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.

IntroductionOrally bioavailable anticoagulants include, Apixaban, Betrixaban, Edoxaban, and Rivaroxaban which are anti Xa drugs approved for various indications. Dabigatran is an anti‐IIa drug which is primarily approved for atrial fibrillation. While these drugs are claimed to have targeted actions toward Factor Xa or IIa, current data suggests that their actions may involve other target sites. This study was designed to compare the relative anticoagulant effects of each of these drugs in whole blood and retrieved plasma, using PT, aPTT and TT tests. Additionally the neutralization of these drugs by an activated prothrombin complex concentrate FEIBA was also investigated.MaterialsPowdered forms of Apixaban, Betrixaban, Edoxaban, Rivaroxaban and Dabigatran were obtained from commercial sources. All drugs were dissolved at a stock concentration of 1 mg/mL in saline. Working concentrations were prepared at 100 ug/mL and 10 ug/mL. Freshly drawn citrated whole blood from healthy volunteers (n=10) was supplemented with each of these agents at a graded concentration of 0 to 1 ug/mL. Whole Blood, PT and aPTT were measured using the ST4 (Diagnostica Stago) instrument. Whole blood supplemented with each of these drugs was centrifuged to obtain retrieved plasma from each of these agents. The retrieved plasma was also analyzed for PT and aPTT. All results were compiled in terms of mean and standard deviation. These studies were repeated in the presence of 0.1 U/mL of FEIBA to determine the relative neutralization of their anticoagulant effects.ResultsAll of the five drugs produced concentration dependent anticoagulant effects in both the PT and aPTT tests. In the whole blood, the PT prolongation was different for each agent, with the rank order Dabigatran > Edoxaban > Betrixaban > Rivaroxaban > Apixaban > Saline. The prolongation of the aPTT in the whole blood followed the rank order Betrixaban > Dabigatran > Rivaroxaban > Edoxaban = Apixaban > Saline. All five drugs produced an increase in the PT in the retreat plasma with rank order Dabigatran > Edoxaban > Apixaban > Rivaroxaban > Betrixaban > Saline. In the aPTT assay the increase in clotting times followed the rank order Dabigatran > Betrixaban > Rivaroxaban > Edoxaban > Apixaban. FEIBA supplementation at a 0.1 U/ml to each of the drugs supplemented at 1 ug/ml markedly neutralized their anticoagulant effects in the PT and aPTT assay systems. At this concentration FEIBA did not have any significant effects on the PT and aPTT assays.ConclusionsThese studies suggest that in comparison to the anti‐Xa agents, Dabigatran produces relatively stronger anticoagulant effects in the PT and aPTT assays in both whole blood and retrieved plasma. FEIBA at a relatively low level of 0.1 U/ml was capable of neutralizing the anticoagulant effects of the newer oral anticoagulant drugs and may be helpful in the control of potential bleeding complications with these drugs.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background Pentosan Polysulfate has traditionally been used to treat patients with Interstitial Cystitis and more recently osteoarthritis, cancer, HIV and prion disease. Such widespread clinical application of PPS and the active search for new effective alternatives to traditional heparin compels a comprehensive investigation of the effect of PPS batches on anticoagulation in comparison to Heparin. The purpose of this study is to create a comprehensive profile and comparison of new PPS batches (L‐15, L‐19, L20 and Ba), old PPS batch (PPS Ba), and Heparin. Furthermore, the feasibility of protamine sulfate as a neutralizing agent to reverse the anticoagulant effects of PPS is investigated. Materials and Methods Various batches of PPS at 1 mg/ml were prepared in saline. For whole blood clotting profile, thromboelastography (TEG) was carried from 2.5 – 5.0 ug/ml and activated clotting time (ACT) at 25ug/mL. Clotting profile in whole blood, retrieved plasma and supplementation in normal human pooled plasma (NHP) were carried out at 100 – 0.0 ug/ml by using anticoagulant assays. Amydolytic methods were used to measure anti‐Xa/ IIa effects. Thrombin generation inhibition studies (TGA) were carried out in NHP from 100 ‐ 0.0 ug/ml. Heparin supplementation and protamine sulfate neutralization in NHP from 10 ‐0 ug/mL were compared to PPS batches in a logarithmic scale. Results All drugs produced a concentration dependent effect. Similar effects in anticoagulation assays were noted except anti‐Xa assay. In TEG, significant viscoelastic changes were observed at 5ug/mL. Prolongation of ACT was observed at 25ug/mL. No significant differences were noted in between batches of PPS in TEG and ACT studies. In TGA, concentration dependent but no significant difference in between the batches were noted. Neutralization of protamine sulfate was observed up to 12.5 ug/mL in anticoagulant assays and marginal effect on anti‐protease assays. Lastly, logarithmic comparison of the protamine sulfate neutralization indicated that protamine sulfate is a weaker neutralizing agent of PPS compared to heparin. Conclusion These studies suggest that multiple batches of PPS have a similar anticoagulant profile. In comparison to heparin, PPS presents itself as a 10‐fold weaker anticoagulant alternative for use in a variety of clinical applications. Neutralization of PPS at low concentrations by protamine sulfate in anticoagulant assays warrants further investigation on an effective neutralizing concentration for clinical use.

ObjectiveNew direct oral anticoagulants have a safety profile that differentiates them from traditionally used oral anticoagulants. DOACs with the similar mechanisms of action and structural similarities are being marketed by numerous companies under varied brands. Such widespread use warrants differentiation of these DOACs on clot formation and their neutralization profile by antidotes. Fibrinokinetics is a method in which thrombin induced clot formation can be measured. The purpose of this study is to compare the effect of Dabigatran, Rivaroxaban, Edoxaban, Betrixaban and Apixaban on thrombin induced fibrinokinetics in normal human plasma. Furthermore, the feasibility of factor eight inhibitor bypass agent (FEIBA) as an antidote to modulate these DOACs is investigated.Materials and MethodsCitrated pool blood plasma was obtained from Loyola blood bank. Purified thrombin was used as a trigger to generate clot at a concentration of 5U/ml. Powdered forms of Dabigatran, Rivaroxaban, and Apixaban were obtained commercially and were used at a working dilution of 1ug/mL. Fibrinokinetics was measured in microtiter plates using 175 microliter of plasma supplemented with the drugs to be tested at a concentration of 25uL of 5U of thrombin followed by 25uL of 0.025M of CaCl2. For the control well plates 25uL of saline was added. To study the FEIBA neutralization, 25uL of FEIBA at a concentration of 0.1U/mL was added in the other set of experimental well plates. The rate of clot formation was monitored for 30 minutes at A45 nm. Both kinetic and endpoint methods were used to calculate the rate of clot formation.ResultsIn the saline control system the time dependent increase in the clot formation was observed for a period of 30 minutes reaching the maximum of 1. Dabigatran, Edoxaban, Rivaroxaban produced a concentration dependent inhibition of clot formation whereas Apixaban and Betrixaban did not produce any significant inhibition of clot formation at concentration up to 1ug/mL. FEIBA supplementation at a final concentration 0.1U/mL produced an increase in clot density in the case of Betrixaban and Apixaban whereas FEIBA reversed the effect of Rivaroxaban, Edoxaban and Dabigatran at a lower concentration up to 0.125U/mL.ConclusionsThree groups can be stipulated from the results of this experiment. The first group includes Dabigatran and has the strongest effect on clot formation and experienced significant inhibition by FEIBA. The second group includes Rivaroxaban and Edoxaban with a strong factor Xa inhibitory activity and moderate neutralization by FEIBA. The third group includes Betrixaban and Apixaban with a weak factor Xa activity and insignificant modulation by FEIBA. The successful modulation by FEIBA on certain DOACs indicate that antidotes for these drugs in the future can stem from procoagulants that are already in use such as FEIBA.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.