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

Abstract

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 (Max p 1 and Max n 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 Max n 1. Roughly biphasic effects of rivaroxaban and dabigatran but not apixaban or edoxaban on fibrinolysis were observed through Max n 1 and the fibrinolysis peak time, which was defined as a time length from the time when Max p 1 (Max p 1 time) to the time when Max n 1 appears (Max n 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. K E Y W O R D Sactivated partial thromboplastin time, clot-fibrinolysis waveform analysis, direct oral anticoagulants, maximum fibrinolysis velocity, tissue plasminogen activator

Simultaneous assessment of DOACs effect on clot formation and fibrinolysis with the FibWave

“…The DOACs are also known to impact coagulation assays and enhance factor X (FX)-dependent fibrinolysis and plasmin generation. 1 Three methods including the thromboelastometry (TEG/ ROTEM), the thrombin generation assay (TGA), and the clot waveform analysis (CWA) are able to monitor the formation of thrombin or fibrin and recognized to reflect complete processes of coagulation and/or fibrinolysis. 1,2 Lately, the clot-fibrinolysis waveform analysis (CFWA), an extension of conventional CWA using tPA, allows the simultaneous analysis of clotting and fibrinolysis processes.…”

“…1 Three methods including the thromboelastometry (TEG/ ROTEM), the thrombin generation assay (TGA), and the clot waveform analysis (CWA) are able to monitor the formation of thrombin or fibrin and recognized to reflect complete processes of coagulation and/or fibrinolysis. 1,2 Lately, the clot-fibrinolysis waveform analysis (CFWA), an extension of conventional CWA using tPA, allows the simultaneous analysis of clotting and fibrinolysis processes. In most studies, coagulation phase was triggered by using activated partial thromboplastin time (aPTT) reagent and CaCl 2 .…”

“…In most studies, coagulation phase was triggered by using activated partial thromboplastin time (aPTT) reagent and CaCl 2 . 1,3 On the other hand, the FibWave, a new developed coagulation assay based on the analysis of the clot formation kinetics, assesses TA B L E 1 Clot formation and fibrinolysis for three pooled plasmas Note: The table reports data for parameters of clot formation and fibrinolysis for 3 pooled plasmas (men, women no CHC, and women CHC). Significant differences were observed between pooled plasmas † but not with tPA ‡.…”

“…As mentioned by Oka et al, the preparation of t-PA solution was followed by addition of the samples for CFF as immediately as possible. 1 The impact of tPA was assessed on parameters of the clot formation by 3 independent runs on 3 separated pooled plasma (men, women not using hormonal contraceptives (no CHC), and women using hormonal contraceptives (CHC)). Wilcoxon test was used to assess significant impact of tPA.…”

“…Both techniques use tPA to trigger the fibrinolytic process but in the CFWA, activated partial thromboplastin time (aPTT) reagent and CaCl 2 containing tPA were used whereas with the FibWave, ±5 pM TF containing tPA were used to trigger coagulation and the subsequent fibrinolysis. 1,3 The physiological coagulation starts when small amounts of TF (sTF) and activated factor VII (FVIIa) activate factor IX (FIX) on the phospholipids (PLs) of the platelet membrane. APTT reagents, including high PL content and contact activator, cannot sufficiently reflect the FIX activation by the extrinsic tenase complex and effect of platelets on physiological clotting, compared with "CWA/ sTF."…”