0.15 and 0.25 μg/ml). 2- Clot permeability was calculated by measuring the flow rate of liquid through the clot. It was expressed as Darcy constant. 3- Clot degradability was evaluated by D dimers generation during clot perfusion with plasminogen and tissue-type plasminogen activator (t-PA). Results: 1- In the absence of Rivaroxaban, WB clots had a lower porosity than that of corresponding plasma clots: Darcy constant of WB clots was 3.1 –fold lower than that of plasma clots. This decreased porosity of WB clots leads to thrombolysis resistance by preventing access of fibrinolytic enzymes to fibrin network: D dimers generation in t-PA-perfused clots for 60 min was 38 -fold lower in WB clots compared to plasma clots. 2- Rivaroxaban increased the permeation rate of WB clots and thrombolysis by t-PA: the addition of Rivaroxaban at 0.15 μg/ml in WB (corresponding in fact to plasma concentration of 0.25 μg/ml), increased the Darcy constant by 5.5 –fold and the clot degradability in 60 min by 108 -fold. These effects of Rivaroxaban were higher in WB clots than in corresponding plasma clots, as Rivaroxaban at 0.25 μg/ml in plasma clots increased the Darcy constant by 2.5-fold and clot degradation by 9.6-fold. In the presence of Rivaroxaban, the Darcy constant and the degradability of WB clots and of plasma clots were nearly identical. 3- To explain the greater efficacy of Rivaroxaban on WB permeation constants and thrombolysis in comparison to plasma clots a) we tested the possibility for Rivaroxaban to reduce the entrapment of red blood cells (RBC) into the network of fibrin as RBC can be responsible for fibrin pore occlusion. This possibility was excluded since Rivaroxaban had no effect on clot permeation rate in clots formed by clotting purified fibrinogen with thrombin in the presence or in the absence of RBC (condition in which there is no generation of thrombin): RBC induced a 2.5 times decrease in permeation rate due to entrapment of RBC into fibrin network, regardless of presence or absence of Rivaroxaban. b) we analyzed the effect of RBC on thrombin generation and its modification by Rivaroxaban: the addition of 0.1 ml RBC diluted ½ to 0,2 ml plasma increased the thrombin generation (540 % of control without RBC). This is probably due to exposure of phosphatidyl serine at surface of RBC during thrombin generation. The increase in thrombin generation by RBC was reduced to 140 % in presence of Rivaroxaban at 0.15 μg/ml. This is explained by Rivaroxaban's inhibition of factor Xa bound to cells. Conclusion: Thrombin generation was greater in WB than in plasma, leading to a lower porosity and degradability of WB clots as compared to plasma clots. Rivaroxaban, by decreasing thrombin generation, increased clot permeability and degradability to the same level in WB clots and plasma clots. This property of Rivaroxaban may contribute to its antithrombotic effect. This study received a support from Bayer-Schering-Pharma France. Disclosures: No relevant conflicts of interest to declare.

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