Increased tissue factor pathway inhibitor activity is associated with myocardial infarction in young women: results from the RATIO study.

BACKGROUND: The tissue factor pathway inhibitor (TFPI)/protein S anticoagulant system is a potent inhibitor of blood coagulation. TFPI and protein S are major determinants of thrombin generation (TG) tests determined at low tissue factor (TF) and at high TF concentrations in the presence of activated protein C (APC). Both TFPI and protein S protect against venous thrombosis, but the importance of the TFPI/protein S system in arterial thrombosis remains unclear. OBJECTIVES: To investigate the influence of the TFPI/protein S anticoagulant system on the risk of myocardial infarction (MI) in young women. METHODS: The RATIO study is a case-control study in women under 50 years of age, including 205 patients and 638 controls. TFPI and protein S were quantified using ELISA. The TFPI/protein S activity (nTFPIr) and the APC sensitivity ratio (nAPCsr) were determined using TG tests. Odds ratios (ORs) adjusted for putative confounders and corresponding 95% confidence intervals (95% CI) were determined. RESULTS: Women with MI had higher TFPI levels than controls (135.9 ± 40% vs. 124.2 ± 41%), resulting in increased TFPI/protein S activities and increased APC sensitivity. Furthermore, an increased TFPI activity was associated with MI [nTFPIr: adjusted OR Q1 vs. Q4 = 2.1 (95%CI 1.1-4.1)]. Additionally, an increased APC sensitivity was associated with MI [nAPCsr: adjusted OR Q1 vs. Q4 = 1.7 (95% CI 0.9-3.2)] CONCLUSION: Women with MI had increased TFPI levels compared with controls. Consequently, the TFPI/protein S activity and APC sensitivity are increased in women with MI. Whether this increase in TFPI activity acts as a compensating mechanism for an increased procoagulant state or is a marker of endothelial damage remains to be investigated.

Pregnancy-associated changes in the hemostatic system in wild-type and factor V Leiden mice.

BACKGROUND: Pregnancy, oral contraceptive (OC)use and hormone replacement therapy (HRT) are established risk factors for venous thrombosis. Acquired resistance to activated protein C (APC) has been proposed to contribute to the increased thrombosis risk. Mouse models are often used for preclinical testing of newly developed hormone preparations. However, it is not known whether hormone-induced APC resistance is also observed in laboratory animals. OBJECTIVES: To investigate whether hormonal changes modulate APC resistance in mice, we used pregnant mice as a model of hormone-induced APC resistance. The effect of pregnancy on APC resistance was studied in wild-type and factor (F)V Leiden mice. METHODS: APC resistance was determined in mouse plasma using a thrombin generation-based APC resistance test. APC resistance determinants,i.e. prothrombin, FV, FX, antithrombin and protein S levels,and of tissue factor pathway inhibitor (TFPI) activity were evaluated in plasma from non-pregnant and pregnant mice. RESULTS: In contrast to humans, pregnancy induced a decrease in APC resistance in wild-type and in FV Leiden mice.Pregnant mice had higher levels of prothrombin, FV, FX,protein S and TFPI activity as compared with non-pregnant mice. CONCLUSIONS: Pregnancy causes a decrease in APC resistance in mice, which can be explained by the elevation of protein S levels and increased TFPI activity in plasma. Our findings show species specificity in the effects of pregnancy on the major determinants of the protein C system and suggest that protein S and TFPI play an important role in the development of pregnancy-induced APC resistance in humans.

Protein S is a cofactor for tissue factor pathway inhibitor.

Protein S is a vitamin K-dependent protein that acts as a cofactor of the anticoagulant protein APC. However, protein S also exhibits anticoagulant activity in the absence of APC. Thrombin generation experiments in normal plasma and in plasma deficient in tissue factor pathway inhibitor (TFPI) and/or protein S demonstrated that protein S stimulates the inhibition of TF by TFPI. Kinetic analysis in model systems containing purified proteins showed that protein S enhances the formation of the binary FXa:TFPI complex by reducing the Ki of TFPI from approximately 4 nM to approximately 0.5 nM. Enhancement of inhibitory activity of TFPI by protein S is only observed with full-length TFPI and in the presence of a negatively charged phospholipid surface. The Ki decrease brings the TFPI concentration necessary for FXa:TFPI complex formation within range of the plasma TFPI concentration which increases FXa:TFPI complex formation and accelerates feedback inhibition of the TF pathway by enhancing the formation of the quaternary TFPI:FXa:TF:FVIIa complex. Thus, protein S is not only a cofactor of APC, but also of TFPI. A reduced TFPI cofactor activity may contribute to the increased risk of venous thrombosis in protein-S deficient individuals. Using calibrated automated thrombography we have developed two assays that enable quantification of the functional activity of the TFPI/protein S system in plasma. These assays show that the activity of the TFPI/protein S system is greatly impaired in oral contraceptive users.

Development of a calibrated automated thrombography based thrombin generation test in mouse plasma.

BACKGROUND: Mouse models have become increasingly important in thrombosis research. However, only a limited number of assays are available for assessment of the coagulation system in mouse plasma. OBJECTIVES: To quantify tissue factor-initiated thrombin generation in murine platelet-rich and platelet-free plasma and to develop a test for measurement of resistance to activated protein C (APC) in mouse plasma. METHODS: Thrombin generation was monitored with calibrated automated thrombography (CAT) using a low-affinity fluorogenic substrate for thrombin. RESULTS: To overcome the higher activity of coagulation inhibitors in mouse plasma as compared with human plasma, the reaction temperature was lowered to 33 degrees C and the assay was carried out at a 2-fold higher final plasma dilution (1:3) than commonly used for CAT in human plasma. This increased the endogenous thrombin potential (ETP) 4- to 5-fold and enabled reliable measurement of thrombin generation in both platelet-free and platelet-rich mouse plasma. For the APC resistance measurement, the reaction conditions were further optimized with respect to tissue factor, phospholipid, APC and CaCl(2) concentrations. The test was validated using plasma of mice with different genetic background with respect to the factor V Leiden mutation (FV Leiden). Mice homozygous for FV Leiden had higher APC sensitivity ratios (mean 5.46; 95% CI 4.88-6.03) than heterozygous FV Leiden mice (mean 4.21; 95% CI 3.53-4.89) and than wild-type mice (mean 2.71; 95%CI 2.15-3.27). CONCLUSIONS: We have established reaction conditions for measurement of thrombin generation and APC resistance in mouse plasma. This assay enables evaluation of the coagulation system and the function of the protein C system in mouse models.

Expression of the normal factor V allele modulates the APC resistance phenotype in heterozygous carriers of the factor V Leiden mutation.

BACKGROUND: Functional defects of the protein C pathway, detectable in plasma as activated protein C (APC) resistance, are a prevalent risk factor for venous thrombosis. The factor V (FV) Leiden mutation causes APC resistance by interfering with the APC-mediated inactivation of both FVa and FVIIIa. Co-inheritance of FV Leiden and quantitative FV deficiency on different alleles, a rare condition known as pseudo-homozygous APC resistance, is associated with pronounced APC resistance and 50% reduced FV levels, because of non-expression of the non-Leiden FV allele. OBJECTIVES: The role of normal FV in modulating the APC resistance phenotype in carriers of FV Leiden was investigated in patients with pseudo-homozygous APC resistance and in model systems. PATIENTS/METHODS: Four functional plasma assays probing both components of APC resistance (susceptibility of FVa to APC and cofactor activity of FV in FVIIIa inactivation) were employed to compare seven clinically and genetically characterized FV Leiden pseudo-homozygotes to 30 relatives with different FV genotypes (including 12 FV Leiden heterozygotes and seven carriers of FV deficiency) and to 32 unrelated FV Leiden homozygotes. RESULTS AND CONCLUSIONS: All assays consistently indicated that FV Leiden pseudo-homozygotes are significantly more APC-resistant than heterozygotes and indistinguishable from homozygotes. Thrombin generation measurements in FV-deficient plasma reconstituted with purified normal FV and FV Leiden confirmed these observations and showed that the expression of the normal FV allele is an important modulator of APC resistance in FV Leiden heterozygotes. These findings provide an explanation for the higher thrombotic risk of FV Leiden pseudo-homozygotes when compared with heterozygotes.

Determinants of the APTT- and ETP-based APC sensitivity tests.

BACKGROUND: A reduced sensitivity for activated protein C (APC) is associated with an increased risk of venous thrombosis even in the absence of the factor (F)V Leiden mutation. This risk has been demonstrated with two APC sensitivity tests, which quantify the effects of APC on the activated partial thromboplastin time (APTT) and the endogenous thrombin potential (ETP), respectively. OBJECTIVES: We examined determinants of both APC sensitivity tests in the control group of the Leiden Thrombophilia Study (LETS). METHODS: Multiple linear regression analysis was performed with normalized APC-SR(APTT) or APC-SR(ETP) as dependent variable and putative determinants [levels of FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII A subunit, FXIII B subunit, protein S total, protein S free, protein C, tissue factor pathway inhibitor (TFPI) total, TFPI free, antithrombin and fibrinogen] as independent variables. RESULTS AND CONCLUSIONS: The major determinant of the APTT-based test was FVIII level, followed by FII level. The ETP-based test was influenced most by free protein S and free TFPI levels. In both tests FXa formation plays a major role, as the effect of FVIII and TFPI on the tests seems to be executed via FXa. The ETP-based test was also strongly influenced by oral contraceptive use, even when we adjusted for all the clotting factors listed above. This means that the effect of oral contraceptives on the ETP-based test is not fully explained by the changes of coagulation factor levels investigated in this study, and that the molecular basis of acquired APC resistance during use of oral contraceptives remains to be established.

Venous thrombosis and changes of hemostatic variables during cross-sex hormone treatment in transsexual people.

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.

The activated protein C (APC)-resistant phenotype of APC cleavage site mutants of recombinant factor V in a reconstituted plasma model.

Recently, new missense mutations in the activated protein C (APC) cleavage sites of human factor V (FV) distinct from the R506Q (FV Leiden) mutation have been reported. These mutations affect the APC cleavage site at arginine (Arg) 306 in the heavy chain of activated FV. Whether these mutations result in APC resistance and are associated with a risk of thrombosis is not clear. The main objective of the present study was to identify the APC-resistant phenotype of FV molecules with different mutations in APC cleavage sites. To study this, recombinant FV mutants were reconstituted in FV-deficient plasma, after which normalized APC-sensitivity ratios (n-APC-SRs) were measured in activated partial thromboplastin time-based and Russell's Viper Venom time-based APC-resistance tests. The mutations introduced in FV were R306G, R306T, R506Q, R679A and combinations of these mutations. Based on the APC-sensitivity ratios, we conclude that the naturally occurring mutations at Arg306 (i.e. FV HongKong and FV Cambridge) result in a mildly reduced sensitivity for APC (n-APC-SR, 0.74-0.87), whereas much lower values (n-APC-SR, 0.41-0.51) are obtained for the mutation at Arg506 (FV Leiden). No effect on the n-APC-SR was observed for the recombinant FV mutant containing the single Ala679 mutation. Because reduced sensitivity for APC, not due to FV Leiden, is a risk factor for venous thrombosis, these data suggest that mutations at Arg306 might be associated with a mild risk of venous thrombosis.

Effect of in vitro fertilization treatment and subsequent pregnancy on the protein C pathway.

Thirty-three women who were planned for an in vitro fertilization (IVF) cycle donated blood at four time points during treatment: at baseline, after downregulation, hyperstimulation and luteal support. Levels of progesterone, 17beta-oestradiol and indicators of the protein C pathway, i.e. activated protein C sensitivity ratios (APCsr), protein C, protein C inhibitor and protein S were measured. Compared with baseline, oestradiol decreased twofold at downregulation and increased 40-fold at hyperstimulation. Progesterone was elevated 2.5-fold at hyperstimulation and 40-fold at luteal support. The APCsr increased slightly at downregulation, significantly increased during hyperstimulation and remained high during luteal support. The plasma levels of the anticoagulant proteins did not change or changed moderately during treatment. During downregulation, progesterone correlated negatively with APCsr (r = -0.398, P = 0.024). At hyperstimulation oestradiol correlated with the APCsr (r =0.615, P < 0.0005). Moreover, there was a significant correlation (r = 0.599, P < 0.0005) between the difference in baseline and hyperstimulation values of oestradiol (Delta E2 = 6.6 nmol/l) and the APCsr (Delta APCsr = 0.30). Six women who participated in this study became pregnant. Compared with baseline, the APCsr was increased 1.9-fold (Delta APCsr = 1.48) and free protein S free level decreased 30% at 7 weeks of pregnancy. This study demonstrates that despite the considerable changes in endogenous oestradiol and progesterone during an IVF cycle, changes in plasma levels of anticoagulant proteins are moderate. The significant increase in the APCsr during hyperstimulation indicates that acquired APC resistance observed during sex steroid hormone changes in women is at least partially caused by high oestrogen levels. Our findings demonstrate that IVF treatment is accompanied by the development of a mild prothrombotic condition.

Purified protein S contains multimeric forms with increased APC-independent anticoagulant activity.

Protein S, the cofactor of activated protein C (APC), also expresses anticoagulant activity independent of APC by directly inhibiting prothrombin activation via interactions with factor Xa, factor Va, and phospholipids. In different studies, however, large variations in APC-independent anticoagulant activities have been reported for protein S. The investigation presented here shows that within purified protein S preparations different forms of protein S are present, of which a hitherto unrecognized form (<5% of total protein S) binds with high affinity to phospholipid bilayers (K(d) < 1 nM). The remaining protein S (>95%) has a low affinity (K(d) = 250 nM) for phospholipids. Using their different affinities for phospholipids, separation of the forms of protein S was achieved. Native polyacrylamide gel electrophoresis demonstrated that the form of protein S that binds to phospholipids with low affinity migrated as a single band, whereas the high-affinity protein S exhibited several bands that migrated with reduced mobility. Size-exclusion chromatography revealed that the slower-migrating bands represented multimeric forms of protein S. Multimeric protein S (<5% of total protein S) appeared to have a 100-fold higher APC-independent anticoagulant activity than the abundant form of protein S. Comparison of purified protein S preparations that exhibited a 4-fold difference in APC-independent anticoagulant activity showed that the ability to inhibit prothrombin activation correlated with the content of multimeric protein S. Multimeric protein S could not be identified in normal human plasma, and it is therefore unlikely that this form of protein S contributes to the APC-independent anticoagulant activity of protein S that is observed in plasma.

Oral contraceptives, thrombosis and haemostasis.

The use of oral contraceptives is a well-established acquired risk factor for venous thrombosis. In 1995, a number of epidemiological studies were published which suggested that women who use third generation oral contraceptives that contain desogestrel or gestodene as progestagen are exposed to a two- to threefold higher risk for venous thrombosis than women using second generation oral contraceptives which contain levonorgestrel. In this paper, the effects of oral contraceptives on the haemostatic system are discussed. It appears that plasma from oral contraceptive users is resistant to the anticoagulant action of activated protein C (APC). This phenomenon, called acquired APC resistance, is more pronounced in users of desogestrel or gestodene-containing oral contraceptives than in women who use oral contraceptive pills with levonorgestrel. On the basis of these observations, it was proposed that acquired APC resistance may be the mechanistic basis of the increased risk for venous thrombosis during oral contraceptive use and for the further increased thrombotic risk of third generation oral contraceptive users. Furthermore, the results of a recent cross-over study are discussed. This study indicated that a large number of other haemostatic parameters were changed during oral contraceptive use. Some of these changes were more pronounced on desogestrel-containing oral contraceptives. The cross-over study also showed that the increased fibrinolytic activity during OC use is counterbalanced by an enhanced activity of thrombin-activatable fibrinolysis inhibitor (TAFI), a protein that participates in the inhibition of fibrinolysis.

Functional properties of factor V and factor Va encoded by the R2-gene.

Carriership of the factor V (FV) gene marked by the R2-haplotype, a series of linked polymorphisms encoding several amino acid changes in FV, is associated with mild resistance to activated protein C (APC) and with an increased risk of thrombosis. We compared the functional properties of normal FV(a) and R2-FV(a) in model systems and in plasma. FV and R2-FV were equally well activated by thrombin and expressed identical cofactor activities in prothrombin activation. Rate constants of APC-catalyzed inactivation of FVa and R2-FVa were similar both with and without protein S. However, significant differences were observed between haemostatic parameters determined in plasma from homozygous carriers of the R2-gene (n = 5) and age-matched non-carriers (n = 19). Plasma from R2-carriers contained significantly lower FV levels and the ratio of the two FV isoforms (FV1 and FV2) was shifted in favor of FV1. The FV2/FV1 ratio was 1.4 (95% CI = 1.3-1.5) in homozygous carriers of R2 and 2.8 (95% CI = 2.5-3.1) in controls (p < 0.00001). In an APC resistance test which quantifies the cofactor activity of FV in APC-catalyzed FVIII(a) inactivation, homozygous R2-carriers had significantly lower (p < 0.00001) APC sensitivity ratios (APCsr = 1.54, 95% CI = 1.48-1.60) than controls (APCsr = 2.17, 95% CI = 2.05-2.28). This indicates that R2-FV has reduced cofactor activity in APC-catalyzed FVIII(a) inactivation. The changes of the relative amounts of FV1 and FV2 in carriers of the R2-gene will result in increased thrombin formation in the presence of APC and may provide a mechanistic explanation for the increased thrombotic risk associated with the R2-haplotype.

Regulation of platelet factor Va-dependent thrombin generation by activated protein C at the surface of collagen-adherent platelets.

Recent studies have indicated that factor Va bound to activated platelets is partially protected from inactivation by activated protein C (APC). To explore whether this sustained factor Va activity could maintain ongoing thrombin generation, the kinetics of platelet factor Va-dependent prothrombinase activity and its inhibition by APC were studied. In an attempt to mimic physiologically relevant conditions, platelets were adhered to collagen type I-coated discs. These discs were then spun in solutions containing prothrombin and factor Xa either in the absence or presence of APC. The experiments were performed in the absence of platelet-derived microparticles, with thrombin generation and inhibition confined to the surface of the adherent platelets. APC completely inactivated platelet-associated prothrombinase activity with an overall second order rate constant of 3.3 x 10(6) m(-)1 s(-)1, which was independent of the prothrombin concentration over a wide range around the apparent K(m) for prothrombin. Kinetic studies on prothrombinase assembled at a planar phospholipid membrane composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine revealed a similar second order rate constant of inhibition (2.5 x 10(6) m(-1) s(-1)). Collectively, these data demonstrate that ongoing platelet factor Va-dependent thrombin generation at the surface of collagen-adherent platelets is effectively inhibited by APC. No differences were observed between the kinetics of APC inactivation of plasma-derived factor Va or platelet factor Va as part of the prothrombinase associated with, respectively, a planar membrane of synthetic phospholipids or collagen-adherent platelets.

Snake venom activators of factor X: an overview.

Activators of blood coagulation factor X have been described in the venom of many snake species belonging to the genus Viperidae and Crotalidae as well as from a few Elapid species. Based on the structural and functional properties of purified activating principles, factor X activators are either metalloproteases or serine proteases. The best known activator is RVV-X from Russell's viper (Daboia russelli), a metalloprotease consisting of a heavy chain containing the catalytic domain and two light chains which share homology with C-type lectins and which are thought to exert a regulatory function in the Ca(2+)-dependent activation of factor X. This activator is also one of the best examples of the use of exogenous activators in coagulation research and in addition it is used in many diagnostic research kits. In this paper, an overview is given of the structural and functional properties of snake venom factor X activators thus far described in the literature.

Factor V activation and inactivation by venom proteases.

Blood coagulation factor V is a single-chain glycoprotein with M(r) = 330,000 which plays an important role in the procoagulant and anticoagulant pathways. Thrombin activates factor V into factor Va, a two-chain molecule which is composed of a heavy (M(r) = 105,000) and a light chain (M(r) = 71,000/74,000). Factor Va accelerates factor Xa-catalysed prothrombin activation more than 1,000-fold and under physiological conditions the cofactor activity of factor Va in prothrombin activation is down-regulated by activated protein C. Factor V can also be activated by a wide variety of snake venoms (e.g. from Vipera species, Naja naja oxiana, Bothrops atrox) and by proteases present in the bristles of a South American caterpillar (Lonomia achelous). Some venoms, notably of Vipera lebetina turanica and Lonomia achelous, contain proteases that are able to inactivate factor V or factor Va. Venom factor V activators are excellent tools in studying the structure-function relationship of factor V(a) and they are also used in diagnostic tests for quantification of plasma factor V levels and for the screening of defects in the protein C pathway. In this review, the structural and functional properties of animal venom factor V activators and inactivators is described.

C4b-binding protein protects coagulation factor Va from inactivation by activated protein C.

We investigated the effect of C4BP on APC-mediated inactivation of factor Va (FVa) in the absence and presence of protein S. FVa inactivation was biphasic (k(506) = 4.4 x 10(8) M(-)(1) s(-)(1), k(306) = 2.7 x 10(7) M(-)(1) s(-)(1)), and protein S accelerated Arg(306) cleavage approximately 10-fold. Preincubation of protein S with C4BP resulted in a total abrogation of protein S cofactor activity. C4BP also protected FVa from inactivation by APC in the absence of protein S. Control experiments with CLB-PS13, a monoclonal anti-protein S antibody, indicated that inhibition of FVa inactivation by C4BP was not mediated through contaminating traces of protein S in our reaction systems. Protection of FVa was prevented by a monoclonal antibody directed against the C4BP alpha-chain. Recombinant rC4BPalpha comprised of only alpha-chains also protected FVa, but in the presence of protein S, the level of protection was decreased, since rC4BPalpha lacks the beta-chain responsible for C4BP binding to protein S. A truncated C4BP beta-chain (SCR-1+2) inhibited protein S cofactor activity, but had no effect on FVa inactivation by APC in the absence of protein S. In conclusion, C4BP protects FVa from APC-catalyzed cleavage in a protein S-independent way through direct interactions of the alpha-chaims of C4BP with FVa and/or APC.

Effects on coagulation of levonorgestrel- and desogestrel-containing low dose oral contraceptives: a cross-over study.

Combined oral contraceptives (OC) are known to increase the risk of venous thromboembolism. The aim of this randomized, cycle-controlled, cross-over study in 28 healthy volunteers was to assess potential differences between the effects of an OC containing 150 microg levonorgestrel (as representative of the so-called second generation OC) and an OC containing 150 microg desogestrel (as representative of the third generation OC) in combination with 30 microg ethinylestradiol on several coagulation factors and markers of thrombin formation. All participants used each OC for two cycles, and were switched to the other OC after a washout period of two menstrual cycles. The plasma concentrations of factors II, VII, X, and fibrinogen significantly increased during use of both the levonorgestrel- and desogestrel-containing OC's. The plasma concentrations of factor VIII increased, and of factor V decreased, changes which only reached statistical significance during the use of the desogestrel-containing OC. During exposure to the desogestrel-containing OC, as compared with the levonorgestrel-containing OC, both factor VII and factor II showed a greater increase (FVII: 32% and 12% respectively; p <0.0001; FII: 16% and 12% respectively; p = 0.048), whereas factor V showed a greater decrease (-11% and -3% respectively; p = 0.010). Only one of the markers for ongoing coagulation (prothrombin fragment 1+2) showed a significant increase during OC use, whereas concentrations of thrombin-antithrombin complexes and soluble fibrin remained unchanged. For these markers, there was no difference between the tested OC's. We conclude that there are differences between the effects of levonorgestrel and desogestrel-containing OC's on some coagulation factors. Whether these changes provide a biological explanation for the reported differences in venous thromboembolic risk is as yet unclear. The real challenge now becomes to define a pattern of changes in the various systems which, if affected simultaneously, may tip the hemostatic balance towards a prethrombotic state and may lead to overt clinical venous thromboembolism.

Increased fibrinolytic activity during use of oral contraceptives is counteracted by an enhanced factor XI-independent down regulation of fibrinolysis: a randomized cross-over study of two low-dose oral contraceptives.

The effect of oral contraceptives (OC) on fibrinolytic parameters was investigated in a cycle-controlled cross-over study in which 28 non-OC using women were randomly prescribed either a representative of the so-called second (30 microg ethinylestradiol, 150 microg levonorgestrel) or third generation OC (30 microg ethinylestradiol, 150 microg desogestrel) and who switched OC after a two month wash out period. During the use of OC, the levels of tissue-type plasminogen activator (tPA) activity, plasminogen, plasmin-alpha2-antiplasmin complexes and D-dimer significantly increased (by 30 to 80%), while the levels of plasminogen activator inhibitor- (PAI-1) antigen, PAI-1 activity and tPA antigen significantly decreased (25 to 50%), suggesting an increase in endogenous fibrinolytic activity. These OC-induced changes were not different between the two contraceptive pills. TAFI (thrombin-activatable fibrinolysis inhibitor) levels increased on levonorgestrel, and even further increased on desogestrel. A clot lysis assay that probes both fibrinolytic activity and the efficacy of the coagulation system to generate thrombin necessary to down regulate fibrinolysis via TAFI showed no change of the clot lysis time during OC use. This finding suggests that the OC-induced increase in endogenous fibrinolytic activity is counteracted by an increased capacity of the coagulation system to down regulate fibrinolysis via TAFI. Indeed we observed that during OC use there was a significant increase of F1+2 generation during clot formation. When these assays were performed in the presence of an antibody against factor XI, we observed that the clot lysis time was significantly increased during OC use and that the increase in F1+2 generation during OC therapy was due to a factor XI-independent process, which was significantly higher on desogestrel than on levonorgestrel. These data indicate that the OC-induced inhibition of endogenous fibrinolysis takes place in a factor XI-independent way and is more pronounced on desogestrel than on levonorgestrel-containing OC.

A randomized cross-over study on the effects of levonorgestrel- and desogestrel-containing oral contraceptives on the anticoagulant pathways.

The 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 cycle-controlled 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 microg levonorgestrel/30 microg ethinylestradiol) or a third generation OC (150 microg desogestrel/30 microg ethinylestradiol) and who switched to the other OC after a two month wash out period. The anticoagulant parameters determined were: antithrombin (AT), alpha2-macroglobulin (alpha2-M), alpha1-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 alpha2-M, alpha1-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 ETP-based (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.

Mutations in the R2 FV gene affect the ratio between the two FV isoforms in plasma.

Molecular genetics and biochemical studies were performed in homozygotes for the R2 allele (4070G) in the factor V gene, most of them affected by coronary artery disease. Novel polymorphisms (G642T, 156Ser; T1328C, 385Met/Thr), among which a functional candidate (A6755G, 2194Asp/Gly) located in the C2 domain of FV, were identified in the R2 gene. In chromatographic studies R2 FV appeared qualitatively identical to normal FV. However, a relative increase of the more thrombogenic and more glycosylated FV isoform (FV1) was observed in plasma of 2194Gly homozygotes (mean FV1/FV2 ratio 0.71, 95% CI 0.66-0.77) as compared to R2-free controls (0.37, 95% CI 0.34-0.40). We conclude that carriership of the R2 FV gene is associated with an imbalance between the two functionally different FV isoforms, and propose that genetically determined differential glycosylation of FV could represent a novel mechanism of thrombotic disease.