Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg 506 3 Gln (factor V R506Q

Summary.Epidemiological studies have shown that women who use third-generation oral contraceptives (OC) containing desogestrel, gestodene or norgestimate have a higher risk of venous thrombosis than women who use second-generation OC containing levonorgestrel. It is also known that a mutation in factor V (factor V Leiden ), which results in resistance to activated protein C (APC) and which is the most common cause of hereditary thrombophilia, potentiates the prothrombotic effect of OC.Effects of APC on thrombin generation in the plasma of women using OC were compared to the response to APC in non-OC users and in individuals that were heterozygous or homozygous for factor V Leiden . The response towards APC was evaluated on basis of the ratio (APC-sr) of the time integrals of thrombin formation determined in the presence and absence of APC.Compared with women not using OC, women who used OC exhibited a significantly decreased sensitivity to APC (P < 0 . 001), independent of the kind of OC used. Women who used third-generation monophasic OC were significantly less sensitive to APC than women using second-generation OC (P < 0 . 001) and had APC-sr that did not significantly differ from heterozygous female carriers of factor V Leiden who did not use OC. Women who were heterozygous for factor V Leiden and used OC had APC-sr in the range of homozygous carriers of factor V Leiden . Two women who started OC therapy had significantly elevated APC-sr within 3 d.Acquired APC resistance may explain the epidemiological observation of increased risk for venous thrombosis in OC users, especially in women using third-generation OC.

Inactivation of membrane-bound factor Va by activated protein C (APC) proceeds via a biphasic reaction that consists of a rapid and a slow phase, which are associated with cleavages at Arg506 and Arg306 of the heavy chain of factor Va, respectively. We have investigated the effects of protein S and factor Xa on APC-catalyzed factor Va inactivation. Protein S accelerates factor Va inactivation by selectively promoting the slow cleavage at Arg306 (20-fold). Factor Xa protects factor Va from inactivation by APC by selectively blocking cleavage at Arg506. Inactivation of factor VaR506Q, which was isolated from the plasma of a homozygous APC-resistant patient and which lacks the Arg506 cleavage site, was also stimulated by protein S but was not affected by factor Xa. This confirms that the target sites of protein S and factor Xa involve Arg306 and Arg506, respectively. Factor Xa completely blocked APC-catalyzed cleavage at Arg506 in normal factor Va (1 nM) with a half-maximal effect (K1/2Xa) at 1.9 nM factor Xa. Expression of cofactor activity of factor Va in prothrombin activation required much lower factor Xa concentrations (K1/2Xa = 0.08 nM). When the ability of factor Xa to protect factor Va from inactivation by APC was determined at low factor Va concentrations during prothrombin activation much lower amounts of factor Xa were required (K1/2Xa = 0.03 nM). This indicates 1) that factor Va is optimally protected from inactivation by APC by incorporation into the prothrombinase complex during ongoing prothrombin activation, and 2) that the formation of a catalytically active prothrombinase complex and protection of factor Va from inactivation by APC likely involves the same interaction of factor Xa with factor Va. In accordance with the proposed mechanisms of action of protein S and factor Xa, we observed that the large differences between the rates of APC-catalyzed inactivation of normal factor Va and factor VaR506Q were almost annihilated in the presence of factor Xa and protein S. This observation may explain why, in the absence of other risk factors, APC resistance only results in a weak prothrombotic condition.

Objective-Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo. Methods and Results-High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated ␣IIb␤3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active ␣IIb␤3. Conclusions-These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication. Key Words: aggregation Ⅲ coagulation Ⅲ microdomains Ⅲ platelets Ⅲ integrin activation A ctivated platelets have a dual role in hemostasis and thrombosis. They form the building blocks of a thrombus and provide the membrane surface for coagulation factor activation, which results in thrombin and fibrin formation. 1 Once formed, thrombin greatly enhances platelet activation and aggregation. Given the strong interdependency of thrombin generation and platelet activation, it is intuitively assumed that those platelets that participate in aggregate formation are also involved in the coagulation process, but this has not been investigated.The mechanism(s) by which platelets contribute to thrombin generation and coagulation have been investigated for decades. Kinetic evidence shows that collagen/thrombin-activated platelets expose phosphatidylserine (PS) at their outer surface and then bind Gla domain-containing coagulation factors, mediating factor Xa and thrombin generation. 2 However, even after activation with strong agonists, only a subpopulation of the platelets tends to expose PS. 3 Conversely, it has been argued that PS exposure alone is insufficient to explain the procoagulant contribution of platelets. 4,5 There is also evidence that subfractions of activated platelets have different roles in the coagulation process. Several reports indicate an imperfect relation of PS exposure and the binding of coagulation factors Va, VIIIa, IXa, and Xa to the platelet surface. 6 -8 Another report characterizes a subfraction of activated platelets according to their so-called SCIP morphology (for susta...

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Summary. Activated protein C (APC) resistance, determined with a thrombin-generation-based APC resistance test, may explain risk differences of venous thrombosis in users of second-and third-generation oral contraceptives (OC). To clinically validate this test, we analysed the Leiden thrombophilia case-control study (474 patients with a first episode of deep vein thrombosis and 474 age-and sexmatched control subjects). Data for men and women were analysed separately. As hormonal status in women is known to strongly influence the APC sensitivity ratio (APCsr), additional strata (OC use and menopausal state) were defined. The APCsr was higher in all patients than in control subjects. Odds ratios (OR), using the 90th percentile of all control subjects (APCsr > 4AE5) as cut-off, were: 7AE5 [95% confidence interval (CI) 1AE6-33AE8] for men, 3AE0 (95% CI 1AE0-8AE8) for premenopausal women not using OC, 4AE8 (95% CI 1AE6-14AE7) for premenopausal women using OC and 4AE7 (95% CI 1AE4-15AE6) for postmenopausal women. After excluding the carriers of factor V Leiden, the OR became infinite for men (no control had an APCsr > 4AE5), 1AE4 (95% CI 0AE2-8AE2) for premenopausal women not using OC, 3AE4 (95% CI 1AE1-10AE8) for premenopausal women using OC and 3AE6 (95% CI 0AE6-20AE5) for postmenopausal women. A high APCsr, determined with the thrombin-generation-based APC resistance test, predicts venous thrombotic risk, in populations with and without factor V Leiden. In addition, acquired APC resistance resulting from OC use predicts an increased risk for venous thrombosis independent of factor V Leiden.

The incidence of venous thrombosis associated with estrogen treatment in male-to-female (M-->F) transsexuals is considerably higher with administration of oral ethinyl estradiol (EE) than with transdermal (td) 17-beta-estradiol (E(2)). To find an explanation for the different thrombotic risks of oral EE and td E(2) use, we compared the effects of treatment of M-->F transsexuals with cyproterone acetate (CPA) only, and with CPA in combination with td E(2), oral EE, or oral E(2) on a number of hemostatic variables [activated protein C (APC) resistance and plasma levels of protein S, protein C, and prothombin], all of which are documented risk factors for venous thrombosis. APC resistance was determined by quantification of the effect of APC on the amount of thrombin generated during tissue factor-initiated coagulation; plasma levels of total and free protein S were determined by standard ELISA; and levels of prothrombin and protein C were determined with functional assays after complete activation of the zymogens with specific snake venom proteases. CPA-only, td-E(2)+CPA, or oral-E(2)+CPA treatment produced rather small effects on hemostatic variables, whereas oral EE treatment resulted in a large increase in APC resistance from 1.2 +/- 0.8 to 4.1 +/- 1 (P < 0.001), a moderate increase in plasma protein C (9%; P = 0.012), and a large decrease in both total and free plasma protein S (30%; P < 0.005). The large differential effect of oral EE and oral E(2) indicates that the prothrombotic effect of EE is due to its molecular structure rather than to a first-pass liver effect (which they share). Moreover, these differences may explain why M-->F transsexuals treated with oral EE are exposed to a higher thrombotic risk than transsexuals treated with td E(2). Testosterone administration to female-to-male transsexuals had an antithrombotic effect.

In a study population consisting of healthy men (n = 8), women not using oral contraceptives (OC) (n = 28) and women using different kinds of OC (n = 187) we used calibrated automated thrombography (CAT) in the absence and presence of added activated protein C (APC) to compare parameters that can be obtained from thrombin generation curves, i.e. lag time, time to peak, peak height and endogenous thrombin potential (ETP). Both with and without APC, plasmas of OC users exhibited the shortest lag time and time to peak, and the highest peak height and ETP. In the absence of APC none of these parameters differed between users of OC containing different progestogens. In contrast, in the presence of APC shorter lag times and time to peak, and higher peak height and ETP were observed in plasma of users of gestodene-, desogestrel-, drospirenone- and cyproterone acetate-containing OC than in plasma of users of levonorgestrel- containing OC. The ETP determined in the absence of APC (ETP(-APC)) had no predictive value for the APCsr (r = 0.11; slope 0.9 x 10(-3); 95% CI: -0.1 x 10(-3) to 2.0 x 10(-3)) whereas the ETP measured in the presence of APC (ETP+APC) showed an excellent correlation with the APCsr (r = 0.95; slope 6.6 x 10(-3); 95% CI: 6.3 x 10(-3) to 6.9 x 10(-3)) indicating that the APCsr is entirely determined by the ETP+APC. In conclusion, OC use increases thrombin generation, but differential effects of second and third generation OCs on the protein C system likely determine the differences in the risk of venous thrombosis between these kinds of OC.