Reduction of intracerebral hemorrhage by rivaroxaban after tPA thrombolysis is associated with downregulation of PAR-1 and PAR-2.

This study aimed to assess the risk of intracerebral hemorrhage (ICH) after tissue-type plasminogen activator (tPA) treatment in rivaroxaban compared with warfarin-pretreated male Wistar rat brain after ischemia in relation to activation profiles of protease-activated receptor-1, -2, -3, and -4 (PAR-1, -2, -3, and -4). After pretreatment with warfarin (0.2 mg/kg/day), low-dose rivaroxaban (60 mg/kg/day), high-dose rivaroxaban (120 mg/kg/day), or vehicle for 14 days, transient middle cerebral artery occlusion was induced for 90 min, followed by reperfusion with tPA (10 mg/kg/10 ml). Infarct volume, hemorrhagic volume, immunoglobulin G leakage, and blood parameters were examined. Twenty-four hours after reperfusion, immunohistochemistry for PARs was performed in brain sections. ICH volume was increased in the warfarin-pretreated group compared with the rivaroxaban-treated group. PAR-1, -2, -3, and -4 were widely expressed in the normal brain, and their levels were increased in the ischemic brain, especially in the peri-ischemic lesion. Warfarin pretreatment enhanced the expression of PAR-1 and PAR-2 in the peri-ischemic lesion, whereas rivaroxaban pretreatment did not. The present study shows a lower risk of brain hemorrhage in rivaroxaban-pretreated compared with warfarin-pretreated rats following tPA administration to the ischemic brain. It is suggested that the relative downregulation of PAR-1 and PAR-2 by rivaroxaban compared with warfarin pretreatment might be partly involved in the mechanism of reduced hemorrhagic complications in patients receiving rivaroxaban in clinical trials. © 2016 Wiley Periodicals, Inc.

Hemostatic therapy in experimental intracerebral hemorrhage associated with rivaroxaban.

BACKGROUND AND PURPOSE: Rivaroxaban has recently been approved for stroke prevention in atrial fibrillation. However, lack of an effective antidote represents a major concern in the event of intracerebral hemorrhage (ICH). The aims of the present study were to establish a murine model of ICH associated with rivaroxaban, and to examine the effectiveness of different hemostatic factors in preventing excess hematoma expansion. METHODS: In C57BL/6 mice receiving 10 or 30 mg/kg rivaroxaban by gastric gavage, plasma concentration, prothrombin time, and coagulation factor activities were measured repeatedly. Thirty minutes after inducing ICH by intrastriatal collagenase-injection, mice received an intravenous injection of either saline, prothrombin complex concentrate (100 U/kg), murine fresh frozen plasma (200 µL), or recombinant human Factor VIIa (1 mg/kg). ICH volume was quantified on brain cryosections and using hemoglobin spectrophotometry 24 hours later. RESULTS: Rivaroxaban in 30 mg/kg dose substantially increased the hematoma volume in ICH induced by 0.060 U collagenase. Prothrombin complex concentrate, fresh frozen plasma, or Factor VIIa prevented excess hematoma expansion caused by anticoagulation. Prevention of hematoma expansion by prothrombin complex concentrate was dose-dependent. None of the 3 agents completely corrected the prolonged prothrombin time, although they restored the activities of deficient FII and X. CONCLUSIONS: Prothrombin complex concentrate, Factor VIIa, and fresh frozen plasma prevent excess intracerebral hematoma expansion in a murine ICH model associated with rivaroxaban. The efficacy and safety of this reversal strategy must be further evaluated in clinical studies.

Laboratory assessment of rivaroxaban: a review.

Research into new anticoagulants for preventing and treating thromboembolic disorders has focused on targeting single enzymes in the coagulation cascade, particularly Factor Xa and thrombin, inhibition of which greatly decreases thrombin generation. Based on the results of phase III clinical trials, rivaroxaban, a direct Factor Xa inhibitor, has been approved in many countries for the management of several thromboembolic disorders. Owing to its predictable pharmacokinetic and pharmacodynamic characteristics, fixed-dose regimens are used without the need for routine coagulation monitoring. In situations where assessment of rivaroxaban exposure may be helpful, anti-Factor Xa chromogenic assays (in tandem with standard calibration curves generated with the use of rivaroxaban calibrators and controls) could be used. It is important to note that test results will be affected by the timing of blood sampling after rivaroxaban intake. In addition, the anti-Factor Xa method measures the drug concentration and not the intensity of the drug's anticoagulant activity, and a higher than expected rivaroxaban plasma level does not necessarily indicate an increased risk of bleeding complications. Therefore, clinicians need to consider test results in relation to the pharmacokinetics of rivaroxaban and other patient risk factors associated with bleeding.

Rivaroxaban: a new oral factor Xa inhibitor.

Rivaroxaban is a direct inhibitor of factor Xa, a coagulation factor at a critical juncture in the blood coagulation pathway leading to thrombin generation and clot formation. It is selective for human factor Xa, for which it has >10 000-fold greater selectivity than for other biologically relevant serine proteases (half-maximal inhibitory concentration [IC(50)], >20 micromol/L). Rivaroxaban inhibits factor Xa in a concentration-dependent manner (inhibitory constant [K(i)], 0.4 nmol/L) and binds rapidly (kinetic association rate constant [k(on)], 1.7x10(7) mol/L(-1) s(-1)) and reversibly (kinetic dissociation rate constant [k(off)], 5x10(-3) s(-1)). By inhibiting prothrombinase complex-bound (IC(50), 2.1 nmol/L) and clot-associated factor Xa (IC(50), 75 nmol/L), rivaroxaban reduces the thrombin burst during the propagation phase. In animal models of venous and arterial thrombosis, rivaroxaban showed dose-dependent antithrombotic activity. In healthy individuals, rivaroxaban was found to have predictable pharmacokinetics and pharmacodynamics across a 5- to 80-mg total daily dose range, inhibiting factor Xa activity and prolonging plasma clotting time. In phase III clinical trials, rivaroxaban regimens reduced rates of venous thromboembolism in patients after total hip or knee arthroplasty compared with enoxaparin regimens, without significant differences in rates of major bleeding, showing that rivaroxaban has a favorable benefit-to-risk profile.

Comparison of unfractionated heparin, low-molecular-weight heparin, low-dose and high-dose rivaroxaban in preventing thrombus formation on mechanical heart valves: results of an in vitro study.

Thromboembolism and bleeding after mechanical heart valve replacement are still unsolved problems, particularly for patients requiring anticoagulative bridging therapy. The aim of this study was to investigate whether rivaroxaban, a new oral selective and direct coagulation factor Xa inhibitor, is as effective as enoxaparin and unfractionated heparin (UFH) in preventing thrombus formation on mechanical heart valves using an in vitro system. Blood from healthy male donors was anticoagulated with either UFH, enoxaparin, rivaroxaban at 300 ng/ml, (n = 10 each), or rivaroxaban at 30 ng/ml (n = 3). Mechanical aortic valve prostheses were placed into the in vitro testing system THIA II and exposed to the anticoagulant blood mixtures at a pulsatile flow for 60 min. Overall thrombus weight, coagulation parameters, and electron microscopic features of thrombus formation on the valve surface were quantified as endpoints. The mean thrombus weights were 163 ± 64 mg for group 1 (UFH), 341 ± 63 mg for the group 2 (enoxaparin), 238 ± 83 mg for group 3 (rivaroxaban 300 ng/ml) and 1.739 ± 16 mg for group 4 (rivaroxaban 30 ng/ml). Whereas high-dosed rivaroxaban showed no significant differences compared to UFH or enoxaparin, low-dosed rivaroxaban generated a massive thrombus generation, thus differing significantly from all other treatment groups regarding the thrombus weight. We hypothesize that high-dose rivaroxaban is a competitive oral available alternative to UFH and LMWH's, that might be a worthwhile alternative for patients in need of anticoagulative bridging therapy. Prospective studies have to evaluate if rivaroxaban might even overcome the limitations of OAC in patients after implantation of artificial heart valves.

Prevention and treatment of experimental thrombosis in rabbits with rivaroxaban (BAY 597939)--an oral, direct factor Xa inhibitor.

Current anticoagulant therapies for the prevention and treatment of thromboembolic disorders have many drawbacks: vitamin K antagonists interact with food and drugs and require frequent laboratory monitoring, and heparins require parenteral administration. Oral rivaroxaban (BAY 597939) is a new, highly selective and potent direct factor-Xa (FXa) inhibitor with a predictable pharmacodynamic and pharmacokinetic profile and could therefore be an attractive antithrombotic drug. It was the objective of this study to investigate the antithrombotic efficacy of oral rivaroxaban in two rabbit models of experimental venous thrombosis. In the venous stasis (prevention) model, animals were randomized to receive oral rivaroxaban 0.3, 1.0, 3.0 or 10.0 mg/kg or vehicle control. Thrombosis was induced by jugular vein stasis and injection of thromboplastin into the ear vein. In the venous thrombosis (treatment) model, intravenous (1.0 and 3.0 mg/kg) and oral (3.0 mg/kg) rivaroxaban was compared with intravenous nadroparin (40 U bolus and 20 U/h), fondaparinux (42 microg/kg) and vehicle control. Thrombus growth was assessed by measuring the accretion of radiolabelled fibrinogen into preformed clots in the jugular veins. Bleeding was assessed using an ear bleeding model. In the prevention model, rivaroxaban reduced thrombus formation dose-dependently (calculated ED(50) 1.3 mg/kg). In the treatment model, oral rivaroxaban (3.0 mg/kg) reduced thrombus growth to a similar extent to intravenous rivaroxaban (1.0 mg/kg), nadroparin and fondaparinux. Oral rivaroxaban did not prolong bleeding time. In conclusion, the orally available selective, direct FXa inhibitor rivaroxaban is effective in the prevention and treatment of venous thrombosis in two well-established models of experimental thrombosis.

Expression of pro-inflammatory genes in human endothelial cells: Comparison of rivaroxaban and dabigatran.

INTRODUCTION: In addition to its central role in coagulation, thrombin is involved in non-hemostatic activities such as inflammation. Direct inhibition of thrombin activity (e.g. with dabigatran) or reducing its generation by inhibition of Factor Xa (e.g. with rivaroxaban) may therefore have anti-inflammatory effects. MATERIALS AND METHODS: Microarray experiments were performed to identify transcriptome-wide changes in mRNA expression levels induced by thrombin in the presence and absence of the PAR-1 antagonist vorapaxar in primary human umbilical vein endothelial cells (HUVECs). On this basis, HUVECs were incubated with recalcified plasma, with or without rivaroxaban (0.3-3000nM), dabigatran (0.3-10,000nM), or vorapaxar (0.3-10nM). Expression levels of preselected pro-inflammatory genes were quantified by real-time PCR. RESULTS: Vorapaxar abolished 67 of the 69 transcripts altered by more than twofold on addition of thrombin to HUVECs. ELAM-1, VCAM-1, ICAM-1, MCP-1, IL-8, CXCL1, and CXCL2 were among the genes most strongly induced by thrombin. Inflammatory gene expression after stimulation of thrombin generation was concentration-dependently suppressed by vorapaxar, dabigatran, and rivaroxaban. However, dabigatran at low concentrations (3-300nM) increased significantly the expression levels of CXCL1, CXCL2, IL-8, ELAM-1, MCP-1, and tissue factor. CONCLUSION: In HUVECs, plasma-induced transcriptional changes are mediated by thrombin-induced PAR-1 activation. Rivaroxaban downregulated the expression of pro-inflammatory markers and tissue factor to a similar extent to dabigatran.

Discovery of the novel antithrombotic agent 5-chloro-N-({(5S)-2-oxo-3- [4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene- 2-carboxamide (BAY 59-7939): an oral, direct factor Xa inhibitor.

Despite recent progress in antithrombotic therapy, there is still an unmet medical need for safe and orally available anticoagulants. The coagulation enzyme Factor Xa (FXa) is a particularly promising target, and recent efforts in this field have focused on the identification of small-molecule inhibitors with good oral bioavailability. We identified oxazolidinone derivatives as a new class of potent FXa inhibitors. Lead optimization led to the discovery of BAY 59-7939 (5), a highly potent and selective, direct FXa inhibitor with excellent in vivo antithrombotic activity. The X-ray crystal structure of 5 in complex with human FXa clarified the binding mode and the stringent requirements for high affinity. The interaction of the neutral ligand chlorothiophene in the S1 subsite allows for the combination of good oral bioavailability and high potency for nonbasic 5. Compound 5 is currently under clinical development for the prevention and treatment of thromboembolic diseases.

Effects of Rivaroxaban on Platelet Activation and Platelet-Coagulation Pathway Interaction: In Vitro and In Vivo Studies.

INTRODUCTION: Activation of coagulation and platelets is closely linked, and arterial thrombosis involves coagulation activation as well as platelet activation and aggregation. In these studies, we investigated the possible synergistic effects of rivaroxaban in combination with antiplatelet agents on thrombin generation and platelet aggregation in vitro and on arterial thrombosis and hemostasis in rat models. MATERIALS AND METHODS: Thrombin generation was measured by the Calibrated Automated Thrombogram method (0.5 pmol/L tissue factor) using human platelet-rich plasma (PRP) spiked with rivaroxaban (15, 30, or 60 ng/mL), ticagrelor (1.0 µg/mL), and acetylsalicylic acid (ASA; 100 µg/mL). Tissue factor-induced platelet aggregation was measured in PRP spiked with rivaroxaban (15 or 30 ng/mL), ticagrelor (1 or 3 µg/mL), or a combination of these. An arteriovenous (AV) shunt model in rats was used to determine the effects of rivaroxaban (0.01, 0.03, or 0.1 mg/kg), clopidogrel (1 mg/kg), ASA (3 mg/kg), and combinations on arterial thrombosis. RESULTS: Rivaroxaban inhibited thrombin generation in a concentration-dependent manner and the effect was enhanced with ticagrelor and ticagrelor plus ASA. Rivaroxaban and ticagrelor also concentration-dependently inhibited tissue factor-induced platelet aggregation, and their combination increased the inhibition synergistically. In the AV shunt model, rivaroxaban dose-dependently reduced thrombus formation. Combining subefficacious or weakly efficacious doses of rivaroxaban with ASA or ASA plus clopidogrel increased the antithrombotic effect. CONCLUSION: These data indicate that the combination of rivaroxaban with single or dual antiplatelet agents works synergistically to reduce platelet activation, which may in turn lead to the delayed/reduced formation of coagulation complexes and vice versa, thereby enhancing antithrombotic potency.

Cardiovascular actions of a novel NO-independent guanylyl cyclase stimulator, BAY 41-8543: in vivo studies.

BAY 41-8543 is a novel non-NO-based stimulator of sGC. This study investigates the acute effects of BAY 41-8543 on haemodynamics in anaesthetized rats and dogs, its long-term effects in conscious hypertension rat models and its antiplatelet effects. In anaesthetized dogs, intravenous injections of BAY 41-8543 (3 - 100 microg kg(-1)) caused a dose-dependent decrease in blood pressure and cardiac oxygen consumption as well as an increase in coronary blood flow and heart rate. In anaesthetized normotensive rats, BAY 41-8543 produced a dose-dependent and long-lasting blood pressure lowering effect after intravenous (3 - 300 microg kg(-1)) and oral (0.1 - 1 mg kg(-1)) administration. A dose-dependent and long-lasting decrease in blood pressure was also observed in conscious spontaneously hypertensive rats with a threshold dose of 0.1 mg kg(-1) p.o. After 3 mg kg(-1) the antihypertensive effect lasted for nearly 24 h. After multiple dosages, BAY 41-8543 did not develop tachyphylaxis in SHR. BAY 41-8543 prolonged the rat tail bleeding time and reduced thrombosis in the FeCl(3) thrombosis model after oral administration. In a low NO, high renin rat model of hypertension, BAY 41-8543 prevented the increase in blood pressure evoked by L-NAME and reveals a kidney protective effect. In this model, the overall beneficial effects of BAY 41-8543 manifested as both antiplatelet effect and vasodilatation were reflected in a significant reduction in mortality. The pharmacological profile of BAY 41-8543 suggests therefore that this compound has the potential to be an important research tool for in vivo investigations in the sGC/cGMP field and it also has the potential of being a unique clinical utility for treatment of cardiovascular diseases.

Pharmacological actions of a novel NO-independent guanylyl cyclase stimulator, BAY 41-8543: in vitro studies.

BAY 41-8543 is a novel, highly specific and so far the most potent NO-independent stimulator of sGC. Here we report the effects of BAY 41-8543 on the isolated enzyme, endothelial cells, platelets, isolated vessels and Langendorff heart preparation. BAY 41-8543 stimulates the recombinant sGC concentration-dependently from 0.0001 microM to 100 microM up to 92-fold. In combination, BAY 41-8543 and NO have synergistic effects over a wide range of concentrations. Similar results are shown in implying that BAY 41-8543 stimulates the sGC directly and furthermore makes the enzyme more sensitive to its endogenous activator NO. In vitro, BAY 41-8543 is a potent relaxing agent of aortas, saphenous arteries, coronary arteries and veins with IC(50)-values in the nM range. In the rat heart Langendorff preparation, BAY 41-8543 potently reduces coronary perfusion pressure from 10(-9) to 10(-6) g ml(-1) without any effect on left ventricular pressure and heart rate. BAY 41-8543 is effective even under nitrate tolerance conditions proved by the same vasorelaxing effect on aortic rings taken either from normal or nitrate-tolerant rats. BAY 41-8543 is a potent inhibitor of collagen-mediated aggregation in washed human platelets (IC(50)=0.09 microM). In plasma, BAY 41-8543 inhibits collagen-mediated aggregation better than ADP-induced aggregation, but has no effect on the thrombin pathway. BAY 41-8543 is also a potent direct stimulator of the cyclic GMP/PKG/VASP pathway in platelets and synergizes with NO over a wide range of concentrations. These results suggest that BAY 41-8543 is on the one hand an invaluable tool for studying sGC signaling in vitro and on the other hand its unique profile may offer a novel approach for treating cardiovascular diseases.

NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle.

1. Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (alpha/ss) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. 2. Through a high-throughput screen we identified BAY 58-2667, an amino dicarboxylic acid which potently activates sGC in an NO-independent manner. In contrast to NO, YC-1 and BAY 41-2272, the sGC stimulators described recently, BAY 58-2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem deficient. 3. Binding studies with radiolabelled BAY 58-2667 show a high affinity site on the enzyme. 4. Using photoaffinity labelling studies we identified the amino acids 371 (alpha-subunit) and 231 - 310 (ss-subunit) as target regions for BAY 58-2667. 5. sGC activation by BAY 58-2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not influenced by nitrate tolerance. 6. BAY 58-2667 shows a potent antihypertensive effect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic effects of BAY 58-2667 and GTN are very similar on the arterial and venous system. 7. This novel type of sGC activator is a valuable research tool and may offer a new approach for treating cardiovascular diseases.

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; 0.2-1.0U/mL), activated PCC (aPCC; 0.2-1.0U/mL) and recombinant activated factor VII (rFVIIa; 5-50µg/mL) on rivaroxaban-induced (200-1000ng/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.

Measurement and reversal of prophylactic and therapeutic peak levels of rivaroxaban: an in vitro study.

BACKGROUND: Rivaroxaban (Xarelto, Bayer HealthCare, Leverkusen, Germany) is a new oral anticoagulant drug. Anticoagulants may cause bleeding, thereby requiring reliable monitoring and efficient therapy. We investigated thromboelastometry versus routine coagulation tests to measure prophylactic and therapeutic concentrations of rivaroxaban and their reversal with prothrombin complex concentrate (PCC) and activated recombinant factor VII (rFVIIa) in vitro. METHODS: Rivaroxaban was solubilized, and PCC and rFVIIa were added in 2 concentrations to the rivaroxaban-spiked blood samples, and thromboelastometry and measurements were performed. RESULTS: Rivaroxaban increased tissue factor-activated clotting time (CT(ExTEM)) dose dependently. Activated partial prothrombin time (aPTT), prothrombin time ratio (PTR), and prothrombin time (PT) were changed significantly in both concentrations. Reversal with PCC in both dosages caused no significant change in the measured parameters. For prophylactic rivaroxaban dosage, rFVIIa changed the PT significantly but not CT(ExTEM), aPTT, and PTR. For therapeutic rivaroxaban dosage, the CT(ExTEM) was significantly reduced. The other parameters remained unaffected. CONCLUSIONS: Thromboelastometry can detect rivaroxaban effects. In vitro rFVIIa seems highly effective for reversal in contrast to PCC.

The discovery of rivaroxaban: translating preclinical assessments into clinical practice.

Direct oral anticoagulants that target a single coagulation factor (such as factor Xa or thrombin) have been developed in recent years in an attempt to address some of the limitations of traditional anticoagulants. Rivaroxaban is an oral, direct factor Xa inhibitor that inhibits free and clot-bound factor Xa and factor Xa in the prothrombinase complex. Preclinical studies demonstrated a potent anticoagulant effect of rivaroxaban in plasma as well as the ability of this agent to prevent and treat venous and arterial thrombosis in animal models. These studies led to an extensive phase I clinical development program that investigated the pharmacological properties of rivaroxaban in humans. In these studies, rivaroxaban was shown to exhibit predictable pharmacokinetics and pharmacodynamics and to have no clinically relevant interactions with many commonly prescribed co-medications. The pharmacodynamic effects of rivaroxaban (for example, inhibition of factor Xa and prolongation of prothrombin time) were closely correlated with rivaroxaban concentrations in plasma. The encouraging findings from preclinical and early clinical studies were expanded upon in large, randomized phase III studies, which demonstrated the clinical efficacy and safety of rivaroxaban in a broad spectrum of patients. This article provides an overview of the discovery and development of rivaroxaban, describing the pharmacodynamic profile established in preclinical studies and the optimal translation to clinical studies in healthy subjects and patient populations.

Whole blood clots are more resistant to lysis than plasma clots--greater efficacy of rivaroxaban.

INTRODUCTION: Defective thrombolysis, a thrombotic risk factor, can be attributed to the formation of a compact clot poorly accessible to fibrinolytic enzymes. Venous thrombi, rich in red blood cells (RBCs), and arterial thrombi containing various amounts of RBCS, plasma and whole blood (WB) clot permeability and degradability were compared. The effect of rivaroxaban, a potent direct factor Xa inhibitor, was also evaluated. MATERIALS AND METHODS: Fibrin permeability was determined by flow measurement through the clot. Clot degradability was evaluated by the amount of D-dimer generated by clot perfusion with plasminogen and tissue plasminogen activator. Fibrin clot structure was assessed by confocal microscopy. RESULTS: WB clot permeability (KS) and degradability were 6.7- and 38-fold lower, respectively, compared with plasma clots. This is attributed to 1) occlusion of fibrin pores by RBCs and 2) a consistent increase in thrombin generation due to platelets and RBCs inducing formation of a tighter clot. Rivaroxaban added to plasma or WB before clotting, in reducing thrombin generation, led to the formation of a looser clot that is more degradable by fibrinolytic enzymes. Permeability and degradability of whole blood clots formed in the presence of rivaroxaban were very similar to those of plasma clots. CONCLUSION: The resistance to fibrinolysis of WB clots was reduced considerably when clots were formed with rivaroxaban. These results may have implications for the development of antithrombotic agents.

Reversal of rivaroxaban anticoagulation by haemostatic agents in rats and primates.

Rivaroxaban is an oral, direct factor Xa inhibitor for the management of thromboembolic disorders. Despite its short half-life, the ability to reverse rivaroxaban anticoagulation could be beneficial in life-threatening emergencies. The potential of prothrombin complex concentrate (PCC; Beriplex®), activated PCC (aPCC; FEIBA®) or recombinant activated factor VII (rFVIIa; NovoSeven®) to reverse rivaroxaban in rats and baboons was investigated. Anaesthetised rats pre-treated with intravenous rivaroxaban (2 mg/kg) received intravenous rFVIIa (100/400 µg/kg), PCC (25/50 U/kg) or aPCC (50/100 U/kg) after initiation of bleeding. Clotting times and bleeding times (BTs) were recorded. Rivaroxaban was administered as an intravenous 0.6 mg/kg bolus followed by continuous 0.6 mg/kg/hour infusion in baboons. Animals received intravenous aPCC 50 U/kg (2 U/kg/minute) or rFVIIa 210 µg/kg. BT and clotting parameters were measured. In rats pretreated with high-dose rivaroxaban, PCC 50 U/kg, aPCC 100 U/kg and rFVIIa 400 µg/kg significantly reduced BT vs rivaroxaban alone (5.4 ± 1.4-fold to 1.5 ± 0.4-fold [p<0.05]; 3.0 ± 0.4-fold to 1.4 ± 0.1-fold [p<0.001]; and 3.5 ± 0.7-fold to 1.7 ± 0.2-fold [p<0.01] vs baseline, respectively). In baboons pre-infused with rivaroxaban and then given aPCC, BT increased by 2.0 ± 0.2-fold and aPCC returned BT to baseline for the duration of its infusion. rFVIIa reduced BT from 2.5 ± 0.3-fold over baseline to 1.7 ± 0.3-fold over baseline. Prolongation of prothrombin time was reduced by PCC, aPCC and rFVIIa in both species. Rivaroxaban reduced thrombin-antithrombin levels; application of PCC and aPCC, but not rFVIIa, increased these levels. In conclusion, PCC, aPCC or rFVIIa have the potential to reverse the anticoagulant and anti-haemostatic effects of rivaroxaban.

Rivaroxaban: a novel oral anticoagulant for the prevention and treatment of several thrombosis-mediated conditions.

The development of rivaroxaban (XARELTO®) is an important new medical advance in the field of oral anticoagulation. Thrombosis-mediated conditions constitute a major burden for patients, healthcare systems, and society. For more than 60 years, the prevention and treatment of these conditions have been dominated by oral vitamin K antagonists (such as warfarin) and the injectable heparins. Thrombosis can lead to several conditions, including deep vein thrombosis, pulmonary embolism, myocardial infarction, stroke, and/or death. Prevention and treatment of thrombosis with an effective, convenient-to-use oral anticoagulant with a favorable safety profile is critical, especially in an aging society in which the risk of thrombosis, and the potential for bleeding complications, is increasing. Rivaroxaban acts to prevent and treat thrombosis by potently inhibiting coagulation Factor Xa in the blood. Factor Xa converts prothrombin to thrombin, which initiates the formation of blood clots by converting fibrinogen to clot-forming fibrin and leads to platelet activation. After a large and novel clinical development program in over 75,000 patients to date, rivaroxaban has received approval for multiple indications in the United States, European Union, and other countries worldwide to prevent and treat several thrombosis-mediated conditions. This review will highlight some of the unique aspects of the rivaroxaban development program.

Evaluation of the anti-factor Xa chromogenic assay for the measurement of rivaroxaban plasma concentrations using calibrators and controls.

Rivaroxaban is an oral, direct factor Xa inhibitor. Routine coagulation monitoring is not required, but a quantitative determination of rivaroxaban concentrations might be useful in some clinical circumstances. This multicentre study assessed the suitability of the anti-factor Xa chromogenic assay for the measurement of rivaroxaban plasma concentrations (ng/ml) using rivaroxaban calibrators and controls, and the inter-laboratory precision of the measurement. Twenty-four centres in Europe and North America were provided with sets of rivaroxaban calibrators (0, 41, 209 and 422 ng/ml) and a set of rivaroxaban pooled human plasma controls (20, 199 and 662 ng/ml; the concentrations were unknown to the participating laboratories). The evaluation was carried out over 10 days by each laboratory using local anti-factor Xa reagents as well as the centrally provided reagent, a modified STA® Rotachrom® assay. A calibration curve was produced each day, and the day-to-day precision was evaluated by testing three human plasma controls. When using the local anti-factor Xa reagents, the mean rivaroxaban concentrations (measured/actual values) were: 17/20, 205/199 and 668/662 ng/ml, and the coefficient of variance (CV) was 37.0%, 13.7% and 14.1%, respectively. When the modified STA Rotachrom method was used, the measured/actual values were: 18/20, 199/199 and 656/662 ng/ml, and the CV was 19.1%, 10.9% and 10.0%, respectively. The results suggest that, by using rivaroxaban calibrators and controls, the anti-factor Xa chromogenic method is suitable for measuring a wide range of rivaroxaban plasma concentrations (20-660 ng/ml), which covers the expected rivaroxaban plasma levels after therapeutic doses.

Evaluation of the prothrombin time for measuring rivaroxaban plasma concentrations using calibrators and controls: results of a multicenter field trial.

This study evaluated the prothrombin time (PT) assay for the measurement of plasma concentrations of rivaroxaban using calibrators and controls. The intra- and interlaboratory precision of the measurement was investigated in a field trial involving 21 laboratories. Each laboratory was provided with rivaroxaban calibrators and control plasma samples containing different concentrations of rivaroxaban, and PT reagents. The evaluation was carried out over 2 consecutive weeks using centrally provided and local PT reagents. A calibration curve was produced each day (for inter-run precision), and day-to-day precision was evaluated by testing 3 control plasma samples. A large interlaboratory variation (in seconds) was observed with local PT reagents. The results were less variable when expressed as rivaroxaban concentrations (ng/mL) or when central PT reagent was used (STA Neoplastine CI Plus). The widely available PT assay, in conjunction with rivaroxaban calibrators, may be useful for the measurement of peak plasma levels of rivaroxaban.