In Vitro Effect of Danaparoid Sodium (Orgaran®) on Thrombin Generation after Minimal Tissue Factor Pathway Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4151-4151
Author(s):  
Ismail Elalamy ◽  
Anna D. Petropoulou ◽  
Mohamed Hatmi ◽  
Meyer M. Samama ◽  
Grigoris T. Gerotziafas
Keyword(s):  
Molecular Weight ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Lag Time ◽  
Dependent Manner ◽  
Pathway Activation ◽  
Coagulation Tests ◽  
Vitro Effect ◽  
Routine Coagulation

Abstract Introduction: Orgaran® (Org 10172) is a low molecular weight heparinoid which consists of natural sulphated glycosaminoglycans (heparan, dermatan, chondroitin sulphate). It has a mean molecular weight of approximately 6 kDa (4–10 kDa), an excellent bioavailability following subcutaneous administration and an anti-Xa/anti-IIa activity ratio superior to 22. It is the anticoagulant of choice in patients developping Heparin-Induced Thrombocytopenia (HIT), whereas its’ use is also proposed for surgical thromboprophylaxis. Orgaran® has no effect on routine coagulation tests (aPTT, PT, TT). Thrombin generation test(TG) is a global clotting assay proven to be sensitive to the anticoagulant effect of LMWHs and specific FXa inhibitors (i.e. fondaparinux and BAY-597939). In this in vitro study, we determined the tissue factor (TF)-induced TG inhibition potency of Orgaran® using the Thrombogram-Thrombinoscope® assay. Materials and Methods: TG was assessed after TF pathway activation in Platelet Rich Plasma (PRP) (1.5x105 platelets/μl) using diluted thromboplastin (Dade Innovin®, 1:1000 final dilution). The clotting process is provoked by a physiologically relevant TF concentration. Orgaran® was added to control plasma from 8 healthy volunteers at five different final concentrations (0.2, 0.4, 0.6, 0.8 and 1IU anti-Xa/ml). TG was initiated by adding the triggering solution containing CaCl2 and the fluorogenic substrate. The analyzed TG parameters are the lag time, the maximal concentration of thrombin (Cmax), the time to reach Cmax (Tmax), the TG velocity and the endogenous thrombin potential (ETP). Results: Orgaran® prolonged significantly the lag time and the Tmax at a concentration over 0.40 IU anti-Xa/ml (p<0.05). At the lowest studied concentration (0.20 IU anti-Xa/ml), lag time and Tmax were only prolonged by 12%, whereas their maximal prolongation (around 50%) was observed at 1IU anti-Xa/ml. Furthermore, Orgaran® inhibited ETP, Cmax and TG velocity in an almost linear dose dependent manner. A significant inhibition of ETP, Cmax and TG velocity was obtained at concentrations superior to 0.20 IU anti-Xa/ml. (p<0.05). At the highest studied concentration (1IU anti-Xa/ml) Orgaran® suppressed all TG parameters by about 80% (Table 1). Conclusion: Orgaran® exhibited a significant inhibitory activity of in vitro TG. At concentrations achieved in clinical practice (prophylactic or therapeutic dose), Orgaran® modified in vitro TG profile while it has no effect on routine coagulation tests. Thus, TG assay is a sensitive method for monitoring Orgaran® and this test requires a clinical prospective evaluation. Table 1. Determination of IC20 and IC50 anti-Xa inhibitory concentrations of Orgaran® on TG parameters Lag Time Tmax ETP Cmax Velocity IC: Inhibitory Concentration * or Concentration increasing 20% and 50% the lag time and the Tmax respectively IC 20 (IU/ml) 0.30 0.30 0.18 0.18 0.15 IC 50 (IU/ml) 0.83 >1 0.30 0.50 0.35 1IU anti-Xa/ml 53% 47% 68% 76% 84%

The Influence of Lepirudin, Bivalirudin and Dabigatran on the Calibrated Automated Thrombogram (CAT)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4055-4055
Author(s):  
Barry J Woodhams ◽  
Audrey Carlo ◽  
Lena Le Flem ◽  
Celine Guinet ◽  
Francois Depasse ◽  
...  
Keyword(s):  
The Netherlands ◽  
Tissue Factor ◽  
Therapeutic Range ◽  
Thrombin Generation ◽  
Lag Time ◽  
Peak Height ◽  
Vitro Effect ◽  
Peak Increase ◽  
Normal Pool Plasma

Abstract In a two centre study (laboratories in Diagnostica Stago and Biomnis) we compared the in vitro effect on thrombin generation (TG) of Dabigatran and Bivalirudin (reversible direct anti-IIa inhibitors) with that of Lepirudin (an irreversible direct anti-IIa inhibitor) spiked into normal pool plasma. The effect of Lepirudin, Bivalirudin and Dabigatran were evaluated in both centres using the CAT (Diagnostica Stago, France) TG method in a concentration ranges up to 5, 20 and 1 μg/mL respectively. Testing was done in triplicate and repeated over 2 days. To reduce assay variability both centres used the same reagents lots and the same normal pool plasma (George King, USA). The range of each drug tested extended well above the therapeutic range concentrations normally found in patient plasma (0.5 to 1.0 μg/mL, 5 to 10 μg/mL and 0.1 to 0.3 μg/mL respectively for Lepirudin, Bivalirudin and Dabigatran). To see the effect of increasing activation forces, TG was performed at 3 different final concentrations of Tissue Factor (TF) - 1, 5 and 20 pM. All reagents were used as recommended by the manufacturer (Thrombinoscope, The Netherlands). A prolongation in the lag time (LT) is observed with all 3 drugs with all 3 concentrations of TF, but this is more marked for Lepirudin and Bivalirudin than it is for Dabigatran. In the therapeutic range Dabigatran (at 5pM TF) shows both an increase in LT and a decrease in peak thrombin and the ETP. At low concentration of Bivalirudin or Lepirudin, there is a paradoxical increase in peak height, which is even more pronounced at low TF concentration. At 1pM TF, this paradoxical peak increase is also observed with Dabigatran. Results obtained in both laboratories are similar and complement our previous results and those reported elsewhere (1–4). The effect of Lepirudin and Bivalirudin on TG is different from that of Dabigatran. We also note that at lower TF concentration the anticoagulant effect on TG initiation is more intense but the test becomes less reproducible.

Reversal of Anticoagulation as Assessed by Thrombin Generation Measurement.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 876-876
Author(s):  
Alex Gatt ◽  
Joost van Veen ◽  
Peter Cooper ◽  
Steve Kitchen ◽  
Michael Makris
Keyword(s):  
Thrombin Generation ◽  
Fresh Frozen Plasma ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Protamine Sulphate ◽  
Reversal Agents ◽  
Time To Peak ◽  
High Doses ◽  
Vitro Effect

Abstract Antidotes to the ever-growing number of anticoagulants are always desirable in order to placate bleeding in emergencies. In general, the older anticoagulants like coumarins and unfractionated heparin (UFH) have proven reversal agents, whereas the newer ones do not. We studied the in vitro effect of 5 different heparinoids (UFH, Tinzaparin, Enoxaparin, Fondaparinux and Danaparoid) on the calibrated automated thrombogram. The assay uses a fluorogenic substrate that is cleaved by the thrombin formed after the addition of plasma to 5pM tissue factor, 4μM phospholipids and calcium chloride. We investigated all the parameters generated by dedicated software (Thrombinoscope™) ie the lag time (LT), the time to peak (ttpeak), the endogenous thrombin potential (ETP) and the peak thrombin. All the five anticoagulants tested inhibited thrombin generation (TG) in a concentration dependent manner. We subsequently analysed the in vitro effect of different concentrations of six potential reversal agents on correcting TG parameters of maximally inhibited plasma for each anticoagulant. These were protamine sulphate at 2.5, 5 & 8μg/ml, activated FVIIa (Novoseven®) at 5, 10 & 50μg/ml, FEIBA® at 0.5,1 & 2U/ml, Beriplex® at 0.3, 0.6 & 1.2U/ml, Prothromplex® TIM4 at 0.4, 0.8 & 1.8U/ml and fresh frozen plasma (FFP) at 250, 500 & 750μl/ml. The three concentrations reflect the recommended therapeutic doses for each agent together with lower and higher doses than normally used. As predicted, UFH (final concentration 0.527U/ml) was completely reversed with a standard protamine concentration of 5μg/ml. However, the highest dose of protamine gave slightly lower TG, indicating that higher concentrations of protamine sulphate can have a paradoxical ‘anticoagulant’ effect. High doses of FEIBA (2U/ml) and FVIIa (50μg/ml) restored ~50% of thrombin generation parameters. Tinzaparin (at 1antiXaUnit/ml) was also completely neutralised by protamine. However, a higher concentration of 8μg/ml protamine was required. This effect was not seen with Enoxaparin with this higher concentration of protamine reversing only ~40% of the ETP, 21% of the peak thrombin, 71% of ttpeak and 72% of the LT. There was no positive effect of protamine on Fondaparinux (3μg/ml) and Danaparoid (1antiXa U/ml)-treated plasma. Whereas Danaparoid seemed relatively resistant to all six reversal agents, Fondaparinux effect was completely neutralised by FVIIa at concentrations between 10–50μg/ml. This study highlights the differences in neutralisation of different low molecular weight heparins and UFH. In particular, Tinzaparin was much more readily reversed with protamine sulphate than Enoxaparin. It also indicates that high doses of FVIIa could completely reverse Fondaparinux anticoagulation.

scholarly journals Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4235-4235
Author(s):  
Paula Acuña ◽  
Elena Monzón Manzano ◽  
Elena G Arias-Salgado ◽  
María Teresa Alvarez Román ◽  
Mónica Martín ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Platelet Function ◽  
Research Funding ◽  
Thrombin Generation ◽  
Bleeding Tendency ◽  
Contact Activation ◽  
Normal Range ◽  
Coagulation Tests ◽  
In Vitro Treatment

Abstract Introduction: Hematologists frequently face a percentage of patients with a mild bleeding tendency due to a haemostatic abnormality that cannot be identified with conventional laboratory techniques. Such patients are termed as having an unclassified bleeding disorder (UBD). A good diagnosis is important in order to prevent bleedings during invasive processes and/or childbirth by choosing the optimal therapeutic treatment. We aimed to investigate hemostatic parameters that may be altered in patients with UBD in order to determine the cause of their bleeding symptoms. In particular, possible defects in the tissue factor (TF)-mediated regulation of coagulation or in the plasmin generation during the fibrinolysis, as well as the possible beneficial effects of treatment with antibodies blockers of TFPI. Methods: This is a single-centre, case-control, non-interventionist, prospective study. During an 8 months-period, 40 patients with bleeding symptoms (evaluated with ISTH-BAT score) were studied. Routine coagulation tests (aPTT and PT) and platelet function testing [aggregometry, PFA-100, flow cytometry and Total Thrombus-formation Analysis System (T-TAS; Zarcos, Japan)] were performed. In 17 patients, no abnormalities were detected in platelet function and/or in coagulation tests; so the following procedures were performed: Thrombin generation test by Calibrated automated thrombography (CAT) in samples of platelet poor plasma with corn trypsin inhibitor (CTI), an inhibitor of contact activation phase, using a low amount of TF (1 pM TF and 4 µM phospholipids) as a trigger to allow the evaluation of the TF-dependent pathway. Plasmin generation (PG) test with a kit from Synapse Research Institute (Maastricht, The Netherlands), using Thrombinoscope software. TFPI activity in plasma, measured with ACTICHROME® TFPI kit (Biomedica Diagnostics, USA). The effects of rFVIIa (Novoseven, NovoNordisk; 90 µg/kg) and of a human Anti-TFPI recombinant Ab (clon mAb2021, Creative Biolabs; 400 ng/ml) were tested in CAT, PG and TFPI activity tests. Results: Those patients with aPTT, PT and a platelet function within normal range were further studied performing thrombin generation, plasmin generation and TFPI activity tests. Table 1 shows the results obtained. Samples from patients 1, 2, 4, 7, 8, 9 and 10 had a diminished generation of thrombin, and in vitro treatment with anti-TFPI and rFVIIa only ameliorated thrombin generation in samples from patients 4, 7, 8 and 9. Plasma from patients 8 and 10 had increased activity of TFPI. Generation of thrombin in samples from patients 3, 5, 6 and 11 was within normal range. Plasmin generation was increased and not modified by in vitro treatment with anti-TFPI and rFVIIa in samples 3 and 11; whereas samples 5 (with normal plasmin generation) and 6 (with no data of plasmin generation due to lack of enough sample) had a high TFPI activity in plasma that was inhibited by anti-TFPI. Normal values in all these parameters evaluated were found in six patients, indicating the involvement of different mechanisms that are still unknown. Conclusions: UBD have a diverse pathological basis for the bleeding. So, a single laboratory test to make a correct diagnosis of this pathology cannot be recommended. In accordance with this fact, a personalized treatment should be applied for each patient. Non-conventional laboratory tests need to be standardized and included for studying possible defects in the regulation of TF and/or plasmin pathways that can be involved in very rare mild bleeding phenotypes. TFPI inhibition might emerge as a good therapy for some of these patients. Failure to detect the bleeding cause in some of these patients, suggests the need to perform further studies in this field. This work was supported by Novo Nordisk Pharma S.A. Table 1- Thrombin and plasmin generation and TFPI activity in samples of patients with UBD. Results out of normal range are shown in red. LT: lagtime; ETP: endogenous thrombin potential; EPP: endogenous plasmin potential; TFPI: Tissue factor pathway inhibitor. Figure 1 Figure 1. Disclosures Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Martín: Novo Nordisk: Speakers Bureau; Pfizer: Speakers Bureau. Jiménez-Yuste: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Canales: Eusa Pharma: Consultancy, Honoraria; Sandoz: Honoraria, Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy; Gilead/Kite: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

scholarly journals Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement

2020 ◽  
Vol 39 (3) ◽  
pp. 207-217
Author(s):  
F. Poitout-Belissent ◽  
D. Culang ◽  
D. Poulin ◽  
R. Samadfan ◽  
S. Cotton ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
In Vivo Studies ◽  
Coagulation Cascade ◽  
Dependent Manner ◽  
Kinetic Assay ◽  
Thrombin Generation Assay ◽  
Coefficients Of Variation

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin–antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

scholarly journals A Neutralizing Monoclonal Antibody (PF-06741086) Against Tissue Factor Pathway Inhibitor in Combination with Activated Prothrombin Complex Concentrate Increases Hemostasis in Inhibitor Hemophilia Plasmas without Excessive Thrombin Generation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3779-3779
Author(s):  
Swapnil Rakhe ◽  
Sheryl Bowley ◽  
John E. Murphy ◽  
Debra D Pittman
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Prothrombin Complex Concentrate ◽  
Hemophilia A ◽  
Lag Time ◽  
Hemophilia B ◽  
Activated Prothrombin Complex Concentrate ◽  
Tissue Factor Pathway

Abstract Hemophilia A and B are hereditary bleeding disorders caused by intrinsic coagulation pathway deficiencies of Factor VIII or Factor IX, respectively. Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. PF-06741086 is a fully human monoclonal antibody which binds the Kunitz-2 domain and neutralizes the inhibitory activity of human tissue factor pathway inhibitor and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. Activated prothrombin complex concentrate (aPCC) is used as bypass treatment for the resolution of bleeding in some hemophilia patients with inhibitors. Hemophilia inhibitor patients receiving PF-06741086 have a possibility to also receive treatment with aPCC. The aim of the current study was to assess the potential additive effect of PF-06741086 with aPCC added in vitro to Hemophilia A and B inhibitor plasmas using a thrombin generation assay (TGA). Thrombin generation in the presence of 1 pM tissue factor and 4 µM phospholipid, was measured using the calibrated automated thrombogram (CAT) system in citrated platelet poor hemophilia A inhibitor (88-160 Bethesda Units) donor plasma or hemophilia B inhibitor (FIX immune-depleted and spiked with FIX neutralizing antibody, 14 Bethesda Units) plasma following the addition of PF-06741086 or aPCC (FEIBA) either alone or in combination. All donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone or spiked in vitro with 16 µg/mL of PF-06741086 was also included in the analysis. Non-hemophilic plasma would have the full complement of coagulation factors. Dose-dependent increases in peak thrombin were observed with the addition of aPCC alone or PF-06741086 alone to the hemophilia plasmas. For combination studies, the aPCC concentration of 1 Unit/mL was selected to correspond to plasma levels that could be achieved clinically post-dosing. The concentration of PF-06741086 at 16µg/mL in these studies was chosen to approximate the Cmax concentration following a single 300 mg subcutaneous dose. Both PF-06741086 (16 µg/mL) and aPCC (1 Unit/mL) decreased the lag time in hemophilia plasma, however, there was not an additive decrease in the lag time with the combination of PF-06741086 and aPCC. The addition of PF-06741086 in combination with aPCC to hemophilia plasma resulted in an increase in thrombin generation including a higher peak thrombin concentration compared to the addition of either alone, but was within the range reported in studies for non-hemophilic normal plasma. To summarize, the addition of aPCC (1 Unit/mL) in combination with PF-06741086 (16µg/mL) in vitro resulted in increased thrombin generation in hemophilia A and hemophilia B inhibitor plasmas without inducing excessive coagulation. Disclosures Rakhe: Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

In-vitro evaluation of anti-factor IXa aptamer on thrombin generation, clotting time, and viscoelastometry

2009 ◽  
Vol 101 (05) ◽  
pp. 827-833 ◽  
Author(s):  
Kenichi Tanaka ◽  
Fania Szlam ◽  
Christopher Rusconi ◽  
Jerrold Levy
Keyword(s):  
Thrombin Generation ◽  
Lag Time ◽  
Clot Formation ◽  
Dependent Manner ◽  
Plasma Samples ◽  
Clotting Time ◽  
Concentration Dependent Manner ◽  
Factor Ixa ◽  
Prolonged Aptt

SummaryThe REG1 system consists of factor IXa inhibitor, RB006, an ap-tamer-based anticoagulant and its antidote, RB007. The optimal use of RB006 can be facilitated by understanding its effect on the formation of thrombin and fibrin, and other standard tests of coagulation. Blood from consented volunteers was drawn into 3.2% citrate (9:1 v/v) and either used immediately or centrifuged to obtain platelet-poor plasma. Increasing concentrations of ap-tamer (6–24 μg/ml) alone or in combination with heparin (0.1 U/ml) or lepirudin (0.2 μg/ml) were added to blood and plasma samples. Activated clotting times (ACT+, low range-ACT), thrombelastometry (ROTEM™) or thrombelastography (TEG®) were performed in recalcified whole blood samples. Thrombin generation, prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in plasma samples. To some samples the antidote RB007 was added to neutralise the anticoagulation activity of RB006. In all experiment the ratio of RB006 to RB007 was kept 1:2. RB006 dose-dependently prolonged aPTT and low range-ACT, but, as expected, had no effect on PT. RB006 prolonged the lag time and decreased the peak of Actin-triggered thrombin generation. Thrombin-activated TEG demonstrated that RB006 decreases the rate of clot formation. These effects were potentiated when RB006 was combined with heparin or lepirudin. In all experiments RB007 reversed the effects of RB006 back to baseline. In conclusion, RB006 inhibits thrombin generation and clot formation in a concentration-dependent manner. It is feasible to monitor RB006 and its reversal with RB007 using aPTT, low range-ACT, and thrombin-activated TEG.

Plasma Microparticles Have Different Effects on Thrombin Generation in Platelet-Rich and Platelet-Poor Plasma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1760-1760
Author(s):  
Gleb E. Ivanov ◽  
N. Macartney ◽  
E. Stephens ◽  
N. Bowen ◽  
S. Lees ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Maximal Velocity ◽  
Physiological Level ◽  
Wide Range ◽  
Coagulation Tests ◽  
Platelet Concentration ◽  
Vitro Effect ◽  
Free Plasma

Abstract Circulating peripheral blood microparticles (MPs) of various cell origin have been described and measured in physiological and a wide range of pathological conditions. MPs are likely to play a role in coagulation either by exposure of procoagulant phospholipids or expression of tissue factor (TF), but the degree of this contribution to global haemostasis is not yet clear. We studied thrombin generation (TG) parameters (lag, peak thrombin, initial velocity (Vini) and maximal velocity (Vmax) in platelet-free (PFP) and platelet-rich plasma (PRP) of normal volunteers (n=9) in presence of corn trypsin inhibitor, using calibrated automated thrombography (CAT). MP-rich plasma was prepared by ultracentrifugation of PFP and reconstitution of pelleted MPs in a reduced volume of autologous MP-free plasma. TG was also measured in MP-depleted supernatant and platelet-free plasma (PFP) filtered through 0.1 μm filter. In MP-rich plasma, triggered with 5pM TF, with no addition of exogenous phospholipids, we found significantly increased peak TG, compared with PFP and supernatant (70.8 +/− 6.3 vs 51.4 +/− 5.0 vs 28.4 +/− 2.2 nM/L thrombin, p=0.024 and p<0.0001 respectively). MP-rich fraction also produced raised Vini (10.3 +/− 0.9 vs 5.0 +/− 0.6 thrombin nM/L/min, p=0.019) and Vmax (18.3 +/− 2.4 vs 6.8 +/− 1.0 thrombin nM/L/min, p=0.004) compared with MP-depleted supernatant. Ultracentrifugation resulted in reduction of peak TG almost by half, compared with native PFP. The augmenting effect of MP-rich plasma on thrombin peak and velocity was shown to be abolished by filtration. In our experiments removal of MPs by filtration of PFP did not affect routine clinical coagulation tests, but resulted in a significant reduction of peak TG (from 51.4 +/− 5.0 to 23.9 +/− 1.4 thrombin nM, p=0.0002), Vini (from 10.2 +/− 0.4 to 5.6 +/− 0.6, p=0.02) and Vmax (from 15.2 +/− 1.8 to 5.9 +/− 0.2, p=0.02) as compared to PFP. In order to assess the contribution of MPs to TG in presence of platelets, MP-rich plasma was added to various dilutions of PRP, using low concentration of TF (0.5pM) as a trigger. Interestingly, addition of MP-rich fraction only marginally augmented PRP with a platelet concentration of 150x109/L, but the enhancement of peak and velocity of TG became more pronounced when platelet concentration was reduced to 1.5x109/L. In a separate set of experiments, we studied TG in PRP in which MP concentration was reduced by dilution with filtered MP-free plasma as compared to PRP diluted with MP-containing PFP. Reduction in PRP MP content did not lead to a significant decrease in TG even at a low platelet concentration (1.5x109/L), when MP concentration was reduced to about 100 times below the physiological level. Our results indicate that MPs contained in PFP of normal donors significantly affect thrombin generation peak and velocity when compared to PFP in which MPs were eliminated by either ultracentifugation or filtration. The in vitro effect of an increased number of MPs on TG is less noticeable in presence of near-physiological platelet count, but contribution of MPs to TG at low platelet concentrations may potentially protect from bleeding in thrombocytopenic states and explain differences in bleeding phenotype. CAT measurement of TG in MP-rich vs MP-poor plasma could serve as a useful tool in assessing these differences.

Inhibition of In Vitro Thrombin Generation: Another Parameter Reinforcing the LMWH Heterogeneity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 912-912 ◽  
Author(s):  
Grigoris T. Gerotziafas ◽  
Anna D. Petropoulou ◽  
Mohamed Hatmi ◽  
Meyer M. Samama ◽  
Ismail Elalamy
Keyword(s):  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
In Vitro Study ◽  
Lag Time ◽  
Inhibitory Effect ◽  
Therapeutic Concentration ◽  
Unfractioned Heparin ◽  
Concentration Peak ◽  
Pathway Activation

Abstract Introduction: Low molecular weight heparins (LMWH) are derived from unfractioned heparin (UFH) by depolymerization. Thus, they present biochemical and pharmacological differences and the ratio of anti-Xa/anti-IIa activities varies from one product to another. In this study, we compared in vitro the Thrombin Generation (TG) inhibition potency of various LMWHs and UFH using the Thrombogram-Thrombinoscope® assay. Materials and Methods: TG was assessed after Tissue Factor (TF) pathway activation in Platelet Rich Plasma (PRP) (1.5x105 platelets/μl) using diluted thromboplastin (Dade Innovin®, 1:1000 final dilution). We studied five different LMWHs (Enoxaparin, Dalteparin, Nadroparin, Tinzaparin and Bemiparin), as well as UFH at five different prophylactic and therapeutic anti-Xa final concentrations. These agents were added to control plasma from 14 healthy volunteers with equivalent anti-Xa concentrations. TG was initiated by adding the triggering solution containing CaCl2 and the fluorogenic substrate. The analyzed TG parameters are the lag time, the maximal concentration of thrombin (Cmax), the time to reach Cmax (Tmax), the TG velocity and the endogenous thrombin potential (ETP). Results: Bemiparin had almost no effect on TG, with concentrations below 0.60 IUanti-Xa/ml. Dalteparin, Nadroparin and Enoxaparin showed a similar potency in inhibiting TG at equal anti-Xa concentrations. Tinzaparin proved to be the most active LMWH in inhibiting TG and had a similar potency to UFH. Tinzaparin and UFH, with the lowest anti-Xa/anti-IIa ratio, exerted their inhibitory effect mostly by prolonging lag time and Tmax and by reducing TG velocity, especially at concentration below 0.40 IU anti-Xa/ml. Besides, UFH totally inhibited TG, as expressed by ETP, at a concentration over 0.40 IU anti-Xa/ml. For a given anti-Xa/anti-IIa ratio characterizing each LMWH the IC50 for each parameter was different. The IC50 for the reduction of TG velocity was lower in comparison to the IC50 for the other parameters (Table 1). Conclusion: Our study reinforces the concept of LMWH heterogeneity and the important effect exerted by the additional anti-IIa activity combined with anti-Xa activity. Thus, their characterization can be made through their ability to inhibit TG and not only their anti-Xa/anti-IIa ratio. Nevertheless, in vitro study ignores pharmacokinetic characteristics which are important in clinical practice. Thus, Enoxaparin, at validated prophylactic concentrations (0.20–0.40 IU anti-Xa/ml) had a weak effect on TG parameters, whereas, at peak therapeutic concentrations, it showed an important inhibitory activity similar to Tinzaparin. (Table 2).The use of TG test for the biological monitoring of LMWH requires further evaluation. Table 1. The IC50 for each parameter of TG (anti-Xa IU/ml) Lag time Tmax ETP Cmax Velocity Bemiparin >1 >1 0.98 0.85 0.68 Enoxaparin 0.62 0.58 0.55 0.45 0.38 Nadroparin 0.80 0.75 0.55 0.45 0.38 Dalteparin 0.65 0.65 0.50 0.42 0.38 Tinzaparin 0.35 0.28 0.35 0.25 0.18 UFH 0.05 0.10 0.30 0.25 0.18 Table 2. Effect of Enoxaparin and Tinzaparin on TG at therapeutic concentration peak in vitro Therapeutic Concentration Peak (anti-Xa) Lag Time Tmax ETP Cmax Velocity Enoxaparin 1IU/ml 91% 94% 72% 82% 91% Tinzaparin 0.85 IU/ml >100% >100% 82% 90% 97%

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 - -

Sign in / Sign up

Export Citation Format

Share Document