TY - JOUR
T1 - Distinct features of bivalent direct thrombin inhibitors, hirudin and bivalirudin, revealed by clot waveform analysis and enzyme kinetics in coagulation assays
AU - Wakui, Masatoshi
AU - Fujimori, Yuta
AU - Nakamura, Shoko
AU - Kondo, Yoshino
AU - Kuroda, Yuko
AU - Oka, Shusaku
AU - Nakagawa, Terumichi
AU - Katagiri, Hisako
AU - Murata, Mitsuru
N1 - Funding Information:
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.
Funding Information:
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.
Publisher Copyright:
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
PY - 2019/12/1
Y1 - 2019/12/1
N2 - 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.
AB - 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.
KW - APTT assay
KW - bivalent direct thrombin inhibitors
KW - clauss fibrinogen assay
KW - clot waveform analysis
KW - enzyme kinetics
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U2 - 10.1136/jclinpath-2019-205922
DO - 10.1136/jclinpath-2019-205922
M3 - Article
C2 - 31366633
AN - SCOPUS:85075665600
VL - 72
SP - 817
EP - 824
JO - Molecular pathology : MP
JF - Molecular pathology : MP
SN - 0021-9746
IS - 12
ER -