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Assessment of in vitro effects of direct thrombin inhibitors and activated factor X inhibitors through clot waveform analysis
  1. Masatoshi Wakui1,
  2. Yuta Fujimori2,
  3. Hisako Katagiri2,
  4. Shoko Nakamura2,
  5. Yoshino Kondo2,
  6. Yuko Kuroda2,
  7. Terumichi Nakagawa2,
  8. Nobuko Shimizu2,
  9. Mitsuru Murata1
  1. 1 Department of Laboratory Medicine, Keio University School of Medicine, Tokyo, Japan
  2. 2 Clinical Laboratory, Keio University Hospital, Tokyo, Japan
  1. Correspondence to Dr Masatoshi Wakui, Department of Laboratory Medicine, Keio University School of Medicine , Tokyo 160-8582, Japan; wakuism{at}


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.

  • APTT
  • clot waveform analysis
  • direct FXa inhibitors
  • direct thrombin inhibitors
  • thrombin positive feedback

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  • Handling editor Mary Frances McMullin.

  • Contributors MW and YF equally contributed to this work. MW conceived the study. MW and YF designed the study, analysed and interpreted the data, and wrote the manuscript. YF, HK, SN, YK and YK performed the experiments. MW, YF, HK, SN, YK, YK, TN, NS and MM discussed the data and critically reviewed and revised the manuscript. All authors have given final approval for this version of the manuscript to be published.

  • Funding This work was supported by the BMS/Pfizer Japan Thrombosis Investigator Initiated Research Program (JRISTA). YF was supported by a grant from the Charitable Trust Laboratory Medicine Research Foundation of Japan.

  • Competing interests The BMS/Pfizer Japan Thrombosis Investigator Initiated Research Program (JRISTA), which supported this work, is administered by the companies, Bristol-Myers Squibb and Pfizer.

  • Patient consent for publication Not required.

  • Ethics approval All the experiments were carried out in accordance with the institutional ethical committee requirements, given the approval numbers 20100224 and 20160291.

  • Provenance and peer review Not commissioned; externally peer reviewed.