SummaryThe use of oral contraceptives (OC) causes disturbances of the procoagulant, anticoagulant and fibrinolytic pathways of blood coagulation which may contribute to the increased risk of venous thrombosis associated with OC therapy. Here we report the results of a cyclecontrolled randomized cross-over study, in which we determined the effects of so-called second and third generation OC’s on a number of anticoagulant parameters. In this study, 28 non-OC using women were randomly prescribed either a second generation (150 µg levonorgestrel/30 µg ethinylestradiol) or a third generation OC (150 µg desogestrel/30 µg ethinylestradiol) and who switched to the other OC after a two month wash out period. The anticoagulant parameters determined were: antithrombin (AT), α2-macroglobulin (α2-M), α1-antitrypsin, protein C inhibitor (PCI), protein C, total and free protein S and activated protein C sensitivity ratios (APC-sr) measured with two functional APC resistance tests which quantify the effect of APC on either the activated partial thromboplastin time (aPTT) or on the endogenous thrombin potential (ETP). During the use of desogestrel-containing OC the plasma levels of αc2-M, α1-antitrypsin, PCI and protein C significantly increased, whereas AT and protein S significantly decreased. Similar trends were observed with levonorgestrel-containing OC, although on this kind of OC the changes in AT, PCI and protein S (which was even slightly increased) did not reach significance. Compared with levonorgestrel, desogestrel-containing OC caused a significant decrease of total (p <0.005) as well as free protein S (p <0.0001) and more pronounced APC resistance in both the aPTT (p = 0.02) and ETPbased (p <0.0001) APC resistance tests. These observations indicate that the activity of the anticoagulant pathways in plasma from users of desogestrel-containing OC is more extensively impaired than in plasma from users of levonorgestrel-containing OC.

Activation of human platelets considerably enhanced their ability to accelerate factor Va inactivation by activated protein C (APC). The anticoagulant activity of platelet suspensions was markedly dependent on the kind of agonist used to activate platelets. APC-catalyzed factor Va inactivation in free solution was characterized by an apparent second-order rate constant of 2 x 10(5) (mol/L)-1 (seconds)-1. Nonstimulated platelets (2.4 x 10(8)/mL) and platelets stimulated with adenosine diphosphate or adrenalin accelerated factor Va inactivation fourfold. Rates of factor Va inactivation were increased 11-fold by thrombin-stimulated platelets, 29-fold after platelet stimulation with the Ca(2+)-ionophore A23187. At low platelet concentrations (3 x 10(7)/mL) only background levels of anticoagulant activity were observed in platelet suspensions that were nonstimulated or stimulated with thrombin or collagen. However, when such reaction mixtures were stirred during the activation procedure, platelet anticoagulant activity was increased more than 10-fold. Independent of platelet stimulation and stirring conditions, exogenously added purified plasma protein S increased platelet-dependent factor Va inactivation approximately twofold. Addition of a neutralizing antiprotein S antibody had little effect on the anticoagulant activity of platelets. This indicates that, under the reaction conditions tested, platelet- released protein S did not contribute to factor Va inactivation. Approximately 25% of the anticoagulant activity of stimulated platelet suspensions appeared to be associated with microparticles that were released on platelet activation. Such microparticles may provide an important source of anticoagulant activity. A similar distribution of procoagulant, ie, prothrombinase, activity between platelets and microparticles was observed for the same platelet suspensions. Because platelet stimulation and stirring also had the same overall effects on the ability of platelets and platelet microparticles to promote prothrombin activation and factor Va inactivation, it appears likely that the generation of potential platelet anticoagulant and procoagulant activities is coupled to the same platelet stimulation reactions.

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.

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