Svetlana N. Tchaikovski

Mechanisms of Estrogen-Induced Venous Thromboembolism

The use of oral contraceptives (OC) is a well established risk factor for venous thrombosis. It has been known for many years that almost all haemostatic parameters i.e. plasma levels of coagulation factors, anticoagulant proteins and proteins involved in the fibrinolytic pathway change during OC use. The discovery of several risk factors of venous thrombosis in the 1990s shed new light on the association between the effects of OC on the haemostatic system and the increased risk of venous thrombosis. In this review, we summarize the current knowledge on the effects of different kinds of hormonal contraceptives (OC, transdermal contraceptives, vaginal ring and levonorgestrel-releasing intrauterine device) on haemostatic variables and the relationship between the changes of these variables and the risk of venous thrombosis.

Effects of oral and transdermal hormonal contraception on vascular risk markers: a randomized controlled trial

OBJECTIVE: To compare the effects of oral and transdermal contraceptives containing similar hormone formulations on vascular risk markers. METHODS: We conducted a randomized, investigator-blinded, crossover, clinical trial with 24 healthy women, aged 18–35 years, who received 2 months of transdermal or oral contraceptive, 2 months washout, then 2 months of the alternative medication. The transdermal contraceptive contained 0.75 mg ethinyl estradiol and 6 mg norelgestromin. The oral contraceptive contained 35 mcg ethinyl estradiol and 250 mcg norgestimate. Blood samples taken before and after each treatment were analyzed in batch for D-dimer, von Willebrand factor, factor VIII, total and free protein S, antithrombin, fibrinogen, C-reactive protein, and normalized activated protein C sensitivity ratio (nAPCsr) determined with two thrombin generation-based assays, the &agr;2macroglobulin-thrombin end point method (&agr;2M-IIa) and calibrated automated thrombinography. Repeated measures analysis of variance was used for analysis. RESULTS: For both contraceptives (transdermal, oral) there were significant declines in free (19%, 11%) and total protein S (19%, 13%) and antithrombin (13%, 10%); increases in fibrinogen (8%, 10%), C-reactive protein (220%, 292%), nAPCsr &agr;2M-IIa (81%, 61%), and nAPCsr calibrated automated thrombinography (102%, 68%), all P<.05. Transdermal contraceptives had a greater effect than oral contraceptives on free protein S (P=.07), nAPCsr &agr;2M-IIa (P=.06), and nAPCsr calibrated automated thrombinography (P=.03). CONCLUSION: Oral and transdermal contraception with similar hormones had similar adverse effects on vascular risk markers. This suggests that this transdermal contraceptive has at least a similar thrombosis risk as its oral counterpart. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00554632 LEVEL OF EVIDENCE: I

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.

Effects of the contraceptive patch, the vaginal ring and an oral contraceptive on APC resistance and SHBG: A cross-over study

INTRODUCTION The transdermal patch (20 microg ethinylestradiol+150 microg norelgestromin daily) and the vaginal ring (15 microg ethinylestradiol+120 microg etonogestrel daily) are new contraceptives, designed to deliver a low dose of hormones, suggesting a low exposure. However, few data are available about their risk of venous thrombosis. The objective was to investigate the effect of the patch, the ring, and an oral contraceptive (30 microg ethinylestradiol+150 microg levonorgestrel daily) on activated protein C sensitivity ratio (APC-sr) and on sex hormone-binding globulin (SHBG) levels in plasma. MATERIALS AND METHODS After a two month wash-out, 13 volunteers were randomly assigned to either the patch followed by the oral contraceptive or vice versa, or the ring followed by the oral contraceptive or vice versa. All treatments lasted two cycles and were separated by a wash-out of two cycles. APC-sr and SHBG levels were determined on day 18-21 of the second cycle of the wash-out and of each treatment period. RESULTS Compared to the oral contraceptive, both the patch and the ring led to higher APC resistance (mean difference APC-sr 1.1; 95% CI 0.67-1.52 and 0.55; 95% CI 0.11-1.00, respectively) and higher SHBG levels (mean difference 210 nmol/l; 95% CI 134-286 and 148 nmol/l; 95% CI 48-248, respectively). CONCLUSION The activity of the protein C system in plasma was impaired more by contraceptive patch and vaginal ring than by an oral contraceptive containing the second generation progestagen levonorgestrel.

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

Summary.  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 °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 CaCl2 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.

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.

The effect of the levonorgestrel-releasing intrauterine system on the resistance to activated protein C (APC)

Exogenously administered estrogens and progestogens as during combined oral contraceptive use increase the risk of venous thrombosis. The thrombin generation-based APC resistance assay is a global coagulation test that enables quantification of the net prothrombotic effect of combined oral contraceptives and that predicts the risk of thrombosis. The thrombotic risk of the levonorgestrel-releasing intrauterine system is unknown. It was the objective of this study to evaluate the thrombotic risk by comparing the APC resistance before and after insertion of a levonorgestrel-releasing or a copper-containing intrauterine device. We measured normalized APC-sensitivity ratios (nAPCsr) before and three months after insertion of the levonorgestrel-intrauterine system in 56 women and the copper-intrauterine device in 18 women. In women without hormonal contraceptive use or a pregnancy in the three months before collection of the baseline samples, nAPCsr were lower three months after insertion of the levonorgestrel-intrauterine system than at baseline (difference -0.29; 95% CI -0.04 to -0.53) and hardly changed after insertion of the copper-intrauterine device (difference -0.11; 95% CI -1.03 to 0.82). In women who switched from a combined oral contraceptive to the levonorgestrel-system the difference was more pronounced (-1.48; 95% CI -0.85 to -2.11). In this study we observed that the levonorgestrel-intrauterine system decreases the resistance to APC which indicates that the levonorgestrel-intrauterine system does not have a prothrombotic effect.

Active site-labeled prothrombin inhibits prothrombinase in vitro and thrombosis in vivo

Mouse and human prothrombin (ProT) active site specifically labeled with d-Phe-Pro-Arg-CH2Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProTS195A mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼10-fold more potent anticoagulant activity compared with ProTS195A as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProTS195A, the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH2Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.

Venous thrombosis and oral contraceptives: current status.

The use of oral contraceptives is associated with an increased risk of venous thrombosis. It is now generally accepted that women who use oral contraceptives that contain so-called third-generation progestins (desogestrel or gestodene) are exposed to a twofold higher risk of venous thrombosis than women who use oral contraceptives that contain the second-generation progestin levonorgestrel. Coagulation studies demonstrated that oral contraceptives increase the plasma level of prothrombin, decrease the level of protein S and induce acquired activated protein C resistance. The changes in hemostatic parameters can explain why women who use oral contraceptives are exposed to an increased risk of venous thrombosis and why the risk is further increased in third-generation oral contraceptive users.

Obesity and thrombin-generation profiles in women with venous thromboembolism.

Obesity is a known risk factor for venous and arterial thrombosis but the mechanisms are still unclear. In women, obesity is correlated with low-grade inflammation and recent data show that BMI is positively associated with thrombin generation. We explored the correlations between obesity, inflammation and thrombin generation in women with increased thrombotic risk by looking at a cohort of women with prior venous thrombosis. One hundred and fifty-six women age 18–65 years were enrolled at diagnosis of first venous thromboembolism (VTE). Plasma samples were obtained at least 3 weeks after cessation of anticoagulant treatment. Thrombin generation was determined with the calibrated automated thrombography (CAT) assay and the Innovance ETP assay. Thrombin generation started later but was more pronounced with higher endogenous thrombin generation potential (ETP) determined with CAT in patients with obesity. The Innovance ETP assay showed results consistent with CAT. Furthermore, patients with obesity had significantly higher levels of fibrinogen, C-reactive protein and plasminogen activator inhibitor-I (PAI-I) than patients without obesity. Increased levels of fibrinogen were the main determinant of the prolonged lag-time in patients with obesity whereas higher levels of prothrombin could account for the difference in the ETP between the groups. We found an association between BMI and ETP values using two different methods to measure thrombin generation. Obesity correlated with increased thrombin generation in women with VTE and the main determinants of this hypercoagulable state were increased levels of fibrinogen and prothrombin. This shows a possible link between obesity, low-grade inflammation and increased thrombin generation in women at increased risk for future thrombosis.