A PROTHROMBINASE-BASED ASSAY FOR DETECTION OF RESISTANCE TO ACTIVATEDPROTEIN-C

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 th e effect of APC an the cofactor activity of plasma factor Va. The fact or V present in a diluted plasma sample was activated with thrombin an d its sensitivity towards APC was subsequently determined by incubatio n with phospholipids and APC; The loss of factor Va cofactor activity was quantified in a prothrombinase system containing purified prothrom bin. factor Xa and phospholipid vesicles and using a chromogenic 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 pl asmas towards ATC 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 mu M phospholipid vesicles to a 1000-fold diluted sample of thrombin-activated normal plasma resulted in loss of mon th an 85% of the cofactor activity factor Va within 6 min. Under the same conditions, APC inactivated similar to 60% and similar to 20% of the factor Va present in plasma samples from APC-resistant individuals tha t were heterozygous or homozygous for the mutation Arg(506)-->Gln in f actor V, respectively. Discrimination between the plasma samples from normal and heterozygous and homozygous APC-resistant individuals was f acilitated by introduction of the so-called APC-sensitivity ratio (APC -sr). The APC-sr was defined as the ratio of the factor Va cofactor ac tivities determined in thrombin-activated plasma samples after 6 min i ncubation with or without 0.2 nM APC and was multiplied by as 100 to o btain integers (APC-sr = {factor Va(+APC)/factor Va(-APC)} x 100). Cle ar differences were observed between the APC-sr of plasmas from normal healthy volunteers (APC-sr: 8-20, n = 33) and from individuals that w ere 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 resi stant individuals (p < 0.0001), In all cases our test gave the same re sult as the DNA-based assay. Since the test is performed on a highly d iluted plasma sample there is no interference by conditions that affec t APC resistance tests that are based on clotting time determinations (e.g. coagulation factor deficiencies, oral anticoagulation, heparin t reatment. the presence of lupus anticoagulants, pregnancy or the use o f oral contraceptives). Furthermore, we show that part of the factor V a assay can be performed on an autoanalyzer which increases the number of plasma samples that can be handled simultaneously.