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.

In vitro effects of human neutrophil cathepsin G on thrombin generation: Both acceleration and decreased potential

2010 ◽  
Vol 104 (09) ◽  
pp. 514-522 ◽  
Author(s):  
Thomas Lecompte ◽  
Agnès Tournier ◽  
Lise Morlon ◽  
Monique Marchand-Arvier ◽  
Claude Vigneron ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Time Course ◽  
Anticoagulant Activity ◽  
Cathepsin G ◽  
Coagulation Cascade ◽  
Clotting Factor ◽  
Factor V ◽  
Clotting Factors

SummaryCathepsin G (Cath G), a serine-protease found in neutrophils, has been reported to have effects that could either facilitate or impede coagulation. Thrombin generation (CAT method) was chosen to study its overall effect on the process, at a plasma concentration (240 nM) observed after neutrophil activation. Coagulation was triggered by tissue factor in the presence of platelets or phospholipid vesicles. To help identify potential targets of Cath G, plasma depleted of clotting factors or of inhibitors was used. Cath G induced a puzzling combination of two diverging effects of varying intensities depending on the phospholipid surface provided: accelerating the process under the three conditions (shortened clotting time by up to 30%), and impeding the process during the same thrombin generation time-course since thrombin peak and ETP (total thrombin potential) were decreased, up to 45% and 12%, respectively, suggestive of deficient prothrombinase. This is consistent with Cath G working on at least two targets in the coagulation cascade. Our data indicate that coagulation acceleration can be attributed neither to platelet activation and nor to activation of a clotting factor. When TFPI (tissue factor pathway inhibitor) was absent, no effect on lag time was observed and the anticoagulant activity of TFPI was decreased in the presence of Cath G. Consistent with the literature and the hypothesis of deficient prothrombinase, experiments using Russel’s Viper Venom indicate that the anticoagulant effect can be attributed to a deleterious effect on factor V. The clinical relevance of these findings deserves to be studied.

The Impact of Factor IX Level on Thrombin Generation and Bleeding During Warfarin Anticoagulation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 541-541
Author(s):  
Yesim Dargaud ◽  
Maureane Hoffman ◽  
Claude Negrier ◽  
Leana Lefrapper ◽  
Dougald M. Monroe
Keyword(s):  
Tissue Factor ◽  
Vitamin K ◽  
Intracranial Haemorrhage ◽  
Thrombin Generation ◽  
Factor Ix ◽  
Target Range ◽  
Significant Difference ◽  
Generation Capacity ◽  
The Impact

Abstract Abstract 541 Bleeding occurs in from 10 – 16% of warfarin-treated patients. Having a PT-INR in the target range is associated with better outcomes. However, even patients with an INR in the target range of 2–3 can suffer bleeding, suggesting that INR does not perfectly reflect the therapeutic effect of warfarin. The goal of our studies was to determine whether the level of specific coagulation factors could predict the risk of bleeding while the INR was in the target range. We modeled warfarin anticoagulation in our previously published in vitro cell based-model by adjusting the levels of vitamin K-dependent factors to those of patients with an INR of 2–3. We then examined the effect of variations in the level of FIX. The cogulation reactions were initiated by monocyte-expressed tissue factor (assayed at 1pM). Variation in FIX had a marked effect on thrombin generation. However, in plasma with the same levels of factors, as expected, variations in FIX had no effect on the PT-INR. Thus, we hypothesized that a subject with a lower FIX level than average may have a lower level of thrombin generation than is indicated by the INR. The INR might, therefore, underestimate the level of anticoagulation in such a subject. If s/he is maintained in the “therapeutic range” as measured by the INR, s/he will actually be over-anticoagulated and prone to hemorrhage. A prospective, single centre clinical study has been carried out to test this hypothesis in warfarinized patients. Between October 2010 and June 2011, 312 consecutive patients admitted to the emergency department of Edouard Herriot Hospital in Lyon, with an INR between 1.8 and 3.2, were included in the study after obtaining informed consent. Twenty six patients were admitted for a bleeding episode, 18 for recurrent thrombosis and 268 for other medical reasons. Patients presenting with bleeding, 17 males and 9 females, were aged 74±14 years old compared to the rest of the patients aged 76±14. Among the 26 bleeders, 7 had a spontaneous intracranial haemorrhage, 2 had a trauma-induced intracranial haemorrhage, 12 presented a gastrointestinal bleeding and 5 exhibited muscle hematomas, severe epistaxis or urinary tract bleeding. PT-INR and vitamin K-dependent factor levels were determined in all patients. Thrombin generation capacity in platelet poor plasma was measured using Calibrated Automated Thrombin generation assay (Thrombinoscope bv, Maastricht, The Netherlands), with tissue factor 1pM and phospholipids PC:PS:PE 4μM. No statistically significant difference was observed in the PT-INR of bleeding patients (INR=2.4±0.4) and those having a thrombosis (INR=2.5±0.5) or patients admitted for other reasons (INR=2.6±0.2). Plasma prothrombin and factor × levels were also similar in all three groups. However, a statistically lower plasma factor IX activity was observed in bleeders (p=0.01, Mann Whitney test) compared to other groups, 47.6±20 IU/dL vs. 63±33 IU/dL. In all the warfarinized subjects with an INR between 1.8 and 3.2, no correlation was found between thrombin generation capacity and PT-INR results (p=0.85, Spearman correlation test). However, a statistically significant correlation was observed between thrombin generation capacity and factor IX levels (p=0.0002). In patients, presenting with warfarin-related haemorrhage, the endogenous thrombin potential (ETP) was significantly lower at 340±335 nM.min (p=0.05) then that of warfarinized subjects who did not suffer bleeding (ETP 406±215 nM.min). These data support our hypothesis based on our in vitro results and show that patients who bleed when their PT-INR is in the target range 2 – 3 might have defective thrombin generation related to a lower level of factor IX than expected. Thus, our results suggest that the appropriate target INR level might not be the same for all patients. Those with factor IX levels that differ significantly from the mean of the population might be managed best by selecting a target INR that is based on the level of thrombin generation. Of course, a “target range” for parameters of thrombin generation during warfarin therapy would need to be developed if the assay were to be used for this purpose. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Factor XI contributes to thrombin generation in the absence of factor XII

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Dmitri V. Kravtsov ◽  
Anton Matafonov ◽  
Erik I. Tucker ◽  
Mao-fu Sun ◽  
Peter N. Walsh ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Xii ◽  
Factor Xi ◽  
Factor Xii Deficiency ◽  
Low Concentrations

Abstract During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or α-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

scholarly journals The Relationship between Thrombin Generation Assay and FVIII Levels in Patients with Mild to Moderate Haemophilia (A)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2454-2454
Author(s):  
Pu-Lin Luo ◽  
Steven K. Austin ◽  
Kiran Parmar ◽  
Dan P Hart ◽  
Michael Laffan
Keyword(s):  
Research Funding ◽  
Thrombin Generation ◽  
Haemophilia A ◽  
Contact Activation ◽  
Extrinsic Pathway ◽  
Two Stage ◽  
One Stage ◽  
Thrombin Generation Assay ◽  
The Relationship

Abstract Introduction Haemophilia A (HA) phenotypes (mild, moderate and severe) are based on the baseline FVIII levels, however considerable variation in the bleeding phenotype exists between patients with similar FVIII level. Moreover, approximately 40% of patients with mild HA have large discrepancies between FVIII measured by one stage (FVIII:C1) and two stage methods (FVIII:Chr2) and it is unclear which method correlates best with in vivo FVIII function and bleeding phenotype. The Thrombin Generation assay (TGA), a global measure of haemostasis may be a better predictor of bleeding phenotype but pre-analytical factors such as contact activation can confound the results. Choice of initiating conditions may also be critical in determining sensitivity: recent studies have suggested that initiation with FIXa rather than tissue factor (TF) in detecting low levels of FVIII:C in severe HA, however its utility in mild to moderate HA patients has yet to be determined. The aim of this study is to establish the relationship between FVIII:C and TGA and the influence of contact factor activation in TF and FIXa triggered TGA in patients with mild to moderate HA. Methods This is a prospective cohort study. Patients aged >18 with known congenital HA and FVIII:C 0.01- 0.2 iu/ml were recruited from 3 Haemophilia Comprehensive Care Centres in London. Peripheral blood was drawn into citrate Vacutainer tubes containing 0.106M trisodium citrate (1:9 volume) and Vacutainer tubes preloaded with CTI (50µg/ml). Samples underwent double centrifugation (2500g) to obtain platelet free plasma. Thrombin generation assay, using a standard calibrated automated thrombogram was triggered with either TF (1pmol) or FIXa (5nM). Factor FVIII levels were assessed by one stage APTT based (FVIII:C1) and two stage chromogenic (FVIII:Chr2) methods. Mutation analysis was carried out in all patients. Results 40 patients were recruited in the study. Five patients (13%) had standard FVIII discrepancy (FVIII:C1/FVIII:Chr2>1.5) with 4 different FVIII mutations located on the inter-domain surface of the A2 domain (p.Tyr683Ser, p.Arg550Cys, p.Gly498Arg, p.MET681.Le). One patient had reverse FVIII discrepancy. In TF triggered TGA, the presence of CTI resulted in significant reduction in mean ETP (nmol .min)(455. vs 278, p<0.01, 95% CI 104-243), mean Peak thrombin (nM) (37.81 vs 16.54, t(6.6) p<0.01 95%CI 14.7-27.3), and mean Velindex (nM/min) (4.86 vs 1.29 t(7.0), p<0.01, 95% CI2.3-4.19) and a longer mean ttPeak (min) (14.26 vs 16.22, t(-3.2) p=0.02 95% CI-3.1- -0.76). In contrast, the presence of CTI did not affect ETP (1143 vs 1042, p=0.19 95% CI -54-256), mean Peak thrombin (252 vs 251, p=0.6 95%CI 27-46) or Velindex (118.54 vs 119.15 p= 0.95, 95%CI -23-12.9) in FIXa triggered TGA. There was a good correlation between FVIII:Chr2 and ETP (r=0.56, p=<0.001) Peak (r=0.6, p=<0.001) and Velindex (r=0.7, p=<0.001) in TF(CTI-) triggered TGA, however no relationship was seen between FVIII:C and TG parameters (ETP r=-0.01 p=0.9, Peak r=-0.003, p=0.97 and Velindex r=0.018, p=0.9) in TF(CTI+) triggered TGA. In both FIXa(CTI-) and FIXa (CTI+) triggered TGA, there was a good correlation seen between Lagtime (r=-0.6 p=<0.01), Peak (r=0.4-0.6, p=<0.01) ttpeak (r= -0.6, p=<0.01) and Velindex (r=0.69 <0.01) with FVIII:Chr2 but not with ETP. In patients with standard FVIII discrepancy (n=5), their ETP and Peak levels in TF and FIXa triggered TGA were in keeping with the ETP and Peak levels of non-discrepant patients with similar FVIII:C2 and significantly lower than that of non-discrepant patients with similar FVIII:C1. Conclusions Our study confirms that at low TF triggered TG, contact factor activation in vitro is an important preanalytical variable. Curiously any TG correlation with FVIII level is lost once the contact pathway is inhibited suggesting that TG remains largely determined by the extrinsic pathway in this system. In contrast, factor FIXa triggered TG is unaffected by inhibition of contact activation and demonstrates a good correlation to FVIII:C with or without CTI. This can be explained by suggesting that the supply of FIXa negates any effect of XIa from contact activation and that TG by this route is more completely dependent on FVIII. Therefore a FIXa triggered TGA may offer a better alternative in the assessment of haemophilia and further studies are underway to determine whether this is a better predictor of bleeding phenotypes. Disclosures Luo: Pfizer: Research Funding. Austin:Pfizer: Research Funding. Laffan:Pfizer: Honoraria; Roche: Consultancy, Speakers Bureau.

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.

Depolymerized Holothurian Glycosaminoglycan Inhibits Plasma Thrombin Generation Via Interaction with the Factor IXa Heparin-Binding Exosite

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3081-3081
Author(s):  
Buyue Yang ◽  
John P. Sheehan
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Fold Increase ◽  
Factor Ix ◽  
Heparin Binding ◽  
Thrombin Inhibition ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Recombinant Factor Ix ◽  
Inhibition Of Thrombin

Abstract Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties in rodent thrombosis and dog hemodialysis models. DHG demonstrates significantly less bleeding in template or tail transection assays than therapeutically equivalent doses of heparins. Several potential in vitro mechanisms have been described for DHG, including acceleration of thrombin inhibition by heparin cofactor II (HCII), inhibition of factor VIII activation by thrombin, and inhibition of factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). The relevant mechanism(s) for inhibition of tissue factor (TF) induced plasma thrombin generation by DHG were examined in HCII or mock-immunodepleted, and factor-deficient human plasmas, using selected recombinant factor IX(a) with mutations in the heparin-binding exosite. Plasma thrombin generation was detected by fluorogenic substrate cleavage in the presence of corn trypin inhibitor to block contact activation, and compared to a standard curve generated with α2-macroglobulin-thrombin complex. The dose-dependent decrease in velocity index, a parameter reflecting the rate of thrombin generation between lag phase and peak thrombin concentration, was used to compare DHG potency. When triggered by 0.2 pM TF, the EC50 for inhibition of thrombin generation by DHG was 0.16 ± 0.01 μM in both HCII-depleted and mock-depleted plasma, suggesting that DHG acts independently of HCII. When triggered by excess (4 pM) TF, plasma thrombin generation was independent of factors VIII and IX. Under these conditions, the EC50 for DHG inhibition of thrombin generation was increased 13-fold in mock-depleted plasma (2.02 ± 0.09 μM) and 28-fold in HCII-depleted plasma (4.31 ± 0.23 μM). These results suggest that components of the intrinsic tenase complex contribute to inhibition of plasma thrombin generation by DHG, and HCII contributes only at high tissue factor concentrations. In the presence of 0.2 pM TF, Western blotting under nonreducing conditions showed preservation of the prothrombin/meizothrombin band and delayed/reduced thrombin generation in the presence of 0.5 μM DHG, confirming that the inhibition involves reduced prothrombin activation rather than accelerated thrombin inhibition. When triggered by 0.2 pM TF in factor VIII-deficient plasma supplemented with 700 pM factor VIII or thrombin-activated factor VIIIa, the EC50 for inhibition by DHG was 0.41 ± 0.02 μM and 0.44 ± 0.05 μM, respectively. Similarly, the EC50 for DHG inhibition of thrombin generation in factor IX deficient plasma supplemented with 0.2 pM TF and 100% plasma-derived factor IX (90 nM), or 100 pM plasma-derived factor IXa alone, was 0.36 ± 0.01 μM and 0.34 ± 0.02 μM, respectively. Thus, activation of factors VIII and IX do not contribute significantly to the inhibition mechanism for DHG. The contribution of intrinsic tenase activity to DHG inhibition of plasma thrombin generation was assessed using recombinant factor IX(a) mutants with moderate (R170A) or marked (R233A) reductions in heparin affinity. Factor IX deficient plasma was supplemented with 0.2 pM TF and 100% recombinant factor IX, or 100 pM factor IXa, with increasing concentrations of DHG. Similar to plasma-derived factor IX(a), DHG demonstrated an EC50 of 0.38 ± 0.01 μM for inhibition of thrombin generation in the presence of factor IX(a) wild type (WT) zymogen or protease. In the presence of factor IX(a) R170A, the EC50 for DHG was 0.86 ± 0.06 μM and 1.02 ± 0.02 μM, respectively, a 2–3 fold increase relative to WT (P ≤ 0.01). For factor IX(a) R233A, the EC50 for DHG was 3.55 ± 0.47 μM for zymogen and 2.98 ± 0.64 μM for protease, an 8–9 fold increase relative to WT (P ≤ 0.01). Thus, mutations in the factor IXa heparin-binding exosite induced resistance to DHG inhibition of thrombin generation as follows: factor IX(a) R233A&gt; R170A&gt; WT. These findings are consistent with the common mechanism for intrinsic tenase inhibition demonstrated for heparin and DHG in purified systems, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition of plasma thrombin generation by DHG. This antithrombin-independent mechanism likely mediates the antithrombotic efficacy of DHG and related glycosaminoglycans, and may represent a novel therapeutic target with lower bleeding risk.

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.

Impact of Factor IXa Content On Function, Safety and Efficacy of Recombinant Factor IX Products.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2233-2233
Author(s):  
Wilfried Auer ◽  
Hanspeter Rottensteiner ◽  
Gerald Schrenk ◽  
Werner Hoellriegl ◽  
Alexandra Schiviz ◽  
...  
Keyword(s):  
Factor Ix ◽  
Hemophilia B ◽  
In Vitro Assays ◽  
Safety And Efficacy ◽  
Clinical Dose ◽  
Thrombin Generation Assay ◽  
Recombinant Factor Ix ◽  
Thrombogenic Potential ◽  
Individual Scores

Abstract Abstract 2233 Baxter has developed a new recombinant factor IX (rFIX) product (BAX 326) for treating patients with hemophilia B. The aim of the presented studies was to evaluate the function, safety and efficacy of BAX 326 compared to a commercially available rFIX regarding differences in activated FIX (FIXa) content. The FIXa concentration of BAX 326 was about 10 fold less than that of a commercial rFIX product. To address the influence of FIXa on functional FIX in vitro assays, the FIXa concentration of BAX 326 was increased. Samples were analyzed by one-stage clotting assay, non-activated partial thromboplastin time assay and thrombin generation assay. In all three assays, FIXa affected the potency determination for FIX, indicating that rFIX products should preferentially contain a low FIXa content. The thrombogenic potential of BAX 326 was assessed in rabbits using a modified Wessler Test at 750 IU/kg (10-fold human clinical dose). No thrombogenic potential was observed with BAX 326 (individual scores of 0), whereas the mean score for the commercially available rFIX was 0.5. After increasing the FIXa concentration of BAX 326 to equalize it with the commercially available rFIX, a mean score of 0.42 (individual scores 0 – 0.5) was determined. These data strongly suggest that the differences in preclinical thrombogenicity were caused by the higher FIXa content of the commercially available rFIX, thereby confirming earlier findings [1]. Efficacy of the rFIX products was studied in hemophilia B (FIX ko) mice that received prophylactic treatment with 75 IU/kg of BAX 326 or the commercially available rFIX. Treated animals were analyzed in two primary pharmacodynamic models: a carotid occlusion model and one using thrombelastography. In both models, results for the two groups were similar, demonstrating that despite its lower FIXa content, BAX 326 was as efficacious as a commercially available rFIX at 75 IU/kg (p<0.0076). Disclosures: Auer: Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Schrenk:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Schiviz:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.

scholarly journals Factor IX from prothrombin complex concentrate augments low dose tissue factor-triggered thrombin generation in vitro

2018 ◽  
Vol 121 (4) ◽  
pp. 936-943 ◽  
Author(s):  
S. Hasan ◽  
E. Abuelkasem ◽  
B. Williams ◽  
R. Henderson ◽  
M.A. Mazzeffi ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Prothrombin Complex Concentrate ◽  
Low Dose ◽  
Factor Ix
Sign in / Sign up

Export Citation Format

Share Document