Guido Tans

Low-dose oral contraceptives and acquired resistance to activated protein C: a randomised cross-over study

BACKGROUND We have reported previously that, compared with use of second-generation oral contraceptives, the use of third-generation oral contraceptives is associated with increased resistance to the anticoagulant action of activated protein C (APC). Owing to the cross-sectional design of that study, these observations may have been subject to unknown bias or uncontrolled effects of the menstrual cycle. We aimed to overcome these sources of bias by doing a cycle-controlled randomised cross-over trial. METHODS The response to APC in plasma was assessed in 33 women who received two consecutive cycles of a second-generation oral contraceptive (150 microg levonorgestrel and 30 microg ethinyloestradiol) or a third-generation oral contraceptive (150 microg desogestrel and 30 microg ethinyloestradiol), and who switched preparations after two pill-free cycles. Normalised APC sensitivity ratios were calculated by measurement of the effect of APC on thrombin generation in the plasma of these women and in pooled plasma from 90 controls. FINDINGS Of the 33 women, five were excluded because not all required plasma samples were available. In the remaining 28 women, the normalised APC sensitivity ratio increased during treatment with both preparations. Compared with levonorgestrel, desogestrel-containing oral-contraceptive treatment caused a highly significant (p<0.0001) additional increase in normalised APC sensitivity ratio (0.51 [95% CI 0.37-0.66]). Normalised APC sensitivity ratios during oral-contraceptive treatment correlated with the values before oral-contraceptive use. INTERPRETATION Oral-contraceptive treatment diminishes the efficacy with which APC down-regulates in-vitro thrombin formation. This phenomenon, designated as acquired APC resistance, is more pronounced in women using desogestrel-containing oral contraceptives than in women using levonorgestrel-containing preparations. Whether acquired APC resistance induced by oral contraceptives explains the increased risk of venous thromboembolism in oral-contraceptive users remains to be established.

Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C

Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg506 → Gln (factor VR506Q). Factor VR506Q was converted by thrombin into factor Va which was further purified yielding a factor Va preparation that had the same cofactor activity in prothrombin activation as normal factor Va. Inactivation of low concentrations of normal factor Va (<5 nM) by 0.15 nM APC in the presence of phospholipid vesicles proceeded via a biphasic reaction that consisted of a rapid phase (k = 4.3 × 107M−1s−1), yielding a reaction intermediate with reduced cofactor activity that was fully inactivated during the subsequent slow phase (k = 2.3 × 106M−1s−1). Inactivation of factor VaR506Q proceeded via a monophasic reaction (k = 1.7 × 106M−1s−1). Immunoblot analysis showed that APC-catalyzed inactivation of factor Va occurred via peptide bond cleavages in the heavy chain. The rapid phase of inactivation of normal factor Va was associated with cleavage at Arg506 and full inactivation of factor Va required subsequent cleavage at Arg306. The slow monophasic inactivation of factor VaR506Q correlated with cleavage at Arg306. Cleavage at Arg506 in normal factor Va resulted in accumulation of a reaction intermediate that exhibited 40% cofactor activity in prothrombin activation mixtures that contained a high factor Xa concentration (5 nM). Compared with native factor Va, the reaction intermediate retained virtually no cofactor activity at low factor Xa concentrations (0.3 nM). This demonstrates that factor Va that is cleaved at Arg506 is impaired in its ability to interact with factor Xa. Michaelis-Menten kinetic analysis showed that cleavage at Arg506 in membrane-bound factor Va was characterized by a low Km for factor Va (20 nM) and kcat = 0.96 s−1. For cleavage at Arg306 in factor VaR506Q the kinetic parameters were Km= 196 nM and kcat = 0.37 s−1. This means that differences between APC-catalyzed inactivation of factors Va and VaR506Q become much less pronounced at high factor Va concentrations. When factor VaR506Q was inactivated by APC in the absence of phospholipids, cleavage at Arg679 of the heavy chain also contributed to factor Va inactivation. Comparison of rate constants for APC-catalyzed cleavage at Arg306, Arg506, and Arg679 in the absence and presence of phospholipids indicated that phospholipids accelerated these cleavages to a different extent. This indicates that the binding of factor Va to phospholipids changes the accessibility of the cleavage sites and/or the sequence of peptide bond cleavage by APC.

Activated protein C resistance determined with a thrombin generation-based test predicts for venous thrombosis in men and women

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 sex‐matched 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 > 4·5) as cut‐off, were: 7·5 [95% confidence interval (CI) 1·6–33·8] for men, 3·0 (95% CI 1·0–8·8) for premenopausal women not using OC, 4·8 (95% CI 1·6–14·7) for premenopausal women using OC and 4·7 (95% CI 1·4–15·6) for postmenopausal women. After excluding the carriers of factor V Leiden, the OR became infinite for men (no control had an APCsr > 4·5), 1·4 (95% CI 0·2–8·2) for premenopausal women not using OC, 3·4 (95% CI 1·1–10·8) for premenopausal women using OC and 3·6 (95% CI 0·6–20·5) 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.

Factor XIIa regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin

Recent data indicate an important contribution of coagulation factor (F)XII to in vivo thrombus formation. Because fibrin structure plays a key role in clot stability and thrombosis, we hypothesized that FXII(a) interacts with fibrin(ogen) and thereby regulates clot structure and function. In plasma and purified system, we observed a dose-dependent increase in fibrin fiber density and decrease in turbidity, reflecting a denser structure, and a nonlinear increase in clot stiffness with FXIIa. In plasma, this increase was partly independent of thrombin generation, as shown in clots made in prothrombin-deficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and α-FXIIa, but not β-FXIIa, bound to purified fibrinogen and fibrin with nanomolar affinity. Immunostaining of human carotid artery thrombi showed that FXII colocalized with areas of dense fibrin deposition, providing evidence for the in vivo modulation of fibrin structure by FXIIa. These data demonstrate that FXIIa modulates fibrin clot structure independently of thrombin generation through direct binding of the N-terminus of FXIIa to fibrin(ogen). Modification of fibrin structure by FXIIa represents a novel physiologic role for the contact pathway that may contribute to the pathophysiology of thrombosis.

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

Summary.  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‐SRAPTT or APC‐SRETP 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.

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 Ca2+-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.

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.

Acquired resistance to activated protein C in breast cancer patients.

Summary. In 56 women with a lymph‐node‐positive breast carcinoma and 28 matched healthy control subjects, the sensitivity to activated protein C (APC‐sr) was determined with an APC resistance test that quantifies the effect of APC on thrombin generation initiated via the extrinsic coagulation pathway. Carriers of the Factor V Leiden mutation were excluded from the study. Significant resistance to APC was found in the breast cancer patients: median APC‐sr 2·02 vs 1·03 in the healthy control subjects (P < 0·001). No difference in APC‐sr was found between patients with metastases and without metastases. In patients with metastases, protein S levels were significantly elevated compared with patients without metastases and healthy control subjects: 108·0%vs 96·0% and 94·5% (P = 0·008 and P = 0·007). The APC‐sr correlated with protein S in the control subjects and in patients without metastases but not in patients with metastases. The disturbance of the haemostatic balance probed by the tissue‐factor‐based APC resistance test might contribute to the cancer‐related hypercoagulability.

Effects of oral contraceptives on hemostasis and thrombosis

OBJECTIVE The object of the study was to determine the effects of oral contraceptives on blood coagulation, in particular on the protein C pathway. STUDY DESIGN Plasma samples from healthy men, from healthy female users and nonusers of oral contraceptives, and from heterozygous and homozygous male and female carriers of the factor V Leiden mutation (some of whom used oral contraceptives) were tested for their sensitivity to activated protein C by means of a new activated protein C resistance test developed in our laboratory. This assay is based on measurement of the effect of activated protein C on the endogenous thrombin potential, the time integral of thrombin generation initiated in plasma through the extrinsic coagulation pathway. RESULTS The normalized activated protein C sensitivity ratio ([ETP+APC/ETP-APC]plasma/[ETP+APC/ETP-APC]normal plasma, where ETP is endogenous thrombin potential, +APC is with activated protein C, and -APC is without activated protein C) of men was lower than that of healthy female nonusers of oral contraceptives. The normalized activated protein C sensitivity ratio of the users of oral contraceptives was significantly higher than that of nonusers of oral contraceptives. The normalized activated protein C sensitivity ratio of women who were using oral contraceptives with third-generation progestogens was higher than that of users of oral contraceptives with second-generation progestogens. Furthermore, the normalized activated protein C sensitivity ratio of 80% of the users of third-generation preparations fell within the 5th to 95th percentile of the normalized activated protein C sensitivity ratio of female carriers of factor V Leiden, a mutation that is associated with hereditary resistance to activated protein C and with an increased risk of venous thromboembolism. CONCLUSION Acquired activated protein C resistance may explain the increased risk of venous thromboembolism among users of oral contraceptives reported in epidemiologic studies and the higher risk of venous thromboembolism among users of oral contraceptives with third- versus second-generation progestogens.

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, 17β‐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 (ΔE2 = 6·6 nmol/l) and the APCsr (ΔAPCsr = 0·30). Six women who participated in this study became pregnant. Compared with baseline, the APCsr was increased 1·9‐fold (ΔAPCsr = 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.