scholarly journals Evaluation of the Antithrombin Potential in a Thrombin Generation Assay Performed at the Surface of Endothelial Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4168-4168
Author(s):  
Béatrice Catieau ◽  
Sami Chtourou ◽  
Jean-Luc Plantier
Keyword(s):  
Endothelial Cells ◽  
Thrombin Generation ◽  
Peak Height ◽  
Factor Ix ◽  
Flat Bottom ◽  
Thrombin Generation Assay ◽  
Heparan Sulfates ◽  
Vitro Effect ◽  
Control Plasma

Abstract Thrombin generation assay (TGA) was recently evaluated on a living endothelial-derived cell line (Coll et al. J. Thromb. Haemost. 2013; 11, 1916). This innovative assay brought into an hemostasis assay the cellular components of the anticoagulation pathway (APC and TFPI pathways) as well as a activated cell surface. It might help elucidate the relationship between hemostasis and inflammation in a more complex system. In the aim of evaluating the potential of antithrombin (AT) connecting both processes we set-up a similar assay on human vein endothelial cells (HUVEC). We first demonstrated that thrombin generation can be measured in flat-bottom 96-wells in factor IX-or factor VIII-deficient plasma substituted by either 0.1 or 1 U/ml of FIX or FVIII, respectively. Next, HUVEC were grown and expanded in a complete commercial medium (EndoGRO-LS, Millipore) for no more than 6 passages. Wells were then coated with gelatin 1% and cells seeded at 10,000 cells/well. The binding of plasma-derived AT (Aclotine ®, LFB; France dialyzed in cell culture medium) to HUVEC was demonstrated as being dose- (0.5; 1; 2.5 and 5 U/ml) and time- (0-6 hours) dependent. Saturating conditions were found using 2.5 U/ml AT for a 2h incubation. We also showed that the binding was moderately affected in the presence of heparin at concentrations up to 50 U/ml (loss of 19% of the signal) and not at all following an heparanase I+II+III treatment suggesting that another receptor(s) than cellular heparan sulfates being responsible for this interaction. The effect of AT on coagulation was then compared in the presence of cells or not. To do this cells were grown to confluence, washed with non-supplemented medium and incubated in the presence of the TGA mix (plasma containing AT or not, 0.5 pM Tissue Factor, 4 µM Phospholipids). The reaction was initiated by injection of the FluCa kit thrombin substrate (Stago). In the presence of HUVEC, the efficiency of thrombin generation from a control plasma (Unicalibrator, Stago) was decreased with a lag time increased (from 5.67 min to 6.83 min), the peak height diminished from 204.4 nM thrombin to 150.4 nM and the velocity from 55.8 nM/min to 33.4 nM/min. However, the overall amount of thrombin generated was less affected, diminishing from 1515.5 nM to 1482 nM. These data confirms that the presence of the HUVEC anticoagulants pathways can effectively diminish the thrombin generation. Without cells, the presence of 0.5, 1 or 2 U/ml AT dose-dependently decreased the generation of thrombin from the control plasma. The velocity was decreased by 23.2%, 57.6% and 75.5% and the peak height by 33.5%, 61.5% and 78.8%, respectively. When the same experiment was performed in the presence of HUVEC cells, the concentrations of AT similarly decreased the velocity by 34.2%, 54% and 70 % and the peak height by 39%, 59.1% 74.3%, respectively. There was no difference in the TGA parameters if AT was pre-incubated at the surface of the cells for up to 2h prior the TGA or if it was added extemporaneously. These results indicate that the presence of HUVEC did not modulate the in vitro effect of AT during coagulation. The effect of AT on the cell response during this process are in the process of being investigated with a particular focus on the anti-inflammatory properties of AT. Disclosures Catieau: LFB Biotechnologies: Employment. Chtourou:LFB Biotechnologies: Employment. Plantier:LFB Biotechnologies: Employment.

Reverting Rivaroxaban Effect By Activated Factor X: Assessment Using Thrombin Generation Assay

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2868-2868 ◽  
Author(s):  
Dominique Grenier ◽  
Meyer Michel Samama ◽  
Sami Chtourou ◽  
Jean-Luc Plantier
Keyword(s):  
Thrombin Generation ◽  
Lag Time ◽  
Peak Height ◽  
Factor X ◽  
Thrombin Generation Assay ◽  
Lag Times ◽  
Dose Dependent ◽  
Control Plasma

Abstract Specific anti-activated factor X molecules are currently used for the prevention and the treatment of various thromboembolic disorders. However, despite a growing use of these molecules, they are still devoid of a reliable antidote. Rivaroxaban is a specific anticoagulant targeting activated factor X (FXa). Its potential in inhibiting FXa in vitro and in vivo was demonstrated during the characterization of the molecule. However, the use of FXa to revert the effect of Rivaroxaban in plasma was never studied. To do so the measurement of thrombin generation (TG) using the calibrated automatic thrombinoscope was performed. The ability of purified human FXa (Haematologic Technologies at 10, 50, 100, 500 and 1000 ng/ml) to induce TG in a platelet-poor plasma (PPP) without the induction of the coagulation was first evaluated. There was a FXa dose-dependent TG. The TG profile at concentrations up to 50 ng/ml of FXa was similar than the control profile obtained by a PPP activated by tissue-factor (0.5 pM) and phospholipids. Above 50 ng/ml FXa, the lag time decreased and the endogeneous thrombin potential (ETP) increased with the dose. This pattern revealed the thrombogenic potential of FXa and demonstrated that a dose of 50 ng/ml (or ≈1 nM) FXa was the maximum safer dose identified by this assay. A similar experiment was performed following the activation of plasma with 0.5 pM Tissue-Factor (TF) and 4 µM phospholipids (PL) and adding FXa at 31, 62, 125, 250 and 500 ng/ml. The kinetics of TG in the presence of the different amounts of FXa differed less than when coagulation was not induced. The lag times varies from 3 to 1.83 min with the increasing concentrations of FXa and the peak heights from 120 to 212 nM, being the two most affected parameters. Following the addition of 62 ng/ml (or ≈1.25 nM) FXa, the TG was more effective than a control plasma identically stimulated. Rivaroxaban was then spiked in the PPP at the therapeutic dose of 0.35 µg/ml (or 0.8 µM). Following 0.5 pM TF/4 µM PL stimulation, this dosage completely inhibits the TG. Increasing doses of FXa (31, 62, 125, 250 and 500 ng/ml) were then added and dose-dependently restores the TG. All the parameters of the TG profile were affected by the presence of FXa. The normalization was attained at the dose of 250 ng/ml (or 5 nM) FXa. A similar set of experiment was repeated by activating the plasma with cephalin, used as a model to mimic the initiation of the contact phase coagulation. The pattern of TG was different than following FT/PL activation. With cephalin and for all FXa concentrations identical peak aspects (velocity, ETP and peak height) were obtained differing only by their lag times and times-to-peak. Lag times and times to peak were shortened by the addition of FXa from 10.7 to 3.7 min and 13.2 to 6 min respectively. Plasma were then spiked by Rivaroxaban (0.35 µg/ml) and activated by cephalin in the presence of various concentrations of FXa (31, 62, 125, 250 and 500 ng/ml). A dose-dependent TG was demonstrated with the ETP, the peak height and the velocity increasing with the amount of FXa spiked whereas the lag time and time to peak were shortened. Following the induction by cephalin, the presence of FXa systematically shortened the TG when Rivaroxaban was present or not, when compared to the TG from control plasma. This work aimed to establish the antidote potential of the natural substrate of the anti-Xa molecules and limiting the risk in promoting a thrombotic response. The calibrated thrombin generation assay was used to determine the in vitro efficiency of FXa to induce a normal thrombin generation without primary induction or following an induction by TF/PL or cephalin. The doses of FXa required to normalize coagulation in the presence of Rivaroxaban and following induction were identified. These conditions will now be assessed in vivo in Rivaroxaban treated-mice. In addition of establishing the antidote properties of FXa, this data paved the way to compare its capacities, which are optimal to inhibit such inhibitor, to further antidote in development. Disclosures Grenier: LFB BIotechnologies: Employment. Chtourou:LFB Biotechnologies: Employment. Plantier:LFB Biotechnologies: Employment.

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.

Characterization of the Procoagulant Activity of Two Different Microparticle Species.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1143-1143
Author(s):  
Ludwig Traby ◽  
Marietta Kollars ◽  
Khalid Shoumariyeh ◽  
Ansgar Weltermann ◽  
Paul Alexander Kyrle ◽  
...  
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Thrombin Generation ◽  
Procoagulant Activity ◽  
Clot Formation ◽  
Coagulation Activation ◽  
Thrombin Generation Assay ◽  
Normal Plasma ◽  
Activation Assay

Abstract Abstract 1143 Background: Microparticles (MP) exhibit procoagulant activity by exposure of tissue factor (TF) and consecutive factor (F) × activation via binding to activated FVII (FVIIa). We have shown that TF-negative MP derived from endothelial cells (EMP) after exposure to cisplatin induce thrombin generation in vitro in a TF-independent manner (Lechner et al., J. Thromb. Haemost. 2007). The procoagulant properties of TF-negative MP are not well characterized. We therefore aimed to investigate the mechanisms of coagulation activation by TF-negative EMP (obtained from human pulmonary microvascular endothelial cells - HMVEC-L) in comparison to TF-abundant MP derived from a cancer cell line (A431-MP). Methods: MP were obtained by ultracentrifugation (100,000 × g for 1 h) of cell culture supernatant. The procoagulant activity was measured in normal plasma and in plasmas deficient in coagulation factors VII, VIII, IX, X, XI, and XII, respectively, by an in vitro thrombin generation assay (Technothrombin TGA, Technoclone, Austria), by activated partial thromboplastin (aPTT), prothrombin time (PT) and a chromogenic FIX activation assay. Antibodies to TF and FVII (both American Diagnostica, USA) were used. Plasmas used in the thrombin generation assay were MP-depleted by ultracentrifugation before use. Results: EMP and A431-MP induced in vitro thrombin generation in normal plasma. In vitro thrombin generation induced by EMP was abolished in plasmas deficient in FVIII or FIX, and was markedly reduced in FXI-deficient plasma. In FVII-deficient plasma, normal thrombin generation after addition of EMP was seen. In contrast, A431-MP-triggered thrombin generation was abolished in FVII-deficient plasma, but was not influenced by depletion of FVIII, FIX or FXI. In FX-deficient plasma, thrombin generation could not be triggered by addition of either EMP or A431-MP. Thrombin generation was normal after addition of EMP or A431-MP to FXII-deficient plasma (Figure). In a modified aPTT system using kaolin as surface activator, EMP and A431-MP induced clot formation in normal plasma. In a modified PT system only A431-MP but not EMP induced clot formation. We then investigated whether MP can directly activate FIX. In a plasma-free environment FIX activation by A431-MP was much more pronounced than by EMP (14-fold vs. 2.4 fold increase compared to a control experiment with cell culture medium only). FIX activation by A431-MP was blocked by the addition of antibodies to TF and FVII, while no such effect was seen after addition to EMP. Conclusions: Our findings show that both TF-positive and TF-negative MP exhibit procoagulant activity. If TF is expressed (A431-MP), coagulation activation is triggered via FVII/FVIIa, which is abolished in FVII-deficient plasma or by the addition of antibodies to TF or FVII. TF-negative EMP induce thrombin generation via the intrinsic coagulation pathway by activating FXI and FIX, as shown by the generation of activated FIX in a FIX activation assay. EMP are also able to induce clot formation in an aPTT based system. We surmise that the procoagulatory effect of EMP may be due to their high phospholipid content or to a specific phospholipid surface composition. The results of our FIX activation assay also confirm a direct FIX activation by TF/FVIIa, which can be abolished by addition of antibodies to TF or FVII. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mechanistic Studies of Thrombin Generation Assay to Evaluate Procoagulant Potential of Fitusiran

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-6
Author(s):  
Sravya Kattula ◽  
Ane Salvador ◽  
Shaobin Wang ◽  
Ayman Ismail ◽  
Arjan van der Flier ◽  
...  
Keyword(s):  
Complex Formation ◽  
Thrombin Generation ◽  
Active Role ◽  
Peak Height ◽  
Publicly Traded ◽  
Time To Peak ◽  
Thrombin Generation Assay ◽  
Hemostatic Activity ◽  
The Impact

Introduction: Fitusiran is a once-monthly subcutaneously administered RNA interference therapeutic targeting antithrombin III (AT) to rebalance hemostasis in patients with hemophilia A and B with or without inhibitors. As for other non-FVIII therapies in development, there is a need to better assess the procoagulant potential of fitusiran. Using thrombin generation assays (TGA), we studied the hemostatic activity in hemA plasma with low AT levels in the presence and absence of varying factor VIII (FVIII) levels to mimic conditions associated with breakthrough bleeds. To better understand the impact of AT depletion in the assay, we monitored multiple TGA parameters as well as the levels of several coagulation components potentially influencing the reactions. Methods: Pooled HemA plasma was applied to an AT affinity resin to remove AT. Residual AT activity was 5% and additional AT was spiked back to produce HemA plasmas with various amounts of AT (5, 10, 20 and 100%). FVIII was spiked into the 4 plasmas at 0, 5, 10, 20, 30, 50, and 100 IU/dL and all samples were analyzed by Calibrated Automated Thrombography (Stago). Thrombin generation parameters were calculated by thrombinoscope software.After the TGA reaction, samples were further analyzed by immunoblot for evaluation of thrombin-AT (TAT) and alpha2-macroglobulin-thrombin (a2M:T) complex formation, as well as remaining (pro)thrombin, AT and a2M levels . In parallel, a2M:T complex levels were assessed by ELISA capture of a2M followed by addition of thrombin substrate to directly measure the thrombin activity stemming from a2M-inhibited thrombin. . Results: TGA parameters for FVIII titrations in HemA plasmas with reduced AT were calculated and compared to the FVIII standard curves generated in HemA plasma with normal AT levels for each TGA parameter (lag time, time to peak, peak height, ETP and velocity index). As expected, a greater reduction of AT activity in hemA plasma correlated with increased thrombin generation compared to hemA plasma (with 100% AT). The apparent FVIII-like activity observed at low AT conditions in HemA plasma was dependent on the TGA parameter evaluated, with velocity index suggesting the lowest and ETP the highest FVIII-like activity. Furthermore, particularly at lower starting levels of AT, AT is depleted during the TGA reaction and leads to enhanced levels of a2M:T complex as observed by immunoblot or a2M:T chromocapture assays. Importantly, the a2M:T complex is capable of cleaving the assay substrate and contributing to the TGA signal. Typically, the a2M:T generated signal is calculated and substracted by the thrombinoscope software. However, under low AT conditions, increased levels of a2M-T lead to substrate consumption at later stages of the reaction, resulting in erroneous underestimation of the a2M:T activity that does not agree with direct measurement of a2M: T levels. These limitations need to be taken into consideration when interpreting TGA data under conditions of low AT activity. While this remains an area of investigation, parameters derived earlier in the reaction prior to AT depletion and significant levels of a2M:T complex formation may better correlate with the true hemostatic potential associated with AT inhibition. Conclusion: Our results show that TGA can be a valuable tool to measure the hemostatic potential associated with fitusiran therapy. We demonstrate that reduced levels of AT are associated with enhanced thrombin generation, but that activity comparison to FVIII varies significantly depending on the TGA parameter analyzed. In addition we show that under low AT conditions, AT can become depleted leading to a2M:T complex accumulation which may lead to overestimation of some TGA parameters. This overestimation is likely an artifact of the TGA method as it is a closed in vitro system that can be influenced by the depletion of components or accumulation of products. Our results suggest that TGA parameters calculated early in the reaction are less influenced by some of these limitations and may be better predictors of the true hemostatic activity of fitusiran. Furthermore, a2M may play a more active role during fitusiran treatment, ensuring free thrombin does not migrate outside of the site of injury. Disclosures Kattula: Sanofi: Current Employment, Current equity holder in publicly-traded company. Salvador:Sanofi: Current Employment, Current equity holder in publicly-traded company. Wang:Sanofi: Current Employment, Current equity holder in publicly-traded company. Ismail:Sanofi: Current Employment, Current equity holder in publicly-traded company. van der Flier:Sanofi: Current Employment, Current equity holder in publicly-traded company. Leksa:Sanofi: Current Employment, Current equity holder in publicly-traded company. Salas:Sanofi: Current Employment, Current equity holder in publicly-traded company.

Thrombin generation as an intermediate phenotype for venous thrombosis

2010 ◽  
Vol 103 (01) ◽  
pp. 114-122 ◽  
Author(s):  
Olivier Segers ◽  
René van Oerle ◽  
Hugo ten Cate ◽  
Jan Rosing ◽  
Elisabetta Castoldi
Keyword(s):  
Genetic Variation ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Coagulation Factors ◽  
Peak Height ◽  
Intermediate Phenotype ◽  
Nucleotide Polymorphisms ◽  
Thrombin Generation Assay

Summary In vitro thrombin generation, which reflects an individual’s plasma coagulation potential and has been shown to correlate with the risk of venous thromboembolism (VTE), might represent a useful intermediate phenotype for the genetic dissection of VTE. As a proof of principle, we have investigated whether the thrombin generation assay can detect changes in the haemostatic balance associated with common genetic variation affecting the level or function of coagulation factors and inhibitors. The study population consisted of 140 healthy individuals. Plasma levels of coagulation factors and inhibitors and thrombin generation parameters determined at low tissue factor (TF) ± thrombomodulin (TM) and at high TF ± activated protein C (APC) were available from a previous study. All individuals were genotyped for F5 Leiden, F2 G20210A and 19 additional single nucleotide polymorphisms (SNPs) in haemostasis-related genes. The association of each SNP with plasma levels of the corresponding proteins and with thrombin generation parameters (lag time, peak height and endogenous thrombin potential [ETP]) was evaluated by statistical analysis. Not only F5 Leiden and F2 G20210A, but also several other common SNPs, significantly affected thrombin generation parameters. In particular, FGA A1069G (Thr312Ala) decreased the ETP-APC, F2 A19911G increased the ETP-APC, F10 IVS2 C+517G decreased the ETP+APC, F12 C-46T decreased peak height at low TF, and TFPI T-287C and TFPI IVS7 T-33C decreased the ETP+APC. These results indicate that the thrombin generation assay is sensitive to genetic variation in haemostasis-related genes, which makes it a promising tool to identify novel genetic risk factors of VTE.

The Effect of Factor IXa on Thrombin Generation Activity Determination: rFIXFc Vs. BeneFIX®

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2266-2266
Author(s):  
Yang Buyue ◽  
Ekta Seth Chhabra ◽  
Li Wang ◽  
Jurg M Sommer
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Time Course ◽  
Drug Product ◽  
Factor Ix ◽  
One Stage ◽  
Thrombin Generation Assay ◽  
Factor Ixa ◽  
Recombinant Factor Ix

Abstract Abstract 2266 Recombinant Factor IX Fc fusion (rFIXFc) was designed to be a long-lasting version of recombinant FIX that has the potential to provide less frequent dosing. This may be applicable to prophylaxis and on-demand therapy of hemophilia B and for control of bleeding during surgery. The potency of the rFIXFc drug product, as well as the commercially available recombinant factor IX, BeneFIX®, is assigned by a one-stage clotting assay. The thrombin generation assay (TGA), a global hemostasis assay that monitors the amount of active thrombin produced in patient plasma after recalcification, represents a useful indication in the evaluation of coagulation capacity of hemophilic plasma. When equal units of rFIXFc and BeneFIX, as determined by the one stage assay, were spiked into hemophilic plasma and their coagulation capacity was assessed by the TGA, BeneFIX generated 2-fold higher peak thrombin and significantly left-shifted thrombin curve relative to rFIXFc in the presence of limiting tissue factor and 4 μM phospholipids. In an assay control without tissue factor triggering, BeneFIX demonstrated considerable thrombogenic activity, whereas rFIXFc was essentially inactive. Since BeneFIX was observed to have a markedly higher level of FIXa impurity than rFIXFc in a factor IXa ELISA, and since factor IXa protease is known to be highly thrombogenic in an in vivo model, we hypothesized that the enhanced in vitro thrombin generation profile of BeneFIX may be due to the presence of excess factor IXa. BeneFIX was incubated overnight with a serine protease active site blocker, EGR-chloromethyl ketone, and dialyzed by extensive buffer exchange. The FIXa-blocked BeneFIX showed very similar thrombin generation profile (ETP, peak thrombin, time course and slope) to rFIXFc, confirming the role of FIXa in thrombin generation by BeneFIX. To quantify the amount of active factor IXa in BeneFIX and rFIXFc, a plasma-derived factor IXa (pFIXa) standard curve was constructed by spiking increasing concentrations of factor IXa (0–100 pM) into human factor IX-deficient plasma in the presence of 4 μM phospholipids. Prior to starting the measurement, 5 nM thrombin was added to the assay in order to improve sensitivity. A dose response was observed with a detection limit as low as 0.5 pM pFIXa in FIX-deficient plasma. BeneFIX, FIXa-blocked BeneFIX and rFIXFc of equal potency (1 IU/mL by the one-stage clotting assay) generated thrombin responses comparable to 20 pM, 1 pM and 2 pM pFIXa, respectively, indicating the amount of FIXa present in each FIX product. In a regular thrombin generation assay triggered with limiting tissue factor, 1 IU/mL rFIXFc supplemented with 20 pM pFIXa demonstrated an equal peak thrombin and velocity index to 1 IU/mL BeneFIX. These data suggest that: 1) Minor amounts of FIXa protease in a FIX drug product (0.1%) can trigger significant thrombin generation in global hemostasis assays 2) Thrombin generation assay could be used to evaluate FIXa level in FIX products with high sensitivity (0.5 pM FIXa per IU/ml FIX) 3) the higher peak thrombin and shortened time course in the thrombin generation profile for BeneFIX relative to rFIXFc is caused entirely by the presence of factor IXa in the drug product 4) Discounting the rFIXa impurities in these drug products, BeneFIX and rFIXFc have equivalent in vitro thrombin generation activity per unit of FIX activity. Disclosures: Buyue: Biogen Idec Hemophilia: Employment. Seth Chhabra:Biogen Idec Hemophilia: Employment. Wang:Biogen Idec Hemophilia: Employment. Sommer:Biogen Idec Hemophilia: Employment.

Effects of argatroban, danaparoid, and fondaparinux on trombin generation in heparin-induced thrombocytopenia

2013 ◽  
Vol 109 (03) ◽  
pp. 504-509 ◽  
Author(s):  
Marion Combe ◽  
Michèle Piot ◽  
Céline Chapelle ◽  
Majid Akrour ◽  
Bernard Tardy ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Study Objective ◽  
Heparin Induced Thrombocytopenia ◽  
Thrombin Generation Assay ◽  
Vitro Effect ◽  
Vitro Data ◽  
In Vitro Data

SummaryThere is no in vitro data on the comparison of the effects of danaparoid, argatroban and fondaparinux on thrombin generation in patients with heparin-induced thrombocytopenia. It was the study objective to compare the in vitro anticoagulant potential of argatroban, danaparoid and fondaparinux using a thrombin generation assay TGA on a mixture of control platelet-rich plasma (PRP) and HIT patient platelet-poor plasma (PPP). The plasma of seven patients with a clear HIT diagnosed at our institution was selected. Mixtures of donor PRP and patient PPP were incubated with unfractionated heparin 0.2 U.mL-1, argatroban at 600 ng.mL-1, argatroban at 400 ng.mL-1, danaparoid at 0.65 IU.mL-1 and fondaparinux at 1 [uni03BC]g.mL-1. Thrombin generation was assessed by calibrated thrombinography. The percentage of inhibition of the endogenous thrombin potential observed with argatroban at 600 ng.mL-1 was statistically significantly higher compared with those observed with fondaparinux (median: 53.6% vs. 3.9%; p = 0.031) but not compared with argatroban at 400 ng.mL-1 and danaparoid. The percentage of inhibition of the thrombin peak observed with argatroban at 600 ng.mL-1 was statistically significantly higher compared with those observed with danaparoid (median: 71.2 vs. 56.8; p = 0.031) and fondaparinux (mean: 71.2 vs. 30; p = 0.031) but not with argatroban at 400 ng.mL-1. In conclusion, the in vitro effect of argatroban and danaparoid on thrombin generation seems to corroborate the results of clinical studies of these drugs in the treatment of HIT in term of efficiency. Fondaparinux showed a very small effect on thrombin generation evaluated by calibrated thrombinography.

scholarly journals The in Vitro Effect on Thrombin Generation of Adding rFVIII or rFIX to Hemophilia a or b Plasma in the Absence or Presence of Concizumab

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Marianne Kjalke ◽  
Søren Andersen
Keyword(s):  
Drug Interaction ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Plasma Samples ◽  
Publicly Traded ◽  
Plasma Pool ◽  
Generation Capacity ◽  
Drug Drug Interaction ◽  
Vitro Effect

Introduction: Lack of factor VIII/IX (FVIII/FIX) in hemophilia A/B (HA/HB), respectively, results in reduced thrombin generation, leading to recurrent/spontaneous bleeds. Concizumab is an anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody, currently under clinical investigation for subcutaneous prophylaxis of HA/HB patients with/without inhibitors. Breakthrough bleeds occurring in HA/HB patients while on concizumab prophylaxis may be treated with FVIII/FIX. We aimed to compare the in vitro effect of recombinant FVIII (rFVIII) and FIX (rFIX) in HA and HB plasma, respectively, in the absence or presence of concizumab. Methods: rFVIII/rFIX was added to HA/HB pooled plasma at 0.25, 0.5 or 1 IU/mL (corresponding to post-administration plasma concentrations of 12.5, 25 and 50 IU/kg rFVIII and 12.5−25, 25−50 and 50−100 IU/kg rFIX) in the absence or presence of concizumab (1,500, 4,500 or 15,000 ng/mL). In a separate experiment, 33 plasma samples from eight HA patients, who were on concizumab prophylaxis as part of the phase 2 explorer5 trial (NCT03196297), were spiked with 0.5, 1 and 1.5 IU/mL rFVIII. Pre-dose samples (before concizumab prophylaxis) from seven of these patients were also included. Thrombin generation was measured after initiation with 1 pM tissue factor (PPP-Low, Thrombinoscope). Statistical analysis of the effects conferred by each (combination of) drug(s) was performed by ANOVA analyses. Results: A significant (p<0.001) and concentration-dependent increase in thrombin peak was observed when HA plasma pool samples were spiked with rFVIII, both in the absence and presence of concizumab. Likewise, concizumab increased the thrombin peak both in the absence and in presence of rFVIII. Increasing concizumab from 1,500 to 4,500 and 15,000 ng/mL only slightly increased the thrombin peak further, demonstrating that a close-to-maximal effect on thrombin peak was achieved at 1,500 ng/mL concizumab. The effects of concizumab and rFVIII were mainly additive with an up to 20% additional effect caused by drug-drug interaction. The addition of rFVIII to explorer5 patient plasma samples resulted in a significant and concentration-dependent increase in thrombin peak. The effects observed for rFVIII and concizumab were exclusively additive. The thrombin peak obtained with 1.0 IU/mL rFVIII before concizumab administration was lower than with 0.5 IU/mL rFVIII in the presence of concizumab. This suggests that a 2-fold reduced rFVIII dose may be sufficient to achieve the same plasma thrombin generation capacity as with the standard rFVIII dose in the absence of concizumab. The addition of rFIX to a HB plasma pool increased the thrombin peak significantly (p<0.001) and in a concentration-dependent manner both in the absence and presence of concizumab (1,500 ng/mL). Likewise, concizumab increased the thrombin peak at all rFIX concentrations (p<0.001). Increasing concizumab from 1,500 to 4,500 and 15,000 ng/mL had no or limited further effect. The effects of concizumab and rFIX were mainly additive with an up to 10% effect conferred by negative drug-drug interaction for 1 IU/mL rFIX combined with concizumab >1,500 ng/mL and 0.5 IU/mL rFIX combined with 15,000 ng/mL concizumab, i.e., a 10% smaller effect of rFIX was observed in the presence of concizumab than in its absence. The thrombin peak obtained upon adding 1.0 IU/mL rFIX to plasma without concizumab was similar to the thrombin peak in the presence of concizumab and 0.5 IU/mL rFIX. This suggests that in the presence of concizumab, a 2-fold reduced dose of rFIX would be sufficient to obtain the same plasma thrombin generation capacity as with 1.0 IU/mL rFIX in the absence of concizumab. Conclusion: rFVIII/rFIX increased the thrombin peak in HA and HB plasma, respectively, both in the absence and presence of concizumab. The combined effects of rFVIII/rFIX with concizumab were mainly additive with an up to 20% additional effect caused by drug-drug interaction with rFVIII and a 10% reduction with rFIX. No signs of exaggerated thrombin generation were observed by combining concizumab with rFVIII/rFIX. Therefore, the data support rFVIII/rFIX use for bleed treatment in patients on concizumab prophylaxis. As rFVIII/rFIX and concizumab have additive effects in terms of thrombin generation capacity, data suggest that clinical effectiveness could be achieved with rFVIII/rFIX doses in the lower range recommended for such products. Disclosures Kjalke: Novo Nordisk A/S: Current Employment, Current equity holder in publicly-traded company. Andersen:Novo Nordisk A/S: Current Employment, Current equity holder in publicly-traded company.

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