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Distinct features of bivalent direct thrombin inhibitors, hirudin and bivalirudin, revealed by clot waveform analysis and enzyme kinetics in coagulation assays
  1. Masatoshi Wakui1,
  2. Yuta Fujimori2,
  3. Shoko Nakamura3,
  4. Yoshino Kondo3,
  5. Yuko Kuroda3,
  6. Shusaku Oka3,
  7. Terumichi Nakagawa3,
  8. Hisako Katagiri3,
  9. Mitsuru Murata1
  1. 1 Department of Laboratory Medicine, Keio University School of Medicine, Tokyo, Japan
  2. 2 Office of Clinical Laboratory Technology, Keio University Hospital, Tokyo, Japan
  3. 3 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 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.

  • APTT assay
  • clot waveform analysis
  • clauss fibrinogen assay
  • enzyme kinetics
  • bivalent direct thrombin inhibitors

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  • Handling editor Prof 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 and SN performed the experiments. MW, YF, SN, YKo, YKu, SO, TN, HK 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.

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

  • Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.