Thromb Haemost 2020; 120(01): 055-064
DOI: 10.1055/s-0039-1700516
Coagulation and Fibrinolysis
Georg Thieme Verlag KG Stuttgart · New York

Development of a Plasma-Based Assay to Measure the Susceptibility of Factor V to Inhibition by the C-Terminus of TFPIα

Peter van Doorn
1  Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
,
Jan Rosing
1  Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
,
Elena Campello
2  Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy
,
Saskia Middeldorp
3  Amsterdam UMC, University of Amsterdam, Department of Vascular Medicine, Amsterdam Cardiovascular Sciences, Amsterdam, The Netherlands
,
Paolo Simioni
2  Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy
,
Joost C. M. Meijers
4  Amsterdam UMC, University of Amsterdam, Department of Experimental Vascular Medicine, Amsterdam Cardiovascular Sciences, Amsterdam, The Netherlands
5  Department of Molecular and Cellular Hemostasis, Sanquin, University of Amsterdam, Amsterdam, The Netherlands
,
Tilman M. Hackeng
1  Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
,
Elisabetta Castoldi
1  Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
› Author Affiliations
Funding This work was supported by grant 2014–1 from the Dutch Thrombosis Foundation. J. Rosing is Van de Laar Professor of Coagulation Enzymology.
Further Information

Publication History

12 April 2019

29 August 2019

Publication Date:
08 November 2019 (online)

Abstract

Background Factor V (FV) is proteolytically activated to FVa, which assembles with FXa in the prothrombinase complex. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) inhibits both the activation and the prothrombinase activity of FV(a), but the pathophysiological relevance of this anticoagulant mechanism is unknown. FV Leiden (FVL) is less susceptible to inhibition by TFPIα, while overexpression of FV splicing variants with increased affinity for TFPIα (FV-short) causes bleeding.

Objective This study aims to develop a plasma-based assay that quantifies the susceptibility of FV(a) to inhibition by the TFPIα C-terminus.

Materials and Methods FV in highly diluted plasma was preactivated with FXa in the absence or presence of the TFPIα C-terminal peptide. After adding prothrombin, thrombin formation was monitored continuously with a chromogenic substrate and prothrombinase rates were obtained from parabolic fits of the absorbance tracings. TFPI resistance was expressed as the ratio of the prothrombinase rates with and without peptide (TFPIr).

Results The TFPIr (0.25–0.34 in 45 healthy volunteers) was independent of FV levels. The TFPIr increased from normal individuals (0.29, 95% confidence interval [CI] 0.28–0.31) to FVL heterozygotes (0.35, 95% CI 0.34–0.37) and homozygotes (0.39, 95% CI 0.37–0.40), confirming TFPI resistance of FVL. Two individuals overexpressing FV-shortAmsterdam had markedly lower TFPIr (0.16, 0.18) than a normal relative (0.29), in line with the high affinity of FV-short for TFPIα.

Conclusion We have developed and validated an assay that measures the susceptibility of plasma FV to the TFPIα C-terminus. Once automated, this assay may be used to test whether the TFPIr correlates with thrombosis or bleeding risk in population studies.

Supplementary Material