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

J Clin Pathol. 2019 Dec;72(12):817-824. doi: 10.1136/jclinpath-2019-205922. Epub 2019 Jul 31.


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.

Keywords: APTT assay; bivalent direct thrombin inhibitors; clauss fibrinogen assay; clot waveform analysis; enzyme kinetics.

Publication types

  • Comparative Study

MeSH terms

  • Antithrombins / pharmacology*
  • Biomarkers / blood
  • Blood Coagulation / drug effects*
  • Dose-Response Relationship, Drug
  • Fibrinogen / metabolism
  • Hirudins / pharmacology*
  • Humans
  • Kinetics
  • Models, Biological
  • Partial Thromboplastin Time*
  • Peptide Fragments / pharmacology*
  • Recombinant Proteins / pharmacology
  • Thrombin / antagonists & inhibitors*
  • Thrombin / metabolism


  • Antithrombins
  • Biomarkers
  • Hirudins
  • Peptide Fragments
  • Recombinant Proteins
  • Fibrinogen
  • Thrombin
  • bivalirudin