A Prothrombinase-based Assay for Detection of Resistance to Activated Protein C

Gerry A F Nicolaes; M Christella; L G D Thomassen; Rene van  Oerle; Karly Hamulyak; H Coenraad Hemker; Guido Tans; Jan Rosing

doi:10.1055/s-0038-1650591

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1996; 76(03): 404-410
DOI: 10.1055/s-0038-1650591

Original Article

Schattauer GmbH Stuttgart

A Prothrombinase-based Assay for Detection of Resistance to Activated Protein C

Gerry A F Nicolaes

¹The Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University Hospital, University of Limburg, Maastricht, The Netherlands

,

M Christella

¹The Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University Hospital, University of Limburg, Maastricht, The Netherlands

,

L G D Thomassen

¹The Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University Hospital, University of Limburg, Maastricht, The Netherlands

,

Rene van Oerle

²The Department of Hematology, University Hospital, University of Limburg, Maastricht, The Netherlands

,

Karly Hamulyak

²The Department of Hematology, University Hospital, University of Limburg, Maastricht, The Netherlands

,

H Coenraad Hemker

¹The Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University Hospital, University of Limburg, Maastricht, The Netherlands

,

Guido Tans

¹The Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University Hospital, University of Limburg, Maastricht, The Netherlands

,

Jan Rosing

¹The Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University Hospital, University of Limburg, Maastricht, The Netherlands

› Author Affiliations

Abstract

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Summary

In this paper we present a new method for the detection of resistance to activated protein C (APC) that is based on direct measurement of the effect of APC on the cofactor activity of plasma factor Va. The factor V present in a diluted plasma sample was activated with thrombin and its sensitivity towards APC was subsequently determined by incubation with phospholipids and APC. The loss of factor Va cofactor activity was quantified in a prothrombinase system containing purified prothrombin, factor Xa and phospholipid vesicles and using a chromogen-ic assay for quantitation of thrombin formation. The reaction conditions were optimized in order to distinguish normal, heterozygous and homozygous APC-resistant plasmas. Maximal differences in the response of these plasmas towards APC were observed when factor Va was inactivated by APC in the absence of protein S and when the cofactor activity of factor Va was determined at a low factor Xa concentration (0.3 nM).

Addition of 0.2 nM APC and 20 μM phospholipid vesicles to a 1000-fold diluted sample of thrombin-activated normal plasma resulted in loss of more than 85% of the cofactor activity factor Va within 6 rnin. Under the same conditions, APC inactivated ∼ 60% and ∼20% of the factor Va present in plasma samples from APC-resistant individuals that were heterozygous or homozygous for the mutation Arg⁵⁰⁶⟶Gln in factor V, respectively. Discrimination between the plasma samples from normal and heterozygous and homozygous APC-resistant individuals was facilitated by introduction of the so-called APC-sensitivity ratio (APC-sr). The APC-sr was defined as the ratio of the factor Va cofactor activities determined in thrombin-activated plasma samples after 6 min incubation with or without 0.2 nM APC and was multiplied by 100 to obtain integers (APC-sr = {factor Va_+APC/factor Va_−APC} × 100). Clear differences were observed between the APC-sr of plasmas from normal healthy volunteers (APC-sr: 8-20, n = 33) and from individuals that were heterozygous (APC-sr: 35-50, n = 17) or homozygous APC resistant (APC-sr: 82-88, n = 7). There was no mutual overlap between the APC-sr of normal plasmas and plasmas from heterozygous or homozygous APC resistant individuals (p <0.0001). In all cases our test gave the same result as the DNA-based assay. Since the test is performed on a highly diluted plasma sample there is no interference by conditions that affect APC resistance tests that are based on clotting time determinations (e.g. coagulation factor deficiencies, oral anticoagulation, heparin treatment, the presence of lupus anticoagulants, pregnancy or the use of oral contraceptives). Furthermore, we show that part of the factor Va assay can be performed on an autoanalyzer which increases the number of plasma samples that can be handled simultaneously.

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References

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