Resistance to activated protein C and impaired TFPI activity in women with previous hormone-induced venous thromboembolism.

INTRODUCTION: Hormonal contraception is a well-known risk factor for venous thromboembolism (VTE). APC resistance and impaired functions of protein S and TFPI are thought to play an important role in the pathogenesis of hormone-related VTE. It is unknown, whether women, who develop VTE during hormonal contraception possess a vulnerability in these pathways, making them susceptible to thrombosis. MATERIALS AND METHODS: Plasma samples were obtained from 57 premenopausal women in average 15.3 years after hormone-associated VTE and from 31 healthy controls. Thrombin generation at high tissue factor (TF) in the absence and in the presence of activated protein C (APC) and at low TF without and with inhibiting anti-protein S- and anti-TFPI-antibodies was measured via calibrated automated thrombography. RESULTS: Women with previous hormone-related thrombosis had higher thrombin generation at low TF, higher APC resistance, protein S- and TFPI ratios, differences: 219.9 nM IIa.min (95%CI:90.4 to 349.3); 1.88 (95%CI:0.71 to 3.05); 0.13 (95%CI:0.01 to 0.26) and 0.19 (95%CI:0.08 to 0.30), respectively. Thrombin generation at high TF without APC did not differ between the groups. Smoking decreased thrombin generation at low TF by -222.6 nM IIa.min (95%CI: -381.1 to -64.1), the APC sensitivity ratio by -2.20 (95%CI: -3.63 to -0.77) and the TFPI ratio by -0.16 (95%CI: -0.29 to -0.03), but did not influence thrombin generation at high TF. DISCUSSION: We demonstrated impairment of the protein S/TFPI system and increased APC resistance in women with previous hormone-induced VTE. Smoking decreased thrombin generation at assay conditions, dependent on the function of the TFPI system.

Effect of blood loss during caesarean section on coagulation parameters.

INTRODUCTION: Venous thrombosis is the leading cause of pregnancy-related maternal morbidity and mortality. The thrombosis risk is increased by caesarean section and blood loss, though underlying mechanisms of these prothrombotic changes remain unknown. MATERIALS AND METHODS: This prospective study recruited 50 pregnant women at term undergoing elective caesarean section at University Hospital Magdeburg, Germany. Blood loss during surgery was correlated with the changes in total protein S, full length TFPI (TFPIfl), prothrombin, the endogenous thrombin potential (ETP) and resistance to activated protein C (APCsr) determined via calibrated automated thrombography. RESULTS: Mean blood loss was 506 ml (95%CI: 456 to 557 ml). Total protein S was 0.63 (95%CI: 0.60 to 0.67) U/ml preoperatively, decreased by 14.8% after caesarean section and almost normalised five days later. TFPIfl was 0.47 (95%CI: 0.41 to 0.53) U/ml before, remained unchanged immediately after and increased by 11.5% five days after surgery. Prothormbin was 1.10 (95%CI: 1.03 to 1.16) U/ml preoperatively, reduced by 10.4% immediately after and increased again five days after caesarean section, exceeding the preoperative values by 4.4% (-0.7 to 9.6). The ETP decreased by 3.9%, whereas the APCsr increased by 37.0% immediately after caesarean section. The changes in total protein S, prothrombin, thrombin generation and APC resistance showed a trend to be more pronounced in the subgroups with higher blood loss. DISCUSSION: Moderate blood loss during caesarean section hardly reduces thrombin generation but aggravates pregnancy-induced APC resistance and combined deficiency of TFPI and protein S, which can account for the increased thrombosis risk in early puerperium.

Differential effects of high prothrombin levels on thrombin generation depending on the cause of the hyperprothrombinemia.

BACKGROUND: Hyperprothrombinemia, resulting from the prothrombin G20210A mutation or other causes, is associated with activated protein C (APC) resistance and increased thrombosis risk. When high prothrombin levels are a result of increased hepatic biosynthesis, these effects may be counteracted by concomitantly increased levels of the anticoagulant factors (particularly protein S). Differently, in prothrombin G20210A carriers only prothrombin levels are elevated. OBJECTIVE: To investigate whether prothrombin G20210A carriers have a more severe hypercoagulable state than non-carriers with comparable prothrombin levels. PATIENTS/METHODS: Coagulation factor levels, thrombin generation (Calibrated Automated Thrombogram in the presence and absence of APC) and APC resistance were measured in normal (n = 132), heterozygous (n = 167) and homozygous (n = 3) individuals. RESULTS: Prothrombin levels, thrombin generation and APC resistance were higher in carriers of the prothrombin G20210A mutation (especially those who had experienced venous thrombosis) than in non-carriers, whereas protein S and antithrombin levels were similar among genotype groups. Because individuals with high prothrombin levels in the absence of the prothrombin G20210A mutation tend to have all liver-synthesized factors elevated, carriers of the mutation had lower protein S and antithrombin levels than non-carriers with equally high prothrombin levels. Accordingly, they also generated more thrombin and showed a tendency toward higher APC resistance. Analogous effects, but less pronounced, were observed in homozygotes for the prothrombin A19911G polymorphism, which also upregulates prothrombin levels. CONCLUSIONS: Individuals with hyperprothrombinemia as a result of prothrombin gene mutations generate more thrombin and tend to be more APC-resistant than individuals with comparable prothrombin levels because of other causes.

Effect of oral contraceptives on thrombin generation measured via calibrated automated thrombography.

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.

New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma.

Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation. SUMMARY: Background Protein S plays an important role in the down-regulation of coagulation as cofactor for activated protein C (APC) and tissue factor pathway inhibitor (TFPI). Aim To develop functional assays to quantify the APC- and TFPI-cofactor activities of protein S in plasma. Methods APC- and TFPI-cofactor activities of protein S in plasma were measured using calibrated automated thrombography in protein S-depleted plasma supplemented with a small amount of sample plasma either in the presence of anti-TFPI antibodies and APC (APC-cofactor activity) or at excess full-length TFPI without APC (TFPI-cofactor activity). Total and free protein S levels in plasma were measured by ELISAs. Results Average APC-cofactor activities of protein S were 113%, 108% and 89% in plasma from normal individuals (n = 15), FV Leiden heterozygotes (n = 14) and FV Leiden homozygotes (n = 7), respectively, whereas the average APC-cofactor activity of protein S in plasma from heterozygous protein S-deficient individuals (n = 21) was significantly lower (55%). Similar trends were observed for the TFPI-cofactor activity of protein S, with averages of 109%, 115% and 124% in plasma from individuals with normal protein S levels and different FV Leiden genotypes, and 64% in plasma from protein S-deficient patients. APC-cofactor activities of protein S correlated significantly with free and total protein S antigen levels, whereas TFPI-cofactor activities correlated less with protein S antigen levels. Conclusion We have developed functional protein S assays that measure both the APC- and TFPI-cofactor activities of protein S in plasma, which are hardly if at all affected by the FV Leiden mutation.

Tissue factor-independent inhibition of thrombin generation by tissue factor pathway inhibitor-α.

BACKGROUND: Tissue factor pathway inhibitor-α (TFPIα) inhibits factor Xa by forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. TF-FVIIa forms a quaternary complex with TFPIα and FXa, which shuts off the initiation of coagulation via the extrinsic pathway. AIM: To investigate whether direct inhibition of FXa by TFPIα independently of TF plays a role in downregulating coagulation. METHODS: Inhibition of FXa by TFPIα in plasma was determined by measuring thrombin generation triggered with FXa, the FX activator from Russell's viper venom (RVV-X), FXIa, or FIXa. TF-independent anticoagulant activities of TFPIα and its cofactor, protein S, were quantified: (i) after neutralization of TFPIα and protein S with anti-TFPI or anti-protein S antibodies; and (ii) in TFPI-depleted or protein S-depleted plasmas supplemented with varying amounts of TFPIα or protein S. RESULTS: Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation in plasma triggered with RVV-X, FXa, FIXa, or FXIa. Anti-TFPI and anti-protein S antibodies decreased the lag time and increased the peak height of thrombin generation to the same extent, indicating that inhibition of FXa by TFPIα requires the presence of protein S. TFPIα and protein S titrations in TFPI-depleted or protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF also revealed TF-independent anticoagulant activity of TFPIα, which was completely dependent on the presence of protein S. CONCLUSION: Direct inhibition of FXa by TFPIα contributes to the downregulation of coagulation.

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.

Changes in haemostatic parameters during the menstrual cycle and subsequent use of drospirenone-containing oral contraceptives.

INTRODUCTION: Oral contraceptives (OC) increase the risk of venous thromboembolism that depends on the OC formulation and could at least partially be explained by impaired function of the protein C-system (APC resistance) and the tissue factor pathway inhibitor (TFPI)-system. There is limited information available on the effects of OC, containing a newer progestogen- drospirenone (DRSP-OC) on these two major anticoagulant pathways, thrombin generation, reflecting the overall state of coagulation, and other coagulation parameters. METHODS: In a study population consisting of 14 healthy women (age 21-33 years) we investigated the effect of the menstrual cycle and subsequent use of DRSP-OC on APC resistance, the function of the TFPI-system, thrombin generation and on their major determinants, i.e. prothrombin, antithrombin, FV, FX, FVIII, protein C, protein S(total and free) and TFPI(full-length and free). RESULTS: All studied parameters remained unchanged during the menstrual cycle. During DRSP-OC use we observed a significant increase in APC resistance (~2.4-fold), thrombin generation measured at low (~2.2-fold) and high tissue factor concentrations (~1.4-fold), plasma concentrations of prothrombin (19%), FX (31%), FVIII (17%) and protein C (43%). DRSP-OC use impaired the function of the TFPI-system and decreased plasma levels of antithrombin (-6%), FV (-22%), protein Stotal (-21%), protein Sfree (-20%), TFPIfull-length (-36%) and TFPIfree (-46%). CONCLUSIONS: DRSP-OC caused procoagulant changes in all studied haemostatic parameters. The impairment of the protein C- and TFPI-systems was more pronounced than the impairment of the coagulation pathways and can at least partially account for the increased risk of venous thromboembolism in users of DRSP-OC.

Role of protein S and tissue factor pathway inhibitor in the development of activated protein C resistance early in pregnancy in women with a history of preeclampsia.

Pregnancy increases the risk of venous thromboembolism. Particularly in early pregnancy, the thrombosis risk can be attributed to the changes in coagulation. Elevated thrombin generation and resistance to activated protein C (APC) are likely to contribute to the increased thrombosis risk during pregnancy. We studied changes and the determinants of thrombin generation and APC resistance in the first 16 weeks of gestation in women with history of preeclampsia. Additionally, we investigated the influence of pregnancy-induced haemodilution on the coagulation system. We measured thrombin generation, APC resistance and plasma levels of prothrombin, factor V, factor X, protein S and tissue factor pathway inhibitor (TFPI) in 30 non-pregnant and 21 pregnant women at 8, 12 and 16 weeks of gestation. All participants shared a history of a hypertensive complication in the preceding pregnancy. Thrombin generation and APC resistance were higher at eight weeks of pregnancy than in the non-pregnant state, and progressively increased between eight and 16 weeks of gestation. Changes in the TFPI and protein S levels accounted for ~70% of pregnancy-induced APC resistance. Interestingly, a significant correlation (slope 2.23; 95%CI: 1.56 to 2.91; r= 0.58) was observed between protein Stotal or protein Sfree levels and haematocrit. In conclusion, pregnancy induces a decrease of TFPIfree and protein Sfree levels that attenuates the function of the TFPI and protein C systems and results in elevated thrombin generation and increased APC resistance. Besides, our data suggest that pregnancy-dependent haemodilution may contribute to the decreased peripheral protein S levels.

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.