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.

The in vivo and in vitro study of polysaccharides from a two-herb formula on ulcerative colitis and potential mechanism of action

2014 ◽  
Vol 153 (1) ◽  
pp. 151-159 ◽  
Author(s):  
Linjing Zhao ◽  
Hongbing Wu ◽  
Aihua Zhao ◽  
Huili Lu ◽  
Wei Sun ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Mechanism Of Action ◽  
In Vitro Study ◽  
Vitro Study ◽  
Potential Mechanism

Abciximab Does Not Inhibit the Increase of Thrombin Generation Produced in Platelet-Rich Plasma In Vitro by Sodium Arachidonate or Tissue Factor

2005 ◽  
Vol 11 (3) ◽  
pp. 271-277 ◽  
Author(s):  
Raul Altman ◽  
Alejandra Scazziota ◽  
Silvina Santoro ◽  
Claudio Gonzalez
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Coronary Intervention ◽  
Inhibitory Effect ◽  
Platelet Membrane ◽  
Time To Peak ◽  
Coronary Events

Aspirin and platelet membrane glycoprotein (GP) IIb/IIIa blockers are currently used for acute coronary events, and in percutaneous coronary intervention for preventing further coronary outcomes, because they inhibit platelet function. Aspirin also inhibits thrombin generation (TG) in platelet-rich plasma (PRP) activated by sodium arachidonate (AA). The effect of the platelet membrane GP IIb-IIIa (integrin αIIbβ3) blocker abciximab on thrombin generation was studied in vitro using PRP. Thirty healthy volunteers taking no medication, and 28 volunteers who had taken aspirin (160 mg/day for 3-4 days), were included in the protocol. Control or in vivo aspirinated PRP, stimulated or not by AA or tissue factor (TF), was investigated for the inhibitory effect of abciximab pre-incubated for 3 minutes. AA and TF added in vitro activated non-aspirinated PRP: lag-time (LT) and time to peak (TTP) were significantly shortened. Peak TG (PTG) and endogenous thrombin potential (ETG) were increased by AA but not TF; thus, AA seems to be more efficient than TF for TG in this system. Abciximab added in vitro to non-activated, non-aspirinated PRP had no effect on LT, TTP, or ETP, but caused a decrease in PTG that was not statistically significant. Abciximab (3 or 4 μg/mL) added in vitro to AA or TF-activated, non-aspirinated PRP produced no effect on TG, although in aspirinated platelets both LT and time to peak were prolonged. AA as well as TF added in vitro to PRP or in vivo aspirinated PRP increased TG, although AA seems to be more efficient in our assay system. Abciximab, which affects nonaspirinated, nonactivated PRP weakly, has no effect on AA or TF in activated control PRP or in vivo aspirinated PRP.

Thrombin Generation Test in Patients with Hemophilia and Inhibitors Treated with by-Passing Agents: Results From In Vivo Studies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1216-1216
Author(s):  
Maria Elisa Mancuso ◽  
Veena Chantarangkul ◽  
Armando Tripodi ◽  
Marigrazia Clerici ◽  
Licia Padovan ◽  
...  
Keyword(s):  
Drug Administration ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Elective Surgery ◽  
Standard Dose ◽  
In Vivo Studies ◽  
Time To Peak ◽  
Coagulation Assay

Abstract Abstract 1216 Background: The development of inhibitors is the most serious complication of modern hemophilia therapy. Patients with inhibitors are often treated with by-passing agents (namely, aPCC and rFVIIa) whose haemostatic efficacy is not always predictable even in the same subject. Indeed clinical response to by-passing therapies may vary between patients and the lack of a specific laboratory test aimed at monitoring their ability in triggering blood coagulation and at correlating clotting activation to clinical outcome renders the management of these drugs somehow empirical. The thrombin generation (TG) test is a global coagulation assay that may serve as a candidate in this setting. Recently, dose tailoring of by-passing agents was performed using in vitro spiking experiments in order to establish the most adequate doses to cover elective surgery in hemophilic patients with inhibitors. Methods: In this study TG capacity was evaluated for the first time in vivo by drawing plasma samples from hemophilic patients with inhibitors treated either with aPCC or rFVIIa in a non-bleeding state after a minimum wash-out period of 3 days from the last infusion. TG test was performed in platelet-rich (PRP) and platelet-poor (PPP) plasma with the addition of corn trypsin inhibitor (CTI, final concentration 18.3 mcg/ml). Blood was drawn at baseline, 30 minutes, 3 hours (if rFVIIa), 6 hours (if aPCC) and 24 hours after drug administration. Four parameters of the TG curve were evaluated: lagtime, endogenous thrombin potential (ETP), peak and time to peak. Patients were defined as responders to by-passing agents when able to control mild/moderate bleeding episodes by home treatment with those drugs. Results: Eight patients with hemophilia A and high-responding inhibitors (historical peak above 5 BU/ml) with a median age of 33 years (IQR: 20–38) received rFVIIa at a standard dose of 90–120 mcg/kg and aPCC at a dose of 80 IU/kg. In 6 patients the TG test was performed also after the administration of a rFVIIa dose of 270 mcg/kg. Four patients were responders to aPCC, one to rFVIIa, 2 to both drugs and one was non-responder to both. Median values of the TG curve observed at baseline and 30 minutes after drug administration are shown in the Table. Similar variations in the TG curve were observed after administration of either rFVIIa or aPCC or after the administration of rFVIIa at the 2 different dosages. By evaluating only the patients responders to aPCC (n=4), there was no difference in the TG curves obtained after the administration of aPCC or rFVIIa. Conclusions: Our preliminary results show that the in vivo administration of by-passing agents causes an increase of TG capacity in hemophilic patients with inhibitors. However, the extent of such increase does not seem to be related neither with the type nor with the dose of these drugs. The evaluation of TG curve in response to by-passing agents will be performed also in haemophilic patients with inhibitors who underwent or will undergo major surgical procedures. Disclosures: No relevant conflicts of interest to declare.

Thrombin Generation by Fetoscopic Trauma to the Fetal Membranes: An in vivo and in vitro Study

2015 ◽  
Vol 39 (4) ◽  
pp. 261-268 ◽  
Author(s):  
Alexander C. Engels ◽  
Dries Bauters ◽  
Rita Rynkevic ◽  
Savitree Pranpanus ◽  
Jute Richter ◽  
...  
Keyword(s):  
Amniotic Fluid ◽  
Thrombin Generation ◽  
In Vitro Study ◽  
Vitro Study ◽  
Thrombin Inhibitor ◽  
Thrombin Receptor ◽  
Fetal Membranes

Objective: We first aimed to investigate in vivo thrombin generation induced by fetoscopy, and second we used term membrane explants for measurement of thrombin generation, thrombin receptor location and induction of selected matrix metalloproteinases (MMPs) in tissue culture. Materials and Methods: In vivo study (37 cases): samples of amniotic fluid were taken at the beginning and end of fetoscopy (mean gestational age 26.7 weeks) and analyzed by ELISA for thrombin-antithrombin complexes. In vitro study: fetal membranes were put in culture and punctured for measurement of thrombin generation by calibrated automated thrombography and ELISA. Induction of MMP-9 and MMP-2 was analyzed by zymography. PAR-1 was localized by immunohistochemistry. Results: No significant increase in thrombin-antithrombin was measured in amniotic fluid obtained during fetoscopy. In vitro, thrombin generation induced by needle trauma of membrane cultures is correlated to the amount of plasma. Activity of MMP-9 but not MMP-2 was elevated in cultured membranes but could not be inhibited by a thrombin inhibitor. On histology, the thrombin receptor PAR-1 was located in the chorion and decidua, but not in the amnion. Discussion: Despite the influence of thrombin on punctured fetal membranes in vitro, the role of thrombin in iatrogenic preterm premature rupture of membranes is questionable.

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.

Antidotal Effects of rFVIIa and FEIBA on Anticoagulant-Induced Inhibition of Thrombin Generation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4124-4124
Author(s):  
Daniele Pillitteri ◽  
Wolfgang Wegert ◽  
Thomas Scholz ◽  
Carl M. Kirchmaier
Keyword(s):  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Dose Range ◽  
Inhibitory Effect ◽  
Thrombin Inhibitors ◽  
In Vitro Assays ◽  
Recombinant Hirudin ◽  
Thrombin Inhibition ◽  
Time To Peak

Abstract Recombinant FVIIa (NovoSeven®, N7) has been demonstrated to antagonize the antihemostatic effects of anticoagulants such as anti-Xa-agents like LMWH or Fondaparinux. Whether this effect, which can be monitored using cell-based in vitro assays including platelets, can also be observed when trying to antagonize thrombin inhibitors and heparinoids, is still insufficiently known. We used a fluorometric thrombin generation test (TGT) to assay N7 efficacy in platelet-rich plasma (PRP) on eight concentrations of Argatroban (Argatra®), Lepirudin (Refludan®) and Danaparoid (Orgaran®) between 0 and 10 μg/ml resp. 0–10 anti-Xa units/ml) including their therapeutic ranges. Blood samples used for spiking were taken from eight healthy male volunteers who had taken no antihemostatic medication 14 days before sampling. TF was used as initiator of coagulation (final conc. 60 fM). FEIBA (factor eight inhibitor bypassing activity) was also tested as a potential antidote in this experimental setting. N7 doses used for in-vitro spiking of PRP samples were betweeen 2.4 (corresponding to clinically used high doses) and 9.6 (Argatroban, Fondaparinux) resp. 24 μg/ml (Lepirudin), FEIBA doses were 0.5, 1 and 2 U/ml. The TGT parameters evaluated were ETP, PEAK, LAG TIME (LT) and TIME TO PEAK (TTP). While the Argatroban effect on thrombin generation could not be reversed up to 9.6 μg/ml of N7, for FEIBA the thrombin activity increased half-logarithmic-like with linear rises for doubled FEIBA doses. The inhibitory effect of Lepirudin on TGT parameters was neutralized by 2.4 μg/ml rFVIIa, and the lowest FEIBA concentration (0.5 U/ml) was also sufficient. For Danaparoid, the reversal of its inhibitory effects in the TGT was more marked for supratherapeutical concentrations, but 2.4 μg N7/ml worked as well as 24. The antidote effect of FEIBA on Danaparoid concentrations increased with FEIBA concentrations, but seemed to be less strong than that of N7. Fondaparinux (Arixtra®), which was run as control substance in our assay and antagonized with N7 2.4 μg/ml (9.6 μg/ml had no stronger effect) and FEIBA in the intermediate concentration of 1U/ml, showed analogous changes to the results published by Lisman et al., JTH 2003. Regarding the TGT parameters, there seem to be “cut-offs” between 2 and 5μg/ml for the inhibitors without antidotes where ETP and PEAK are reduced to zero. Inhibitor-induced prolongation of LT and TTP showed similar characteristics with and without antidotes, but on different levels, i. e. the antidotes shortened these time values over the whole inhibitor concentration range. Higher concentrations of antidotes were more effective on high inhibitor concentrations. Argatroban could be antagonized with FEIBA only, whereas N7 was also effective on Lepirudin and Fondaparinux, and even better on Danaparoid. The N7 reversal of Lepirudin-induced irreversible thrombin inhibition seems to be contradictory to the missing effect on the reversible Argatroban-induced effect but could be due to the (slow) binding kinetics (Elg et al Thromb Haemost 1997) of the recombinant hirudin. The assay could be used to determine or predict the antihemostatic effects of anticoagulants over a dose range and the potential efficacy of antidotes for overdosing with anticoagulants. Furthermore, it may be a tool for a personalized medicine approach.

BCH-2763, a Novel Potent Parenteral Thrombin Inhibitor, Is an Effective Antithrombotic Agent in Rodent Models of Arterial and Venous Thrombosis – Comparisons with Heparin, r-Hirudin, Hirulog, Inogatran and Argatroban

1998 ◽  
Vol 79 (02) ◽  
pp. 431-438 ◽  
Author(s):  
Carolyn Finkle ◽  
Annie Pierre ◽  
Lorraine Leblond ◽  
Isabelle Deschenes ◽  
John DiMaio ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Arterial Thrombosis ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Direct Thrombin Inhibitors ◽  
Antithrombotic Agent ◽  
Plasma Clot ◽  
Antithrombotic Efficacy

SummaryCurrent clinical use of heparin as an antithrombotic agent is limited by suboptimal efficacy and safety considerations. Thrombin’s central role in thrombosis makes it an attractive target to develop more effective and safer antithrombotic agents. BCH-2763 is a novel, potent (Ki: 0.11 nM), low molecular weight (1.51 kDa), bivalent direct thrombin inhibitor. The antithrombotic efficacy of BCH-2763 in vivo following i.v. bolus plus infusion in rats was compared in arterial and venous thrombosis models with two other bivalent direct thrombin inhibitors, r-hirudin and hirulog, with two catalytic site-directed thrombin inhibitors, inogatran and argatroban, and with heparin. In vivo efficacy was related to inhibition in vitro of fibrin clot formation, thrombin-induced aggregation of rat or human washed platelets and activity of free and plasma clot-bound thrombin. All the direct thrombin inhibitors were effective on both arterial and venous thrombosis at markedly lower fold aPTT increases than heparin. The antithrombotic doses of all inhibitors against venous thrombosis were less than against arterial thrombosis. The rank order of potency based on doses (mg/kg/h) required for full efficacy against arterial thrombosis was BCH-2763 (1.2) inogatran (1.5) r-hirudin (1.8) hirulog (3.3) argatroban ( 3.0); heparin required a markedly higher dose (5.7). In venous thrombosis the doses required for full efficacy were substantially lower for the bivalent (BCH-2763: 0.12; r-hirudin: 0.12; hirulog: 0.18) than for the catalytic site-directed (inogatran: 0.48; argatroban: 0.90) thrombin inhibitors; the dose required for heparin was 0.19. All the direct thrombin inhibitors caused similar shifts in aPTT at doses required to inhibit arterial thrombosis, but BCH-2763 inhibited venous thrombosis at lower aPTT fold increases. In vivo antithrombotic efficacy of direct thrombin inhibitors correlated with their inhibitory activity in vitro against fibrin clot formation and platelet aggregation. In contrast to heparin, all the direct thrombin inhibitors inhibited plasma clot-bound thrombin, but the relative IC50s did not correlate with their antithrombotic efficacy. In summary, direct thrombin inhibitors are more effective than heparin in inhibiting arterial and venous thrombosis in rats with less aPTT increases. BCH-2763 is effective at lower doses than the other direct thrombin inhibitors and for venous thrombosis at a smaller aPTT increase. BCH-2763 may offer an improved therapeutic index in the treatment of thromboembolic complications over heparin and other direct thrombin inhibitors.

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.

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