Prothrombinase-induced Clotting Time Research Articles

Prothrombinase-Induced Clotting Time to Measure Drug Concentrations of Rivaroxaban, Apixaban, and Edoxaban in Clinical Practice: A Cross-Sectional Study.

Prothrombinase-induced clotting time (PiCT) is proposed as a rapid and inexpensive laboratory test to measure direct oral anticoagulant (DOAC) drug levels. In a prospective, multicenter cross-sectional study, including 851 patients, we aimed to study the accuracy of PiCT in determining rivaroxaban, apixaban, and edoxaban drug concentrations and assessed whether clinically relevant drug levels could be predicted correctly. Citrated plasma samples were collected, and the Pefakit® PiCT was utilized. Ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to measure drug concentrations. Cut-off levels were established using receiver-operating characteristics curves. We calculated sensitivities and specificities with respect to clinically relevant drug concentrations. Spearman’s correlation coefficient between PiCT and drug concentrations was 0.85 in the case of rivaroxaban (95% CI 0.82, 0.88), 0.66 for apixaban (95% CI 0.60, 0.71), and 0.78 for edoxaban (95% CI 0.65, 0.86). The sensitivity to detect clinically relevant drug concentrations was 85.1% in the case of 30 µg L−1 (95% CI 82.0, 87.7; specificity 77.9; 72.1, 82.7), 85.7% in the case of 50 µg L−1 (82.4, 88.4; specificity 77.3; 72.5, 81.5), and 85.1% in the case of 100 µg L−1 (80.9, 88.4; specificity 73.2%; 69.1, 76.9). In conclusion, the association of PiCT with DOAC concentrations was fair, and the majority of clinically relevant drug concentrations were correctly predicted.

The mode of action of sugammadex on coagulation.

To explore the mode of action (MoA) by which sugammadex interferes with coagulation. The effect of sugammadex on various steps in the coagulation cascade including thrombin generation, factor Xa activity, and factor Xa generation was explored in human plasma. Sugammadex did not affect a conventional thrombin generation test (TGT), while it prolongs activated partial thromboplastin time (APTT) and prothrombin time (PT). However, a customized TGT with PT reagent revealed sugammadex effects. In addition, sugammadex prolonged a one-step prothrombinase-induced clotting time (PiCT) using human factor Xa. Furthermore, sugammadex interfered with factor Xa generation induced by an intrinsic and not by an extrinsic activator, nor by Russell's viper venom factor X (RVV-X). Adapted, rather than standard, experiments show that sugammadex is likely to decrease factor Xa activity in the common pathway and activation of factor X specifically in the intrinsic pathway.

Validation of the Bioequivalence of USP Potency Adjusted Porcine, Ovine, and Bovine Heparins

Introduction: Currently, there is a shortage of porcine heparin due to several factors such as limited availability of porcine mucosa, supply chain issues, and increased usage due to COVID-19. This has warranted the development of heparin from alternate sources such as bovine and ovine mucosa which is abundantly available for this purpose. On a mass basis, commercially available porcine heparins exhibit a similar potency (200 units/mg) to their ovine counterpart (190 units/mg) and a higher potency in contrast to their bovine counterpart (130-150 units/mg). Therefore, at gravimetric levels, the porcine heparins exhibit stronger biochemical and pharmacological effects in various laboratory assays in comparison to bovine heparin and similar effects in comparison to ovine heparins. Since heparin is standardized in biologic units and cross referenced against USP or EP Standard, it is hypothesized that potency equated porcine, ovine, and bovine heparin will exhibit similar biologic activities in laboratory assays carried out in the in vitro setting. The purpose of this study is to compare the biologic properties of the porcine, ovine, and bovine heparin at USP potency equated levels in standardized laboratory assays. Materials and Methods: Active pharmaceutical ingredients (API) of porcine mucosal heparin (200 units/mg) of U.S. origin was commercially obtained from Medefil Inc. (Glendale Heights, IL). Ovine heparin was obtained from Ronnsi Pharmaceutical (Jiangsu, China). Bovine heparin (140 units/mg) was obtained from Kin Master Pharmaceuticals (Posso Fundo, Brazil). All heparins were diluted at a concentration of 100 units/mL in saline. The anticoagulant effect of all heparins were evaluated using the whole blood clotting assays such as the ACT and thromboelastographic methods. Heparins were diluted in citrated human plasma yielding a final concentration range of 0-1 unit/mL. Clot based assays such as aPTT, TT, and prothrombinase induced clotting time (PiCT) were measured. Thrombin generation inhibition assay was carried out using a kinetic assay (CAT system, Diagnositca Stago, Paris, France). Protamine and heparinase neutralization profiles of these agents were also investigated in the plasma-based systems. These assays were then repeated at gravimetric dosages at final concentrations of 0-10 ug/mL. The results collected from these trials were then mathematically converted to units and compared to the results collected from the potency adjusted trials. All results were tabulated and compared, and applicable statistical methods were applied. Results: The USP potency adjusted heparin exhibited comparable anticoagulant effects in both the ACT and TEG assays. At equigravimetric levels porcine and ovine heparins produced comparable anticoagulant effects and bovine heparin produced weaker anticoagulant effect in both assays. In the citrated plasma supplementation studies, all drugs produced similar anticoagulant effects at potency adjusted dosages. In the chromogenic anti-Xa and anti-IIa assays, the behaviors of the agents were also comparable. In the thrombin generation assays, in terms of peak thrombin generation, area under the curve, and lag time, the porcine, ovine, and bovine heparins showed comparable effects. The protamine neutralization profiles of the porcine, ovine, and bovine heparin exhibited variable assay dependent results. Potency adjusted bovine heparin required higher amount of protamine for the complete neutralization of the biologic effects in comparison to the porcine heparin. At gravimetric concentrations, bovine heparins exhibited lower potencies than both the porcine and ovine heparins, which produced similar results. Summary and Conclusion: These results show that at potency adjusted concentrations, the porcine, ovine, and bovine heparin exhibit comparable biochemical and anticoagulant responses in the plasma-based systems. Therefore, the hypothesis that potency equated porcine, ovine, and bovine heparins exhibit comparable biochemical and anticoagulant activities is validated. Thus, the proposed approach to standardize heparins against a common standard in a biologic assay such as the USP method is valid. Furthermore, these results warrant regulatory considerations to fast track the review process for the re-introduction of bovine heparin and approval of bovine heparin as a biosimilar anticoagulant to porcine heparin. Disclosures No relevant conflicts of interest to declare.

Resourcing of Porcine Unfractionated and Low‐Molecular Weight Heparins from Ovine, and Bovine Sources

IntroductionThe current shortage of porcine mucosal heparin supply warrants the need for exploring alternate sources to manufacture heparins. UFHs and LMWHs can also be derived from ovine and bovine sources, which could provide substitute for porcine derived anticoagulants. The purpose of this study is to compare the biologic properties of UFHs and LMWHs from porcine, ovine, and bovine sources at gravimetric concentrations.Materials and MethodsUSP standard (100 ug/ml), NIBSC (100 U/mL), APTT reagents (Triniclot), Spectrozymes Xa and IIa (Sekisui), Bovine Factor Xa (Enzyme Research, USA), Thrombin (Enzyme Research, USA), and blood bank plasma. The following agents were tested: porcine UFH (Medefil 13353), porcine LMWH (Enoxaparin 7L010), ovine UFH (lot# 1001), ovine LMWH (Enoxaparin lot# 404D), bovine UFH (GAG 25), and bovine LMWH (Enoxaparin lot# 003). All agents were diluted at a stock solution of 100 ug/mL in saline. Working dilutions were prepared at 100 ug/mL. Whole blood ACT was measured using a Hemochron instrument at a fixed concentration of 10 ug/mL and 25 ug/mL. Prothrombinase induced clotting time (PiCT), TT, aPTT, anti‐Xa, and anti‐IIa activities were measured in plasma supplemented with each of these agents at a concentration range of 0–10 ug/mL using the ACL Elite instrument. The USP potency assay was carried out using chromogenic antiprotease assays (Biophen Kits). The inhibitory concentrations at 50% (IC‐50) were measured using a purified antithrombin biochemical system employing a kinetic method. Thrombin generation inhibition assay was carried out using a CAT method (Diagnositca Stago, Paris, France).ResultsIn the ACT method, ovine UFH showed the highest value followed by porcine UFH, bovine UFH, ovine LMWH, porcine LMWH, then bovine LMWH. The PiCT results showed ovine UFH with the strongest anticoagulant activity followed by porcine UFH and bovine UFH. All three LMWH demonstrated comparable PiCT results and were significantly lower than their UFH counterparts. In the plasma supplementation studies, a similar trend was seen in the aPTT, TT, factor Xa, and factor IIa studies: ovine UFH demonstrated the strongest anticoagulant and antiprotease effects followed by porcine UFH, bovine UFH, ovine LMWH, porcine LMWH, then bovine LMWH. The IC‐50 values for the anti‐Xa ranged from 0.71–2.12 ug/mL for the UFH and from 2.23–3.92 for the LMWH, whereas the anti‐IIa values ranged from 3.91–8.37 ug/mL for the UFH and were greater than 10 ug/mL for the LMWH. Ovine UFH and LMWH had the lowest IC‐50 values for anti‐Xa in their respective categories while bovine UFH and LMWH had the highest. In the thrombin generation assays, the ovine UFH showed slightly stronger activities followed by porcine UFH, bovine UFH, ovine LMWH, porcine LMWH, then bovine LMWH in all three parameters including peak thrombin, lag time, and percent inhibition of area under the curve.

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

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

Potency Equated Porcine and Bovine Mucosal Heparin Are Bioequivalent in Terms of Biochemical and Pharmacological Effects

Introduction: Currently, there is a shortage of porcine heparin due to limited availability of porcine mucosa and supply chain issues. Bovine heparin has been used for clinical purposes globally and is being considered for reintroduction in the U.S. On a mass basis, commercially available porcine heparins exhibit a higher potency (180-220 units/mg) than their bovine counterpart (130-150 units/mg). Therefore, at gravimetric levels, the porcine heparins exhibit stronger biochemical and pharmacological effects in comparison to bovine heparin. Since heparin is standardized in biologic units relative to the USP or EP Standard, it is hypothesized that potency equated porcine and bovine heparin will exhibit similar biologic activities in laboratory assays. The purpose of this study was to compare the biologic properties of the porcine and bovine heparin at USP potency equated levels in standardized laboratory assays used for measuring and monitoring heparins. Materials and Methods: Active pharmaceutical ingredient (API) porcine mucosal heparin (200 units/mg) of U.S. origin was obtained from Medefil Inc. (Glendale Heights, IL). Bovine heparin (140 units/mg) was obtained from KinMaster Produtos Químicos (Passo Fundo, Brazil). Whole blood Activated Clotting Time (ACT) and Thrombelastographic analyses were performed. Clot based assays such as aPTT, TT, and prothrombinase induced clotting time (PiCT) were performed using citrated human plasma supplemented with heparins up to 1 anti-Xa unit/mL. Anti-Xa and anti-IIa activities were determined using chromogenic antiprotease assays (Biophen Kits). In a purified antithrombin-supplemented system, the inhibitory effects of these agents were measured in terms of anti-Xa and anti-IIa activities and expressed as IC50 values. Thrombin generation inhibition was measured using a kinetic assay (CAT system, Diagnostica Stago, Paris, France). Protamine and platelet factor 4 neutralization profiles were investigated in plasma-based systems. The heparin induced thrombocytopenia antibody (HIT) interaction profile was assessed using platelet aggregometry. Results: Both heparins produced comparable prolongation of the ACT to a range of 200-250 seconds. The thrombelastographic profile was comparable at concentrations of 0.125 unit/mL and 2.5 units/mL. Whole blood and plasma clotting times (aPTT and TT assays) were comparable at concentrations up to 1 unit/mL. In the chromogenic anti-Xa and anti-IIa assays, the behavior of both agents was also comparable. In the purified system, the IC50 values for both agents for the anti-Xa activity ranged from 0.33-0.40 unit/mL, whereas the anti-IIa values ranged from 0.38-0.42 unit/mL; no significant differences were noted between the porcine and bovine heparins. In the thrombin generation assays, in terms of peak thrombin generation, area under the curve, and lag time, both the porcine and bovine heparins showed comparable effects. The protamine neutralization profiles of the porcine and bovine heparin exhibited variable assay dependent results. Potency adjusted bovine heparin required higher amount of protamine for the complete neutralization of the biologic effects in comparison to the porcine heparin. Similar results were obtained in the platelet factor 4 neutralization studies. In the functional HIT screening assay, both heparins exhibited a similar concentration dependent aggregation of human platelets. These results are summarized in the Table. Summary and Conclusion: These results show that at potency adjusted concentrations, porcine and bovine heparin exhibit comparable biochemical and anticoagulant responses in whole blood, plasma based, and purified biochemical assays. Most notably, both the porcine and bovine heparin produced comparable HIT antibody mediated aggregation responses. Thus, the proposed approach to standardize heparins against a common standard using a biologic assay such as the USP method is valid. These results warrant additional validation studies to reintroduce bovine heparin for clinical use. Table Disclosures Jeske: KinMaster Produtos Quimicas: Research Funding.

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

The currently available oral anti-Xa agents are claimed to produce their anticoagulant and antithrombotic effects solely by the inhibition of factor Xa. This study profiled various anti-Xa drugs in routinely used laboratory assays to demonstrate that their effects are not solely related to the anti-Xa activities. Apixaban, betrixaban, edoxaban, and rivaroxaban were obtained commercially. Native and citrated whole blood was used for the activated clotting time (ACT) and thromboelastography (TEG). Citrated plasma was used for monitoring the prothrombin time (PT), activated partial thromboplastin time (aPTT), Heptest, and prothrombinase-induced clotting time (PiCT) tests. An amidolytic method was used for the determination of anti-Xa effects. Thrombin-induced fibrinokinetics was monitored optically. Thrombin generation studies were carried out using the calibrated automated thrombogram. All of the anti-Xa agents produced concentration- and assay-dependent effects. In the ACT at 2.5 μg/mL and TEG at 1.0 μg/mL, edoxaban exhibited the strongest anticoagulation effect. In the PiCT, PT, and aPTT assay at 1 μg/mL, edoxaban showed stronger effects than other agents. The half maximal inhibitory concentration of these agents for the inhibition of factor Xa ranged from 340 to >1000 ng/mL. In the thrombin generation inhibition assay, apixaban showed the strongest activity. In the fibrinokinetics, different anti-Xa agents produced varying degrees of inhibition. These results demonstrate that the measured anti-Xa activity alone does not fully reflect the overall biologic spectrum of these agents.

Accuracy, reproducibility and costs of different laboratory assays for the monitoring of unfractionated heparin in clinical practice: a prospective evaluation study and survey among Swiss institutions

ObjectivesTo investigate the accuracy, reproducibility and costs of different laboratory assays for the monitoring of unfractionated heparin (UFH) in clinical practice and to study test utilisation in Switzerland.DesignProspective evaluation study...

The PiCT® test is a reliable alternative to the activated partial thromboplastin time in unfractionated heparin therapy management: results from a multicenter study

Background Unfractionated heparin (UFH) is still a commonly used anticoagulant for prevention and treatment of thromboembolism in a variety of situations. Increasingly, chromogenic anti-Xa assays are used for UFH monitoring given the high variability of the activated partial thromboplastin time (APTT) in this setting. On the other hand, and despite the known variability, the APTT test remains the most frequently used monitoring tool in UFH therapy because of its broad availability, lower costs and wide acceptance. Various guidelines continue to recommend the use of the APTT as an anti-Xa surrogate, but this approach remains controversial. Objective To assess the prothrombinase-induced clotting time (PiCT® ) test, reported in seconds, as an alternative to the APTT in the management of UFH-mediated anticoagulation. Methods Plasma samples from patients receiving UFH were obtained in three different centers in the USA and Europe. Samples were analyzed for PiCT, APTT and anti-Xa activities with conditions set to allow comparability. Target-ranges in seconds for PiCT and APTT were established for a UFH concentration of 0.3-0.7 IU mL-1 , derived from anti-Xa results as suggested by the ACCP guidelines. Results PiCT demonstrated better correlation with anti-Xa IU mL-1 than APTT, higher ability to identify samples within target range and, importantly, comparable target-ranges between different centers. Conclusion Accuracy and reliability of PiCT are significantly better than those of APTT in monitoring UFH for anticoagulant therapy.

A Simultaneous Monitoring of Coagulation Time and Fibrinogen via PiCT on QCM-D

‘Prothrombinase induced Clotting Time’ (PiCT) has potential to detect all anticoagulants in clinics. In the present study, PiCT has been used as a tool for anticoagulant detection in human plasma on quartz crystal microbalance with dissipation (QCM-D) technique. QCM-D technique enables monitoring of PiCT point, total coagulation and fibrinogen concentration from frequency and dissipation curves in single set of measurements. This is impossible on mechanical coagulometer (which is considered as 'gold standard') technique, and it cannot yield coagulation and fibrinogen concentration from a single set of measurements. It requires additional Clauss method or modified Clauss method to calculate fibrinogen by employing different reagents and experimental setups. Additionally, the present report utilizes the lowest sample volume (and each reagent volume) consumptions of 1.66 µL. The sample/reagent volume consumption of 1.66 µL on QCM-D is 30 times lower in comparison with mechanical coagulometer’s that uses 50 µL for laboratory experiments for PiCT. This element is crucial for application of spot test via QCM-D in laboratory and clinics for Point of Care (POC) settings. Different doses of anticoagulant in 20 human plasma samples on QCM-D technique have been studied and compared in parallel to ‘gold standard’. PiCT on QCM-D technique yielded precise and accurate data. Additionally, both techniques produced % RSD values between 3 and 8.5 with slight fluctuations on both sides for PiCT points. The %RSD data for both techniques has lower variability for danaparoid. Furthermore, QCM-D technique enables monitoring of substantial fibrinogen concentrations (i.e. 1 - 6 g/L) with outstanding R2 value of 0.99 on the calibration curve. PiCT-QCM-D technique proved superior at all concentrations of fibrinogen in standard reference plasma for PiCT range (precision) on comparing to that of 'gold standard'.

Oral Factor Xa Inhibitors and Clinical Laboratory Monitoring

Oral anticoagulation therapy is currently undergoing great changes with the development and use of several new medications. The newer drugs have a more specific mechanism of action for inhibiting coagulation and do not require continuous monitoring like their predecessor. Their efficacy and safety has been proven, but problems arise in the clinical laboratory with developing accurate procedures to measure the therapy when needed. Methods being used are the prothrombin time and anti-factor Xa chromogenic assay which require calibration with the drug being measured. The prothrombinase-induced clotting test shows promise as a measure for Xa inhibitors. The article reviews the oral Factor Xa inhibitor9s clinical usefulness, effects on current laboratory coagulation tests and methods for measuring their anticoagulant activity. <b>ABBREVIATIONS:</b>CYP – cytochrome; DOAC – direct oral anticoagulant; DVT – deep venous thrombosis; INR – international normalized ratio; PE – pulmonary embolism; PiCT – prothrombinase-induced clotting time; PT – prothrombin time; APTT – activated partial thromboplastin time.

PiCT: 1st Recognition for Human Whole Blood on QCM-D Platform

Instruments for point-of-care (POC) coagulation monitoring for whole blood could reduce fundamental limitations of routine coagulation tests. ‘Prothrombinase induced Clotting Time’ (PiCT) is considered a universal assay for anticoagulants monitoring in laboratory and clinics but it has not been evaluated yet. It could reduce the inherited drawbacks of highly variability of results among different individuals in the activated partial thromboplastin time (aPTT) which is the most applied and clinical standard assay. In this report, PiCT for whole blood on quartz crystal microbalance with dissipation (QCM-D) platform for anticoagulant monitoring has been recognized for the first time. The present study demonstrates the lowest historical sample volume of human whole blood (as well as each reagent) consumption of 1.66 µL employed ever for coagulation. This is substantial support for launching spot test via QCM-D in laboratory and clinics and ultra-refining of POC settings. Different doses of danaparoid anticoagulant in blood samples (n=20) have been studied on QCM-D platform in parallel to ‘gold standard’. Both techniques demonstrated lower variability for anticoagulant with %RSD values between 3 and 8.5 depending on different anticoagulant doses. Data could be considered as precise and accurate for an anticoagulant recognition directly in blood using a clotting assay on QCM-D platform. Present study is crucial in the perspectives of robustness due to direct whole blood method on QCM-D platform and its cost-effectiveness due to lowest sample (as well as reagents) volume consumption.

From laboratory to clinical practice: Dabigatran effects on thrombin generation and coagulation in patient samples

IntroductionDabigatran (Dabi) is not routinely monitored. However, in emergency cases quantitative assessment is required and laboratories must provide suitable tests at all hours. Little is known about Dabi effects on thrombin generation. Materials and methodsPatient samples (n=241) were analyzed for functional Dabi concentrations (Dabi-TT) using a combination of the Hemoclot Thrombin Inhibitors assay (HTI®) and, for samples with low levels, undiluted thrombin time (TT). Results were compared to prothrombin time (PT) and activated partial thromboplastin time (APTT). In 49 samples Dabi effects were further investigated with Calibrated Automated Thrombogram (CAT®) for thrombin generation and with Russell's viper venom time (RVVT), prothrombinase-induced clotting time (PiCT®), chromogenic Anti-IIa® and ecarin clotting assay (ECA®). Fibrinogen and D dimer were assessed to reflect the coagulation status of the patient. A subset of these samples (n=21) were also analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). ResultsDabi-TT correlated with RVVT (R2=0.49), PiCT® (R2=0.73), ECA® (R2=0.89), Anti-IIa® (R2=0.90) and LC-MS/MS (R2=0.81). APTT correlated curvi-linearly with Dabi-TT (R2=0.71), but was normal in many cases (18/70) despite Dabi-TT>40ng/mL. There was no association between Dabi-TT and fibrinogen or D dimer levels. Increasing Dabi concentrations prolonged lag time (R2=0.54) and, surprisingly, elevated the ETP and Peak of CAT® (p<0.001). ConclusionsThrombin-specific tests measure Dabi accurately, whereas coagulation time based assays depend more on other factors. The enhanced thrombin generation in Dabi-treated patients may predict clinically relevant hypercoagulability and warrants further investigation.

Circulating Levels of Rivaroxaban Provide Different Results in Different Clot Based and Amidolytic Assays. a Study on Patients Treated with Two Different Dosages of Rivaroxaban

Introduction:Rivaroxaban is an orally active anti-Xa agent which is approved for various clinical indications including DVT/ PE management. Although monitoring of this agent is not commonly required, in certain clinical conditions circulating anti-Xa levels may be useful for the optimal management of patients treated with this drug. The circulating plasma levels of anti-Xa activity can be obtained by using amidolytic methods; however, their relevance to observed bleeding and antithrombotic effects is not clear. Plasma based assays, such as the PT/APTT, are largely dependent on other plasma components and population based differences are noted. Heptest is a clot based method measuring Xa mediated clotting activity in plasma. The prothrombinase induced clotting time (PICT) and diluted Russell’s Viper Venom time (DRVVT) tests are also useful in measuring Xa mediated clot based activity. In order to compare the amidolytic method with some of these assays, a study was undertaken to analyze plasma samples from patients treated with two dosages of Rivaroxaban.Materials:Citrated plasma samples from 106 patients treated with Rivaroxaban at 15mg BID or 20mg OD were collected in a University Medical Center (Ljubljana, Slovenia) at varying times and duration of treatment. These samples were frozen at -80 C and analyzed in batches. Commercially available Rivaroxaban was used to prepare the calibration curves in normal human pool plasma (n=20) in a range of 0-1000ng/ml.Methods:The frozen plasma samples along with the calibration curve were analyzed in the following assays; PT – prothrombin time, innovin (Dade Behring, Marburg, Germany), aPTT – activated partial thromboplastin time (Tcoag Ireland Limited, Wicklow, Ireland), Heptest time (Heptest Laboratories Inc, St. Louis, MO), DRVVT – diluted russel’s viper venom time (Instrumentation Laboratory, Bedford, MA), Prothrombinase induced clotting time (PiCT) (Pentapharm, Basel, Switzerland) and Coamatic anti-Xa assay (Instrumentation Laboratory, Bedford, MA). PT, aPTT and heptest were performed on an ACL centrifugal analyzer. DRVVT and PiCT tests were measured on a Stago ST4 instrument. Coamatic anti-Xa assay was carried out on a microtiter plate monitoring optical density at 405nm. Tissue factor pathway inhibitor (TFPI) levels were measured using commercially based ELISA method (Diagnostica Stago, Paris France).Results:Wide variations in the results obtained by different methods were noted. The coamatic anti-Xa method provided a mean 173±87ng/ml, which was comparable to the results obtained by DRVVT (170±42ng/ml). All of the clot based assays resulted in a markedly higher level of Rivaroxaban, ranging from 238-458ng/ml. The heptest (453±288ng/ml) and PICT (458±433ng/ml), were markedly higher than the results from PT (341±299ng/ml) and aPTT (238±306ng/ml). The correlation between PICT and other tests ranged from 0.04 to 0.47. The PICT test exhibited relatively better correlation with the Coamatic assay (r squared=0.47). When the Coamatic results are correlated with various tests, the correlation ranged from 0.13 to 0.47. TFPI levels ranged from 60-100ng/ml (71+16ng/ml) and did not correlate with any of the tests used in this study. There was no Rivaroxaban dosage dependance on TFPI levels.Discussion:These results show that the available clot based and amidolytic methods provide widely variable, assay dependent results for the circulating levels of Rivaroxaban. The DRVVT and Coamatic results were much lower than other test results. The Heptest and PICT test provided much higher levels of Rivaroxaban, whereas the PT and aPTT gave relatively lower results. While the amidolytic methods measure the direct inhibitory effects of Rivaroxaban on factor Xa, the clot based assays measure the cumulative effects including the amplification responses resulting in a higher level of Rivaroxaban. Thus, while the amidolytic method measures the absolute drug level, the clot based methods take into account the pharmacodynamic effects, which may also be dependent on other patient based predispositions. Furthermore, TFPI levels were not found to be affected by rivaroxaban treatment and did not contribute to the modulation of any of the tests included in this study. DisclosuresNo relevant conflicts of interest to declare.

Laboratory-monitored fondaparinux and coagulation activity in association with total hip replacement

Fondaparinux, indirect factor Xa (FXa) inhibitor, is recommended for thromboprophylaxis for high-risk patients undergoing major orthopedic surgery. We evaluated the prothrombotic state and anticoagulant intensity of fondaparinux (2.5 mg daily) after total hip replacement (THR). Twenty patients underwent THR - seven bilateral and 13 unilateral. Blood samples were collected preoperatively and at 6 h, 8 h (2 h after fondaparinux), 1 day (12-14 h after fondaparinux), and 4 weeks (12-14 h after fondaparinux) postoperatively. Antithrombin (AT), fibrinogen, factor VIII activity, coagulation times, thrombin-AT (TAT) complex, D-dimer, C-reactive protein, prothrombinase-induced clotting time (PiCT) and anti-Xa activity were measured. The latter two were also tested after plasma spiking with fondaparinux 0-1.25 μg/ml. In spiked prophylactic fondaparinux samples (0-0.25 μg/ml), PiCT and anti-Xa activity correlated (r = 0.84) better than in the patient samples (r = 0.35). On the first day, anti-Xa activity and PiCT dissociated, and PiCT lost sensitivity for fondaparinux. AT decreased but stayed within the normal range, whereas TAT complex and D-dimer peaked at 6 h as signs of thrombin generation. On the first postoperative day, TAT and D-dimer halved. Bilateral THR associated with higher TAT and D-dimer levels up to 4 weeks. Perioperative FVIII levels were not affected, but were elevated in both groups (range 191-211%) after 4 weeks. Anti-Xa activity detected prophylactic fondaparinux with higher sensitivity than PiCT in vitro, but even more so in vivo. Thus, PiCT is not the method of choice to assess fondaparinux at least in association with THR. THR, bilateral more than unilateral, increased thrombin generation and D-dimer 7-11-fold early after surgery. Factor VIII activity and D-dimer remained elevated even after 4 weeks despite the compliant thromboprophylaxis with fondaparinux.

Pharmacodynamics and pharmacokinetics during the transition from warfarin to rivaroxaban: a randomized study in healthy subjects

AimsThis study investigated relevant pharmacodynamic and pharmacokinetic parameters during the transition from warfarin to rivaroxaban in healthy male subjects.MethodsNinety-six healthy men were randomized into the following three groups: warfarin [international normalized ratio (INR) 2.0–3.0] transitioned to rivaroxaban 20 mg once daily (od; group A); warfarin (INR 2.0–3.0) followed by placebo od (group B); and rivaroxaban alone 20 mg od (group C) for 4 days. Anti-factor Xa activity, inhibition of factor Xa activity, prothrombin time (PT), activated partial thromboplastin time, HepTest, prothrombinase-induced clotting time, factor VIIa activity, factor IIa activity, endogenous thrombin potential and pharmacokinetics were measured.ResultsAn additive effect was observed on the PT and PT/INR during the initial transition period. The mean maximal prolongation of PT was 4.39-fold [coefficient of variation (CV) 18.03%; range 3.39–6.50] of the baseline value in group A, compared with 1.88-fold (CV 10.35%; range 1.53–2.21) in group B and 1.57-fold (CV 9.98%; range 1.37–2.09) in group C. Rivaroxaban had minimal influence on the PT/INR at trough levels. Inhibition of factor Xa activity, activated partial thromboplastin time and endogenous thrombin potential were also enhanced, but to a lesser extent. In contrast, the effects of rivaroxaban on anti-factor Xa activity, HepTest and prothrombinase-induced clotting time were not affected by pretreatment with warfarin.ConclusionsChanges in pharmacodynamics during the transition from warfarin to rivaroxaban vary depending on the test used. A supra-additive effect on PT/INR is expected during the initial period of transition, but pretreatment with warfarin does not influence the effect of rivaroxaban on anti-factor Xa activity.

Comparison of the aPTT With Alternative Tests for Monitoring Direct Thrombin Inhibitors in Patient Samples

The activated partial thromboplastin time (aPTT) test has been used for years to monitor parenteral direct thrombin inhibitors (DTIs) and unfractionated heparin. Because the aPTT correlates poorly with unfractionated heparin levels, we hypothesized that the aPTT may not be the best test for monitoring parenteral DTIs. Using 235 excess plasma specimens from 82 adult patients receiving treatment with DTIs (argatroban, bivalirudin, or dabigatran), we compared the aPTT with the ecarin chromogenic assay (ECA), the dilute thrombin time (dTT) test, and the prothrombinase-induced clotting time (PiCT) test. The aPTT correlated poorly with each of the other tests in both bivalirudin- and argatroban-containing samples (r(2) = 0.04-0.23). The ECA and dTT exhibited the best correlations (r(2) = 0.66-0.93). Intermediate correlations were seen when the results of the PiCT were plotted against the dTT or ECA (r(2) = 0.46-0.58). Nineteen specimens obtained from six patients receiving dabigatran showed a good correlation between the dTT and the ECA (r(2) = 0.92). The aPTT does not correlate well with other tests that might be used to monitor parental DTI administration. Further studies are needed to evaluate the clinical usefulness of alternative tests and their correlation with clinical outcomes.

Population pharmacokinetics and pharmacodynamics of rivaroxaban in patients with non‐valvular atrial fibrillation: Results from ROCKET AF

Two once-daily rivaroxaban dosing regimens were compared with warfarin for stroke prevention in patients with non-valvular atrial fibrillation in ROCKET AF: 20 mg for patients with normal/mildly impaired renal function and 15 mg for patients with moderate renal impairment. Rivaroxaban population pharmacokinetic (PK)/pharmacodynamic (PD) modeling data from ROCKET AF patients (n = 161) are reported and are used to confirm established rivaroxaban PK and PK/PD models and to re-estimate values of the models' parameters for the current AF population. An oral one-compartment model with first-order absorption adequately described rivaroxaban PK. Age, renal function, and lean body mass influenced the PK model. Prothrombin time and prothrombinase-induced clotting time exhibited a near-linear relationship with rivaroxaban plasma concentration; inhibitory effects were observed through to 24 hours post-dose. Rivaroxaban plasma concentration and factor Xa activity had an inhibitory maximum-effect (Emax ) relationship. Renal function (on prothrombin time; prothrombinase-induced clotting time) and age (on factor Xa activity) had moderate effects on PK/PD models. PK and PK/PD models were shown to be adequate for describing the current dataset. These findings confirm the modeling and empirical results that led to the selection of doses tested against warfarin in ROCKET AF.

Prothrombinase Induced Clotting Time (PICT) and Commercially Available Diluted Russell's Viper Venom Times For The Monitoring Of New Oral Anticoagulants

The new oral anticoagulants such as Rivaroxaban (Bayer Healthcare) (R) Apixaban (BMS/Pfizer) (A) and Dabigatran (Boehringer Ingleheim) (D) have been approved for Several indications in the US and Europe. Initially it was suggested that these agents did not require monitoring at the approved dosages for specific indications, however there have been reported bleeding complications with some of these agents that warrants the monitoring of these drugs to optimize therapy in some patient populations. A new clot-based assay, the prothrombinase induced clotting time (PICT) (Pentapharm, Basal, Switzerland) has been developed to monitor the anticoagulant effect of these new oral anticoagulants. In the assay, plasma is mixed with FXa, phospholipids, calcium and RVV-V from the venom of the Daboia russelli. The prothrombinase complex formed and the Xa and IIa generated is inhibited by the test agent. Two other forms of diluted Russell's Viper Venom time, namely DRVVT (screen), and DRVVT (confirm) are also available for the laboratory diagnosis of the lupus anticoagulant. The purpose of this study is to compare the PICT teste with the two versions of DRVVT test times for the monitoring of newer anticoagulants. MaterialsCitrated blood was drawn from 15 donors and spun at 800 rpm to obtain platelet rich plasma (PRP). The PRP was removed and spun at 3000 rpm to obtain platelet poor plasma (PPP). Both PRPand PPP were frozen at -80ºC for 24 hours. Thawed PPP and PRP were supplemented with A, R and D in a concentration range of 0-2.5 ug/ml. The plasma samples were analyzed using 3 PT/INR reagents (Innoven, Dade-Behring, Germany; Recombiplastin, Instrumentation Laboratories, Bedford, MA; Neoplastin, Stago, Parsippany, NJ), two APTT reagents (Platelin, TCoag, Ireland; Actin FSL, Instrumentation Laboratories, Bedford, MA), Heptest, the one stage PICT and the two versions of DRVVT. All assays were performed on the ACL 300 Plus (Instrumentation Laboratories, Bedford, MA) with the exception of the PICT and DRVVTs which were run on the ST4 (STago, Parsippany, NJ). ResultsIn the PPP system, the A, R and D showed assay differences in the clotting times which demonstrated good sensitivity to D and R compared to A. The PICT and the two versions of DRVVT showed much higher sensitivity and linearity. The relative sensitivity to DRVVTs were higher than PICT. In the PT/INR assay, all reagents were sensitive to D and the Innovin and recombiplastin were sensitive to R and showed a weaker response to A. Similar responses were observed in the APTT and Heptest assay. In the PICT and DRVVTs, all drugs showed a concentration dependent increase. D was strongest followed by R, and A showed the weakest effect. In the PRP supplemented system, all agents showed a weaker effect on the clotting times in all tests, however PICT and DRVVTs demonstrated a concentration dependent response for all agents. ConclusionsThese results demonstrated that neither the PT/INR, APTT nor heptest can be solely used to monitor the effects of all of the new oral anticoagulants. The one stage PICT is a simple, fast, automated or semi-automated test which can be performed on any mechanical or optical coagulation analyzer to monitor the anticoagulant effects of these agents. Similarly the two versions of DRVVT can also be performed on these instruments. The one stage PICT and DRVVT tests can also be used to monitor the effects of these agents in other matrices such as PRP. These studies warrant clinical validation of this test in patients treated with the newer oral anticoagulant drugs. Disclosures:No relevant conflicts of interest to declare.

Variations in the Circulating Heparin Levels During Maintenance Hemodialysis in Patients With End-Stage Renal Disease

Unfractionated heparin has remained the anticoagulant of choice in patients undergoing hemodialysis. However, wide variations in the heparinization responses have been observed in patients anticoagulated with this drug. The purpose of this investigation was to measure circulating heparin levels in patients with end-stage renal disease (ESRD) prior to and after maintenance hemodialysis. This study included 119 patients with ESRD undergoing maintenance hemodialysis who received heparin during dialysis. Citrated blood samples were collected prior to and immediately after the dialysis session and analyzed utilizing clot-based methods such as activated partial thromboplastin time (APTT), Heptest, and prothrombinase-induced clotting time (PiCT). Circulating anti-Xa levels, antithrombin III levels, and thrombin generation (TG) were also measured. The circulating heparin levels ranged from 0 to 1.08 IU/mL with a mean of 0.07 ± 0.11 for the APTT and a range of 0 to 1.98 for the Heptest with a mean of 0.09 ± 0.26 U/mL. There was no significant difference in circulating levels of heparin between pre- and post-hemodialysis samples using APTT, Heptest, and PiCT, whereas the TG and anti-Xa tests showed a statistically significant P value <0.05 when comparing the 2 groups. The presence of detectable levels of heparin in the predialysis plasma samples for almost two-thirds (87 of 119) of the patients suggests that residual heparin circulates in these patients for a longer period of time. In all, 5% of postdialysis samples, 6 of 119, contained >0.25 U/mL of heparin, which may be related to a central catheter vascular access flushed with heparin. These findings suggest that patients on maintenance hemodialysis may accumulate a detectable amount of heparin due to the decreased renal clearance.