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.

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.

Additive effects of anticoagulants: recombinant human activated protein C and heparin or melagatran, in tissue factor-activated umbilical-cord plasma

2005 ◽  
Vol 94 (07) ◽  
pp. 69-74 ◽  
Author(s):  
Siegfried Gallistl ◽  
Wolfgang Muntean ◽  
Bettina Leschnik ◽  
Peter Fritsch ◽  
Gerhard Cvirn ◽  
...  
Keyword(s):  
Severe Sepsis ◽  
Tissue Factor ◽  
Umbilical Cord ◽  
Protein C ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Thrombin Inhibitor ◽  
Human Umbilical Cord ◽  
Cord Plasma

SummarySevere sepsis in children or adults may cause a life-threatening coagulopathy, with widespread consumption of activated protein C (APC); recombinant human APC (rhAPC) is a promising candidate anticoagulant treatment. We investigated the effects of rhAPC and other anticoagulants on coagulation triggered by adding small quantities of lipidated tissue factor to human umbilical-cord plasma in vitro. rhAPC, unfractionated heparin (UH),and melagatran (a direct thrombin inhibitor) were studied individually, and in combinations of rhAPC with either UH or melagatran. rhAPC alone dose-dependently prolonged the activated partial-thromboplastin time (aPTT) but not the prothrombin time (PT), and dose-dependently suppressed two indices of thrombin generation, namely prothrombin fragment F 1.2 (F 1.2) generation and thrombin–antithrombin (TAT) complex formation. UH alone dose-dependently prolonged the aPTT but not the PT, while melagatran alone dose-dependently prolonged both the aPTT and the PT. Adding either UH or melagatran dose-dependently augmented the capacity of rhAPC to suppress F 1.2 generation (with addition of UH showing a greater effect) and TAT formation (with addition of melagatran showing a greater effect). Both the capacity of UH to prolong the aPTT and the capacity of melagatran to prolong the aPTT and the PT were augmented by adding rhAPC. In our in-vitro study, adding either UH or melagatran augmented the capacity of rhAPC to suppress thrombin generation in human umbilical-cord plasma, with the anticoagulant effect of melagatran being more predictable than that of UH. Hence, combining rhAPC with melagatran might be a valuable therapeutic option in patients with severe sepsis.

Reversal of Novel Oral Anticoagulant (NOAC)-Induced Bleeding in Mice By Engineered superfactor Va

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 695-695 ◽  
Author(s):  
Vikas Bhat ◽  
Andrew J. Gale ◽  
John H. Griffin ◽  
Laurent O. Mosnier ◽  
Annette von Drygalski
Keyword(s):  
Blood Loss ◽  
San Diego ◽  
Thrombin Generation ◽  
Direct Thrombin Inhibitor ◽  
University Of California ◽  
Thrombin Inhibitor ◽  
Scripps Research Institute ◽  
Research Institute

Abstract Introduction and Objectives: Novel oral anticoagulants (NOACs), such as Factor (F) Xa inhibitors (Rivaroxaban and Apixaban) or the direct thrombin inhibitor (Dabigatran), are used for prevention of venous thromboembolism and ischemic strokes. However, conventional hemostatic reversal strategies in case of bleeding or surgery are ineffective, posing a major unmet clinical need. We recently demonstrated that superFVa, a novel FVa variant engineered with mutations of 3 activated protein C (APC) cleavage sites and a disulfide bond between the A2 and A3 domains, has enhanced biological activity and APC resistance. SuperFVa provided efficient normalization of hemostasis in hemophilia A mice and in animal models of APC-induced bleeding. Moreover, superFVa synergistically reduced bleeding in combination with recombinant human (rh) FVIIa in hemophilia mice. Thus, superFVa fits the criteria for a prohemostatic biologic. Therefore, the in vitro and in vivo effects of superFVa alone and in combination with other prohemostatic agents such as rhFVIIa or 4-Factor prothrombin complex concentrates (4F-PCC) as a novel hemostatic reversal strategy for NOAC-induced bleeding were determined. Materials and Methods: In vitro procoagulant properties of superFVa alone and in combination with rhFVIIa or 4F-PCC were studied using thrombin generation assays in normal human plasma (NHP) in the presence of FXa inhibitors (Rivaroxaban, Apixaban) or the direct thrombin inhibitor (Dabigatran, active form). In vivo prevention of blood loss by superFVa after intravenous injection of Rivaroxaban, Apixaban, or Dabigatran was studied using the tail clip model in BalbC mice. Results: Rivaroxaban and Apixaban each dose-dependently reduced thrombin generation in NHP and reduced the Endogenous Thrombin Potential (ETP) by ~60% at therapeutic concentrations (200 nM). Addition of either superFVa (50 nM) or rhFVIIa (40 nM, equivalent to a 90 µg/kg therapeutic dose in hemophilia patients), increased thrombin generation to some extent. However, ETP and thrombin peak height increased dose-dependently when increasing concentrations of superFVa (6.25 to 400 nM) were added with rhFVIIa (40 nM). superFVa effects appeared synergistic, with a plateau reached at 25-50 nM superFVa and ETP restored to ~93% of normal. Synergistic effects of superFVa were also present and even more pronounced in combination with 4F-PCC (1.35 U/ml; therapeutic plasma concentration). In the presence of Dabigatran (1 µM), superFVa (0.1 nM-100 nM) in combination with rhFVIIa (40 nM) or 4F-PCC (1.35 U/ml) showed a concentration dependent reduction of the lag time of thrombin generation. However, in contrast to the FXa inhibitors, in the presence of Dabigatran, no effects on ETP and thrombin peak height were observed for the addition of superFVa or combinations of superFVa and rhFVIIa or 4F-PCC. The in vivo efficacy of superFVa to reduce NOAC-induced bleeding was determined by blood loss after tail transection in BalbC mice injected i.v. with Rivaroxaban (40 mg/kg), Apixaban (20 mg/kg), or Dabigatran (0.4 mg/kg). Mean blood loss in mice injected with Rivaroxaban (16 µL/g), Apixaban (16.5 µL/g) or Dabigatran (14.5 µL/g) was significantly higher than baseline bleeding (4 µL/g, p<0.001). superFVa reduced blood loss after Rivaroxaban or Apixaban administration significantly in a dose dependent manner, e.g., superFVa at 40 U/kg significantly reduced bleeding to the baseline control level (5 µL/g). RhFVIIa (1 mg/kg) was able to reduce bleeding caused by Rivaroxaban but not by Apixaban. Neither superFVa nor rhFVIIa was able to reduce bleeding caused by Dabigatran. Conclusion: superFVa alone or in combination with rhFVIIa significantly improved in vitro thrombin generation in the presence of FXa inhibitors and to some extent in the presence of a direct thrombin inhibitor. SuperFVa alone consistently reduced bleeding in mice treated with FXa inhibitors, whereas a mixed response dependent on the type of FXa inhibitor was obtained with rhFVIIa. None of the agents was able to decrease bleeding in mice induced by Dabigatran. Because superFVa exerts a potent class effect as a hemostatic reversal strategy for FXa inhibitor-induced bleeding, it deserves further consideration for potential development as a hemostatic agent. Disclosures Gale: University of California, San Diego: University of California, San Diego Patents & Royalties. Griffin:The Scripps Research Institute: The Scripps Research Institute Patents & Royalties. Mosnier:The Scripps Research Institute: The Scripps Research Institute Patents & Royalties. von Drygalski:University of California, San Diego: University of California, San Diego Patents & Royalties.

scholarly journals Effect of a Direct Thrombin Inhibitor Compared with Dalteparin and Unfractionated Heparin on Human Osteoblasts

2011 ◽  
Vol 5 (1) ◽  
pp. 52-58 ◽  
Author(s):  
Tobias Winkler ◽  
Carsten Perka ◽  
Dörte Matziolis ◽  
Georg Matziolis
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Cell Number ◽  
Direct Thrombin Inhibitor ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Direct Thrombin Inhibitors ◽  
Type I ◽  
Human Osteoblasts

Purpose:Osteoporosis is a relevant problem after long term administration of unfractionated heparin (UFH) and low molecular weight heparin. Melagatran is a representative of a new group of direct thrombin inhibitors with comparable data in the prevention of thromboembolic events after orthopaedic surgery. The aim of ourin vitrostudy was to investigate the effect of a direct thrombin inhibitor compared with dalteparin and UFH on human osteoblasts.Materials and Methods:Melagatran, dalteparin and UFH were added to primary osteoblast cultures in their therapeutic range and two decimal powers below and above. Cell number, protein synthesis, mitochondrial and alkaline phosphatase activity and collagen type I synthesis were evaluated.Results:Melagatran showed the least influence on protein synthesis and cell proliferation with a reduction of cell number to 83.5 ± 9% (p = 0.027) of the control group only in the highest investigated concentration after 15 days of incubation.Mitochondrial and alkaline phosphatase activity and collagen type I synthesis in osteoblasts incubated with melagatran and dalteparin showed similar patterns. UFH showed the most pronounced influence on cellular metabolism.Conclusions:Melagatran showed less inhibitory in vitro effects on human osteoblasts than dalteparin or UFH. The presented study gives first hints that direct thrombin inhibitors may help prevent heparin-induced negative effects on bone metabolism.

Abstract 264: Differential Profiles of Thrombin Inhibitors (Dabigatran, Hirudin, Bivalirudin and Heparin) in the Thrombin Generation Assay (TGA) and Thromboelastography (TEG) in Vitro

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Yiming Xu ◽  
Weizhen Wu ◽  
Andrew S Plump ◽  
Madhu Chintala ◽  
Martin L Ogletree ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Rank Order ◽  
Therapeutic Index ◽  
Lag Time ◽  
Thrombin Inhibitors ◽  
Bleeding Complications ◽  
Thrombin Inhibitor ◽  
Direct Thrombin Inhibitors ◽  
Therapeutic Benefits

Thrombin is a central enzyme in haemostasis and thrombosis, and a proven target for anticoagulant therapies. Different classes of thrombin inhibitors, while exerting therapeutic benefits in most clinical trials, have different indications, dosing regimens, and bleeding complications. To gain more insight into the underlying mechanisms for their differential clinical profiles, we compared four marketed and representative agents, including dabigatran, hirudin, bivalirudin (direct thrombin inhibitors, DTIs), and heparin (an indirect thrombin inhibitor), in two in vitro spike-in assays with concentration titrations covering their therapeutic ranges. The two assays were the Thrombinoscope TGA with plasma, triggered by low tissue factor (1 nM TF), and TEG with whole blood, triggered by 1:8000 Recombiplastin (equivalent to low TF), with or without a threshold level of tPA to induce fibrinolysis. In TGA, the largest effect was prolongation of lag time, with the potency of the three DTIs rank-ordered as hirudin>dabigatran>bivalirudin; regarding peak, slope, and ETP, while complete inhibition was achieved with 1-2 μM dabigatran or hirudin, bivalirudin had no effect even at 4 μM, possibly due to its short half life in plasma. In TEG, the three DTIs prolonged clotting time (R) in the same rank order as TGA; for clot strength (MA), while all four agents reduced MA in synergy with tPA, only hirudin reduced MA without tPA, likely due to its highest potency. With tPA-induced fibrinolytic activity (Ly30), dabigatran and bivalirudin enhanced Ly30 (dabigatran>bivalirudin), but hirudin and heparin did not. This contrast might involve differential access to clot-bound thrombin. Heparin had a steep dose-response curve for both lag time in TGA and R in TEG, which is in line with its very narrow therapeutic index. All three DTIs, but not heparin, displayed the previously reported paradoxical increase in peak and slope in TGA in the low concentration range, suggesting this is indeed a class effect of DTI. In summary, our observations highlight the distinct features of each agent in thrombin generation, coagulation, and fibrinolysis. These results in combination with known clinical properties are informative on efforts to define the optimal profiles of new anticoagulants.

The Thrombin Activation Pathway Modulates the Assembly, Structure and Lysis of Human Plasma Clots In Vitro

1995 ◽  
Vol 73 (05) ◽  
pp. 785-792 ◽  
Author(s):  
Jim Torbet
Keyword(s):  
Human Plasma ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Phase Factor ◽  
Factor X ◽  
Contact Phase ◽  
Factor Ixa ◽  
Factor Xia ◽  
Thrombin Activation

SummaryThrombin activation of the soluble plasma protein fibrinogen is vital for successful haemostasis. Thrombin is generated from prothrombin by the prothrombinase complex which also includes factor Xa, factor Va, Ca2+ and a procoagulant membrane surface. Factor X activation is catalysed in a complex including either factor Vila and tissue factor, or factor IXa and factor Villa. Factor IXa can be generated either by the factor Vlla/tissue factor complex or by factor XIa which is in turn produced by the contact phase reactions in vitro. Once activated, fibrinogen develops into the fibrin polymeric matrix at the site of injury. It is not known to what extent the properties of this haemostatic plug are sensitive to the pathway leading up to thrombin generation. Here static human plasma is studied in vitro using magnetically induced birefringence. It is shown that the contact phase/factor XIa pathway gives rise to linear fibrin assembly progress curves whereas the factor Vlla/tissue factor activation of factor X provokes largely sigmoid assembly. The latter pathway also causes the formation of significantly thicker fibres even although assembly is more rapid. This result is the inverse of that anticipated from the study of simple model systems. Whilst the streptokinase activated lysis of both types of clot exhibits similar biphasic kinetics, an exponential main phase followed by a sigmoidal tailing off, the data suggest that clots produced by the contact phase/factor XIa pathway are more recalcitrant to lysis. These results demonstrate that the profile of thrombin generation not only determines the kinetics of assembly but also influences the rate of lysis and structure of the haemostatic plug.

A direct thrombin inhibitor studied by dynamic whole blood clot formation

2006 ◽  
Vol 96 (10) ◽  
pp. 446-453 ◽  
Author(s):  
Jørgen Ingerslev ◽  
Benny Sørensen
Keyword(s):  
Whole Blood ◽  
Plasma Concentrations ◽  
Direct Thrombin Inhibitor ◽  
Clot Formation ◽  
Thrombin Inhibitors ◽  
Bleeding Complications ◽  
Thrombin Inhibitor ◽  
Direct Thrombin Inhibitors ◽  
Dose Dependent

SummaryDirect thrombin inhibitors have proven efficacious in prevention of venous thromboembolism. Bleeding complications are rare, but in case of acute serious bleeding, an effective and instant haemostatic intervention may be required. In the present study it was demonstrated that the direct thrombin inhibitor melagatran induces dose-dependent abnormalities in whole blood (WB) clotting profiles as recorded bya recently described modified thrombelastographic model, and that rFVIIa or APCC are capable of improving the haemostatic capacity. Experiments were performed using WB from 30 healthy males. In-vitro titration experiments (n=10) with addition of melagatran to WB corresponding to plasma concentrations ranging from 0 to 5.0 µM (12 steps) showed a dose-dependent prolongation of the clot initiation and characteristic decrease of the maximum rate of clot propagation. In-vitro intervention studies (n=20) were completed with four different concentrations of melagatran as well as addition of four different levels of rFVIIa or APCC. At all tested concentrations of melagatran, rFVIIa significantly shortened the melagatran-induced prolonged clot initiation but induced only minor improvements of the reduced clot propagation. In contrast, APCC significantly and dose-dependently shortened the clot initiation and accelerated the clot propagation. In conclusion, our thrombelastographic model appears useful for evaluating the effect of direct thrombin inhibitors on dynamic WB clot formation and rFVIIa, but especially APCC significantly improved theWB clot formation. The pronounced stabilizing effect of APCC may be caused by its content of prothrombin and activated coagulation factors.

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