Differential Neutralization of Apixaban, Betrixaban, Edoxaban, and Rivaroxaban By Andexanet Alfa As Measured By Whole Blood Thromboelastographic Analysis

Introduction/Background: 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 as an antidote to neutralize the bleeding effects of oral FXa inhibitors such as, Apixaban and Rivaroxaban. The relative biological effect of these drugs have been investigated using various clot based and amidolytic methods for FXa inhibition. This Factor Xa inhibitory activity acts as a surrogate marker for the circulating level of these agents. We have recently reported that the FXa activity of these Anti-Xa agents does not fully reflect their biologic spectrum (JCath 25,1-11,2019). Whole blood assays such as thromboelastographic analysis represent a global assay which takes into account both the plasmatic and cellular components of blood and provides a more physiologic endpoint to study the anticoagulant effects of these drugs. The purpose of this study was to investigate the anticoagulant effects of currently available oral Anti-Xa agents such as Apixaban, Betrixaban, Edoxaban, and Rivaroxaban and their relative neutralization by AA in terms of such thromboelastographic parameters as R, K, Angle and MA. Materials and Methods: Analysis was carried out in whole blood using thromboelastography (TEG) using the TEG 5000 Hemostasis System (Haemonetics Corp, Massachusetts). Blood was drawn from healthy donors in individual groups (n=5-10) into 3.2% citrated tubes. In the TEG cup for testing, 0.2 M CaCl2, saline (with a filler and control), each of the individual FXa Inhibitors at a final concentration(FC) of 1 ug/mL, AA at FC of both 100 ug/mL and 50 ug/mL were tested for the relative neutralization of the anticoagulant effects. TEG parameters such as R-time, K-time, angle and MA were measured. All results were compiled individually for the saline control, 100 ug/mL AA and 50 ug/mL AA supplemented systems. Statistical analysis was carried out via an F test for equality of variances followed by the appropriate t test for equal or unequal variances. Results: When comparing the anticoagulants directly to one another, it was observed that Edoxaban shows the strongest anticoagulant effects in both R and K time followed by Betrixaban, then Rivaroxaban which were very similar in their anticoagulative effects, with Apixaban showing the weakest anticoagulant effect as shown in Table 1A. In the reversal studies as shown in Table 1B, as measured by various TEG parameters, R-Time, AA (FC=100 ug/mL) showed full neutralization effects in Apixaban (p=.027), Betrixaban(p=<.01), Edoxaban(p<.01), and Rivaroxaban(p<.01). In K-time, Betrixaban and Edoxaban were fully neutralized (respectfully p=.049 and p=.035) with partial neutralizations of Apixaban and Rivaroxaban. No significant neutralization was noted in the Angle and MA. AA at 50 ug/mL showed full neutralizations, in R-time, in Betrixaban and Rivaroxaban (Betrixaban[p<.01] and Rivaroxaban[p=.0287]), AA at this concentration showed partial neutralizations of Apixaban and Edoxaban. In K-time, AA showed full neutralization of Betrixaban and Edoxaban (Betrixaban[p=<.01] and Edoxaban[p<.01]). Apixaban and Rivaroxaban saw no neutralization effect by AA at FC=50 ug/mL in K-time. AA did not exhibit any significant neutralization effects in the Angle or MA parameters. Summary and Conclusion: All of the 4 Agents produced measurable anticoagulative activities at 1 ug/mL as measured by the TEG parameters. Edoxaban exhibited the strongest anticoagulative effect followed by Betrixaban and Rivaroxaban whereas Apixaban showed much weaker anticoagulant effects. AA FC=100 ug/mL showed much stronger, consistent, and complete neutralization effects of all of the 4 FXa Inhibitors when compared to AA at FC=50 ug/mL. These results strongly suggest that regardless of the variable anticoagulative effect exhibited by the FXa Inhibitors, AA at FC=100ug/mL fully neutralized the anticoagulant effects of this agent as measured by the TEG parameters. AA is shown to be the most effective in neutralizing Betrixaban in R-Time and K-Time at both concentrations of AA. AA was seen to neutralize Apixaban the least. It can be concluded that the effect of AA as a neutralizing agent is both drug and donor dependent and therefore dosage adjustment may be needed for the optimal clinical outcome with this antidote. 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.

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Prothrombinase Induced Clotting Time Is More Sensitive then aPTT and PT and Can be Used for the Monitoring of Anti-Xa Agents in Whole Blood and Plasma

Blood ◽

10.1182/blood-2019-131221 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3374-3374

Author(s):

Debra Hoppensteadt ◽

Emily Bontekoe ◽

Fakiha Siddiqui ◽

Ambar Farooqui ◽

Omer Iqbal ◽

...

Keyword(s):

Whole Blood ◽

Research Funding ◽

Anticoagulant Activity ◽

Surrogate Marker ◽

Factor Xa ◽

High Sensitivity ◽

Relative Sensitivity ◽

Inhibitory Effect ◽

Clotting Time ◽

Higher Sensitivity

Introduction: Currently there are four commercially available oral anti-Xa agents namely apixaban, betrixaban, edoxaban and rivaroxaban available for indication specific clinical use. All agents used are at a fixed dosage and their use may be associated with potential hemorrhagic complications. Although factor Xa inhibitory effect is considered to be a surrogate marker for the biologic action of these drugs we have demonstrated that factor Xa inhibitory profile of apixaban, betrixaban, edoxaban, and rivaroxaban does not fully reflect their biologic spectrum (https://doi.org/10.1177/1076029619847524). Furthermore the prothrombin time (PT) and activated partial thromboplatin time (aPTT) methods are of limited sensitivity and are dependant on several other enodogenous factors. Prothrombinase induced clotting time (PiCT), (Pentaharm, Basel, Switzerland) is a sensitive test for the global monitoring of anticoagulant drugs including heparins and parenteral oral anti-Xa and anti-IIa agents. The test is based on the RVV venom activation of endogenous FVa which forms prothrombinase complex with phospholipids (PL), FXa and calcium. The clot-based end point is proportional to the activities of FXa and FIIa. This study is designed to compare the relative responses of PT, aPTT and PiCT test in normal human blood, retrieved plasma and agents supplemented plasma with the oral anti-Xa agents to demonstrate the relative sensitivity of this assay in comparison to the PT and aPTT. Materials and Methods: Active pharmaceutical ingredient versions of apixaban, betrixaban, edoxaban and rivaroxaban were obtained from commercial sources. All agents were prepared at a stock concentration of 100ug/ml in saline and diluted to a working solution of 10ug/ml. Serial dilutions were prepared in various matrices. Citrated plasma from normal individuals (n=50) was obtained from a commercial source (George King Biomedical, Overland Park, Kansas). Whole blood and plasma samples (n=20) were supplemented at a concentration of 0-1000 ng/ml. Whole blood samples were also centrifuged to obtain retrieved plasma. These samples were analyzed using a single stage PiCT, aPTT and PT. Whole blood and retrieved plasma studies were carried out by using ST4 (Diagnostica Stago). PiCT were measured by using Pefakit PiCT. aPTT measurements were made by using Tcoag, TriniCLOT aPTT (Diagnostica Stago, Paris, France). For PT, HemosIL TM (Instrumentation Laboratory, MA, USA) was used. All results were compiled in individual groups as mean + SD. Results: All of these drugs produced a concentration dependent anticoagulant effects in the PiCT, aPTT and PT tests in both the whole blood and plasma systems. PiCT consistently showed much higher sensitivity in comparison to other tests. When compared at 1000ng/ml the anticoagulant effects of these drugs were stronger in plasma systems as shown in the table. PiCT consistently showed higher sensitivity in comparison to PT and aPTT in the whole blood, retrieved plasma and agent supplemented plasma. Edoxaban showed the strongest anticoagulant activity measured by PiCT in comparison to the other agents. Matrix based variations in the PiCT results were observed. Interestingly, the retrieved plasma from whole blood showed weaker anticoagulant effects in comparison to the directly supplemented plasma systems. Discussion: In comparison to PT and aPTT, the PiCT test was found to be the most sensitive in the three matrices studied. In the whole blood and plasma-based systems PiCT test showed linearity and high sensitivity for all of the four anti-Xa agents. In the PiCT test, consistently drug supplemented plasma showed the highest response in comparison to retrieved plasma and whole blood, suggesting differential binding of these drugs to cells. These results indicate that the PiCT test can be reliably used for the monitoring of anti-Xa agents. PiCT test can also be performed on currently available optical and mechanical instrument used for clotting studies. Owing to rapid turnaround time, high sensitivity, and lower cost PiCT can be used for the routine monitoring of oral anti-Xa agents. Table 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.

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

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029619863493 ◽

2019 ◽

Vol 25 ◽

pp. 107602961986349 ◽

Cited By ~ 3

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.

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Assay Dependent Variations in the Anticoagulant and Protamine Sulfate Neutralization Profiles of Generic Copies of Enoxaparin.

Blood ◽

10.1182/blood.v108.11.908.908 ◽

2006 ◽

Vol 108 (11) ◽

pp. 908-908 ◽

Cited By ~ 2

Author(s):

Walter P. Jeske ◽

Jeanine M. Walenga ◽

Paul D. Ackerman ◽

Debra A. Hoppensteadt ◽

Curtis Vandenberg ◽

...

Keyword(s):

Whole Blood ◽

Factor Xa ◽

Protamine Sulfate ◽

Inhibitory Effects ◽

Clotting Time ◽

Fda Approval ◽

Potential Safety ◽

Branded Product ◽

Generic Products ◽

The Individual

Abstract Currently several generic versions of a branded low molecular weight heparin (LMWH; enoxaparin, Sanofi-Aventis, Paris, France) have become available for clinical use in several countries. Such products include Cutenox (Gland Pharma, Hyderabad, India), Dripanina (Ariston, São Paulo, Brazil) and Clenox (Pharmayect, Barranquilla, Columbia). Although these products are not currently approved for use in the U.S., several other manufacturers have sought FDA approval for their products. Due to a lack of specifications and guidelines, this approval is still pending. In order to compare the relative potency of different anti-factor Xa U/ml adjusted generic preparations, studies were designed to compare each of the individual generic LMWHs with the branded product. Additionally, multiple batches of some of the generic products were also profiled. All of the agents were tested in human whole blood and citrated plasma over a concentration range of 0.15 to 10 U/ml. Whole blood activated clotting time (ACT) and thrombelastography (TEG) measurements along with fibrinopeptide A (FPA) generation were compared. The plasmatic tests included anti-Xa and anti-IIa activity by amidolytic assay and aPTT, Heptest and PiCT clotting time assays. In addition, protamine sulfate neutralization profiles for these agents were investigated at fixed protamine concentrations of 12.5 and 25 μg/ml. In the whole blood assays, at concentrations < 2.5 U/ml, no significant differences were observed between the branded and potency adjusted generic LMWHs. However, in the plasma-based systems, assay-dependent variations were observed which were more obvious at concentrations > 1.25 U/ml (aPTT, anti-IIa, anti-Xa; p<0.05). Similarly, product and assay based variations were also observed in the protamine neutralization profile of these LMWHs. Moreover, marked differences in some assays were observed when different batches of the generic copies of LMWHs were tested. Additional studies carried out to profile the oligosaccharide composition also showed product and batch-dependent variations. The relative amounts of antithrombin affinity components among the different generic products and within product batches also exhibited some variations. These studies clearly demonstrate that some of the generic copies of enoxaparin may not produce comparable anticoagulant and thrombin generation inhibitory effects at anti-Xa potency adjusted doses. Such differences may not be clinically relevant in the prophylactic indications (dosages ≤ 40 mg O.D). However, in therapeutic or interventional indications (IV or SC dosage > 100 mg), these products may exhibit differential safety/efficacy profiles. These observations underscore the importance of clear guidelines on the chemical and biologic specifications for the acceptance of generic versions of LMWHs. Such measures are crucial to avoid any potential safety/inefficacy issues particulatly in indications where these drugs are used at higher dosages.

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Factor Xa Inhibitors Can Be Differentiated in Xa, IIa and Microparticle Generation and Other Whole Blood Based Assays.

Blood ◽

10.1182/blood.v110.11.931.931 ◽

2007 ◽

Vol 110 (11) ◽

pp. 931-931 ◽

Cited By ~ 1

Author(s):

Omer Iqbal ◽

Cafer Adiguzel ◽

Debra Hoppensteadt ◽

Josephine Cunannan ◽

Jawed Fareed

Keyword(s):

Tissue Factor ◽

Whole Blood ◽

Thrombin Generation ◽

Factor Xa ◽

Final Concentration ◽

Factor Xa Inhibitors ◽

Saline Control ◽

Functional Assay ◽

Control Value ◽

Anticoagulant Agents

Abstract Currently used oral anticoagulants such as Vitamin K antagonists have drawbacks, which reportedly limit their safety and efficacy. Oral Factor Xa and IIa inhibitors are claimed to overcome these limitations. Factor Xa inhibitors provide more complete suppression of thrombin generation than Factor IIa inhibitors. Four Factor Xa anticoagulant agents, both direct and indirect, namely A,B,C and D with Ki values 6–200 nM, were studied in various assays at equigravimetric final concentration of 10 ug/ml to determine their relative potencies. Apparent differences in their biochemical profiles were noted in thrombin generation, Factor Xa generation and microparticle generation inhibition assays. Furthermore, the anticoagulant potential of these Xa inhibitors was studied in celite activated clotting time (ACT) and modified celite ACT system using different concentrations of tissue factor. Microparticle generation was performed using agent- supplemented whole blood that was incubated for three minutes with varying concentrations of tissue factor. Following three minutes the reaction was stopped using EDTA stop solution. The functional assay for the determination of microparticle procoagulant activity in the plasma was performed using Zymuphen MP-Activity assay kit from Hyphen BioMed (Neuville-sur-Oise, France). While three of the Xa inhibitors studied showed microparticle generation inhibition levels of 31.37 nM, one agent provided a level of 17.87 nM compared to a saline control value of 39.28nM. Supplementation studies were also carried out in whole blood PT, APTT and Heptest assays in the concentration range of 0 to 10 ug/ml. The whole blood prothrombin time assay showed a maximum of 208.8, 61.3, 77.8 and 15.9 seconds at a final concentration of 10 ug/ml for the respective anticoagulant agents when compared to a normal value of 12.3 seconds. Similarly the whole blood APTT assay showed a maximum of 267.9, 161.8, >300 and 71.9 seconds respectively when compared to a control value of 46.8 seconds. The whole blood Heptest assay showed a varying maximum anticoagulant effect from >300, 42.7, >300 and >300 seconds respectively when compared to a control value of 13.8 seconds. In each of these assays Factor Xa inhibitors showed concentration-dependent effects and had varying potencies. In TF-mediated platelet activation assays the different Xa agents produced varying effects which were not proportionate to their Ki values. Differentiation of Factor Xa inhibitors have a clinical impact in dosage selection, dosage adjustment and their monitoring when given in large dosages. Synthetic factor Xa agents exhibit Ki values of 20–200 nM, while pentasaccharide -AT complex has a Ki value of 60 nM. However, these results do not translate into proportionate anticoagulant effects to their Ki values. Large-scale clinical studies are necessary to validate these preliminary results.

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Towards the Care of Hemophilia a Patients Using Induced Pluripotent Stem Cell (iPSC)-Derived Megakaryocytes (iMks) Expressing Coagulation Factor (F) VIII

Blood ◽

10.1182/blood.v126.23.2266.2266 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2266-2266

Author(s):

Randolph B Lyde ◽

Li Zhai ◽

Karen Vo ◽

Danuta Jadwiga Jarocha ◽

Spencer Sullivan ◽

...

Keyword(s):

Whole Blood ◽

Neutralizing Antibodies ◽

Research Funding ◽

Hemophilia A ◽

Specific Activity ◽

Coagulation Factor ◽

Clot Formation ◽

Patient Specific ◽

Furin Cleavage ◽

Furin Cleavage Site

Abstract We and others have shown that FVIII expressed ectopically in platelets (pFVIII) is stored in α-granules, released at sites of vascular injury and restores hemostasis in FVIIInull mice, even in the presence of neutralizing antibodies to FVIII. These studies support the concept that unlike therapeutic interventions that correct plasma FVIII, pFVIII may be a useful therapy in hemophilia A with intractable inhibitors and significant bleeds. We have also demonstrated this approach has several limitations that may make pFVIII gene therapy bone marrow transplantation (BMT) strategies problematic: 1) pFVIII is not equivalent to plasma FVIII and its efficacy in joint and intracranial bleeds has yet to be shown, especially in the presence of inhibitors, and 2) pFVIII expressed during megakaryopoiesis can cause injury to the Mks, potentially exacerbating post-BMT thrombocytopenia. We propose an alternative strategy: interval prophylactic infusions of FVIII-containing platelets generated from patient-specific iMks expressing either human B-domain-deleted (BDD) FVIII or variants of this FVIII that have greater stability and longer half-lives; making them especially efficacious as pFVIII as we previously demonstrated. iPSCs are a renewable source of cells that can be pre-screened prior to clinical usage for lines that express optimal levels of pFVIII and also release optimal numbers of platelets after differentiation into iMks. Such iPSCs were transfected with a self-inactivating lentivirus containing cDNA for one of three FVIII variants: wildtype BDD FVIII (WT FVIII), R1645H PACE/furin cleavage site FVIII (FVIIIR1645H), and amino acid 1645 to 1648 deletion FVIII (FVIIIΔ). FVIIIR1645H and FVIIIΔ show greater stability and consequently greater specific activity with no increase in injuring Mks. All FVIII variants were expressed using the MK-specific Cxcl4 promoter and were shown to be effective in several bleeding models in FVIIInull mice. Differentiated and transduced iMKs were analyzed for RNA and protein expression. All of the FVIII variant iMKs expressed at least forty-fold higher levels of mRNA compared to the non-transduced control (n=6) and protein was expressed at >550 pg/106 CD42b+ iMKs (n=6). Transduced MKs released FVIII into the supernatant when activated by thrombin showing the pFVIII was likely stored in α-granules. Annexin staining was the same between FVIII-expressing iMKs and control iMks suggesting that the level of pFVIII did not cause the iMks to become apoptotic. To test the ability of FVIII-expressing iMKs to correct the coagulopathy in hemophilia A, 5x105 iMKs were added to FVIIInull murine whole blood and evaluated for clot formation using rotational thromboelastometry (ROTEM). Each FVIII variant showed a decrease in clotting time, clot formation time, and an increase in maximum clot firmness when compared to the non-transduced control (n=4). These data show that iMKs expressing FVIII variants can improve hemostasis in a whole blood clotting assay. Our next goal is to generate sufficient platelets from these iMKs to test for correction of the bleeding diathesis in immunodeficient FVIIInull mice and to determine their efficacy in improving hemostasis in a number of clinically relevant hemostatic models. Disclosures Arruda: Pfizer: Consultancy, Patents & Royalties, Research Funding; Spark Therapeutics: Patents & Royalties. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; NovoNordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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

Blood ◽

10.1182/blood-2019-124596 ◽

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.

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

Blood ◽

10.1182/blood-2019-127072 ◽

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.

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Coagulation Factor Xa (Recombinant), Inactivated-Zhzo (Andexanet Alfa) Hemostatic Outcomes and Thrombotic Event Incidence at an Academic Medical Center

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029619896619 ◽

2019 ◽

Vol 25 ◽

pp. 107602961989661 ◽

Cited By ~ 7

Author(s):

Victoria M. Stevens ◽

Toby Trujillo ◽

Scott W. Mueller ◽

Robert MacLaren ◽

Paul M. Reynolds ◽

...

Keyword(s):

Medical Center ◽

Academic Medical Center ◽

Thrombotic Event ◽

Factor Xa ◽

Coagulation Factor ◽

Thromboembolic Events ◽

Bleeding Events ◽

Major Bleed ◽

Academic Medical ◽

Andexanet Alfa

Andexanet alfa is approved for the reversal of factor Xa inhibitors in patients with major bleeding events. We aimed to review the incidence of effective hemostasis with andexanet alfa in a real-world environment. This retrospective cohort included patients hospitalized for a major bleed that resulted in andexanet alfa administration. The primary outcome was effective hemostasis at 12 hours after andexanet alfa treatment. Thromboembolic events and mortality within 30 days were also assessed. Over a 14-month period, 13 patients received andexanet alfa with a mean age of 69 ± 10 years, 54% male, 69% exposed to apixaban (31% rivaroxaban), and had intracranial (46%) and nonintracranial (54%) bleeding sites. Effective hemostasis was observed in 10 (77%) patients. Four (31%) patients experienced 5 thromboembolic events with a median time to event of 6.5 days (range: 0.5-29). Four thrombotic events occurred during the period in which anticoagulation (prophylaxis or therapeutic) was not restarted. Mortality rate was 15%. Andexanet alfa was effective in obtaining hemostasis in a majority of patients. However, the incidence of thromboembolic events was high and may be attributed to a delay in restarting anticoagulation.

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Gender-Based Differences in Hemostatic Responses. Implications in Effective Anticoagulant Management.

Blood ◽

10.1182/blood.v114.22.2103.2103 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2103-2103

Author(s):

Omer Iqbal ◽

Nasir Sadeghi ◽

Fadi Bakhos ◽

Debra Hoppensteadt ◽

Jawed Fareed

Keyword(s):

Whole Blood ◽

Conflicts Of Interest ◽

Statistical Significance ◽

Factor Xa ◽

The United States ◽

Saline Control ◽

Anticoagulant Drugs ◽

Significant Difference ◽

Males And Females ◽

Gender Based

Abstract Abstract 2103 Poster Board II-80 Abstract: Recent reports from the National Heart, Lung and Blood Institute indicate that as many as 3 million women (particularly young) in the United States suffer from a form of heart disease fundamentally different from that in men, characterized by more even plaque development inside major and smaller blood vessels, posing diagnostic and treatment challenges leading to increased morbidity and mortality. It is hypothesized that women have variable but attenuated hemostatic responses to anticoagulant drugs when compared to men. In order to validate this hypothesis the hemostatic responses in healthy males (n=10) and females (n=10) were evaluated by performing the global clotting assays, fibrinokinetic assays and thrombin generation assays in the presence of Rivaroxaban, an oral Factor Xa inhibitor likely to replace warfarin, Enoxaparin, a low molecular weight heparin and saline as a control. Blood (20 ml) was drawn from healthy volunteers, males (n=10) and females (n=10) and placed into citrated tubes with one part of 3.2% sodium citrate to 9 parts of blood. The citrated whole blood was supplemented with Rivaroxaban (FC=0.3mg/ml), Enoxaparin (FC=5mg/ml) and saline as a control. The samples were analyzed to determine the whole blood APTT and Heptest clotting assays. The remaining citrated blood was centrifuged at 3000 rpm to obtain platelet poor plasma that was aliquoted and kept frozen at -70°C until further analysis. The plasma was then thawed and supplemented with saline, rivaroxaban (FC=0.3mg/ml) and Enoxaparin (FC=5mg/ml). A statistically significant difference between males and females was noted in APTT (p=0.0442)) and Heptest (p=0.0345) assays in the saline control values. However, the anticoagulant response to supplementation of the plasma samples with rivaroxaban at a final concentration of 0.3ug/ml and Enoxaparin at 5 ug/ml showed a statistically significant difference between males and females in the Heptest (P=0.0423) while the APTT assay felt a little short of statistical significance (P=0.0511). Fibrinokinetics was performed and absorbance recorded (405 nm) at every minute for the next 30 minutes. There are gender-based differences in fibrinokinetic responses to anticoagulant drugs with females showing faster fibrin formation than males. The attenuated hemostatic responses observed in women compared to men may interfere in achieving adequate and effective anticoagulation leading to thrombotic complications. Time (min) 0 30 Gender Male ODs Female ODs Male - ODs Female - ODs Male - ODs Female - ODs Saline control 0.857±0.31 0.611±0.22 1.367±0.28 1.214±0.28 1.377±0.26 1.24±0.28 Rivaroxaban (0.3ug/ml) 0.853±0.32 0.602±0.21 1.353±0.27 1.221±0.18 1.363±0.27 1.23±0.18 Enoxaparin (5ug/ml) 0.713±0.35 0.507±0.24 0.794±0.33 0.621±0.23 0.802±0.33 0.629±0.23 Disclosures: No relevant conflicts of interest to declare.

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Andexanet Alfa Corrects the Interference of Direct Factor Xa Inhibitors on Lupus Anticoagulant Testing

Blood ◽

10.1182/blood.v128.22.2626.2626 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2626-2626

Author(s):

Ross I Baker ◽

Grace Gilmore ◽

Scott McGregor

Keyword(s):

Board Of Directors ◽

False Positive ◽

Research Funding ◽

Factor Xa ◽

Plasma Samples ◽

Direct Factor ◽

Advisory Committees ◽

Equity Ownership ◽

Andexanet Alfa ◽

Support Research

Abstract Background Direct Factor Xa (FXa) inhibitors (e.g. rivaroxaban or apixaban) are widely used oral anticoagulants and are known to interfere with dilute Russell's viper venom time (dRVVT) and aPTT-based lupus anticoagulant (LA) testing, often yielding false-positive LA results. A false positive LA result might incorrectly diagnose a patient with the anti-phospholipid antibody syndrome and lead to a wrong decision for long-term anticoagulation to prevent recurrent thrombosis. Andexanet alfa is a recombinant modified human FXa decoy protein that has been specifically designed as an antidote to neutralise the effect of the direct and indirect FXa inhibitors. Aim To determine whether the addition of andexanet alfa can overcome the interference of rivaroxaban and apixaban on LA testing in plasma samples from both LA positive and LA negative patients. Methods After written informed consent, citrated blood (20mL) was collected from 12 patients on therapeutic doses of rivaroxaban (n=6) or apixaban (n=6) with previously known LA status (3/6 LA positive in each group). Samples were double centrifuged at 3,000 x g for 10 minutes and aliquots were frozen at -40oC until analysed. Normal pooled plasma and rivaroxaban and apixaban calibrated plasma was obtained from a commercial source (Diagnostica Stago) or control plasma from healthy volunteers, prepared as above. IgG was purified by a Protein G column from a known LA positive patient and a control healthy volunteer. Rivaroxaban (Bayer) and apixaban (Bristol-Myers Squibb) tablets were crushed and dissolved in DMSO. Andexanet alfa (Portola Pharmaceuticals, Inc.) was dissolved in distilled water and aliquots were frozen at -40oC until used. Plasma samples were spiked with varying concentrations of FXa inhibitors and andexanet alfa. LA-sensitive aPTT, PT, TCT, dRVVT (Siemens Healthcare Diagnostics) and anti-FXa level (Diagnostica Stago) were performed with and without andexanet alfa on the Sysmex CS5100 analyser. Andexanet alfa (at a final concentration of 250µg/mL) was added to LA positive and LA negative plasma samples to assess the impact on the dRVVT normalised ratio. Results Andexanet alfa demonstrated a concentration-dependent increase in LA sensitive aPTT and dRVVT clotting times, with negligible impact on PT or TCT. Andexanet alfa was capable of reversing FXa inhibition in a concentration-dependent manner in plasma samples containing rivaroxaban or apixaban. When rivaroxaban is added to normal plasma it significantly prolongs the dRVVT without phospholipid more than with excess phospholipid leading to a false-positive LA ratio at final concentrations >100ng/mL. This level of rivaroxaban is in the therapeutic range. The addition of andexanet alfa reduced the measurable anti-FXa level and returned the normalised dRVVT ratio to LA negative result (<1.25). In contrast, apixaban and andexanet alfa proportionately prolongs the dRVVT with or without excess phospholipid and even at high concentrations, the normalised LA ratio remains negative (<1.25). Andexanet alfa converted the false positive dRVVT rivaroxaban patient plasmas LA ratio to negative. True LA positive patients remained positive after the addition of andexanet alfa. These results were confirmed by adding LA positive and control purified IgG to known concentrations of rivaroxaban and apixaban (200ng/ml) in normal plasma. Conclusion Andexanet alfa corrects the false positive LA ratio in patients on rivaroxaban without affecting the true diagnosis in LA positive patients. Apixaban and andexanet alfa proportionately prolong the dRVVT with and without phospholipid, so the LA ratio remains negative. For this reason true LA positive patients can be diagnosed whilst on apixaban at therapeutic concentrations without the addition of andexanet alfa. Disclosures Baker: Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Other: Conference travel support; Astellas and CSL Behring: Research Funding; Bristol-Myers Squibb: Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Daiichi Sankyo: Research Funding; Portola Pharmaceuticals: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support; Biogen Idec: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Roche: Other: Conference travel support. McGregor:Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Equity Ownership, Other: Conference travel support; Pfizer: Other: Conference travel support; Roche: Honoraria; Abbvie: Equity Ownership; Portola Pharmaceuticals: Equity Ownership.

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