Thrombin generation-based assays to measure the activity of the TFPI–protein S pathway in plasma from normal and protein S-deficient individuals

Authors

  • L. F. A. MAURISSEN,

    1. Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands
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  • E. CASTOLDI,

    1. Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands
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  • P. SIMIONI,

    1. Department of Cardiologic, Thoracic and Vascular Sciences, 2nd Chair of Internal Medicine, University of Padua Medical School, Padua, Italy
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  • J. ROSING,

    1. Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands
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  • T. M. HACKENG

    1. Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands
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Tilman M. Hackeng, Department of Biochemistry, Cardiovascular Research Institute Maastricht, Academic Medical Centre Maastricht, 6200 MD Maastricht, the Netherlands.
Tel.: +31 43 388 1674; fax: +31 43 388 4159.
E-mail: t.hackeng@bioch.unimaas.nl

Abstract

Summary. Background: Protein S acts as a cofactor for full-length tissue factor pathway inhibitor (TFPI) in the downregulation of thrombin formation. Objective: To develop a functional test to measure the activity of the TFPI–protein S system in plasma. Methods/Patients: Using calibrated automated thrombography, we quantified the activity of the TFPI–protein S system in plasma by measuring thrombin generation in the absence and presence of neutralizing antibodies against protein S or TFPI. Moreover, we designed an enzyme-linked immunosorbent assay (ELISA) to determine the level of full-length TFPI in plasma. The performance of these assays was examined in plasma from 85 normal individuals and from 35 members of protein S-deficient families. Results: The ratio of thrombin peaks determined in the absence and presence of anti-protein S antibodies (protein S ratio = 0.5 in normal plasma) is a measure of the TFPI cofactor activity of protein S, whereas the ratio of thrombin peaks determined in the absence and presence of anti-TFPI antibodies (TFPI ratio = 0.25 in normal plasma) is a measure of the overall activity of the TFPI–protein S system. Protein S and TFPI ratios were elevated in protein S-deficient individuals, indicating an impairment of the TFPI–protein S system. Both ratios correlated well with full-length TFPI levels, which were significantly lower in protein S-deficient patients than in normal family members. Conclusions: Functional assays for the TFPI–protein S system and an ELISA for full-length TFPI were developed. These assays show that the activity of the TFPI–protein S anticoagulant pathway is impaired in individuals with congenital protein S deficiency.

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