Concomitant assessment of rivaroxaban concentration and its impact on thrombin generation.

BACKGROUND: Reliable assays to measure direct oral anticoagulant (DOAC) levels and their activity in critical situations are needed. Drug levels alone are not representative of the effect of DOACs on an individual's coagulation. We developed a technique that provides direct assessment of the global effect of rivaroxaban on the individual's coagulation in addition to plasma concentrations. METHODS: DOAC concentrations were determined in fifty patients using rivaroxaban, with the new assay, Xross-CAT. The effect of rivaroxaban on coagulation (activity) was measured with thrombin generation (TG) in platelet poor plasma using 5 pM tissue factor on the same device. The levels were validated with the Biophen DiXal assay. The prothrombin time (PT) and dilute Russell viper venom time (dRVVT) were performed to estimate the effect on coagulation. RESULTS: The variability of Xross-CAT was below 12%. Xross-CAT correlates well with Biophen DiXaI (rs = 0.885). The bias, determined by Bland-Altman analysis, was 4.9% and the Passing-Bablok equation was y = 1.1x - 2.1. The correlation of plasma levels with TG was moderate (ETP rs = -0.548; Peak rs = -0.559), as for the PT (rs = 0.739) and the dRVVT (rs = 0.692). CONCLUSIONS: Xross-CAT shows a good correlation with Biophen DiXaI that was previously confirmed to accurately assess rivaroxaban levels. Bleeding and thrombotic complications are not necessarily associated with drug levels and could be influenced by concomitant risk factors. The main benefit of Xross-CAT is that it can be performed simultaneously with thrombin generation, providing an overview of the global anticoagulation status of a patient in relation to circulating DOAC levels.

A Hypoxic Environment Attenuates Exercise-Induced Procoagulant Changes Due to Decreased Platelet Activation.

Introduction Although physical exercise is protective against cardiovascular disease, it can also provoke sudden cardiac death (exercise paradox). Epidemiological studies suggest that systemic hypoxia at high altitude is a risk factor for venous thromboembolism. Forthcoming, this study investigated the effect of repeated exercise at high altitude on blood coagulation, platelet function, and fibrinolysis. Methods Six trained male volunteers were recruited. Participants ascended from sea level to 3,375 m altitude. They performed four exercise tests at 65 to 80% of their heart-rate reserve during 2 hours: one time at sea level and three times on consecutive days at 3,375 m altitude. Thrombin generation (TG) was measured in whole blood (WB) and platelet-rich and platelet-poor plasma. Coagulation factor levels were measured. Platelet activation was measured as αIIbß3 activation and P-selectin expression. Fibrinolysis was studied using a clot-lysis assay. Results Normoxic exercise increased plasma peak TG through increased factor VIII (FVIII), and increased von Willebrand factor (VWF) and active VWF levels. Platelet granule release potential was slightly decreased. After repetitive hypoxic exercise, the increase in (active) VWF tapered, and there was no more distinct exercise-related increase in peak. Platelet aggregation potential and platelet-dependent TG decreased at high altitude. There were no effects on fibrinolysis upon exercise and/or hypoxia. Conclusion Strenuous exercise induces a procoagulant state that is mediated by the endothelium, by increasing VWF and secondarily raising FVIII levels. After repetitive exercise, the amplitude of the endothelial response to exercise diminishes. A hypoxic environment appears to further attenuate the procoagulant changes by decreasing platelet activation and platelet-dependent TG.

The anticoagulant effect of dabigatran is reflected in the lag time and time-to-peak, but not in the endogenous thrombin potential or peak, of thrombin generation.

INTRODUCTION: Calibrated automated thrombinography (CAT) is a sensitive method to assess coagulation. Dabigatran inhibits both free thrombin and the α2macroglobulin (α2M)-thrombin complex, which results in an erroneously increased peak and endogenous thrombin potential (ETP) without affecting lag time and time-to-peak. The aim of this study was to elucidate the artefacts in CAT when dabigatran is present. MATERIALS AND METHODS: Thrombin generation (TG) was measured in vitro by using CAT in the presence or absence of 6 µM idarucizumab in plasma spiked with dabigatran. Additionally, ex vivo measurements were performed in plasmas of 63 patients using dabigatran in the presence and absence of idarucizumab. RESULTS: The in vitro experiments confirmed that the ETP, peak and velocity index were artificially increased. This was mainly due to the inhibition of the calibrator by dabigatran and partly due to CAT algorithms. The calibration artefact could be resolved by adding idarucizumab to the calibrator well. However, the second, mathematical artefact remains when dabigatran is present in the TG well. These findings were corroborated by ex vivo experiments i.e. the lag time and time-to-peak were significantly reduced in patients upon addition of idarucizumab, but the ETP and peak were not significantly affected. The velocity index did change significantly, since this is a combination of time-dependent factors and the peak. CONCLUSIONS: The peak, ETP and velocity index do not represent the anticoagulant effect of dabigatran on TG measured with CAT. The lag time and time-to-peak, however, do reflect the effect of dabigatran.

Calibrated Automated Thrombinography (CAT), a Tool to Identify Patients at Risk of Bleeding during Anticoagulant Therapy: A Systematic Review.

Background Bleeding is a feared adverse event during anticoagulant treatment. In patients on vitamin K antagonists, most bleedings occur with the international normalized ratio (INR) in the therapeutic range. Currently, identification of high-risk patients via laboratory methods is not reliable. In this systematic review, we assessed the ability of calibrated automated thrombin generation (CAT-TG) to predict bleeding in patients on anticoagulant treatment. Methods A systematic search was executed in three databases: Medline, Embase, and Cochrane. Results Seven studies were included; two were of good methodological quality. One study showed that patients on warfarin with INRs in range (2-3) admitted for hemorrhage ( n = 28), had lower CAT-TG levels (endogenous thrombin potential [ETP]: 333 ± 89 nM/min) than patients on warfarin admitted for other reasons (ETP: 436 ± 207 nM/min; p < 0.001). Another study found no difference in ETP or peak levels between bleeding and nonbleeding patients in PPP or PRP. When measured in whole blood, both levels were significantly lower in patients with bleeding compared with nonbleeding patients (median [interquartile range, IQR] ETP: 182.5 [157.2-2,847 nM/min] vs. median [IQR] ETP: 256.2 [194.9-344.2 nM/min]; p < 0.001) and median [IQR] peak: 23.9 [19.6-41.8 nM] vs. median [IQR] peak: 39.1 [24.9-53.2 nM]; p < 0.05). From the remaining studies, four suggested that CAT-TG is more sensitive in detecting hemostatic abnormalities than INR and one article found ETP and INR to be equally useful. However, insufficient data were provided to validate these conclusions. Conclusion Studies investigating the direct association between decreased CAT-TG values and hemorrhagic events are rare. Therefore, the clinical consequences of low CAT-TG values remain to be further investigated.

Prediction of bleeding risk in patients taking vitamin K antagonists using thrombin generation testing.

Until recently, vitamin K antagonists (VKAs) were the mainstay of oral anticoagulant treatment with bleeding as the most prevalent adverse effect. One to four percent of patients experience major bleeding episodes, while clinically relevant bleeding occurs in up to 20%. At this moment no laboratory assays are available to identify patients at risk for bleeding. With this study we aimed to investigate whether thrombin generation tests might identify a bleeding risk in patients taking VKAs. This prospective cohort study included 129 patients taking VKAs for more than three months. Calibrated automated thrombinography (CAT) was performed in whole blood, platelet rich and platelet poor plasma. Hematocrit, hemoglobin concentrations and the International Normalized Ratio (INR) were defined and coagulation factor levels were measured. Forty clinically relevant bleeding episodes were registered in 26 patients during follow-up. No differences were found in plasma CAT parameters or INR values. Bleeding was not associated with age, sex, hematocrit, hemoglobin levels or coagulation factor levels. In whole blood a significantly lower endogenous thrombin potential (ETP) and peak were found in patients with bleeding (median ETP: 182.5 versus 256.2 nM.min, p = 0.002; peak: 23.9 versus 39.1 nM, p = 0.029). Additionally, the area under the receiver operating curve (AUC ROC) was significantly associated with bleeding (ETP: 0.700, p = 0.002; peak: 0.642, p = 0.029). HAS-BLED scores were also significantly higher in bleeding patients (3 versus 2, p = 0.003), with an AUC ROC 0.682 (p = 0.004). In conclusion, bleeding in patients taking VKAs is associated with a decreased whole blood ETP and peak as well as with an increased HAS-BLED score.

Determinants of agreement between proposed therapeutic windows of platelet function tests in vulnerable patients.

AIMS: Therapeutic windows for residual platelet reactivity in patients with coronary artery disease on P2Y12 inhibitors were proposed in a consensus document. We aimed to explore the level of agreement between windows for different platelet function tests (PFTs) used to classify patients in low, optimal, and high on-treatment platelet reactivity categories, and to identify variables contributing to the level of agreement. METHODS AND RESULTS: In this explorative clinical study, the VerifyNow P2Y12, Multiplate adenosine diphosphate (ADP), and light transmission aggregometry (LTA) 20 µmol/L ADP were performed simultaneously in 145 consecutive vulnerable patients. Measurements were performed within 6 months of percutaneous intervention. Patients were considered vulnerable if they had ≥2 risk factors for bleeding or ischaemic events. Window-agreement between PFT pairs was slight to moderate. Multiplate-VerifyNow agreed in 72 patients (50%), κ = 0.41; VerifyNow-LTA agreed in 76 patients (52%), κ = 0.36; and LTA-Multiplate agreed in 64 patients (44%), κ = 0.20. Several variables including the type of P2Y12 inhibitor, aspirin, haemoglobin level, platelet count, age, and previous stroke significantly influenced agreement between PFTs. CONCLUSIONS: Our results suggest that the PFTs, with accompanying therapeutic windows, are not interchangeable when determining the response to antiplatelet therapy in vulnerable coronary artery disease patients on P2Y12 inhibitors. Hence, the type of PFT can directly affect the treatment strategy, which may be especially relevant for patients with multiple factors influencing individual PFTs and thereby test agreement.