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

Abstract

SummaryIn 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.

Acquired APC resistance and oral contraceptives: differences between two functional tests

“…The occurrence of heterozygosity for factor V Leiden was established by determination of the sensitivity of plasma factor Va for APC (Nicolaes et al, 1996) or by DNA analysis (Beauchamp et al, 1994).…”

Inherited and Acquired Thrombophilia in Pregnancy

“…The molecular basis for this defect was shown to be a point mutation in the factor V gene located on chromosome 1 (1691 GA) (Bertina et al, 1994;Greengard et al, 1994;Voorberg et al, 1994;Zoller and Dahlback, 1994). This mutation has been coined the factor V Leiden mutation (Aparicio and Dahlback, 1996;Heeb et al, 1995;Nicolaes et al, 1996). The mutant factor V gene causes the replacement of an amino acid arginine by glycine Arg  Gln at a critical cleavage site 506, the site of the first molecular cleavage of factor Va by APC.…”

Less Effect of Intranasal Than Oral Hormone Therapy on Factors Associated With Venous Thrombosis Risk in Healthy Postmenopausal Women

“…All women were tested for the FV Leiden mutation with a functional test that fully discriminates between homozygous, heterozygous, and noncarriers of the mutation. 28 Serum levels of E 2 and of follicle-stimulating hormone were measured with an electrochemiluminiscence immunoassay (Roche Diagnostics), and serum sex hormone binding globulin levels were measured using an immunoradiometric assay (Orion Diagnostica).…”