Comparison of antithrombotic and haemorrhagic effects of edoxaban, an oral direct factor Xa inhibitor, with warfarin and enoxaparin in rats

Introduction

Factor Xa (FXa) is a key serine protease in the coagulation cascade and a promising target for a new antithrombotic agent. Edoxaban is an oral, selective and direct FXa inhibitor. The objective of this study was to compare the antithrombotic and haemorrhagic effects of edoxaban with clinically available anticoagulants, warfarin and enoxaparin, in rat models of thrombosis and haemorrhage.

Methods

Rats were treated with single oral administration of edoxaban, repeated oral dosing of warfarin for 4 days and single subcutaneous administration of enoxaparin before thrombosis or haemorrhage induction. Thrombosis was induced by the insertion of a platinum wire into the inferior vena cava for 60 min. Tail template bleeding time was measured after making an incision on the tail.

Results

Edoxaban at 0.3, 1 and 3 mg/kg exerted dose-dependent and significant inhibition of venous thrombus formation. The 50% thrombus inhibition dose (ED₅₀) was 1.9 mg/kg. At supra-therapeutic doses (10 and 20 mg/kg), edoxaban significantly but moderately (less than 2-fold) prolonged bleeding time. Warfarin and enoxaparin also dose-dependently inhibited venous thrombosis and prolonged bleeding time. The ED₅₀ values of warfarin and enoxaparin were 0.12 mg/kg and 500 IU/kg, and the 2-fold bleeding time prolongation doses (BT2) were 0.16 mg/kg and 1700 IU/kg, respectively. The safety margin (ratio of BT2 to ED₅₀) of edoxaban (> 10.5) was greater than those of warfarin (1.3) and enoxaparin (3.4).

Conclusions

Edoxaban inhibited venous thrombosis comparably to warfarin and enoxaparin, and the attendant bleeding risk of edoxaban was lower than that of warfarin and enoxaparin in rats.     

Comparison of the effect of edoxaban,a direct factor Xa inhibitor,with a direct thrombin inhibitor,melagatran, and heparin on intracerebral hemorrhage induced by collagenase in rats

Introduction

Intracerebral hemorrhage (ICH) is a major clinical concern with anticoagulation therapy. The effect of a new oral direct FXa inhibitor, edoxaban, was determined in a rat model of ICH and compared with a direct thrombin inhibitor, melagatran, and heparin.

Methods

To induce ICH, 0.1 U collagenase type VII was injected into the striatum of male Wistar rats under anesthesia with thiopental or halothane. Immediately after ICH induction, edoxaban, melagatran, or heparin were infused intravenously. Five hours after ICH induction, the brain was removed and ICH size was measured. To estimate the margin of safety, antithrombotic effects were evaluated in a rat venous thrombosis model.

Results

Edoxaban at 6 mg/kg/h significantly increased ICH volume (1.8-fold) and prolonged prothrombin time (PT) 2.8-fold compared to the vehicle group. No deaths were observed with edoxaban. Melagatran at 1 mg/kg/h increased ICH volume at 1 mg/kg/h (2.8-fold) with 6.1-fold PT prolongation. At 3 mg/kg/h, all rats died due to severe ICH (3.9-fold). Heparin at both 100 and 500 U/kg/h significantly increased ICH. At 500 U/kg/h, 5 out of 8 rats died. The doses required for 50% inhibition of thrombosis of edoxaban, melagatran, and heparin were 0.045 mg/kg/h, 0.14 mg/kg/h, and 55 U/kg/h, respectively. The safety margins between antithrombotic and ICH exacerbation effects of these anticoagulants were 133, 7.1, and 1.8, respectively.

Conclusion

The safety margin of edoxaban was wider than that of melagatran or heparin. These results suggest that edoxaban may be preferable from the perspective of ICH exacerbation risk.     

Synergistic effect of a factor Xa inhibitor, TAK-442, and antiplatelet agents on whole blood coagulation and arterial thrombosis in rats

Introduction

Activated platelets facilitate blood coagulation by providing factor V and a procoagulant surface for prothrombinase. Here, we investigated the potential synergy of a potent factor Xa/prothrombinase inhibitor, TAK-442, plus aspirin or clopidogrel in preventing arterial thrombosis and whole blood coagulation.

Methods

Thrombus formation was initiated by FeCl₃-induced rat carotid injury. Bleeding time was evaluated with the rat tail transection model. Whole blood coagulation was assessed by thromboelastographic examination (TEG) for which blood obtained from control, aspirin-, or clopidogrel-treated rats was transferred to a TEG analyzer containing, collagen or adenosine diphosphate (ADP), and TAK-442 or vehicle.

Results

TAK-442 (3 mg/kg, po), aspirin (100 mg/kg, po) or clopidogrel (3 mg/kg, po) alone had no significant effect on thrombus formation, whereas the combination of TAK-442 with aspirin and clopidogrel remarkably prolonged the time to thrombus formation without additional significant prolongation of bleeding time. TEG demonstrated that the onset of collagen-induced blood coagulation were slightly longer in aspirin-treated rats than control; however, when the blood from aspirin-treated rats was subsequently treated in vitro with 100 nM TAK-442, the onset of clotting was significantly prolonged. In contrast, only marginal prolongation was observed with TAK-442 treatment of blood from control animals. The onset time of ADP-induced blood coagulation was slightly longer in clopidogrel-treated rats compared with control, and it was further extended by TAK-442 treatment.

Conclusion

These results demonstrate that blood coagulation can be markedly delayed by the addition of TAK-442 to antiplatelets treatment which could contribute to synergistic antithrombotic efficacy in these settings.     

Comparison of a direct Factor Xa inhibitor, edoxaban, with dalteparin and ximelagatran: A randomised controlled trial in healthy elderly adults

Introduction

Edoxaban (the free base of DU-176b) is a new, oral direct Factor Xa inhibitor. This is the first study to compare the hemostatic response to edoxaban, ximelagatran, and dalteparin in healthy, elderly adults.

Materials and Methods

In this open-label, active-controlled clinical trial, 40 adults (65-75 years), were randomised to: oral edoxaban (60 mg, twice-daily, 7 doses), subcutaneous dalteparin (5000 IU, once-daily, 4 doses), oral ximelagatran (24 mg, twice-daily, 7 doses) or no drug. Blood samples were taken before, and 1.5, 4, 12, 24, 72, 84, 96, 108, 120, and 144 hours after, the first dose. The primary outcomes were changes in thrombin-antithrombin complex, prothrombin fragment 1 + 2 and D-dimer, and adverse events. Additional biomarkers of coagulation, and endothelial cell and platelet activation were compared (ANOVA).

Results

All subjects completed the study. Inhibition of thrombin generation lag time, peak, and constant velocity index were significantly greater, and extended for a longer period of time, following edoxaban administration, compared with dalteparin. We found that the traditional assay for anti-FXa activity was not appropriate for the new anticoagulants. Biomarker changes following edoxaban administration (including prolongation of prothrombin time) reflected inhibition of Factor Xa; there was no effect on platelet, tissue factor or endothelial activation. There were no clinically significant changes in primary outcomes. No serious adverse events were reported.

Conclusion

Oral administration of edoxaban resulted in effective Factor Xa and TG inhibition, and was well-tolerated. Studies are needed to confirm edoxaban (60 mg daily) use in clinical practice.

Sponsorship

Daiichi Sankyo Pharma Development.     

A new plastic collection tube made of polyethylene terephtalate is suitable for monitoring traditional anticoagulant therapy (oral anticoagulant, unfractionated heparin, and low molecular weight heparin)

To improve the safety of blood collection, plastic tubes have been developed but various interactions with the coagulation system and/or antithrombotic drugs were reported with the first generation of such tubes. The aim of this multicentre study was to compare hemostasis test results measured in evacuated plastic tubes made of polyethylene terephtalate (VenoSafe, Terumo Europe) and in siliconized glass tubes containing the same citrate concentration (0.129 M). In addition, the impact of aging of the plastic tube was investigated by collecting blood samples in tubes at 8 months and at 1 month before expiry. Blood was drawn in 3 centres from untreated patients (n=269), patients on oral anticoagulant treatment (OAT, n=221), and patients treated with either unfractionated heparin (UFH, n=73) or a low molecular weight derivative (LMWH, n=48). Prothrombin time (PT) or INR, activated partial thromboplastin time (APTT) and anti-FXa activity were locally performed, when applicable. In untreated patients and in patients on OAT, PT and APTT values were found statistically shorter (p<0.05) when evaluated in plastic tubes than in glass tubes, except when PT was evaluated using a human thromboplastin. Surprisingly, significantly longer APTT and higher anti-FXa activities were obtained when blood from patients on UFH was drawn in plastic than in glass tubes. However, none of the differences had any clinical relevance (Bland-Altman analysis). In patients on anticoagulant treatment, there was no effect of aging of the plastic tubes. These results suggest that the plastic tube VenoSafe is suitable for coagulation testing both in untreated subjects and more interestingly in patients on traditional anticoagulant therapy during the whole shelf life indicated by the manufacturer.     

Heparin-induced thrombocytopenia: a stoichiometry-based model to explain the differing immunogenicities of unfractionated heparin, low-molecular-weight heparin, and fondaparinux in different clinical settings

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4 (PF4)/heparin complexes. The frequency of HIT is highly variable in different clinical settings, and is more frequent with unfractionated heparin (UFH) than with low-molecular-weight heparin (LMWH), despite the in vitro observation that HIT antibodies activate platelets similarly well with LMWH as with UFH. An important difference between UFH, LMWH, and fondaparinux is their widely differing plasma concentrations. We aimed to provide a model that included anticoagulant concentrations and PF4 availability as risk factors influencing the anti-PF4/heparin immune response. MATERIALS AND METHODS: By photon correlation spectroscopy we determined the concentrations at which UFH, LMWH, and fondaparinux form complexes optimally with PF4. Plasma concentrations of UFH and LMWH were calculated based on ex vivo pharmacokinetic data, with information on fondaparinux and PF4 concentrations taken from the literature. RESULTS AND CONCLUSIONS: The main features of our model are: optimal complex formation occurs at prophylactic-dose UFH and high PF4 levels, whereas therapeutic-dose LMWH concentrations are too high for optimal complex formation; in contrast, concentrations of fondaparinux are usually below the optimal stoichiometric range. Thus, immunization should occur more often in situations with major rather than minor platelet activation, and--for a given degree of platelet activation (PF4 availability)--as: prophylactic-dose UFH>therapeutic-dose UFH>prophylactic-dose LMWH, fondaparinux>therapeutic-dose LMWH. Our model provides a framework for explaining empirical observations that LMWH induces less anti-PF4/heparin antibodies than does UFH, and that anti-PF4/heparin antibodies are more often found in patients undergoing major surgery than in medical patients.     

Discovery of glycyrrhetinic acid as an orally active,direct inhibitor of blood coagulation factor xa

Introduction

Factor Xa (FXa) plays an important role in blood coagulation. This study investigated glycyrrhetinic acid, a small molecule derived from Chinese herbs, and whether it has a direct inhibitory effect on FXa to display its anticoagulant activity.

Materials and Methods

Enzyme activities of FXa, plasmin, trypsin and thrombin, inhibition of FXa enzyme kinetics and plasma clotting time by glycyrrhentinic acid were performed in vitro. A rat tail-bleeding model and a rat venous stasis model were also used to evaluate in vivo tail-bleeding time and thrombus formation, respectively.

Results

Glycyrrhetinic acid in vitro directly inhibited FXa uncompetitivly with IC₅₀ of 32.6 ± 1.24 μmol/L, and displayed 2-, 14- and 20-fold selectivity for FXa when compared to plasmin, thrombin and trypsin, respectively. The plasma clotting time was increased in a dose-dependent manner. The prothrombin time doubled (PT₂), when the concentration of glycyrrhetinic acid reached 2.02 mmol/L. During in vivo experiments intragastric administration of glycyrrhetinic acid caused a dose-dependent reduction in thrombus weight on the rat venous stasis model (all P < 0.05). 50 mg/kg glycyrrhetinic acid resulted in 34.8% of venous thrombus weight lost, compared to the control. In addition, 200, 300 and 400 mg/kg doses of glycyrrhetinic acid caused a moderate hemorrhagic effect in the rat tail-bleeding model by prolonging bleeding time 1.1-, 1.5- and 1.9-fold compared to the control, respectively.

Conclusions

Glycyrrhetinic acid is a direct inhibitor of FXa that is effective by oral administration, and with further research could be used to treat blood coagulation disorders.     

Ex vivo reversal of the anticoagulant effects of edoxaban

Introduction

Edoxaban is an oral, once-daily direct factor Xa (FXa) inhibitor. Although rapidly cleared, strategies to reverse edoxaban-mediated effects on anticoagulation are needed in cases of excessive bleeding or emergency. This study evaluated the effect of two prohemostatic agents, recombinant factor VIIa (rFVIIa) and factor VIII inhibitor bypass activity (FEIBA), on the anticoagulatory effects of supratherapeutic concentrations of edoxaban in human whole blood ex vivo.

Materials and Methods

Blood samples were collected from six healthy volunteers. Edoxaban (500 or 1000 ng/mL), alone or followed by rFVIIa (0.8 or 1.8 μg/mL) or FEIBA (0.75 or 1.5 U/mL), was added to an aliquot of each sample. Biomarkers, including prothrombin time (PT), activated partial thromboplastin time (aPTT), extrinsic FXa activity (anti-FXa), intrinsic factor X activity, and D-dimer, were assessed at 0.25, 0.5, 1, 2, and 4 hours after adding rFVIIa or FEIBA.

Results

Decreases in measures of PT (p < 0.0001), aPTT (p < 0.0001), and anti-FXa (p < 0.0001) were observed when rFVIIa or FEIBA was added to edoxaban-containing blood samples. Intrinsic FX activity was increased up to 20% and 31% of normal in the presence of edoxaban by rFVIIa and FEIBA, respectively. The impact of these agents on the anticoagulant effects of edoxaban were observed within 15 minutes and remained relatively unchanged at each timepoint thereafter.

Conclusions

The findings of this ex vivo study suggest that rFVIIa and FEIBA rapidly reversed edoxaban-mediated anticoagulation effects based on PT and aPTT, but had minimal effect based on intrinsic FX activity. No dose response was observed for rFVIIa or FEIBA.     

Quality of anticoagulation with unfractionated heparin plus phenprocoumon for the prevention of thromboembolic complications in cardioversion for non-valvular atrial fibrillation. Sub-analysis from the Anticoagulation in Cardioversion using Enoxaparin (ACE) trial

INTRODUCTION: Anticoagulation in cardioversion for atrial fibrillation is performed using unfractionated heparin and oral anticoagulants. TEE-guided cardioversion, after achievement of therapeutic anticoagulation (1-3 days), may be an alternative to the traditional procedure (3-week anticoagulation followed by cardioversion). The quality of anticoagulation in atrial fibrillation has not been investigated in a randomised trial with TEE-guided cardioversion. We analysed respective data from the ACE trial on the quality of conventional anticoagulation, where most participating centres chose the TEE-guided approach. MATERIALS AND METHODS: In a randomised, prospective, multicentre trial, we analysed the efficacy of unfractionated heparin plus phenprocoumon in 248 patients on an intention-to-treat basis. There were 2373 evaluable anticoagulation measurements (out of 2925 measurements) and 4 categories of anticoagulation quality (under-, target, over- and severe over-anticoagulation). Of patients with evaluable measurements, 88% received short-term anticoagulation (4 weeks) in TEE-guided cardioversion. RESULTS: The median time to achieve therapeutic anticoagulation (aPTT> or =60 and <80 s or INR> or =2 and <3) was 3 days. Anticoagulation values were out of therapeutic range in 69.5% of measurements during 4- or 7-week follow-up, and never within therapeutic range in 10% of patients. Of the 15 primary endpoints observed (death, thromboembolism and major bleeding complications), only 3 were in patients with anticoagulation measurements within therapeutic range. CONCLUSIONS: In this study setting, with predominance of 4 weeks anticoagulation in TEE-guided cardioversion for atrial fibrillation, therapeutic anticoagulation was reached within 3 days using conventional anticoagulation. Despite careful dose adjustments, anticoagulation was out of therapeutic range in almost 70% of total measurements and 80% of primary endpoints.     

Underestimation of unfractionated heparin therapy assessment due to platelet activation when using partial-draw (pediatric) citrate collection tubes

The “so-called” pediatric tubes are often used when collecting smaller blood volume is necessary, particularly in pediatric patients or in case of difficult/recurrent sampling. The aim of this multicenter study was to compare coagulation test results evaluated in evacuated polymer tubes containing 0.109 M citrate (1 vol./9 vol.) specifically designed to allow either a partial (2.0 mL,“pediatric”) or a total (3.5 mL) filling. No significantly relevant discrepancy was found between routine coagulation test results in both tubes collected from untreated patients and from patients on vitamin K antagonist or low molecular weight heparin. In contrast, aPTT was significantly shorter and anti-FXa activity was significantly lower in partial-draw than in full-draw tubes collected from 46 patients receiving unfractionated heparin (UFH). This discrepancy was likely related to increased platelet activation in partial-draw tubes, as suggested by higher platelet factor 4 plasma concentrations and platelet P-Selectin expression in partial-draw than in full-draw citrate tubes. To confirm this hypothesis, we then evaluated partial-draw tubes containing CTAD, a mixture of anticoagulant and antiplatelet agents. In 25 patients on UFH, aPTT and anti-FXa activity were not significantly different in partial-draw CTAD tubes and in full-draw citrate tubes. In conclusion, despite increased platelet activation, samples collected into partial-draw citrate tubes allow accurate routine coagulation testing in all patients but those requiring UFH assessment, in which their use could lead to significant underestimation of anticoagulation. In such cases, partial-draw tubes containing CTAD could be validly used to monitor heparin therapy as well as to perform routine coagulation testing.     

The mechanism of action of rivaroxaban--an oral, direct Factor Xa inhibitor--compared with other anticoagulants

Although results of some phase III clinical trials of new oral anticoagulants are now known, it is important to understand the mechanisms of their actions. These new agents exert their anticoagulant effect via direct inhibition of a single Factor within the coagulation cascade (such as Factor Xa or thrombin). Rivaroxaban – the first oral, direct Factor Xa inhibitor – is a small-molecule oxazolidinone derivative that binds directly and reversibly to Factor Xa via the S1 and S4 pockets. Rivaroxaban competitively inhibits Factor Xa and is more than 10,000-fold more selective for Factor Xa than other related serine proteases, and it does not require cofactors (such as antithrombin) to exert its anticoagulant effect. Unlike indirect Factor Xa inhibitors, rivaroxaban inhibits both free and clot-bound Factor Xa, as well as prothrombinase activity, thereby prolonging clotting times. Dabigatran etexilate is a direct thrombin inhibitor that inhibits both free and fibrin-bound thrombin. Although the mechanism of action differs between the direct Factor Xa and direct thrombin inhibitors, phase III studies of these new agents confirmed that both Factor Xa and thrombin are viable anticoagulation targets.     

The antithrombotic and anti-inflammatory effects of BCX-3607, a small molecule tissue factor/factor VIIa inhibitor

Tissue factor (TF) is a transmembrane glycoprotein that binds its zymogen cofactor, Factor VIIa (FVIIa) on the cell surface. Together (TF/FVIIa) they activate Factor X (FX) and Factor IX (FIX) and start the extrinsic pathway of blood coagulation. As such, the TF/FVIIa complex plays an important role in normal physiology as well as in thrombotic diseases such as unstable angina (UA), disseminated intravascular coagulation (DIC), and deep vein thrombosis (DVT). In addition to its function as an initiator of coagulation, TF/FVIIa plays an important role in inflammation. Expression of TF on the cell surface and its appearance as a soluble molecule are characteristic features of acute and chronic inflammation in conditions such as sepsis and atherosclerosis. Here we demonstrate that BCX-3607, a small molecule potent inhibitor of TF/FVIIa, reduces thrombus weight in an animal model of DVT. BCX-3607 also decreases the level of interleukin-6 (IL-6) in a LPS-stimulated mouse model of endotoxemia. Additionally, in vitro studies indicate that BCX-3607 blocks the generation of TF/FVIIa-induced IL-8 mRNA in human keratinocytes and reduces the TF/FVIIa-mediated generation of IL-6 and IL-8 in human umbilical vein endothelial cells (HUVEC). Therefore, BCX-3607 might block the TF/FVIIa-mediated coagulation and inflammation associated with pathological conditions.     

A comparative study of antithrombotic and antiplatelet activities of different fucoidans from Laminaria japonica

Introduction

Fucoidans extracted from brown algae represent an intriguing group of natural fucose-enriched sulfated polysaccharide, with excellent anticoagulant, antimetastatic, antiangiogenic and anti-inflammatory activities. In the present study, we compared antithrombotic activities of four fucoidan fractions with different molecular weight and sulfated ester content from Laminaria japonica in an electrical induced arterial thrombosis and their potential mechanism underlying such activity.

Results and Conclusions

In vivo middle molecular weight (MMW) fucoidan fractions with molecular weight about 28000 and 35000 exhibited better antithrombotic activity in electrical induced arterial thrombosis than low molecular weight (LMW) fucoidan LF1 and LF2 (Mw 7600 and 3900). Inhibition of arterial thrombosis occurred at dose of 0.1-0.25 mg/kg for MMW fucoidans, accompanied with moderate anticoagulant activity and significant decrease of whole blood viscosity and hematocrit. The antithrombotic effects of MMW Fucoidans might be related with promotion of TFPI content and decrease of TXB2 content, without affecting platelet aggregation and 6-keto-PGF1α content in vivo. In contrast, LMW fucoidans showed a correlation among anticoagulant, antiplatelet and antithrombotic effects in vivo. Antithrombotic action of LF1 and LF2 required high dose of 2.5-10 mg/kg, concomitantly with anticoagulant activity and specific inhibition of platelet aggregation in vivo. Their antithrombotic effect might be related to their promotion of TFPI and 6-keto-PGF1α, down regulation of TXB2, without affecting hemorheology. These findings suggested that fucoidan fractions with different molecular weight acted on the antithrombotic action by different mechanism. By comparison, highly sulfated fucoidan LF2 with molecular weight of 3900 seemed to be a more suitable choice of antithrombotic drug for its antithrombotic activity accompanied with specific inhibitory activity on platelet aggregation, low anticoagulant activity and low hemorrhagic risk in vivo.     

Comparison of a fluorogenic anti-FXa assay with a central laboratory chromogenic anti-FXa assay for measuring LMWH activity in patient plasmas

Introduction

Low molecular weight heparins (LMWHs) are used worldwide for the treatment and prophylaxis of thromboembolic disorders. Routine laboratory tests are not required due to the predictable pharmacokinetics of LMWHs, with the exception of pregnant patients, children, patients with renal failure, morbid obesity, or advanced age. Anti-Factor Xa (anti-FXa) plasma levels are most often employed in the assessment and guidance of accurate dosing in these patient cohorts.

Materials and methods

A LMWH calibration curve was generated using citrated human pooled plasma spiked with pharmacologically relevant concentrations (0–1.2 U/ml) of two low molecular weight heparins; enoxaparin and tinzaparin. Least squares analysis determined the best curve fit for this set of data which returned low sum of squares (SS) values for the log linear fit with an R² value of 0.98. 30 patient samples were tested in the fluorogenic assay and concentrations were determined using the log linear regression equation and correlated with a standard chromogenic assay used for heparin monitoring.

Results

A statistically significant correlation was found between the fluorogenic and the chromogenic anti-FXa assays for 30 patient samples, with a slope of 0.829, offset of 0.258 and an R² value of 0.72 (p < 0.0001).

Conclusions

In the study presented here, a fluorogenic anti-FXa assay was correlated with a standard laboratory chromogenic anti-FXa assay using samples from patients on LMWH therapy. Significant correlations between the values derived by the fluorogenic and chromogenic anti-FXa assays were found for the patient cohort tested in this study.     

Reversal of rivaroxaban-induced anticoagulation with prothrombin complex concentrate,activated prothrombin complex concentrate and recombinant activated factor VII in vitro

Introduction

Anticoagulation therapies carry a risk of bleeding; reversal agents may be beneficial in cases of severe bleeding even for anticoagulants with a relatively short half-life, such as the oral factor Xa inhibitor rivaroxaban.

Materials and Methods

We investigated the in vitro reversal effect of prothrombin complex concentrate (PCC; 0.2-1.0 U/mL), activated PCC (aPCC; 0.2–1.0 U/mL) and recombinant activated factor VII (rFVIIa; 5–50 μg/mL) on rivaroxaban-induced (200–1000 ng/mL) changes in prothrombin time (PT) and thrombin generation (TG) in plasma, and in thromboelastometry (clotting time [CT]) in whole blood from healthy subjects.

Results

All three agents were partially effective in reversing rivaroxaban-induced anticoagulation but showed different profiles. rFVIIa and aPCC were more effective than PCC in reversing prolongations of PT, CT and TG lag time; rFVIIa was more effective than aPCC. However, the reversal effect reached a plateau with a maximal effect of approximately 50%. Inhibition of maximum thrombin concentration was slightly reversed by these agents; aPCC was the most effective. In contrast, inhibition of endogenous thrombin potential (ETP) was strongly reversed by aPCC, with significant increases over baseline at low rivaroxaban concentrations. Compared with aPCC, PCC showed a similar but less effective reversal profile. rFVIIa reversed ETP inhibition by approximately 50%.

Conclusions

The extent of reversal by aPCC, PCC and rFVIIa was dependent on the parameter measured in rivaroxaban-anticoagulated plasma or blood. ETP measurements may have predictive power for assessing the reversal potential of PCC or aPCC and may be used to indicate an increased prothrombotic risk.     

Comparison of the anticoagulant response of a novel fluorogenic anti-FXa assay with two commercial anti-FXa chromogenic assays

Introduction

Fast and accurate monitoring is crucial in the successful regulation of coagulation therapy. For the treatment of venous thromboembolism, both unfractionated heparin (UFH) and low molecular weight heparins (LMWHs) are commonly administered. The chromogenic anti-factor Xa (FXa) assay is currently considered the ‘gold standard’ assay for monitoring LMWH. However different commercial chromogenic methods often differ when tested with the same samples. Fluorogenic anti-FXa assays have the potential to offer greater benefits over chromogenic assays in terms of greater specificity, sensitivity and they are not so influenced by sample opacity or turbidity.

Materials and methods

Commercial plasmas were spiked with pharmacologically relevant concentrations (0–1 U/ml) of UFH, enoxaparin, and tinzaparin. The fluorogenic assay was carried out using previously optimized concentrations of 12 nM FXa and 2.7 μM fluorogenic substrate, in addition to 6 μl of 100 mM CaCl₂ and 44 μl of plasma. The Biophen® and Coamatic chromogenic assays were carried out according to the manufacturer's instructions. Reaction rates and endpoint values were analyzed and statistical analysis by means of one-way analysis of variance (ANOVA) was performed.

Results

The fluorogenic anti-FXa assay was found to have the broadest therapeutic range of 0–1 U/ml with CVs of < 5% for UFH and tinzaparin and CVs < 9% for enoxaparin. Despite their limited measuring range, good assay reproducibility was observed with both chromogenic kits.

Conclusions

This study indicated that the fluorogenic assay is the most sensitive assay with the broadest dynamic range for monitoring LMWH therapy when compared with standard chromogenic assays.     

Safety and efficacy of edoxaban in patients undergoing hip fracture surgery

Introduction

Edoxaban is an oral, direct, once-daily factor Xa inhibitor. This study evaluated the safety and efficacy of edoxaban compared to subcutaneous enoxaparin in Japanese patients undergoing hip fracture surgery.

Materials and methods

In this multicenter, randomized, open-label, active-comparator, phase 3 trial, 92 patients were randomized 2:1 to receive edoxaban 30 mg once daily (n = 62) or enoxaparin sodium (enoxaparin) 2000 IU (equivalent to 20 mg) twice daily (n = 30) for 11 to 14 days. The primary endpoints were the incidence of major or clinically relevant non-major (CRNM) bleeding and incidence of any bleeding events (major, CRNM, or minor bleeding). Secondary efficacy endpoints included the incidence of thromboembolic events, venous thromboembolism-related deaths, and all-cause deaths. Additional adverse events were recorded throughout the study.

Results

In the edoxaban and enoxaparin treatment groups, the incidence of major or CRNM bleeding was 3.4% and 6.9%, respectively, while any bleeding event occurred in 25.4% and 17.2% of patients, respectively. The incidence of thromboembolic events was 6.5% in the edoxaban group and 3.7% in the enoxaparin group. All events were asymptomatic deep vein thrombosis. The incidence of adverse events was 72.9% and 82.8% in the edoxaban and enoxaparin groups, respectively.

Conclusions

Compared to subcutaneous enoxaparin 2000 IU twice daily, oral edoxaban 30 mg once daily demonstrated similar safety and efficacy in the prevention of thromboembolic events in Japanese patients undergoing hip fracture surgery.Clinical trials registration number: NCT01181141.     

Evaluation and performance characteristics of the Q Hemostasis Analyzer, an automated coagulation analyzer

The Q Hemostasis Analyzer (Grifols, Barcelona, Spain) is a fully-automated random-access multiparameter analyzer, designed to perform coagulation, chromogenic and immunologic assays. It is equipped with a cap-piercing system. The instrument was evaluated in a hemostasis laboratory of a University Hospital with respect to its technical features in the determination of coagulation i.e. prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time, fibrinogen and single coagulation factors V (FV) and VIII (FVIII), chromogenic [antithrombin (AT) and protein C activity] and immunologic assays [von Willebrand factor antigen (vWF:Ag) concentration], using reagents from the analyzer manufacturer. Total precision (evaluated as the coefficient of variation) was below 6% for most parameters both in normal and in pathological ranges, except for FV, FVIII, AT and vWF:Ag both in the normal and pathological samples. No carryover was detected in alternating aPTT measurement in a pool of normal plasma samples and in the same pool spiked with unfractionated heparin (> 1.5 IU/mL). The effective throughput was 154 PT, 66 PT/aPTT, 42 PT/aPTT/fibrinogen, and 38 PT/aPTT/AT per hour, leading to 154 to 114 tests performed per hour, depending of the tested panel. Test results obtained on the Q Hemostasis Analyzer were well correlated with those obtained on the ACL TOP analyzer (Instrumentation Laboratory), with r between 0.862 and 0.989. In conclusion, routine coagulation testing can be performed on the Q Hemostasis Analyzer with satisfactory precision and the same apply to more specialized and specific tests.     

A systematic review of rivaroxaban versus enoxaparin in the prevention of venous thromboembolism after hip or knee replacement

Introductions

Rivaroxaban is a novel Xa inhibitor with an encouraging anti-thrombosis effect. The aim of this study is to assess whether rivaroxaban is superior to enoxaparin in venous thromboembolism prevention after knee- or hip-joint replacement.

Materials and Methods

We searched for reports of randomized controlled trials on rivaroxaban versus enoxaparin in venous thromboembolism prophylaxis after knee- or hip-joint replacement in the Cochrane library, Embase, Pubmed, the Ovid database, and Chinese databases including VIP, CNKI, and CBM. Correlated data was extracted and analyzed.

Results

Eight studies involving 15246 patients were included, and all were randomized controlled studies. The methodological quality of six of the trials was generally moderate, while that of the remaining two was considered high quality. 10 mg rivaroxaban daily is more effective than 40 mg/30 mg enoxaparin daily after the joint replacement in respect of the incidence of venous thromboembolism (P < 0.0001, RR = 0.38; P = 0.05, RR = 0.77, respectively). No significant difference between 10 mg rivaroxaban daily and 40 mg/30 mg enoxaparin daily were found in major postoperative bleeding (P = 0.45, RR = 1.31;P = 0.34, RR = 1.61, respectively). With respect to other outcomes, rivaroxaban is not inferior to enoxaparin, while extended therapy with rivaroxaban (> 30 d) is more effective than short-term therapy (< 15 d) in relation to the incidence of venous thromboembolism (1.36% versus 10.13%).

Conclusions

Rivaroxaban is superior to enoxaparin in venous thromboembolism prophylaxis after hip- or knee-joint replacement. Extended therapy – longer than 30 d – is recommended.     

Zucker Diabetic Fatty rats exhibit hypercoagulability and accelerated thrombus formation in the Arterio-Venous shunt model of thrombosis

Introduction

Diabetes is a significant risk factor for thrombosis. The present study aimed at assessing coagulability, platelet reactivity, and thrombogenicity of the diabetic female Zucker Diabetic Fatty (ZDF) rat model and its relevance in studying antithrombotic mechanisms.

Materials and Methods

The basal coagulant state in ZDF rats was evaluated by clotting times, thromboelastography, and thrombin generation assay. A 14-day treatment with dapagliflozin in ZDF rats was pursued to investigate if glycemic control can improve coagulability. Thrombus formation in the Arterio-Venous (A-V) shunt model and the FeCl₃-induced arterial thrombosis model was studied, with the antithrombotic effect of apixaban in the former model further investigated.

Results

ZDF rats exhibited significantly shortened clotting times, enhanced thrombin generation, and decreased fibrinolysis at baseline. Effective glycemic control achieved with dapagliflozin did not improve any of these parameters. ZDF rats displayed accelerated thrombus formation and were amenable to apixaban treatment in the A-V shunt model albeit with less sensitivity than normal rats. ZDF rats exhibited less platelet aggregation in response to ADP, collagen and PAR-4, and attenuated thrombotic response in the FeCl₃ model.

Conclusions

ZDF rats are at a chronic hypercoagulable and hypofibrinolytic state yet with compromised platelet reactivity. They display accelerated and attenuated thrombosis in the A-V shunt and FeCl₃ model of thrombosis, respectively. Results shed new light on the pathophysiology of the ZDF rat model and illustrate its potential value in translational research on anticoagulant agents in diabetics. Caution needs to be exerted in utilizing this model in assessing antiplatelet mechanisms in diabetes-associated atherothrombosis.