A direct thrombin inhibitor suppresses protein C activation and factor Va degradation in human plasma: Possible mechanisms of paradoxical enhancement of thrombin generation

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.

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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

Thrombosis Research ◽

10.1016/j.thromres.2015.06.034 ◽

2015 ◽

Vol 136 (3) ◽

pp. 658-662 ◽

Cited By ~ 6

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

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The inhibitory effect of melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, compared with enoxaparin and r-hirudin on ex vivo thrombin generation in human plasma

Thrombosis Research ◽

10.1016/j.thromres.2004.02.009 ◽

2004 ◽

Vol 113 (1) ◽

pp. 85-91 ◽

Cited By ~ 14

Author(s):

Stig L Boström ◽

Göran F.H Hansson ◽

Troy C Sarich ◽

Michael Wolzt

Keyword(s):

Human Plasma ◽

Thrombin Generation ◽

Ex Vivo ◽

Active Form ◽

Inhibitory Effect ◽

Direct Thrombin Inhibitor ◽

Thrombin Inhibitor ◽

Oral Direct Thrombin Inhibitor

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

Thrombosis and Haemostasis ◽

10.1160/th11-06-0382 ◽

2011 ◽

Vol 106 (12) ◽

pp. 1076-1083 ◽

Cited By ~ 34

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.

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Phospholipid-bound Tissue Factor Modulates both Thrombin Generation and APC-mediated Factor Va Inactivation

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614898 ◽

1999 ◽

Vol 82 (12) ◽

pp. 1673-1679 ◽

Cited By ~ 12

Author(s):

Katalin Váradi ◽

Jürgen Siekmann ◽

Peter Turecek ◽

H. Peter Schwarz ◽

Victor Marder

Keyword(s):

Tissue Factor ◽

Surface Density ◽

Protein C ◽

Thrombin Generation ◽

Feedback Inhibition ◽

Activated Protein C ◽

Reaction System ◽

Inverse Correlation ◽

High Concentration ◽

Factor Va

SummaryHemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.

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C6PS Regulates the Inactivation of Factor Va by Activated Protein C.

Blood ◽

10.1182/blood.v106.11.1023.1023 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1023-1023 ◽

Cited By ~ 1

Author(s):

Rinku Majumder ◽

JinMing Wang ◽

Barry R. Lentz

Keyword(s):

Fluorescence Anisotropy ◽

Binding Sites ◽

Protein C ◽

Thrombin Generation ◽

Activated Protein C ◽

Water Soluble ◽

Amidolytic Activity ◽

Factor Va ◽

Partial Inactivation ◽

Cofactor Activity

Abstract Protein C is a vitamin K-dependent, anti-thrombotic protein that is proteolytically cleaved by thrombin to produce the active serine protease, activated protein C (APC). APC inactivates co-factors Va and VIIIa, leading to down-regulation of thrombin generation. Factor Va requires phosphatidylserine (PS) for full cofactor activity. APC inactivates bovine factor Va by catalyzing cleavage in its heavy-chain at Arg505, Arg 662 and Arg306. The cleavage at Arg 306 is stimulated by PS-containing membranes. In this paper, we use water-soluble 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS) to ask whether a membrane or molecular PS regulates inactivation of factor Va by APC. Synthetic substrate titration suggested that APC has two Ca2+-requiring binding sites for C6PS: one site increased the APC amidolytic activity (Kdeff ~ 1.3 μM) while the other site decreased it (Kdeff ~ 2 μM) in the presence of 2 mM Ca2+. The effect of C6PS on APC amidolytic activity was PS-specific, with C6PE having no effect. However, titration of both intrinsic fluorescence and DEGR ([5-(dimethylamino)-l-naphthalenesulfonyl] glutamylycylarginyl chloromethyl )-labeled APC fluorescence showed only one Ca2+-requiring C6PS binding site (Kdeff ~ 0.8 μM). The fluorescence anisotropy of DEGR-APC in the presence of 200 μM C6PS revealed C6PS-dependent 1:1 binding to both factor Va isoforms (Va1or Va2) with Kdeff of 1.13x10-9 M (Va1) and 0.3x10-9 M (Va2). The inactivation of factors Va1/Va2 by APC was also promoted by C6PS. In the presence of 200 μM C6PS to saturate sites on both APC and factor Va, both factors Va1/Va2 were fully inactivated by APC (indicating cleavage at Arg 306). However, in the absence of C6PS or presence of only 4 μM C6PS (sufficient to saturate sites only on APC), only partial inactivation (48–52%) of factor Va1/Va2 was observed. These results suggest that PS binding to APC may have some effect on cleavage at Arg505 or Arg 662 but that PS binding to factor Va was needed to promote cleavage at Arg 306. Supported by USPHS grant HL072827 to BRL.

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Anticoagulant effects of statins and their clinical implications

Thrombosis and Haemostasis ◽

10.1160/th13-08-0720 ◽

2014 ◽

Vol 111 (03) ◽

pp. 392-400 ◽

Cited By ~ 59

Author(s):

Kathleen Brummel-Ziedins ◽

Kenneth Mann ◽

Anetta Undas

Keyword(s):

Factor Xiii ◽

Observational Studies ◽

Protein C ◽

Thrombin Generation ◽

Clinical Implications ◽

Factor V ◽

Reductase Inhibitors ◽

Factor Va ◽

Factor Expression ◽

Antithrombotic Effects

SummaryThere is evidence indicating that statins (3-hydroxy-methylglutaryl coenzyme A reductase inhibitors) may produce several cholesterol-independent antithrombotic effects. In this review, we provide an update on the current understanding of the interactions between statins and blood coagulation and their potential relevance to the prevention of venous thromboembolism (VTE). Anticoagulant properties of statins reported in experimental and clinical studies involve decreased tissue factor expression resulting in reduced thrombin generation and attenuation of pro-coagulant reactions catalysed by thrombin, such as fibrinogen cleavage, factor V and factor XIII activation, as well as enhanced endothelial thrombomodulin expression, resulting in increased protein C activation and factor Va inactivation. Observational studies and one randomized trial have shown reduced VTE risk in subjects receiving statins, although their findings still generate much controversy and suggest that the most potent statin rosuvastatin exerts the largest effect.

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UPLC MS/MS assay for routine quantification of dabigatran – A direct thrombin inhibitor – In human plasma

Journal of Pharmaceutical and Biomedical Analysis ◽

10.1016/j.jpba.2011.09.018 ◽

2012 ◽

Vol 58 ◽

pp. 152-156 ◽

Cited By ~ 43

Author(s):

Xavier Delavenne ◽

Julie Moracchini ◽

Silvy Laporte ◽

Patrick Mismetti ◽

Thierry Basset

Keyword(s):

Human Plasma ◽

Direct Thrombin Inhibitor ◽

Thrombin Inhibitor

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Inhibition of Thrombin Generation by the Oral Direct Thrombin Inhibitor Ximelagatran in Shed Blood from Healthy Male Subjects

Thrombosis and Haemostasis ◽

10.1055/s-0037-1612989 ◽

2002 ◽

Vol 87 (02) ◽

pp. 300-305 ◽

Cited By ~ 96

Author(s):

Ulf Eriksson ◽

Christer Mattsson ◽

Michael Wolzt ◽

Lars Frison ◽

Gunnar Fager ◽

...

Keyword(s):

Thrombin Generation ◽

Active Form ◽

Direct Thrombin Inhibitor ◽

Thrombin Inhibitor ◽

Healthy Male ◽

Oral Direct Thrombin Inhibitor ◽

Shed Blood ◽

Dose Levels ◽

Male Subjects ◽

Control Water

SummaryXimelagatran, an oral direct thrombin inhibitor, whose active form is melagatran, was studied using a model of thrombin generation in humans. Healthy male volunteers (18 per group) received ximelagatran (60 mg p.o.), dalteparin (120 IU/kg s.c.) or a control (water p.o.). Shed blood, collected after incision of the forearm with standardised bleeding time devices at pre-dose, and at 2, 4 and 10 h post-dosing, was analysed for markers of thrombin generation. Statistically significant reductions (p < 0.05) in levels of prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complex (TAT) in shed blood were detected at 2 and 4 h post-dosing in both the ximelagatran and dalteparin groups. Shed blood F1+2 and TAT levels had returned to pre-dose levels at 10 h post-dosing. Using a shed blood model, we demonstrate that the reversible thrombin inhibitor melagatran and, therefore, oral administration of ximelagatran, inhibits thrombin generation in humans after acute activation of coagulation.

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