scholarly journals Assay Dependent Reversal of the Oral and Parenteral Anti-Xa Agents By Andexanet Alfa

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-40
Author(s):  
Fakiha Siddiqui ◽  
Debra Hoppensteadt ◽  
Jeanine Walenga ◽  
Walter Jeske ◽  
Alfonso J Tafur ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Control Group ◽  
Inhibitory Effects ◽  
Strong Concentration ◽  
Dependent Inhibition ◽  
Ic50 Values ◽  
Andexanet Alfa

Introduction: Andexanet alfa (AA, Portola Pharmaceuticals, San Francisco, USA) is an approved reversal agent for the control of potential bleeding associated with apixaban and rivaroxaban. Beside the oral anti-Xa agents, parenteral forms of the inhibitors of factor Xa such as otamixaban (Sanofi Aventis, Paris, France) and DX9065a (Mitsubishi Pharmaceuticals, Tokyo, Japan) have also been developed. These agents represent synthetic organo-mimerics with comparable selectivity and inhibitory profile to the currently available oral anti-Xa agents. Parenteral anti-Xa agents are considered for clinical development. Andexanet alfa is a broad-spectrum neutralizing agent for anti-Xa drugs including heparin and heparino-mimerics. We hypothesized that andexanet alfa may also reverse the effects of such parenteral anti-Xa agents as otamixaban and DX9065a. This study is designed to compare the neutralization profile of andexanet alfa for apixaban and rivaroxaban with otamixaban and DX9065a in various laboratory assays. Materials and Method: Apixaban, rivaroxaban, otamixaban and DX9065a were commercially obtained in powdered form and diluted in 0.9 % sodium chloride to make stock solution of 1.0 mg/ml. Andexanet alfa was obtained from the hospital pharmacy. Drugs were supplemented in plasma in the concentration range of 0.0 - 1.0 ug/ml. Individual aliquots of samples were supplemented with either saline or andexanet alfa at a final concentration of 100 ug/ml. Factor Xa activity was measured by using an amidolytic method. For clotting profile, prothrombin time (PT) and activated partial thromboplastin time were measured. Thrombin generation studies were carried out using a calibrated automated thrombogram (CAT, Diagnostica Stago, Paris, France). Such parameters as peak thrombin (PT), area under the curve (AUC) and lag time (LT) were measured. The inhibitory effects of each of these agents towards factor Xa were calculated and their reversal by andexanet alfa was determined. Results were compiled as mean SD of 3 individual determination. Result: Both the oral and parenteral anti-Xa agents produced a concentration dependent inhibition of factor-Xa with the IC50 values ranging from 0.17 - 1.1 ug/ml in control group. Supplementation of andexanet alfa at 100 ug/ml resulted in the neutralization of the anti-Xa activities of these agents with the IC50 values ranging from 0.22 - 1.1 ug/ml. Andexanet alfa did not produce any reversal of the anti-Xa activities of DX9065a. In the thrombin generation studies, Apixaban, rivaroxaban and otamixaban produced strong concentration dependent inhibition of thrombin formation. However, DX9065a produced relatively weaker anti-Xa effects. The IC50 values varied with apixaban (0.08 ug/ml), rivaroxaban (0.22 ug/ml), otamixaban (0.6 ug/ml) and DX9065a (>2.5 ug/ml). In the clot-based prothrombin time assay all agents produced a concentration dependent prolongation of PT in the range of 0 - 1 ug/ml. Andexanet alfa at 100 ug/ml produced a complete neutralization of apixaban, rivaroxaban and otamixaban, whereas it partially neutralized the anticoagulant effects of DX9065a in this assay. The parenteral anticoagulants otamixaban and DX9065a produced a much stronger anticoagulant effects in the aPTT assay in comparison to both apixaban and rivaroxaban. Andexanet alfa at 100 ug/ml effectively neutralized the anticoagulant effects of otamixaban in comparison to Apixaban and rivaroxaban. Whereas DX9065a were not neutralized. Table 1 shows the composite results for the neutralization of oral and parenteral anti-Xa agents at 0.5 ug/ml by andexanet alfa at 100 ug/ml. Conclusion: Our results suggest that andexanet alfa is capable of neutralizing the effects of potent parenteral anti-Xa agents such as otamixaban in an assay dependent fashion. The data also points to the varying inhibitory effects of anti-Xa agents which are differentially neutralized by andexanet alfa. These results also underscore that the in-vitro anti-Xa potency of both the oral and parenteral anti-Xa agents does not fully reflect their inhibitory effects on the overall coagulation process. Nevertheless, andexanet alfa may be a useful agent in the neutralization of parenteral anti-Xa agents. Disclosures No relevant conflicts of interest to declare.

scholarly journals Reversal of Factor Xa Inhibitors by Andexanet Alfa May Increase Thrombogenesis Compared to Pretreatment Values

2019 ◽  
Vol 25 ◽  
pp. 107602961986349 ◽  
Author(s):  
Fakiha Siddiqui ◽  
Alfonso Tafur ◽  
Lorenzo Storino Ramacciotti ◽  
Walter Jeske ◽  
Debra Hoppensteadt ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Area Under The Curve ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Inhibitory Effects ◽  
Clotting Time ◽  
Reversal Effect ◽  
Normal Human ◽  
Fxa Inhibitor ◽  
Andexanet Alfa

Recombinant coagulation factor Xa (FXa), inactivated Zh-zo, also known as andexanet alfa (AA), is a modified version of human FXa that has been developed to neutralize FXa inhibitors. We studied the reversal effect of AA for these inhibitors in various anticoagulant and thrombin generation (TG) assays. Individual aliquots of normal human plasma containing 1 µg/mL of apixaban, betrixaban, edoxaban, and rivaroxaban, were supplemented with saline or AA at a concentration of 100 µg/mL. Clotting profiles include prothrombinase-induced clotting time, activated partial thromboplastin time, and prothrombin time. Factor Xa activity was measured using an amidolytic method. Thrombin generation was measured using a calibrated automated thrombogram. Differential neutralization of all 4 anticoagulants was noted in the activated clotting time and other clotting tests. The FXa activity reversal profile varied with an observed decrease in apixaban (22%), betrixaban (56%), edoxaban (28%), and rivaroxaban (49%). Andexanet alfa also led to an increased TG in comparison to saline. The peak thrombin was higher (40%), area under the curve (AUC) increased (15%), whereas the lag time (LT) decreased (17%). Andexanet alfa added at 100 µg/mL to various FXa supplemented systems resulted in reversal of the inhibitory effects, restoring the TG profile; AUC, LT, and peak thrombin levels were comparable to those of unsupplemented samples. Andexanet alfa is capable of reversing anti-Xa activity of different oral FXa inhibitors but overshoots thrombogenesis in both the saline and FXa inhibitor supplemented systems. The degree of neutralization of Xa inhibitor is specific to each agent.

Impact Of Thrombin Generation, Tissue Factor Activity and Thrombomodulin Activity On The Positivity Of Assisted Reproductive Technique In Infertile Women

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3630-3630
Author(s):  
Emmanuelle Mathieu d'Argent ◽  
Patrick Van Dreden ◽  
Marjorie Comtet ◽  
Vassiliki Galea ◽  
Hela Ketatni ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Hormone Treatment ◽  
Control Group ◽  
Significant Finding ◽  
Assisted Reproductive Technique ◽  
Infertile Women ◽  
Reproductive Techniques

Abstract Introduction Women undergoing assisted reproductive techniques (ART) are given gonadotrophins to promote the development of multiple follicles within their ovaries. This treatment may be associated with a risk of ovarian hyperstimulation syndrome and venous or arterial thrombosis. The association of clinical and biological criteria of hypercoagulability might contribute to the identification of patients at risk and probably could predict success of ART. The aim of this study was to evaluate thrombin generation, thrombomodulin activity, tissue factor (TF) activity, and procoagulant phospholipids in selected women undergoing ART. We also assessed the potential correlation between the levels of the studied biomarkers and the outcome of the ART. Material and Method A cohort of 27 infertile women eligible for ART and 30 healthy age matched women was studied. All patients were Caucasian aged 33.75 ± 3.52 years and weight 61.08 ± 8.10 kg. Women included in the study did not present any personal or family history of VTE or known thrombophilia. They did not present any signs of hemorrhagic syndrome and did not suffer from any known autoimmune disease. Blood samples were taken under fasting conditions at the following time-points: at the inclusion (T0), between the 5th and 8th day of ovarian stimulation with gonadotrophines (T1) and at the day of HCG administration (T2). Thrombin generation (TG) in plasma was assessed using the Calibrated Automated Thrombogram assay (using, PPP-Reagent-5pM TF from Diagnostica Stago, France), Plasma levels of thrombomodulin activity (TMa), and TF activity (TFa) were measured by home-made tests, Procoagulant phospholipids (PPL) dependent clotting time was measured using a factor Xa-based assay (STA-R Procoag-PPL, Diagnostica Stago, France). Results The endogenous thrombin potential (ETP), PPL, TMa and TFa were significantly higher in the ART-T0 group as compared to the control group. At ART-T2 a significant increase of TG was observed as compared to ART-T0. At ART-T0 44.5%, 44.4 % and 33.3 % of women had ETP, TFa and TMa higher than the Upper Normal Limit respectively (UNL = mean+2 S.D.). Among with negative ART 89% and 91.7% showed TMa and TFa levels > UNL at ART-T0. At T1 50% of women had a least one parameter of TG higher than the UNL. At ART T2 65.2 % of women had TG > UNL. At the same time, 87.5% and 83.4% of women with negative ART had levels of TMa and TFa > UNL. Conclusion This study analyzed the profile of thrombin generation in infertile women eligible for ART and investigated the influence of hormone treatment with gonadotropins and HCG on TG and levels of TMa and TFa. Hypercoagulability, in terms of increased ETP is present in 46% of infertile women eligible for ART. These women remain in a hypercoagulable state throughout the entire period of hormone treatment. The most significant finding of this study was that 33% of patients show a value superior to the UNL for thrombomodulin and 45% for tissue factor. Interestingly 89%of women with negative ART had TMa higher than the UNL. Respectively 91% of women with negative ART had TFa levels higher than the UNL Disclosures: No relevant conflicts of interest to declare.

Inhibition of Thrombin Generation in Plasma by Inhibitors of Factor Xa

1997 ◽  
Vol 78 (04) ◽  
pp. 1215-1220 ◽  
Author(s):  
D Prasa ◽  
L Svendsen ◽  
J Stürzebecher
Keyword(s):  
Thrombin Generation ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Thrombin Generation Assay ◽  
Ic50 Values ◽  
Fxa Inhibitor ◽  
20 Nm ◽  
Tick Anticoagulant Peptide ◽  
Inhibition Of Thrombin

SummaryA series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations.The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

FXIIa Differentially Regulates Thrombin Generation during Steady State and Vaso-Occlusive Crisis in Sickle Cell Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 162-162 ◽  
Author(s):  
Erica M Sparkenbaugh ◽  
Camille Faes ◽  
Denis Noubouossie ◽  
Daniel K. Kirchhofer ◽  
András Gruber ◽  
...  
Keyword(s):  
Steady State ◽  
Mouse Model ◽  
Vascular Permeability ◽  
Sickle Cell ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Liver And Kidney

Abstract Sickle cell disease (SCD) is associated with chronic activation of coagulation. Previously, we demonstrated that inhibition of tissue factor (TF) attenuates thrombin generation (measured by plasma levels of thrombin-antithrombin complexes [TAT]) in a mouse model of SCD during steady state. Furthermore, we showed that neither inhibition of FXIIa-dependent activation of FXI (using 14E11 antibody) nor FXI deficiency reduces thrombin generation (TG) in sickle mice. In contrast, genetic deficiency of FXII or kininogen (HK) reduced plasma TAT levels. These data suggest that during steady state, FXIIa contributes to TG in sickle mice via activation of the kallikrein/HK pathway, but not FXI. In the present study, we further investigated the mechanisms of HK-induced TG at steady state, and increased TG observed during vaso-occlusive crisis (VOC). All experiments were performed using 4-5 month old Townes SS (sickle) and AA (control) mice. Kallikrein cleaves HK into HK fragments (HKFs) and bradykinin (BK). First, we investigated whether a BK-mediated increase in vascular permeability contributes to TG by exposing perivascular TF. This hypothesis was disproved by data demonstrating no difference in vascular permeability (measured by the extravasation of Evans blue in the heart, lung, liver and kidney) between AA (n=8) and SS (n=10) mice. HKFs were shown to induce leukocyte TF expression in vitro via binding to CD11b/CD18 (Mac-1). Therefore, we investigated whether Mac-1 inhibition affects TG in SS mice. AA and SS mice were treated with an inhibitory anti Mac-1 (M1/70) or IgG control antibody on days 0, 3 and 6 (i.p. 1 mg/kg) and TG was analyzed 1 day after the last injection. In the control group, SS mice demonstrated higher plasma TAT levels compared to AA mice (8.1±1.6 vs 4.2±0.6 ng/mL, n=10-11, p<0.05), but inhibition of Mac-1 significantly reduced plasma TAT levels in SS mice (4.6±0.7 ng/mL, n=11, p<0.05). These data suggest that HK might contribute to TG during steady state via Mac-1-dependent induction of monocyte TF. The steady state of SCD is interspersed with acute periods of VOC. Clinical data demonstrate that compared to the steady state, plasma levels of cell free DNA (cfDNA), activation of the contact system, and TG are further enhanced during VOC. To determine the mechanism of increased TG during VOC, we used the previously characterized mouse model of TNFα -induced VOC. Townes AA and SS mice were injected with recombinant TNFα (2 µg/g body weight) or the same volume of PBS, and plasma was collected 5 hours later. TNFα not only dramatically increased plasma levels of cfDNA in SS mice (14.78 ± 1.64 vs 679 ± 300 ng/mL; p<0.01), but also further increased plasma TAT levels compared to those observed in PBS-treated SS mice (2.9 fold, p<0.001, n=8). Importantly, there was a significant positive correlation between cfDNA and TAT in SS mice (r2 =0.65, p<0.001). Since cfDNA can activate FXII, we determined whether FXIIa-dependent activation of FXI contributes to TG during VOC. AA and SS mice received 14E11 or IgG control (4 mg/kg) 30 minutes before TNFα (2 μg/g) or PBS injection, and plasma TAT was assessed 5 hours later. Strikingly, 14E11 attenuated the increased TAT level in TNFα-treated SS mice, to the level observed in SS mice injected with PBS and IgG (IgG/SS/PBS: 9 ng/mL ± 1.8 vs. IgG/SS/TNF: 18.9 ± 3.6, p<0.001; 14E11/SS/TNF: 9.86 ± 0.72, p<0.05 vs. IgG/SS/TNF). We also determined if TF activity is required for the increased TG observed during VOC. Interestingly, inhibition of TF with an inhibitory 1H1 antibody (25 or 75 mg/kg injected i.p. 1 or 18 hours prior to TNFα, respectively) had no effect on the increased TG observed in TNFα treated SS mice. In aggregate, our data suggest that during the steady state of SCD, FXII-dependent TG is not FXI-dependent, but instead is mediated by a pathway involving HK, Mac-1 integrin and leukocyte TF. Furthermore, we propose that during VOC the massive release of cfDNA results in FXIIa-dependent FXI activation and enhances TG independently of TF. This study provides mechanistic insight into the initiators of TG in SCD. Moreover, it implicates FXIIa as a potential therapeutic target to reduce the prothrombotic state in SCD, during both steady state and VOC. Disclosures No relevant conflicts of interest to declare.

Idarucizumab, a Specific Antidote for Dabigatran, Cross-Reacts with Melagatran and May Also Interact with Other Benzamidine-Containing Compounds

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3836-3836
Author(s):  
Jawed Fareed ◽  
Larissa Reikensmeyer ◽  
Amanda Walborn ◽  
Debra Hoppensteadt ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Whole Blood ◽  
Conflicts Of Interest ◽  
Dabigatran Etexilate ◽  
Factor Xa ◽  
Cross Reactivity ◽  
Thrombin Time ◽  
Fab Fragment ◽  
Strong Concentration ◽  
Procoagulant Effect ◽  
Us Fda

Abstract Introduction: Dabigatran etexilate is a pro-drug which is used to prevent embolic stroke in patients with atrial fibrillation. This oral anticoagulant is also approved for other indications in Europe. As with all anticoagulants, there is a potential for serious hemorrhage with dabigatran usage which may require antidotes to control bleeding. Idarucizumab is an anti-dabigatran Fab fragment (Boehringer-Ingelheim) that binds to the benzamidine group on dabigatran and inhibits its anti-thrombin activity. Idarucizumab has recently been approved by the US FDA for the control of bleeding associated with dabigatran. Materials and Methods: Such antithrombin agents as argatroban, melagatran, hirudin, and bivalirudin, human antithrombin, thrombomodulin, heparin cofactor II, and heparin-AT complex were commercially obtained. Anti-factor Xa agents (rivaroxaban, apixaban and DX-9065a were also obtained from various sources To test the specificity of the inhibitory effects of idarucizumab, each of these agents were supplemented to whole blood and citrated plasma at concentrations ranging from 0.1 to 100 µg/mL. Idarucizumab was added to each mixture at a concentration of 1 mg/mL and anticoagulant activities were assessed using PT, aPTT, thrombin time and chromogenic anti-IIa/Xa and flurometric thrombin generation assays. Results: Idarucizumab itself did not produce any anticoagulant effects on whole blood or plasma clotting profile. However it showed a slight procoagulant effect in the whole blood and plasma based assays. It produced a strong concentration dependant inhibition of both dabigatran and melagatran. The antibody showed strong specificity for the inhibition of dabigatran amd melagatran and did not affect the anticoagulant and other effects of the other synthetic and natural thrombin and FXa inhibitors. The prolongation of the PT, APTT and thrombin time by melagatran was completely inhibited by idarucizumab. Idarucizumab more effectively inhibited the prolongation of thrombin time by dabigatran than the prolongation induced by melagatran. Discussion: The cross-reactivity of idarucizumab with melagatran may result from the presence of a common benzamidine pharmacophore which is present in both of these anticoagulant agents. Since the benzamidine pharmacophore is present in a number of serine protease inhibitors as well as drugs such as pentamidine, propamidine and dibromopropamidine. These observations suggest that simultaneous administration of idarucizumab may compromise the pharmacodynamics profile of benzamidine derived drugs such as the anti-malarials, anti-psychotic, anti-fungal and other compounds. Thus there is a need for a systemic screening of idarucizumab for its potential interactions with drugs containing benzamidine based therapeutic agents. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effects of Andexanet Alfa on Thrombin Generation in Bleeding Associated with Factor Xa Inhibitors

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 711-711
Author(s):  
Michiel Coppens ◽  
Lizhen Xu ◽  
Roisin Bavalia ◽  
Saskia Middeldorp ◽  
Peter Verhamme ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Educational Material ◽  
Factor Xa Inhibitor ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Andexanet Alfa

Introduction Andexanet alfa is a modified recombinant inactive form of human factor Xa developed for reversal of factor Xa inhibitors. In the ANNEXA-4 study, patients with acute major bleeding within 18 h after administration of a factor Xa inhibitor were enrolled and received a bolus of andexanet, followed by a 2-h infusion (Connolly, NEJM 2019;380:1326). In this study, 82% of patients achieved effective hemostasis at 12 h and 10% developed a thrombotic event within 30 days. Anti-Xa activity decreased by 92% after the andexanet bolus but partially recovered after the end of the 2 h infusion. In the present analysis, we evaluated the effect of andexanet alfa on thrombin generation (TG) in patients enrolled in the ANNEXA-4 study and we explored whether TG predicts effective hemostasis or thrombotic events. Methods We included all patients who received andexanet alfa. TG was expressed as the endogenous thrombin potential (ETP) which is the area under the thrombin generation curve. We plotted mean TG at different timepoints between baseline and 30 days after andexanet alfa in patients treated with apixaban and rivaroxaban. We compared the absolute ETP level at 8 h (ETP-8H) after andexanet bolus as this was the first timepoint after the 2 h infusion for which an ETP level was available for most patients. We compared ETP-8H levels between patients with and without effective hemostasis and between those with and without thrombotic events, respectively. ETP-8H was evaluated as a predictor of effective hemostasis and thrombotic events by logistic regression analysis in all patients, and in subgroups of patients with intracranial hemorrhage (ICH) and non-ICH separately. In the ICH subgroups, ETP-8H was also evaluated as a predictor of absolute change in hematoma volume. Results The study population comprised 352 patients (mean age 77.4 years; 47% female) with acute major bleeding (64% ICH, 26% gastrointestinal, 10% other) treated with apixaban (55%), rivaroxaban (36%), enoxaparin (6%), or edoxaban (3%). ETP-8H was available for 327 patients (93%). In patients treated with apixaban or rivaroxaban, andexanet bolus promptly increased mean ETP and this was maintained during infusion. After end of infusion ETP fell but remained in the reference range for at least 18 hours (Figure 1). ETP-8H was similar in patients with or without effective hemostasis (Fig 2a, p = 0.544) and in patients with or without thrombotic complications (Fig 2b, p = 0.610). In the logistic regression analysis, ETP-8H did not predict effective hemostasis (p=0.491) or thrombotic events (p=0.743) (Table), and these results were consistent in ICH and non-ICH patients. ETP-8H did not predict hematoma growth in patients with ICH (p = 0.349). Conclusion A bolus of andexanet alfa, followed by a 2-h infusion in patients with factor Xa inhibitor associated major bleeding promptly restores thrombin generation and this effect is sustained for at least 18 hours. Thrombin generation at 8 h after andexanet bolus did not predict effective hemostasis, intracranial hematoma growth, or thrombotic events. This may be explained by the andexanet dose which was chosen to ensure full reversal of the factor Xa inhibitor in all patients. Disclosures Coppens: Bayer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Daiichi Sankyo: Honoraria, Research Funding; Sanquin Blood Supply: Research Funding; Pfizer: Honoraria; Uniqure: Research Funding; CSL Behring: Honoraria, Research Funding; Portola Pharmaceuticals, Inc: Honoraria; Boehringer Ingelheim: Research Funding. Middeldorp:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Other: honoraria for advisory activities; Aspen: Research Funding; Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: honoraria for advisory activities; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Other: honoraria for advisory activities; Bayer: Membership on an entity's Board of Directors or advisory committees, Other: honoraria for advisory activities, Research Funding; Sanofi: Speakers Bureau; Daiichi Sankyo: Other: honoraria for advisory activities, Research Funding. Verhamme:Portola Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Consultancy, Research Funding, Speakers Bureau; Boehringer Ingelheim: Consultancy, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Research Funding, Speakers Bureau; Leo Pharma: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy. Eikelboom:Heart and Stroke Foundation: Research Funding; Sanofi Aventis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Glaxo Smith Kline: Honoraria, Research Funding; Eli Lilly: Honoraria, Research Funding; Daiichi Sankyo: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Boehringer Ingelheim: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding. Crowther:Bayer: Other: Data and Safety Monitoring Board, Research Funding, Speakers Bureau; BMS Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Other: preparing educational material and/or providing educational presentations; CSL Behring: Other: preparing educational material and/or providing educational presentations; Diagnostica Stago: Other: preparing educational material and/or providing educational presentations, Research Funding; Alnylam: Equity Ownership; Asahi Kasei: Membership on an entity's Board of Directors or advisory committees; Alexion: Speakers Bureau; Shionogi: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees. Lu:Portola Pharmaceuticals: Employment, Equity Ownership. Yue:Portola Pharmaceuticals: Employment, Equity Ownership. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Connolly:Portola Pharmaceuticals: Consultancy, Research Funding; Bayer Healthcare: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding.

scholarly journals Andexanet Alpha Differentially Neutralizes the Anticoagulant, Antiprotease and Thrombin Generation Inhibitory Effects of Unfractionated Heparin, Enoxaparin and Fondaparinux

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1158-1158
Author(s):  
Fakiha Siddiqui ◽  
Alfonso J Tafur ◽  
Debra Hoppensteadt ◽  
Jeanine Walenga ◽  
Walter Jeske ◽  
...  
Keyword(s):  
Research Funding ◽  
Thrombin Generation ◽  
Surrogate Marker ◽  
Factor Xa ◽  
Bleeding Complications ◽  
Dependent Manner ◽  
Thrombin Time ◽  
Inhibitory Effects ◽  
Biologic Effects ◽  
Inhibition Of Thrombin

Introduction: Andexanet Alpha (Coagulation factor Xa recombinant, inactivated Zh-zo; AA, Portola Pharmaceuticals) is a recombinant factor Xa decoy protein which is designed to reverse the effects of apixaban and rivaroxaban and is approved for the control of bleeding complications associated with their use. The molecular modification in this recombinant protein involves the substitution of serine active site by alanine and the removal of the gamma-carboxyglutamic acid (GLA) domain to restrict its assemblage into prothrombinase complex. Beside the reversal of the effects of anti-Xa agents AA is also reported to neutralize the biologic effects of heparin and related drugs. Assay dependent variations in the neutralization profile of various factor Xa inhibitors by andexanet has been recently reported https://doi.org/10.1177/1076029619847524. Since heparin and related drugs also mediate their biologic actions by inhibiting factor Xa via AT complexation, it is hypothesized that AA may also inhibit their biologic effects as measured in various laboratory assays. It is the purpose of this study is to compare the relative neutralization profile of heparin (UFH), a low molecular weight heparin, enoxaparin (E) and a chemically synthetic pentasaccharide, Fondaparinux (F) by AA. Materials and Methods: API versions of UFH, E and F were commercially obtained in powdered forms and dissolved in saline at a working dilution of 1mg/ml. AA was dissolved in saline to obtain a 10mg/ml working solution. The anticoagulant profile of UFH, E and F was studied using the activated partial thromboplastin time (APTT) and thrombin time (TT) in a concentration range of 0 - 10 ug/ml in pooled human plasma. The anti-Xa and anti-IIa studies were carried out in amidolytic assays in the same concentration range. The thrombin generation inhibition was studied using calibrated automated thrombin generation systems (CAT, Diagnostica Stago). The effect of AA on the reversal of the anticoagulant and anti-protease and thrombin generation effects of each of these agents were studied by supplementing this agent at 100 ug/ml. The results are compared to determine the difference between pre and post AA neutralization settings. Results: All agents produce a concentration dependent effect in the anticoagulant and anti-protease assays with the exception of F which showed mild anticoagulant effects, and very weak anti-IIa actions and strong anti-Xa activity. In the anti-Xa assay the IC-50 for UFH was 2.1ug/ml (0.13 um), E 4.3 ug/ml (0.95 um) and F 0.7 ug/ml (0.41 um) upon supplementation of AA the IC50s for UFH was increased to 5 ug/ml (0.31 um) and for E 5 ug/ml (1.11 um). However, there was no neutralization of the anti-Xa effects of the F by AA and the IC50 remained the same for both pre and post andexxa studies. The anticoagulant effects of UFH as measured by aPTT and TT was strongly neutralized whereas E was only partially neutralized in the aPTT assay and almost completely neutralized in the thrombin time assay. At concentrations of up to 10 ug/ml F did not produced any significant anticoagulant effects, both in the presence and absence of AA. In the thrombin generation inhibition assays, UFH produced a complete inhibition of thrombin generation which was completely reversed by AA. Although both E and F produced strong inhibition of thrombin generation, AA did not completely neutralize these effects. The results are tabulated on table 1 for the studies carried out at 10 ug/ml of UFH, E and F. Conclusion: These results indicate that AA is capable of differentially neutralizing anticoagulant and anti-protease effects of UFH in an assay dependent manner. However, AA is incapable of neutralizing the anti-Xa effects of E and F. This may be due to the relatively differential affinities of enoxaparin and fondaparinux AT complex to factor Xa rendering it inhibited in the presence of AA. These studies also demonstrate that the primary surrogate marker anti-Xa activity for measuring the activities of anti-Xa agents is not proportional to the anticoagulant and thrombin generation inhibitory effects of these agents. A global clotting assay may be a better indication of the biologic effects of these agents and their reversal by AA. Disclosures Tafur: Recovery Force: Consultancy; Janssen: Other: Educational Grants, Research Funding; BMS: Research Funding; Idorsia: Research Funding; Daichi Sanyo: Research Funding; Stago: Research Funding; Doasense: Research Funding.

scholarly journals Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3815-3823 ◽  
Author(s):  
GJ Jr Broze ◽  
DA Higuchi
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Feedback Inhibition ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor X ◽  
Dependent Inhibition ◽  
Fibrinolysis Inhibitor ◽  
Normal Hemostasis

Coagulation is initiated by the binding of factor VIIa to tissue factor, with resultant limited factor IX and X activation and thrombin production. Owing to the feedback inhibition of the factor VIIa/tissue factor complex by tissue factor pathway inhibitor (TFPI), additional factor X activation and thrombin generation must proceed through a pathway involving factors VIII, IX, and XI. Experiments designed to elucidate the requirement for amplified factor Xa and thrombin generation in normal hemostasis show that the resistance of plasma clots to tissue plasminogen activator (tPA)- and urokinase-induced fibrinolysis is related to the extent of thrombin generation. Inhibition of fibrinolysis is mediated in part by plasma carboxypeptidase-U ([CPU] carboxypeptidase-R, procarboxypeptidase-B, thrombin-activatable fibrinolysis inhibitor), a proenzyme that is proteolytically activated by thrombin in a process enhanced dramatically by the cofactor thrombomodulin. A clot induced in factor IX-deficient plasma with limited amounts of tissue factor in the presence of urokinase (100 U/mL) lyses prematurely, and this defect is corrected by supplementation of the deficient plasma with factor IX (5 micrograms/mL) or thrombomodulin (20 ng/mL). These additions enhance the rate and extent of CPU activation: in the case of factor IX, presumably by permitting amplified generation of factor Xa and thrombin, and in the case of thrombomodulin, presumably by increasing the degree of CPU activation produced by the low levels of thrombin generated in the absence of factor IX. Pretreatment of the factor IX-deficient plasma with specific anti-CPU antibodies prevents the increased resistance to fibrinolysis produced by addition of factor IX and thrombomodulin. Likewise, when coagulation is induced by thrombin (2 U/mL) in the presence of tPA (60 U/mL), clots formed from plasmas deficient in factors VIII, IX, X, or XI lyse prematurely unless the missing factor is replaced or thrombomodulin (20 ng/mL) is added.

A Novel Mechanism of Action of Rivaroxaban: Inhibition of Monocyte and Macrophage Procoagulant Activity and Consequence On Inflammatory Process.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3124-3124
Author(s):  
Marc Laurent ◽  
Remi Varin ◽  
Ulrich Joimel ◽  
Hong Li ◽  
He Lu ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Procoagulant Activity ◽  
Moderate Increase ◽  
Amidolytic Activity ◽  
Monocytes And Macrophages ◽  
Activated Monocytes ◽  
Prothrombinase Activity

Abstract Abstract 3124 Poster Board III-61 Aim of the study Tissue factor is normally absent from monocytes in circulating blood. It can be induced by inflammatory mediators leading to the formation of monocyte-associated prothrombinase activity, which participates to thrombin generation. This activity appears to be important in both thrombosis (venous and arterial) and in chronic inflammation by inducing the release of inflammatory cytokines. This could be attributed to the activation of PAR-1 and PAR-2 (protease-activated receptors) by factor Xa or by thrombin. In this study, we compared the action of Rivaroxaban and Fondaparinux, two specific factor Xa inhibitors, on the activation of coagulation and on the secretion of inflammatory cytokines in both activated monocytes and activated human monocyte/macrophage cell line THP1. Methods 1-Monocytes were isolated from healthy volunteers and THP-1 cells were used as macrophages. 2- Activation of cells was performed by adding 1 μg/ml LPS for 2 hours at 37°C, in the presence of defibrinated human plasma which provides plasma coagulation factors, in the absence (control) or presence of Fondaparinux (500, 1000 and 1500 ng/ml) or Rivaroxaban (150, 250, 350 ng/ml, final concentrations). After incubation, the cells were isolated and their supernatants collected. 3- The procoagulant activity of cells was tested by measuring their effect on the clotting time of normal plasma in presence of calcium. 4- The release of cytokines was tested by antibody-cytokines array in the supernatants (RayBio®). 5-To test the neutralization of factor Xa bound to monocytes, activated monocytes were incubated with human purified factor Xa (100 ng/ml) for 10 min at room temperature. After isolation of the cells, 250 ng/ml Rivaroxaban or the appropriate solvent was added for 1, 10 and 30 min. The Xa activity associated to monocytes was measured by its amidolytic activity. Results 1- The procoagulant activity of monocytes and macrophages was reduced by Rivaroxaban and not by Fondaparinux. Results of prothrombinase activity in the presence of Rivaroxaban at 150, 250, 350 ng/ml, expressed as percentage of the control value was: 30 ± 3, 16 ± 4 and 12 ± 2 % for monocytes and: 43 ± 2, 24 ±3, 15 ± 1 % for THP-1 cells. In contrast, Fondaparinux did not modify the prothrombinase activity of monocytes (105 ± 12%). It was also shown that in the conditions used, Rivaroxaban (250 ng/ml) inhibited completely the amidolytic activity of factor Xa bound to activated monocytes. 2- Both Rivaroxaban and Fondaparinux modify the profile of chemokines secretion by activated monocytes and THP-1. In monocytes and THP-1 cells, LPS induced an important increase in Il-8 and angiogenin and a moderate increase in MIP-1d (Macrophage Inflammatory Protein-1) and RANTES, a member of the Il-8 superfamily. Both Rivaroxaban and Fondaparinux decreased the secretion of these chemokines to the basal level of secretion by non activated monocytes or THP-1. The secretion of leptin was only induced by LPS- treated THP-1 and was strongly decreased by both FXa inhibitors. In contrast, secretion of EGF (epithelial growth factor) was only induced by activated monocytes and strongly decreased by Rivaroxaban and moderately by Fondaparinux. Discussion and conclusion The results show that 1- Rivaroxaban induced a concentration-dependent inhibition of the procoagulant activity of activated monocytes and macrophages, whereas fondaparinux was devoid of this effect. This difference is attributed to a better access of Rivaroxaban to FXa bound to monocytes, as compared to the Fondaparinux-antithrombin complex. This inhibitory effect of Rivaroxaban could contribute to its antithrombotic activity. 2- Rivaroxaban inhibited the secretion of inflammatory chemokines by activated monocytes and THP-1. This decreased secretion was also observed with Fondaparinux, suggesting that it could be due to the decrease in thrombin generation in plasma, affecting the PAR-1 cell signaling system. As it was reported that that elevated IL-8 is associated with recurrent venous thrombosis and that these inflammatory cytokines are involved in plaque progression and rupture by recruitment of subpopulations of leukocytes and by potent angiogenic activity, this decrease in cytokines could also contribute to the antithrombotic efficacy of Rivaroxaban. This study received a support from Bayer Schering Pharma, France. Equal contribution of Marc Laurent and Rémi Varin Disclosures No relevant conflicts of interest to declare.

Non-Pathogenic Anti-Heparin/PF4 Antibodies Generated by Low Molecular Weight Heparins Mediate Procoagulant Effects.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1091-1091
Author(s):  
Jeanine M. Walenga ◽  
Debra Hoppensteadt ◽  
Evangelos Litinas ◽  
Harry L. Messmore ◽  
Bruce E Lewis ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Biologically Active ◽  
Control Group ◽  
Heparin Group ◽  
Thrombin Generation Assay ◽  
Enoxaparin Group ◽  
Pre Treatment ◽  
Inhibition Of Thrombin

Abstract Abstract 1091 Introduction: While the incidence of symptomatic heparin-induced thrombocytopenia (HIT) is relatively low with the use of low molecular weights heparins (LMWHs), these agents do generate anti-heparin/PF4 antibodies in 10–20% of treated patients. Dosage, duration, and the pathologic predisposition of the patient influence the quantitative and qualitative nature of these antibodies. It has been suggested that these non-pathogenic antibodies (NPAs) which do not produce symptomatic HIT may, nevertheless, be biologically active and mediate thrombogenic responses. The overall pathophysiologic role of NPAs is unknown at this time. Hypothesis: NPAs generated by LMWHs cause coagulation activation and compromise the anticoagulant effects of the administered LMWH. Study Design: Blood plasma samples collected at baseline and day 10 from patients enrolled in orthopedic surgery clinical trials of LMWHs for the prophylactic management of deep vein thrombosis (Lovenox enoxaparin, sanofi-aventis, n=352; Clivarin reviparin, Abbott, n=380) were retrospectively screened for the presence of anti-heparin/PF4 antibodies using the GTI ELISA method (Waukesha, WI). Positive samples were tested by the 14C-SRA to determine if the antibodies were capable of functionally activating platelets. Both ELISA positive and negative samples were evaluated in an assay of thrombin generation (Technothrombin TGA kit, diaPharma, West Chester, OH). Result: In the enoxaparin study, the baseline pre-treatment samples only showed one patient in the heparin control group to be positive by ELISA. On day 10, 11 of 175 (6.3%) enoxaparin patients had a positive ELISA response, whereas 22 of 177 (12.4%) heparin patients were ELISA positive. None of the samples were 14C-SRA positive. In the thrombin generation assay, the ELISA positive samples showed a lesser inhibition of thrombin generation for both the enoxaparin and heparin groups (270 ± 27 nM TGA enoxaparin group; 220 ± 21 nM TGA heparin group) compared to the thrombin generation response of the ELISA negative samples (190 ± 18 nM TGA enoxaparin group; 160 ± 20 nM TGA heparin group). In the reviparin study, none of the patients were ELISA positive at baseline. On day 10, in the reviparin group 19 of 200 (9.5%) patients were ELISA positive, whereas 28 of 180 (15.6%) heparin control patients had a positive ELISA titer. None of the samples were 14C-SRA positive. In comparison to the baseline (pre-treatment), both the reviparin and heparin treated patients showed an inhibition of thrombin generation (410 ± 27 nM TGA baseline vs 180–290 nM with treatment). However, consistent with the above study, those samples that were ELISA antibody positive showed a lesser inhibition of thrombin generation (240 ± 21 nM TGA reviparin group; 210 ± 16 nM TGA heparin group) in comparison to the ELISA negative samples (190 ± 12 nM TGA reviparin group; 180 ± 14 nM TGA heparin group). Interestingly, the D-dimer levels were found to be higher in the ELISA positive samples in all groups for both studies (p<0.05). Conclusion: These studies suggest a potential pathologic role of NPAs. The results of the thrombin generation studies strongly suggest that the generation of NPAs may result in a reduction of the antithrombotic potential of both LMWH and heparin in treated patients. While the exact mechanism of this process is not clear, dosage adjustment may be useful in those patients who generate NPAs. Disclosures: No relevant conflicts of interest to declare.

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