Clot waveform analysis for perioperative hemostatic monitoring in a hemophilia A patient on emicizumab undergoing liver transplantation.

How to optimize perioperative factor VIII (FVIII) replacement through hemostatic monitoring is critically important to manage hemophilia A patients. The bispecific antibody emicizumab binds activated FIX (FIXa) and FX to functionally mimic FVIIIa. While being used for hemostatic control in hemophilia A, this therapeutic antibody inconveniently interferes with coagulation tests using human FIXa and FX, such as activated partial thromboplastin time (APTT) and FVIII activity measurement based on one-stage clotting assays. Clot waveform analysis (CWA) extends the interpretation of measurement curves for coagulation time to provide global information. We performed APTT-CWA to monitor perioperative hemostasis in a hemophilia A patient on emicizumab undergoing liver transplantation. Plasma samples were treated with anti-idiotype monoclonal antibodies against emicizumab to enable accurate coagulation assays. Kinetics of maximum coagulation velocity and acceleration mimicked that of FVIII activity. These CWA parameters better correlated with FVIII activity than APTT. The plateaus of them were observed at FVIII activity of 100% or more, supporting the protocol for perioperative FVIII replacement. Thus, CWA may measure coagulation potential in hemophilia A patients undergoing liver transplantation, aiding in optimizing perioperative hemostasis.

Characterisation of antithrombin-dependent anticoagulants through clot waveform analysis to potentially distinguish them from antithrombin-independent inhibitors targeting activated coagulation factors.

AIMS: While antithrombin (AT)-independent inhibitors targeting thrombin or activated factor X have been assessed through clot waveform (CWA), there are no reports on assessment with respect to AT-dependent anticoagulants. The present study aims to characterise AT-dependent anticoagulants through CWA to distinguish them from AT-independent inhibitors. METHODS: CWA was applied to the activated partial thromboplastin time (APTT) assay of plasma samples spiked with each of AT-dependent drugs (unfractionated heparin, enoxaparin and fondaparinux) and AT-independent drugs (rivaroxaban, apixaban, edoxaban, dabigatran, argatroban, hirudin and bivalirudin), which was performed using the CS-5100 or CN-6000 (Sysmex). The APTT-CWA data were automatically gained by the analyser program. The positive mode of clotting reaction curves was defined as the direction towards fibrin generation. RESULTS: Regarding dose-response curves in AT-dependent anticoagulants, the maximum positive values of the first and secondary derivatives (Max1 and Maxp2, respectively) and the maximum negative values of the secondary derivative (Maxn2) seemed to drop to zero without making an asymptotic line, consistent with the irreversibility. Such a feature was observed also in hirudin, as reported previously. Notably, the symmetric property of Max1 peaks in the waveforms was distorted dose dependently in AT independent but not AT-dependent drugs. A plot of Maxp2 logarithm versus Maxn2 logarithm was linear. The slope was about 1 in AT-dependent drugs while that was more than 1 in AT-independent drugs. These features made it possible to distinguish AT-dependent and AT-independent drugs. CONCLUSIONS: The results aid in further understanding of the pharmacological aspects of anticoagulation and in screening of candidates for novel anticoagulants.

Application of clot-fibrinolysis waveform analysis to assessment of in vitro effects of direct oral anticoagulants on fibrinolysis.

INTRODUCTION: Acceleration of fibrinolysis by direct oral anticoagulants (DOACs) has been reported by several groups, suggesting contribution of not only anticoagulant but also fibrinolytic effects to the therapeutic efficacy. The present study aims to evaluate the usability of clot-fibrinolysis waveform analysis (CFWA) for assessment of in vitro effects of DOACs on fibrinolysis. METHODS: The experimental conditions were optimized according to how t-PA concentrations and a time length after t-PA adjustment affect parameters of CFWA. Addition of the activated partial thromboplastin time (APTT) reagent followed by that of calcium and t-PA was done to obtain clotting and fibrinolytic reaction curves which were mathematically differentiated for CFWA (APTT-CFWA). The positive and negative modes of waveforms were defined as the direction toward fibrin generation and that toward fibrin degradation, respectively. The maximum positive and negative values (Maxp 1 and Maxn 1) correspond to the maximum coagulation velocity and the maximum fibrinolysis velocity, respectively. Plasma spiked with each of DOACs (rivaroxaban, apixaban, edoxaban, and dabigatran) was subjected to APTT-CFWA. RESULTS: Optimization of t-PA use was based on Maxn 1. Roughly biphasic effects of rivaroxaban and dabigatran but not apixaban or edoxaban on fibrinolysis were observed through Maxn 1 and the fibrinolysis peak time, which was defined as a time length from the time when Maxp 1 (Maxp 1 time) to the time when Maxn 1 appears (Maxn 1 time). CONCLUSION: The results suggest the usability of CFWA for assessment of DOAC effects and provide insights into relevance of anticoagulation to therapeutic efficacy and bleeding risk from the perspective of fibrinolysis.

Distinct features of bivalent direct thrombin inhibitors, hirudin and bivalirudin, revealed by clot waveform analysis and enzyme kinetics in coagulation assays.

AIMS: Bivalent direct thrombin inhibitors (DTIs), hirudin and bivalirudin, bind to the active site and exosite 1 of thrombin irreversibly and reversibly, respectively. The present study aims to assess in vitro effects of hirudin and bivalirudin through clot waveform analysis (CWA) and enzyme kinetics in coagulation assays. METHODS: The pooled normal plasma and its dilutions were spiked with hirudin or bivalirudin. The activated partial thromboplastin time (APTT) assay and the Clauss fibrinogen assay were performed using the CS-5100 (Sysmex). The APTT-CWA data were automatically gained by the CS-5100 programme. RESULTS: In APTT-CWA, the maximum coagulation velocity, acceleration and deceleration were decreased dependently on the drug concentrations, demonstrating evidence for the blockade of thrombin-positive feedback by hirudin or bivalirudin. The Hill plot analysis was applied to the dose-dependent curves in bivalirudin. The Hill coefficients were greater than 1, showing positive anticoagulant cooperativity. Regarding the dose-dependent curves in hirudin, all the parameters dropped to almost zero without making an asymptotic line. In the Clauss fibrinogen assay, the Lineweaver-Burk plots demonstrated that both drugs exhibit mixed inhibition mimicking uncompetitive binding. The Dixon plots in bivalirudin were linear and supported the inhibition type described above. The Dixon plots in hirudin were non-linear and inappropriate to use for determination of the inhibition type. In addition, the inverse function of the clotting time appeared to drop to zero without making an asymptotic line, suggesting complete loss of thrombin activity by irreversible binding. CONCLUSIONS: The results provide insights into anticoagulation with bivalent DTIs.

Assessment of in vitro effects of direct thrombin inhibitors and activated factor X inhibitors through clot waveform analysis.

AIMS: Clot waveform analysis (CWA) has been reported to extend the interpretation of clotting time measurement. The parameters obtained from successive derivatives of the clotting reaction curves reflect the rates of activation of individual coagulation factors, theoretically dissecting the cascade pathway. This study aims to assess the in vitro effects of direct thrombin inhibitors (DTIs) and activated factor X (FXa) inhibitors. METHODS: CWA was applied to the activated partial thromboplastin time (APTT) assay of plasma samples spiked with each drug. For CWA of APTT measurement curves (APTT-CWA), the positive mode of clotting reaction curves was defined as the direction towards fibrin generation. RESULTS: All the maximum positive values in the successive derivatives were decreased dependently on the concentrations of each drug. Moreover, the negative values in the second and third derivatives appeared putatively due to consumption of thrombin and factor FXa, respectively, to form complexes with plasma serine protease inhibitors. The decrease of the maximum negative values observed dependently on the concentrations of each drug appeared to be consistent with the decreased generation of thrombin and factor FXa. The analysis of Hill coefficients of each drug in the dose-response of changes in the APTT-CWA parameters revealed a difference in anticoagulant cooperativity between DTIs versus FXa inhibitors. CONCLUSIONS: The APTT-CWA demonstrated evidence for the blockade of thrombin-positive feedback by DTIs and FXa inhibitors and that for the differences in anticoagulant cooperativity between them. The results demonstrate the usability of CWA for assessment of anticoagulation and provide insights into direct anticoagulants.