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.

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.

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.

The direct thrombin inhibitors (argatroban, bivalirudin and lepirudin) and the indirect Xa-inhibitor (danaparoid) increase fibrin network porosity and thus facilitate fibrinolysis

2010 ◽  
Vol 103 (05) ◽  
pp. 1076-1084 ◽  
Author(s):  
Margareta Blombäck ◽  
Niklas Bark ◽  
Hans Johnsson ◽  
N. Hakan Wallen ◽  
Shu He
Keyword(s):  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Thrombin Inhibitors ◽  
Therapeutic Window ◽  
Bleeding Complications ◽  
Direct Thrombin Inhibitors ◽  
Vitro Assay ◽  
Fibrin Network

SummaryThe present study aimed to assess whether the fibrin network structure is modified by the direct thrombin-inhibitors lepirudin, argatroban or bivalirudin and by the indirect Xa-inhibitor danaparoid. Using an in vitro assay that imitates the physiological process of coagulation from thrombin generation to fibrin formation, we examined a normal plasma pool spiked with one of the inhibitors. At concentrations considered to be the plasma levels observed during therapy, almost no influence was detected for lepirudin despite clear-cut effects on “clotting time”. However, argatroban, bivalirudin and danaparoid increased the fibrin gel permeability (Ks) to a similar extent. At concentrations higher than the “therapeutic” levels, the dose-response curve in the Ks assay became very steep for lepirudin while those were shallow for the others. In parallel with the drug-induced increases of Ks, larger network pores in 3D-microscopic images and significant shortenings in “clot lysis time” induced by addition of rtPA were observed. Recombinant factor VIII (rFVIII) added to danaparoid-treated samples profoundly counteracted the increase of Ks but had only a slight or no effect on the other drugs. Thus, in vitro, argatroban, bivalirudin and danaparoid have comparable anticoagulating effects, rendering the fibrin network more permeable and less resistant to fibrinolysis. For lepirudin, the steep dose-response curve supports previous clinical findings, i.e. this thrombin inhibitor has a narrow therapeutic window. Furthermore, our data suggest that the haemostatic agent, rFVIII, might be effective in treatment of bleeding complications induced by danaparoid.

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.

Direct Thrombin Inhibitors, but Not Factor Xa Inhibitors, Enhance Thrombin Formation in Human Plasma by Interfering with the Thrombin–Thrombomodulin–Protein C System

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 528-528 ◽  
Author(s):  
Elisabeth Perzborn ◽  
Michaela Harwardt
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Lag Time ◽  
Thrombin Inhibitors ◽  
Direct Thrombin Inhibitors ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Normal Plasma ◽  
Thrombin Formation

Abstract Activation of protein C is dependent on thrombin complexed with thrombomodulin (TM). Activated protein C (APC), together with its cofactor protein S, degrades coagulation Factors Va and VIIIa, thereby limiting further thrombin formation. Thus, in addition to suppressing the procoagulant effects of thrombin, direct thrombin inhibitors (DTIs) may also downregulate anticoagulant effects of thrombin-mediated feedback mechanisms. By contrast, direct Factor Xa (FXa) inhibitors block the formation of thrombin, but not its actions. The objective of this study was to investigate whether the direct FXa inhibitor, rivaroxaban, and the DTIs, dabigatran and melagatran, inhibit the negative-feedback reaction of the thrombin–TM complex/APC (thrombin–TM/APC) system and thereby increase thrombin formation. Experiments were conducted in plateletpoor plasma from healthy donors (normal plasma) and in pooled protein C-deficient plasma, both substituted with 1.33 μM phospholipids, in the presence or absence of 10 nM TM with increasing concentrations of rivaroxaban, dabigatran, melagatran, or the appropriate vehicles. Thrombin formation was initiated by adding 1.67 pM tissue factor (TF) and assessed by measuring the cleavage of the fluorogenic substrate Z-Gly-Gly-Arg-AMC (Bachem) using the Calibrated Automated Thrombogram (CAT, Thrombinoscope® BV) method. The parameters assessed were lag time, time to peak thrombin generation (tmax), peak thrombin generation (Cmax), and endogenous thrombin potential (ETP). In addition, formation of prothrombin fragments 1+2 (F1+2) was determined by ELISA (Enzygnost® F1+2 monoclonal [Dade Behring]). Rivaroxaban potently inhibited thrombin formation in the absence and presence of TM across all parameters in a concentration-dependent manner in both normal plasma and protein C-deficient plasma (see Table). In the absence of TM, melagatran and dabigatran also inhibited thrombin formation in a concentration-dependent manner, both in normal plasma and protein C-deficient plasma. In the presence of TM, DTIs prolonged lag time and tmax in a concentration-dependent manner. However, only high concentrations of the DTIs reduced ETP, Cmax, and F1+2, In normal plasma,lower concentrations even increased ETP, Cmax, and F1+2. Increased thrombin formation was observed with melagatran 119–474 nM or dabigatran 68–545 nM. DTIs did not increase thrombin formation in protein C-deficient plasma, suggesting that both protein C and TM are needed for the DTI-mediated increase in thrombin formation. The results suggest that low concentrations of DTIs suppress the anticoagulant effects of the thrombin–TM/APC system by inhibiting activation of protein C by the thrombin–TM complex, and thereby enhance thrombin formation. Conversely, rivaroxaban does not increase thrombin formation, suggesting that it does not suppress the negative-feedback reaction by inhibition of protein C activation. This hypothesis is supported by the absence of enhanced thrombin formation in protein C-deficient plasma. Enhanced thrombin formation might explain the hypercoagulation observed with DTIs in a rat model of TF-induced intravascular coagulation (Furugohri, et al. 2005; Morishima, et al. 2005; Perzborn, et al. 2008) and suggests that DTIs could cause activation of coagulation at lower plasma concentrations. Table. Effect of rivaroxaban, dabigatran, and melagatran on peak thrombin formation (Cmax [nM thrombin]) in the absence or presence of thrombomodulin (TM) in normal plasma (NP) from healthy volunteers (n=8–12), and in pooled protein C-deficient plasma (PPC) in the presence of TM (n=3). Results were obtained by the CAT method and are a mean of n plasma samples. Prothrombin fragments F1+2 (nM F1+2) results were obtained in the presence of TM (mean results; n=3 [NP]). Thrombin (nM) in normal and in protein C-deficient plasma n.d:, no data. Rivaroxaban (nM) 0 18 91 182 363 1,090 Cmax: − TM in NP 273 222 113 71 43 15 Cmax: + TM in NP 100 67 32 18 10 3 Cmax: + TM in PPC 290 249 168 117 80 38 F1+2: + TM (in NP) 184 89 43 18 n.d. 2 Dabigatran (nM) 0 68 136 273 545 1,090 Cmax: − TM in NP 261 287 289 282 239 102 Cmax: + TM in NP 81 156 203 240 218 96 Cmax: + TM in PPC 298 301 300 292 253 116 F1+2: + TM in NP 252 n.d. 362 422 351 98 Melagatran(nM) 0 24 119 237 474 948 Cmax: − TM in NP 276 290 297 289 251 127 Cmax: + TM in NP 101 133 215 251 237 113 Cmax: + TM in PPC 294 296 299 292 256 132 F1+2: + TM in NP 213 n.d. 389 427 393 123

Trends in Parenteral Direct Thrombin Inhibitor Use in Pediatric Patients: Analysis of a Large Administrative Database

2014 ◽  
Vol 138 (9) ◽  
pp. 1229-1232 ◽  
Author(s):  
Brady S. Moffett ◽  
Jun Teruya
Keyword(s):  
Pediatric Patients ◽  
Health Information System ◽  
Thrombin Inhibitors ◽  
Administrative Database ◽  
Bleeding Complications ◽  
Thrombin Inhibitor ◽  
Direct Thrombin Inhibitors ◽  
High Morbidity ◽  
Thrombin Time ◽  
Hospital Data

Context.—Parenteral direct thrombin inhibitors (DTIs) may be used in pediatric patients with contraindications to heparin therapy, such as heparin-induced thrombocytopenia. Few data exist regarding the use of DTIs in pediatric patients. Objective.—To characterize the use of DTIs in pediatric patients, including monitoring strategies and bleeding complications. Design.—A retrospective descriptive study was designed and the Pediatric Health Information System database was queried from 2004 to 2011 for pediatric patients receiving a parenteral DTI. Patient demographic and hospital data, mortality, disease state, and procedure information (from International Classification of Diseases, Ninth Revision codes) were collected from the query. DTI monitoring information was also collected. Patients were divided into 2 time periods (2004–2007, 2008–2011) to evaluate trends. Results.—Two hundred eight patients met study criteria (50.9% male, 64.4% white), and children (2–12 years of age) represented 34.6% of the population. Congenital heart disease was present in 43.8% and cardiovascular surgical procedure occurred in 28.4%. Argatroban was most commonly used (73.1%) and bivalirudin use increased (P < .001). Bleeding complications were present in 37.9% of patients and mortality was 19.7%. Bleeding complications were associated with lepirudin (62.5%) and argatroban (41.7%) more often as compared with bivalirudin (18.8%) (P < .001). Activated partial thromboplastin time and prothrombin time were used more often in patients receiving argatroban and lepirudin in comparison with bivalirudin, and thrombin time was used more often in patients receiving lepirudin (P < .001). Activated clotting time use increased over time (5.1% versus 17.5%, P = .02). Conclusion.—Pediatric use of DTIs is infrequent and occurs in patients with high morbidity and mortality.

Differential Inhibition of Thrombin Activity and Thrombin Generation by a Synthetic Direct Thrombin Inhibitor (Napsagatran, Ro 46-6240) and Unfractionated Heparin in Patients with Deep Vein Thrombosis

1999 ◽  
Vol 81 (04) ◽  
pp. 498-501 ◽  
Author(s):  
Henri Bounameaux ◽  
Herbert Ehringer ◽  
Alain Gast ◽  
Johan Hulting ◽  
Herbert Rasche ◽  
...  
Keyword(s):  
Deep Vein Thrombosis ◽  
Unfractionated Heparin ◽  
Thrombin Generation ◽  
Thrombin Inhibitors ◽  
Fixed Dose ◽  
Thrombin Inhibitor ◽  
Direct Thrombin Inhibitors ◽  
Vein Thrombosis ◽  
Thrombin Activity ◽  
Deep Vein

Summary Background: Direct thrombin inhibitors belong to a new class of antithrombotic drugs whose effects on blood coagulation in vivo in patients suffering from acute thrombotic conditions have not yet been fully explored. Methods and Results: One hundred and five patients with acute proximal deep-vein thrombosis were randomized to receive a continuous intravenous infusion of napsagatran, a novel synthetic thrombin inhibitor, at a fixed dose of 5 mg/h (n = 36) or 9 mg/h (n = 25) for five days, or APTT-adjusted unfractionated heparin (UFH, n = 44) for the same time. In these patients, thrombin activity and thrombin generation could be assessed by measuring thrombin-antithrombin III complexes (TAT) and prothrombin fragment 1+2 (F1+2), respectively, on three occasions. At baseline, TAT and F1+2 did not differ among the three groups. On Day 2 (steady state), TAT significantly decreased in all groups, and the decrease was significantly more pronounced in the patients given higher-dose napsagatran. F1+2 decreased significantly only in UFH-treated patients. Two hours after cessation of the infusion, the TAT levels increased in the two napsagatran groups but not in the UFH group, whilst F1+2 went back to the baseline levels in the napsagatran-treated patients but remained low in the UFH-treated patients. There was no rebound effect. Conclusions: The data presented suggest that direct thrombin inhibition with napsagatran at 9 mg/h is more potent than UFH in attenuating thrombin activity, but is less potent than UFH in inhibiting thrombin generation. The real significance of these findings will have to be substantiated in further trials with clinically relevant endpoints.

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.

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

Blood ◽  
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.

Platelets and Heparin Induced Thrombocytopenia Antibodies Do Not Influence the Inhibitory Activity of Argatroban on Thrombin Generation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 929-929
Author(s):  
Grigoris T. Gerotziafas ◽  
Marie-Paule Roman ◽  
Elisabeth Verdy ◽  
Mohamed Hatmi ◽  
Meyer M. Samama ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Lag Time ◽  
Thrombin Inhibitor ◽  
Prospective Clinical Study ◽  
Dependent Manner ◽  
Heparin Induced Thrombocytopenia ◽  
Normal Platelet

Abstract Argatroban is a synthetic, reversible, direct thrombin inhibitor (DTI) used in patients with heparin induced thrombocytopenia (HIT). Argatroban as other DTIs prolongs PT, aPTT and ecarin clotting time. Argatroban added in vitro in normal platelet poor plasma (PPP) inhibits tissue factor (TF) triggered thrombin generation (TG) in a concentration dependent manner. We studied the influence of platelets, HIT antibodies and residual heparin, on the inhibitory effect of argatroban on TG. Argatroban (0 to 2 μM) was added in normal platelet rich plasma (PRP) and PPP, in pool PPP from three consecutive HIT patients (HIT-PPP) and in HIT-PRP prepared by mixing normal PRP with HIT-PPP (v/v). HIT-PRP and HIT-PPP were containing residual heparin (0,035 and 0,07 anti-Xa IU/ml respectively). All experiments were repeated 3 times. TG was triggered in the presence of TF (Dade-Behring Innovin; 1/1000 final dilution in plasma) and assessed with Calibrated Automated Thrombinoscope®. TG in PPP was triggered by adding PPP reagent (Thrombogram-Thrombinoscope®). Lag time (LT), time to peak (ttP), peak (P), endogenous thrombin potential (ETP) and mean velocity index (MVI) of thrombin generation were measured. The concentrations of argatroban which prolonged 2-fold the LT and the ttP and which inhibited 50% (IC50) the P, ETP and MVI were calculated. In the presence of low argatroban concentrations (0,1 an 0,2 μM) an artifactual increase of TG was observed. This is probably due to the interference of alpha2macroglobulin-bound thrombin with the fluorogenic substrate (as shown by Hemker’s group for other reversible DTIs). Argatroban at 1 μM inhibited TG by 50% in both normal PRP and PPP. Argatroban at 1 μM induced a 2-fold prolongation of aPTT (Table 1). HIT antibodies did not modify the inhibitory activity of argatroban on TG in HIT-PRP and HIT-PPP. The presence of traces of heparin in plasma from HIT patients had a synergistic effect with argatroban on the inhibition of TG (Table 1). Our in vitro study shows that argatroban at concentration achieved in clinical practice (about 1 μM) induces 50% inhibition of TG. The inhibitory activity of argatroban on TG is not modified by the presence of platelets or HIT antibodies. In contrast, traces of residual heparin in plasma from HIT patients amplify the inhibition of thrombin generation induced by argatroban. This finding has to be confirmed in a prospective clinical study since it implicates an increased vigilance during the switch from heparin to argatroban in acute HIT patients. Table 1. Inhibitory activity of argatroban on thrombin generation. normal PRP normal PPP HIT-PRP (0,03 aXa/ml) HIT-PPP (0,07 aXa/ml) Lag-time x2 0,7 μM 0,7 μM 0,9 μM 0,1 μM ttPeak x2 1 μM 1 μM 1 μM 0,2 μM Peak IC50 1 μM 1 μM 0,7 μM 0,3 μM ETP IC50 1 μM 1 μM 0,7 μM 0,3 μM MRI IC50 1 μM 1 μM 0,5 μM 0,3 μM PTx2 - 1 μM - - aPTT x2 - 1 μM - -

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