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

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.

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Generic and Branded Versions of Argatroban Exhibit Differential Anticoagulant Effects In Whole Blood, Plasma Based Assays and Thrombin Generation Assays

Blood ◽

10.1182/blood.v116.21.5129.5129 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5129-5129

Author(s):

Jawed Fareed ◽

Debra Hoppensteadt ◽

Omer Iqbal ◽

Jeanine M. Walenga ◽

Bruce E Lewis

Keyword(s):

Protein Interactions ◽

Whole Blood ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Anticoagulant Effect ◽

Thrombin Time ◽

Clotting Time ◽

Generic Products

Abstract Abstract 5129 Several generic versions of argatroban) (Mitsubishi; Tokyo, Japan) have been introduced in Japan (Argaron, Gartban, Slovastan). In addition, other generic versions of argatroban are being considered by the European and North American regulatory bodies. While the generic versions of argatroban exhibit similar antithrombin potency (Ki values), because of the differential compositional variations their anticoagulant effects in whole blood systems may differ due to their cellular and plasmatic protein interactions. Branded and generic versions of argatroban may exhibit differential anticoagulant actions in the whole blood and plasma based assays due to their differential interactions with blood cells, platelets and plasma proteins. Three generic versions of argatroban that are commercially available in Japan namely Argaron, Gartban and Slovastan and a powdered version of generic argatroban (Lundbeck) were compared with the branded argatroban. Native whole blood thrombelastographic (TEG) analysis was carried out at 0.1 ug/mL, the Activated Clotting Time (ACT) assay was carried out in a concentration range of 0–10 ug/mL, and such coagulation tests as the PT/INR, aPTT, Heptest, and calcium thrombin time were performed. Plasma retrieved from the supplemented whole blood was also assayed. Ratios of the clotting time test values from whole blood and plasma were calculated. Retrieved plasma samples were also assayed in the thrombin generation assays (TGA). All of the different versions of argatroban produced a concentration dependent anticoagulant effect in the native whole blood TEG and ACT. In the TEG, while argatroban and Slovastan showed a similar effect, Gartban, Argaron and a powdered generic showed weaker effects. Argatroban was also different in the ACT assay. At a concentration of 5 ug/ml the ACTs were, Arg 340+15.2 secs, S 297+10.5 secs, G 292.0+19.1 secs and A 285.2+21.7 secs. In the citrated whole blood systems, all agents produced a concentration dependent anticoagulant effect; however, the generic versions produced a stronger anticoagulant effect in comparison to branded argatroban (p<0.001). In the PT assay at 5 ug/mL, argatroban showed 32 ± 3 sec vs 40–50 sec for the generic products. Similarly in the aPTT, Heptest and thrombin time tests argatroban was weaker than the generic products. Differences among generic versions were also evident. Similar results were obtained in the retrieved plasma, however the ratio of whole blood over plasma varied from product to product. The IC50 of the generic and branded argatrobans in the TGA were also different. These results show that while in the thrombin inhibition assays generic and branded argatroban may show similar effects, these agents exhibit assay dependent differences in the whole blood and plasma based assays. Such differences may be more evident in the in vivo studirs where endothelial cells and other interactions may contribute to product individuality. Therefore, based on the in vitro antiprotease assays, generic argatrobans may not be considered equivalent and require a multi-parametric study. Currently available generic argatrobans may not be equivalent in the in vivo anticoagulant effects. Therefore, clinical validation of the clinical equivalence for these drugs is warranted. Disclosures: No relevant conflicts of interest to declare.

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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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Lyso-Sulfatide Binds Factor Xa and Potently Inhibits Thrombin Generation.

Blood ◽

10.1182/blood.v116.21.1130.1130 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1130-1130

Author(s):

Subramanian Yegneswaran ◽

Yajnavalka Banerjee ◽

Jose A. Fernandez ◽

Hiroshi Deguchi ◽

John H. Griffin

Keyword(s):

Free Amino ◽

Conflicts Of Interest ◽

Factor Xa ◽

Coagulation System ◽

Amino Group ◽

Thrombin Time ◽

Binding Studies ◽

Binding Interaction ◽

Dependent Inhibition ◽

Gla Domain

Abstract Abstract 1130 Although phospholipids are well-recognized for their effects on coagulation reactions, little is generally known about the effects of sphingolipids on clotting pathways. Negatively-charged sulfatides can potently initiate the intrinsic pathway of coagulation system by binding and autoactivating factor (f) XII. Sphingosine potently inhibits the ability of factor Xa (fXa) to generate thrombin (fIIa) in the prothrombinase complex (II-ase) (fXa/fVa/phospholipids) by interacting directly with fXa's Gla domain. Here we report that lyso-sulfatide (lyso-SF) (sulfogalactosyl sphingosine), a lipid of minor abundance in plasma that is primarily in HDL particles, exhibits potent anticoagulant activity. Lyso-SF dose-dependently prolonged clotting in fXa-1-stage but not thrombin-time clotting assays. Lyso-SF inhibited II-ase activity by > 90 % in purified reaction mixtures (fXa/fVa/II) in the presence of 6 or 30 μM phospholipids (PL). However, lyso-SF did not inhibit fIIa generation by fXa/fVa in the absence of PL, suggesting the absolute requirement of PL for lyso-SF-dependent inhibition of fIIa generation. Lyso-SF inhibited fIIa generation by fXa/PL in the absence of fVa. Additionally, lyso-SF inhibited fIIa generation by Gla-domainless (gd)-fXa in the presence but not in the absence of fVa and PL. Lyso-SF-dependent inhibition of fIIa generation was also observed for fXa/fVa/PL when gd-II was used as the substrate instead of II. However, no inhibition by lyso-SF was observed when using gd-fXa/PL and gd-II/PL in the presence or absence of fVa. Lyso-SF had no effect on fXa or fIIa amidolytic activity. These data plus other studies suggested that ≥ two components of the II-ase complex needed to be PL-bound for potent inhibition of fIIa generation by lyso-SF. PL surfaces bind and assemble each the II-ase protein components; however, PL's and lyso-SF may also alter the conformations of fXa, fVa and II. To gain mechanistic insights for lyso-SF inhibition of II-ase activity, Surface Plasmon Resonance (SPR) and fluorescence spectroscopy were used to define molecular interactions. Remarkably, SPR binding studies showed that lyso-SF binds to immobilized fXa (KD = 83 μM) and gd-fXa (KD = 36 μM). Controls using SPR showed no binding of lyso-SF to immobilized fVIIa or fIXa whereas SPR confirmed the ability of fXa, fVIIa and fIXa to bind PL's. Fluorescence binding assays confirmed SPR data showing that lyso-SF bound to and altered the dansyl fluorescence of dansyl-GluGlyArg-labeled fXa (DEGR-fXa) both in the presence (KD = 50 μM) and absence (KD = 75 μM) of PL and that this binding required calcium ions. Thus, lyso-SF binds fXa outside the Gla domain. Fluorescence monitoring of fVa binding to DEGR-fXa in the presence of PL showed that lyso-SF inhibited this binding interaction. To characterize structure-activity relationships for lyso-SF inhibition of II-ase, different analogs of lyso-SF were tested for their ability to inhibit fIIa generation by gd-fXa/fVa/PL. Psychosine (galactosyl sphingosine), glucosyl sphingosine and lyso-sphingomyelin each inhibited fIIa generation showing that the sulfate ester moiety and the sugar group in lyso-SF were not essential for the anticoagulant effects of lyso-SF. However, acetylation of the free amino group in lyso-SF ablated its inhibition of fIIa generation showing that the free amino group on carbon 2 is essential for the inhibitory activity of lyso-SF. In conclusion, these findings show that lyso-SF and several of its analogs are potent anticoagulant lipids and that the mechanism for inhibition of fXa by lyso-SF may involve its binding to fXa at sites outside fXa's Gla domain. This suggests that certain sphingolipids may exert allosteric downregulation of fXa activity without inhibiting the enzyme's active site or the binding of the Gla domain to PL surfaces. Disclosures: No relevant conflicts of interest to declare.

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Factor Xa Inhibitory Profile of Apixaban, Betrixaban, Edoxaban and Rivaroxaban Does Not Fully Reflect Their Biologic Spectrum

Blood ◽

10.1182/blood-2018-99-116993 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2520-2520

Author(s):

Fakiha Siddiqui ◽

Siddharth Mehrotra ◽

Vishnu Venkitasubramony ◽

Rithik Raina ◽

Lorenzo Storino Ramacciotti ◽

...

Keyword(s):

Whole Blood ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Anticoagulant Activity ◽

Kinetic Method ◽

Factor Xa ◽

Biochemical Properties ◽

Therapeutic Effects ◽

Distinct Individual

Abstract Introduction: There are four oral anti-Xa drugs currently available for clinical use in various indications. These drugs are claimed to mediate their therapeutic effects by solely targeting factor Xa. While these agents are structurally similar, their biochemical properties and their effects on blood coagulation differ. Such differences may impact their safety and efficacy profile. The purpose of this study was to demonstrate the differences among factor Xa inhibitors in terms of their in vitro anticoagulant activity and other biochemical effects. Materials and Methods: Commercially obtained powdered forms of Apixaban, Betrixaban, Edoxaban and Rivaroxaban were profiled in this study. Stock solutions of each drug were prepared at 1mg/ml. To investigate the effect on the whole blood clotting profile, thromboelastographic studies were carried out over a concentration range of 0.5 - 2.5 ug/ml and whole blood activated clotting time (ACT) was measured at 1.0 and 2.5 ug/ml. The anticoagulant profile in citrated human pool plasma was measured at concentrations of 0.062-1.0 ug/ml using such tests as prothrombin time (PT) and activated partial thromboplastin time (aPTT). The anti-Xa effects of each agent were measured using a kinetic amydolytic method. The inhibitory potency was calculated in terms of IC-50. Thrombin generation inhibition studies on each drug were carried out in human pool plasma in a concentration range of 0.0-1.0 ug/ml using calibrated aotomated thrombogram (CAT) assay (Diagnostica Stago, Paris, France). Fibrinokinetics studies were carried out using an optical kinetic method, where thrombin was used to trigger clot formation. All results were compiled in terms of mean + 1 SD of 3-5 replicates. Results: All of the anti-Xa agents produced concentration and assay-dependent effects in these studies. The summary of each agent's effects at selected fixed concentrations and the IC-50 of the anti-Xa activity is given in the Table. In the whole blood ACT at 2.5ug/ml, Edoxaban showed the strongest anti-coagulant effects followed by Rivaroxaban > Betrixaban, whereas Apixaban showed minimal effects. In the TEG analysis at 1ug/ml, Edoxaban exhibited stronger anti-coagulant effects as measured by various TEG parameters, including R-time, K-time, alpha, and MA. Edoxaban and Rivaroxaban showed comparable effects followed by Betrixaban, whereas Apixaban exhibited weaker effects. In the PT assay at 1ug/ml, Edoxaban showed stronger effects, whereas Apixaban, Betrixaban and Rivaroxaban were comparable. aPTT at 1ug/ml revealed that Edoxaban was the strongest anti-Xa inhibitor followed by Betrixaban, whereas Apixaban and Rivaroxaban were comparable. In the anti-Xa assay Edoxaban was stronger (IC-50 = 340ng/ml, 0.62uM) than Apixaban (IC-50 =400ng, 0.87uM), Rivaroxaban (IC-50 = 840ng, 1.9uM) and Betrixaban (IC = >1000ng, >2.22 uM). In the thrombin generation assays at 1ug/ml, Apixaban showed the strongest inhibitory activity (IC-50 = 50ng/ml, 108nm) followed by Edoxaban (IC-50 = 58ng/ml, 108nm), Betrixaban (IC-50 = 60ngml, 133nm) while Rivaroxaban showed relatively weaker activity (IC-50 = 100ng/ml, 299nm). In the fibrinokinetics study at 1ug/ml, the anti-Xa agents produced varying degrees of inhibition with Rivaroxaban (67%), Edoxaban (42%), Apixaban (32%) and Betrixaban (12%). Summary and Conclusion: These results demonstrate that the measured anti-Xa activity alone does not fully reflect the overall biologic spectrum of these agents. Assay dependent variations are exhibited by each of these drugs, revealing distinct individual profiles. Edoxaban was the only anti-Xa agent which consistently exhibited relatively stronger inhibitory profile which was proportional to its anti-Xa activity. These studies indicate that the oral anti-Xa drugs may modulate the hemostatic system through additional mechanisms independent of the inhibition of factor Xa. Table. Table. Disclosures No relevant conflicts of interest to declare.

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USP Standardized Mixtures of Bovine, Ovine and Porcine Heparin Exhibit Comparable Biologic Effects to Referenced Single Sourced Heparins and May be Interchangeable,

Blood ◽

10.1182/blood-2021-147995 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1067-1067

Author(s):

Guy Olson ◽

Walter Jeske ◽

Omer Iqbal ◽

Ambar Farooqui ◽

Fakiha Siddiqui ◽

...

Keyword(s):

Supply Chain ◽

Conflicts Of Interest ◽

Factor Xa ◽

African Swine Fever ◽

Single Species ◽

Protamine Sulfate ◽

Thrombin Time ◽

Plasma Clot ◽

Porcine Tissue ◽

Biologic Effects

Abstract Introduction: Unfractionated heparin (UFH) is the first line anticoagulant for the management of medical indications. UFH complexes with antithrombin to produce strong inhibition of thrombin and factor Xa. The UFHs are standardized using USP compliant amidolytic anti-Xa and IIa methods in defined conditions. Clinically used UFH is solely sourced from porcine mucosal tissue. Because of the shortage of porcine tissue and the African Swine Fever, the supply chain of this anticoagulant is compromised. Thus, there is a need for resourcing of this anticoagulant. Bovine and ovine mucosal sources represent alternate material for production of UFH. Previous studies have shown that bovine and ovine UFH exhibit anticoagulant effects which can be standardized by using the USP method. Additionally, the standardized heparins from various sources can be blended and their potency can be adjusted to exhibit comparable effects as the single sourced UFH. The purpose of this study is to evaluate the pharmacologic profile of the blended heparin and compare these activities to that of the single sourced porcine, ovine and bovine heparins. Methods: Two groups of heparins were evaluated in this study, porcine, ovine, bovine, and the blended heparin in gravimetric measurements (ug/ml) and these same four in potency adjusted measurements (U/ml). The pharmacologic profiles of the heparins in this study were investigated via global anticoagulant assays and anti-protease assays performed in plasma. Clot based assays such as the activated partial thromboplastin time (aPTT) and thrombin time (TT) were used to study the anticoagulant effects of the single source and blended heparins. The amidolytic anti-Xa and IIa assays were used to assess the inhibitory effects of these heparins on these proteases. USP compliant anti-Xa and IIa assays were used to determine potencies of the various heparins. Protamine sulfate (PS) neutralization studies were performed to evaluate the reversal of anticoagulant effects in each of the heparins. Results: The aPTT assay showed that at final concentrations of 5 ug/ml and 2.5 ug/ml porcine heparin significantly (p < .01) prolonged the aPTT compared to ovine, bovine, and blended heparins. When studied with potency adjusted heparins, all heparins demonstrated comparable aPTT values at all concentrations (U/ml). The TT assay showed that porcine and ovine heparins prolonged the TT at 1.25 ug/ml compared to bovine and blended heparins. When studied with potency adjusted heparins, all heparins demonstrated comparable TT values at all concentrations (U/ml). The anti-Xa assay showed that at all final concentrations between 10 ug/ml and 0.625 ug/ml porcine, ovine, and blended heparins produced significantly (p <.001) stronger Xa inhibition than bovine heparin. When studied with potency adjusted heparins, all heparins demonstrated comparable anti-Xa inhibition at all concentrations (U/ml). The anti-IIa assay showed that at final concentrations 2.5 ug/ml, 1.25 ug/ml, and 0.625 ug/ml porcine and ovine heparins produced significantly (p < .05) stronger IIa inhibition than bovine heparin. When studied with potency adjusted heparins, all heparins demonstrated comparable anti-IIa inhibition at all concentrations (U/ml). The USP compliant anti-Xa assay with gravimetric heparins showed potencies of 201, 201, 150, and 184 U for porcine, ovine, bovine, and blended heparins respectively. The USP compliant anti-Xa assay with potency adjusted heparins showed comparable potencies for all four heparins. The USP compliant anti-IIa assay with gravimetric heparins showed potencies of 204, 196, 127, and 167 U for porcine, ovine, bovine, and blended heparins respectively. The USP compliant anti-IIa assay with potency adjusted heparins showed comparable potencies for all four heparins. The protamine sulfate neutralization studies demonstrated complete neutralization at all concentrations for all of the potency adjusted heparins in the aPTT, TT, anti-Xa, and anti-IIa assays. Conclusion: These studies support the hypothesis that a blended heparin product from bovine, ovine, and porcine tissue, when standardized in USP unit-equivalent proportions, exhibits a comparable anticoagulant profile to the single species heparins. These findings suggest that there is a potential for development of blended heparin to stabilize supply chain of this important anticoagulant and warrant clinical validation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Management of Major Bleeding Caused By Rivaroxaban and the Use of Desmopressin

Blood ◽

10.1182/blood.v124.21.5099.5099 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5099-5099

Author(s):

Ahmad Jajeh

Keyword(s):

Major Bleeding ◽

Large Scale ◽

Conflicts Of Interest ◽

Factor Xa ◽

Fresh Frozen Plasma ◽

Thrombin Time ◽

Gi Bleeding ◽

Complex Product ◽

Packed Red Blood Cells ◽

Treatment And Prevention

Abstract Rivaroxaban is a new anticoagulant that is substituted for Coumadin on a large scale in the treatment and prevention of Deep Vein Thrombosis DVT and Pulmonary Embolism PE. It is an oral agent that inhibits Factor Xa. The most attractive attribute of this new anticogulant is the lack of monitoring PT/INR. However, out of many cases put on Rivaroxaban a few reports of major and threatening bleed that could be fatal. Particularly, the the GI bleeding. Unfortunately, no set standard antidote or management is available when such catastrophic bleeds happen. This abstract present our experience with three major bleeding cases that presented with massive GI bleeding. Two are associated with peptic ulcer upon Upper GI endoscopy. Two males and one female age 60, 71 (males) and 71 (female). The first two patients were treated with Prothrombin complex product. The female patient presented with sever anemia of 4 grams of Hb with hematemesis and bright red blood per rectum. The Prothrombin complex product was not readly available . She was given multipe doses of Fresh Frozen Plasma FFP and multiple units of packed red blood cells. She was also given a product Profilnine which contains Factor II, IX and VII. Patient's coagulation profile of PTT, PT and Thrombin time were corrected. However, she continue to have bright blood per NG suction. Upon receiving D-DAVP Desmopressin 0.3 micrograms per Kg she stopped bleeding and EGD was done later with sclerosing treatment of gastric ulcer and ligation. Patient was given later a small dose of Prothrombine complex when was available since the last dose of Rivaroxaban was given less than 13 hours from her presentation to the hospital. All of the mentioned patients had prolongation of PT/INR/PTT at presentation. Thrombin time was monitored in all of them. All patients had survived the magor GI bleeding. D-DAVP were given to all of them. In conclusion D-DAVP Desmopressin should be considered as an adjuvant drug in patient presentong with major GI bleeding secondary to Rivaroxaban. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2020-142653 ◽

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.

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Is Thrombin Time Useful To Guide Peri-Procedural Management For Patients On Dabigatran Etexilate?: An In Vitro Validation Study

Blood ◽

10.1182/blood.v122.21.2388.2388 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2388-2388

Author(s):

François Mullier ◽

Jonathan Douxfils ◽

Helene Baes ◽

Justine Baudar ◽

Sarah Lessire ◽

...

Keyword(s):

Conflicts Of Interest ◽

Dabigatran Etexilate ◽

Invasive Procedure ◽

Bleeding Risk ◽

Heterogeneous Data ◽

Important Variable ◽

Thrombin Inhibitor ◽

Thrombin Time ◽

Biological Test

Abstract Introduction Possibilities to monitor the intensity of dabigatran etexilate (DE) treatment may be valuable before urgent intervention. The Working Group on Perioperative Haemostasis proposed that the drug plasma concentration ([]) should be less or equal to 30ng/mL. However, plasma levels where it is safe to carry out an invasive procedure or surgery have not been confirmed prospectively. In addition, no biological test has been correlated with bleeding risk. Literature showed heterogeneous data regarding the arrest of DE depending on the renal function and the haemorrhagic risk. In addition, physicians request guidance for patients excluded from the clinical trials. Consequently, there is still a need for a rapid and widely available biological test. Some authors make proposals based on activated Partial Thromboplastin Time (aPTT), Hemoclot Thrombin Inhibitor® (HTI) or Thrombin Time (TT). TT displayed several advantages over aPTT. However, TT is affected by numerous analytic variables. Therefore, the objectives of the present study were: 1. To determine the optimal [thrombin] with a variety of instruments and reagents 2. To assess the repeatability of TT at optimized conditions 3. To compare the sensitivity and linearity of TT at residual [dabigatran] (DA) with those of aPTT and HTI 4. To validate the fibrinogen reagent (with heparin inhibitor) for TT experiments Methods DA was spiked at increasing [] in pooled citrated normal human platelet-poor plasma (NPP). The following [DA] were prepared: 0, 5, 10, 20, 30, 40 and 50ng/ml. 1. Optimal [thrombin] determination. The optimal [thrombin] was defined with 2 reagents: bovine thrombin (HemosIL® TT) and human thrombin (STA®-Thrombin) on 4 instruments: STA-R Evolution®, ACLTOP® , CS2000i® and KC10®. TT higher than Tmax is not informative for the physician. Thus the optimized [thrombin] was defined as the maximum [] giving, in a reproducible way, a TT at 50ng/ml lower than Tmax and a TT at 0ng/ml higher than the minimum TT. The Tmax was set arbitrarily at 120 sec. 2. Repeatability The repeatability of optimized TT was assessed by running aliquots of NPP spiked with all [DA] on 10 consecutive days 3. TT (1.5 NIH/ml, STA®-Thrombin), aPTT with SynthasIL® and STA®-C.K.Prest, and HTI were also determined within 1 hour on STA-R® on replicates of all NPP solutions. 4. STA®- Fibrinogen-5 was diluted to the optimized [thrombin] (1.5 NIH/ml) on STA-R® and used as a thrombin reagent. Results 1. The thrombin origin is a more important variable in comparison to the type of coagulometer. At [DA] of 30ng/ml and [thrombin] of 1.5 NIH/ml, the STA®-TT ranges from 56 sec to 74 sec according to the instrument, whereas on a same instrument, the TT varied of minimum 43 sec, depending on the thrombin origin. The optimized [thrombin] are the following : a) STA-R®, STA®-Thrombin : 1.5 NIH/ml, b) STA-R®, HemosIL® TT : 3.8 NIH/ml, c) ACLTOP®, STA®-Thrombin 3.8 NIH/ml, d) ACLTOP®, HemosIL® TT 5.0 NIH/ml, e) KC10®, STA®-Thrombin 1.5 NIH/ml, f) KC10®, HemosIL® TT 5.0 NIH/ml, g) CS2000i®, STA®-Thrombin 1.4 NIH/ml, h) CS2000i®, HemosIL® TT 3.8 NIH/ml. Except for STA®-Thrombin on STA-R®, the [optimized] is not the one recommended by the manufacturer. Figure 1 shows the results of optimized TT according to thrombin origin and coagulometer. 2. Repeatability experiments showed that variability increased with the [DA] (i.e. STA®-Thrombin (STA-R®): coefficient of variation: 8.0% and 28.9% for 0ng/ml and 50ng/ml, respectively) and that the variability depends on the coagulometer and the reagent. 3. Comparison of sensitivity and linearity of TT, aPTT and HTI are presented in Table 1. APTT is not sensitive enough in low [DA], whatever the reagent, whereas HTI is not suitable in [DA] lower than 50ng/ml. 3. TT performed with fibrinogen reagent shows longer times in comparison to thrombin reagent (>120 sec vs 109 sec at 30ng/ml DA). Conclusions TT may be more informative than aPTT and HTI to provide with guidance to carry out an urgent procedure or surgery for patients receiving DE. However, TT is affected by a lot of analytic variables that should be understood by laboratories. Each laboratory should optimize its TT procedure according to its combination coagulometer-reagent. Disclosures: No relevant conflicts of interest to declare.

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The Venous Antithrombotic Profile of Naroparcil in the Rabbit

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648977 ◽

1994 ◽

Vol 72 (06) ◽

pp. 874-879 ◽

Cited By ~ 11

Author(s):

Jean Millet ◽

Jocelyne Theveniaux ◽

Neil L Brown

Keyword(s):

Oral Administration ◽

Bleeding Time ◽

Dermatan Sulfate ◽

Factor Xa ◽

Bleeding Risk ◽

Thrombin Time ◽

Heparin Cofactor Ii ◽

Antithrombotic Activity ◽

Thrombin Inhibition ◽

Maximal Reduction

SummaryThe venous antithrombotic profile of naroparcil or (4-[4-cyanoben-zoyl]-phenyl)-1.5-dithio-β-D-xylopyranoside was investigated in the rabbit following single i. v. and oral administration. Naroparcil attenuated thrombus development in a Wessler stasis model of venous thrombosis (jugular vein) employing bovine factor Xa as a thrombogenic stimulus giving ED50 values of 21.9 mg/kg and 36.0 mg/kg after respectively i. v. and oral administration. Venous antithrombotic activity was maximal 2-3 h after i. v. administration and 4-8 h after oral administration. Four hours after the oral administration of maximal antithrombotic (Wessler model, factor Xa) doses (100 and 400 mg/kg), naroparcil had no significant effect on bleeding time. In platelet poor plasma obtained from animals treated 4 h previously with various doses (25 to 400 mg/kg) of naroparcil, there was no detectable anti-factor Xa nor antithrombin activity. Similarly, naroparcil had no effect on APTT nor on thrombin time. A sensitized thrombin time (to about 35 s) was modestly but significantly increased following oral administration of the compound at 400 mg/kg. However, thrombin generation by the intrinsic pathway was reduced in a dose-related manner, maximal reduction being 65% at 400 mg/kg. The same doses of naroparcil enhanced the formation of thrombin/heparin cofactor II complexes at the expense of thrombin/antithrombin III complexes in plasma incubated with (125I)-human a-thrombin and induced the appearance of dermatan sulfate-like material in the plasma of treated rabbits, as measured by a heparin cofactor II-mediated thrombin inhibition assay. The results suggest that naroparcil could have a safe venous antithrombotic profile following oral administration (antithrombotic effect compared to bleeding risk). It is probable that part of the mechanism of action of the β-D-xyloside, naroparcil, is due to the induction of chondroitin sulfate-like glycosaminoglycan biosynthesis, this material being detectable in the plasma.

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The Measurement of Heparin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649125 ◽

1973 ◽

Vol 30 (03) ◽

pp. 471-479 ◽

Cited By ~ 31

Author(s):

K. W. E Denson ◽

John Bonnar

Keyword(s):

Degradation Products ◽

Factor Xa ◽

Assay Method ◽

Thrombin Time ◽

Fibrinogen Degradation Products ◽

Low Levels

SummaryA method for the measurement of heparin utilising the potentiating effect of heparin on the action of anti-factor Xa is described. The effect on the assay of platelet contamination of plasma, the presence of fibrinogen degradation products and low levels of anti-factor Xa have been studied. The assay method has been compared with the calcium thrombin time method and a group of obstetrical patients have been studied using both methods.

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