Mechanisms Contributing to Acquired Activated Protein C Resistance in Patients Treated with Thalidomide: A Molecular Dynamics Study.

INTRODUCTION: There is a high incidence of venous thromboembolism (VTE) in patients with Multiple Myeloma (MM), however; until now, the exact mechanisms behind VTE in MM are unknown, and some of the elements that may play a significant role are the treatment with an immunomodulator (IMiD) and acquired resistance to activated protein C (APC). OBJECTIVE: The study aims to reveal the possible mechanisms linked to the reduced antithrombotic activity of APC associated with thalidomide. METHODS: The molecular docking approach was used to ascertain the in silico inhibitory potential of thalidomide on the APC protease domain in the architecture of the catalytic triad and its interaction with major substrate binding sites. RESULTS: The coupling showed that the inhibitory activity of thalidomide depends on the induction of structural changes in the protease domain of APC, at the level of the Ser/His/Asp catalytic triad, as a result of a significant increase between the distances of CαAsp102 and Cα Ser195 (11.175 angstroms, increase 14.83%) and between CαSer195 and CαHis57 (9.478 angstroms, increase 13.78 %). This can result in an inefficient transfer of the proton between these residues, the other possible mechanism of inhibition, is a potential reduced binding of the substrate as a result of a direct interaction through a carbon-hydrogen bond on His57, an H-bond on Arg306, and a carbon hydrogen bond on Arg506. CONCLUSION: We demonstrate the in silico inhibitory potential of thalidomide on APC, through two possible inhibition mechanisms, a pathophysiologically relevant finding to understand the factors that can affect the stability and functions of APC in vivo.

Evaluation of the effect of a new oral contraceptive containing estetrol and drospirenone on hemostasis parameters.

OBJECTIVE: To assess the effect on hemostasis parameters of a new combined oral contraceptive (COC). STUDY DESIGN: In this randomized, single centre, open-label, exploratory study, healthy women received either 15 mg estetrol/3 mg drospirenone (E4/DRSP) (n = 39), 30 mcg ethinylestradiol/150 mcg levonorgestrel (EE/LNG) (n = 30), or 20 mcg ethinylestradiol/3 mg drospirenone (EE/DRSP) (n = 32) for six 28-day cycles. Blood was collected at baseline, cycle 3, and cycle 6. Median change from baseline was evaluated for procoagulant, anticoagulant, and fibrinolytic parameters, and for sex hormone-binding globulin (SHBG). RESULTS: Median change of endogenous thrombin potential (ETP) based activated protein C sensitivity resistance (APCr) at cycle 6 was +30% for E4/DRSP, +165% for EE/LNG (p-value <0.05 vs E4/DRSP), and +219% for EE/DRSP (p-value <0.05 vs E4/DRSP). Changes to prothrombin fragment 1 + 2 and SHBG for E4/DRSP, EE/LNG, and EE/DRSP were +23%, +71%, and +64% (p-value <0.05 vs E4/DRSP); and +55%, +74% and +251% (p-value <0.05 vs E4/DRSP), respectively. At cycle 6, changes to other hemostasis parameters for E4/DRSP were similar or smaller than for EE/LNG or EE/DRSP. CONCLUSIONS: In this study, changes in hemostasis parameters after treatment with 6 cycles of E4/DRSP were smaller or similar to those observed for EE/LNG. Similar, but more pronounced changes were also observed versus EE/DRSP, which supports the hypothesis that the effect of COCs on hemostasis parameters is mainly mediated by the estrogenic component. Further studies are needed to provide more insight into the venous thromboembolic risk of E4/DRSP. IMPLICATIONS STATEMENT: This study reports that the effects on hemostasis parameters of a COC containing 15 mg E4/3 mg DRSP are less or similar to those for EE/LNG or EE/DRSP. It also demonstrates that the choice of estrogen modulates the effects of COCs on hemostasis parameters.

DOAC-Remove abolishes the effect of direct oral anticoagulants on activated protein C resistance testing in real-life venous thromboembolism patients.

Background Direct oral anticoagulants (DOACs) may cause false results of activated protein C resistance (APC-R) ratio. DOAC-Remove, a new reagent based on activated carbon, has been designed to eliminate the interference of DOACs on coagulation assays. The aim of the study was to investigate whether the use of DOAC-Remove enables to determine APC-R in patients treated with DOACs. Methods We assessed 74 venous thromboembolism (VTE) patients, including 25 on rivaroxaban, 25 on apixaban and 24 taking dabigatran. APC-R was determined using the Russell Viper Venom Time (RVVT)-based clotting test. APC-R and DOAC concentrations were tested at baseline and following DOAC-Remove. Thrombophilia, including factor V Leiden (FVL) mutation was tested. Results FVL mutation was found in 20 (27%) patients. The APC-R ratio at baseline was measurable in 43 patients (58.1%), including 20 (80%) on rivaroxaban, 19 (76%) on apixaban and four (16.7%) on dabigatran. In patients with measurable APC-R at baseline, the ratio >2.9 was found in 23 patients (53.5%). In 16 (37.2%) subjects APC-R ratio <1.8 suggested FVL mutation which was genetically confirmed. Four (9.3%) FVL carriers on dabigatran showed negative/equivocal APC-R results. In 11 (14.9%) patients taking rivaroxaban or apixaban, in whom blood was collected 2-5 h since the last dose, we observed unmeasurable APC-R. DOAC-Remove almost completely eliminated all plasma DOACs. After addition of DOAC-Remove all APC-R ratios were measurable. In four FVL carriers on dabigatran with false negative APC-R, DOAC-Remove resulted in APC-R ratios <1.8. Conclusions DOAC-Remove effectively reduces DOACs concentration in plasma, which enables FVL testing using APC-R.

Elevated Plasma Factor IXa Activity in Premenopausal Women on Hormonal Contraception.

OBJECTIVE: Combined oral contraceptives induce a reversible hypercoagulable state with an enhanced risk of venous thromboembolism, but the underlying mechanism(s) remain unclear. Subjects on combined oral contraceptives also demonstrate a characteristic resistance to APC (activated protein C) in the thrombin generation assay. Here, we report the potential role of plasma factor IXa (FIXa) as a mechanism for hormone-induced systemic hypercoagulability. APPROACH AND RESULTS: A novel assay was used to determine FIXa activity in plasma samples from volunteer blood donors. Plasma from 36 premenopausal females on hormonal contraception and 35 not on hormonal contraception, 35 postmenopausal females, and 10 males were analyzed for FIXa activity, total PS (protein S), total tissue factor pathway inhibitor (TFPI), and TFPI-α antigen. Premenopausal females on hormonal contraception demonstrated significantly increased FIXa activity and decreased TFPI-α compared with the other groups. Remarkably, FIXa values were not normally distributed in the hormonal contraception group, but skewed toward the high end. Plasma FIXa activity inversely correlated with both TFPI-α and total PS antigen. Ex vivo determination of TF-dependent FIX activation in FV-deficient plasma demonstrated that inhibitory anti-TFPI antibodies enhanced FIXa generation by 2- to 3-fold, whereas addition of 75 nmol/L PS reduced FIXa generation by ≈2-fold. Further, increasing FIXa concentration enhanced APC resistance during TF-triggered plasma thrombin generation. CONCLUSIONS: Elevation of plasma FIXa activity in association with reductions in TFPI-α and PS is a potential mechanism for systemic hypercoagulability and resistance to APC in premenopausal females on hormonal contraception.

Impact of hormone-associated resistance to activated protein C on the thrombotic potential of oral contraceptives: a prospective observational study.

INTRODUCTION: The increased thrombotic risk of oral contraceptives (OC) has been attributed to various alterations of the hemostatic system, including acquired resistance to activated protein C (APC). To evaluate to what extent OC-associated APC resistance induces a prothrombotic state we monitored plasma levels of thrombin and molecular markers specific for thrombin formation in women starting OC use. Elevated plasma levels of thrombin have been reported to characterize situations of high thrombotic risk such as trauma-induced hypercoagulability, but have not yet been studied during OC use. PATIENTS AND METHODS: Blood samples were collected prospectively from healthy women (n = 21) before and during three menstruation cycles after start of OC. APC resistance was evaluated using a thrombin generation-based assay. Plasma levels of thrombin and APC were directly measured using highly sensitive oligonucleotide-based enzyme capture assay (OECA) technology. Thrombin generation markers and other hemostasis parameters were measured additionally. RESULTS: All women developed APC resistance as indicated by an increased APC sensitivity ratio compared with baseline after start of OC (p = 0.0003). Simultaneously, plasma levels of thrombin, prothrombin fragment 1+2, and of thrombin-antithrombin complexes did not change, ruling out increased thrombin formation. APC plasma levels were also not influenced by OC use, giving further evidence that increased thrombin formation did not occur. CONCLUSIONS: In the majority of OC users no enhanced thrombin formation occurs despite the development of APC resistance. It cannot be ruled out, however, that thrombin formation might occur to a greater extent in the presence of additional risk factors. If this were the case, endogenous thrombin levels might be a potential biomarker candidate to identify women at high thrombotic risk during OC treatment. Large-scale studies are required to assess the value of plasma levels of thrombin as predictors of OC-associated thrombotic risk.

Tamoxifen induces resistance to activated protein C.

INTRODUCTION: The estrogen antagonist tamoxifen (TAM) increases the thrombotic risk similar to estrogen containing oral contraceptives (OC). In OC users this risk is attributed to alterations of hemostasis resulting in acquired resistance to activated protein C (APC). TAM-induced APC resistance has not been reported yet. MATERIALS AND METHODS: Blood samples were collected prospectively from women with breast cancer before (n=25) and monthly after start of adjuvant TAM treatment (n=75). APC resistance was evaluated on basis of the effect of APC on the endogenous thrombin generation potential. To detect increased in vivo APC generation APC plasma levels were measured using a highly sensitive oligonucleotide-based enzyme capture assay. Routine hemostasis parameters were measured additionally. RESULTS: APC sensitivity decreased by 41% (p=0.001) compared to baseline after one month of TAM application and remained significantly decreased during the study period. Free protein S increased (p=0.008) while other analyzed procoagulant factors, inhibitors, and activation markers of coagulation decreased or did not change significantly. In five patients the APC concentration increased to non-physiological levels but an overall significant increase of APC was not observed. CONCLUSIONS: This is the first study showing acquired APC resistance under TAM therapy. Acquired APC resistance might explain the increased thrombotic risk during TAM treatment. Observed changes of hemostasis parameters suggest different determinants of TAM-induced APC resistance than in OC-induced APC resistance. The presence of acquired APC resistance in TAM patients warrants further evaluation if these patients may benefit from antithrombotic prophylaxis in the presence of additional thrombotic risk factors.

Thyroid function, activated protein C resistance and the risk of venous thrombosis in users of hormonal contraceptives.

INTRODUCTION: Use of combined hormonal contraceptives is associated with a three- to eight-fold increased risk of venous thrombosis compared with non-use. The thrombotic risk depends on the estrogen dose as well as the progestogen type. Use of hormonal contraceptives leads to resistance to activated protein C (APC), which may serve as marker for the risk of venous thrombosis. Hyperthyroidism is also associated with an increased risk of venous thrombosis, due to increased free Thyroxine (FT4) levels which cause a hypercoagulable state. MATERIALS AND METHODS: The objective of this study was to evaluate the effects of hormonal contraceptives on levels of FT4, thyroid stimulating hormone (TSH) and thyroxine binding globulin (TBG), and to investigate the effects on APC resistance per contraceptive group. We measured FT4, TBG and TSH levels and APC resistance in 231 users of oral contraceptives. RESULTS: Users of the most thrombogenic hormonal contraceptives, i.e. containing desogestrel, cyproterone acetate or drospirenone, had higher TBG levels than users of less thrombogenic hormonal contraceptives, i.e. the levonorgestrel-containing intrauterine device. TSH levels were not significantly elevated and FT4 levels did not change. TBG levels were also associated with APC resistance. CONCLUSION: Use of hormonal contraceptives lead to elevated TBG levels, slightly elevated TSH levels and unchanged FT4 levels without causing a hyperthyroid state. Thus, the increased thrombotic risk during the use of hormonal contraceptives cannot be explained by a hyperthyroid state caused by use of these hormonal contraceptives.

The effect of different hormonal contraceptives on plasma levels of free protein S and free TFPI.

Use of combined oral contraceptives is associated with a three- to six-fold increased risk of venous thrombosis. Hormonal contraceptives induce acquired resistance to activated protein C (APC), which predicts the risk of venous thrombosis. The biological basis of the acquired APC resistance is unknown. Free protein S (PS) and free tissue factor pathway inhibitor (TFPI) are the two main determinants of APC. Our objective was to assess the effect of both hormonal and non-hormonal contraceptives with different routes of administration on free TFPI and free PS levels. We conducted an observational study in 243 users of different contraceptives and measured APC sensitivity ratios (nAPCsr), free TFPI and free PS levels. Users of contraceptives with the highest risk of venous thrombosis as reported in recent literature, had the lowest free TFPI and free PS levels, and vice versa, women who used contraceptives with the lowest risk of venous thrombosis had the highest free TFPI and free PS levels. An association was observed between levels of free TFPI and nAPCsr, and between free PS and nAPCsr. The effect of oral contraceptives on TFPI and PS is a possible explanation for the increased risk of venous thrombosis associated with oral contraceptives.

[Oral contraceptive pill and thrombotic risk: epidemiological studies]. / Pillola e rischio trombotico: gli studi wpidemiogici.

The venous thromboembolism (VTE) is a rare event during childbearing age and during the assumption of combined oral contraceptive. The absolute risk of VTE in users of combined oral contraceptives is 20-30 per 100000 women years. A number of case-control studies published in recent years have shown an apparent increase in the risk of VTE among users of oral contraceptives (OCs) containing desogestrel, gestodene, drospirenone and cyproterone, relative to the use of levonorgestrel. The data derived from these recent studies is of borderline statistical significance because any important factors are not considered to evaluate the real correlation between the assumption of OCs and risk of venous thromboembolism. Among the factors that should be considered, there are: EE dose, duration of use, coexistance of other risk factors of venous thromboembolism (age, BMI, familiarity, surgical interventions) and other prescription bias. The lack of these factors is likely to contribute to the increased risk of venous thromboembolism observed in users of third-generation OCs when compared to that in users of second-generation OCs. To date, because of the inadequacy of epidemiological studies, the data about the correlation between OCs and TVE, are not conclusive and it will be necessary to carry out other studies to clarify this debating point, definitively.

Sex hormone-binding globulin as a marker for the thrombotic risk of hormonal contraceptives.

BACKGROUND: It takes many years to obtain reliable values for the risk of venous thrombosis of hormonal contraceptive users from clinical data. Measurement of activated protein C (APC) resistance via thrombin generation is a validated test for determining the thrombogenicity of hormonal contraceptives. Sex hormone-binding globulin (SHBG) might serve as a marker for the risk of venous thrombosis, and can be easily and rapidly measured in routine laboratories. OBJECTIVE: To determine whether SHBG is a useful marker for the thrombotic risk of hormonal contraceptive users by comparing plasma SHBG levels with normalized APC sensitivity ratio (nAPCsr) values and thrombosis risks reported in the recent literature. METHODS: We conducted an observational study in 262 users of different contraceptives, and measured nAPCsr and SHBG levels. RESULTS: Users of contraceptives with a higher risk of causing venous thrombosis, i.e. combined hormonal contraceptives containing desogestrel, cyproterone acetate or drospirenone, and the transdermal patch, had higher SHBG levels than users of combined hormonal contraceptives containing levonorgestrel, which carry a lower thrombosis risk. Users of the patch had the highest SHBG levels, with a mean difference of 246 nmol L(-1) (95% confidence interval 179-349) from that in users of levonorgestrel-containing combined hormonal contraceptives. SHBG levels were positively associated with both the nAPCsr and the risks of venous thrombosis reported in the recent literature. CONCLUSION: SHBG is a useful marker with which to estimate the thrombotic safety of a preparation.

Effects of switching from oral to transdermal or transvaginal contraception on markers of thrombosis.

BACKGROUND: The study was conducted to determine the impact of switching from oral to transdermal patch or vaginal ring contraception on biomarkers of thrombosis. STUDY DESIGN: Current healthy oral contraceptive (OC) users were randomized to switch to either a contraceptive ring (CR) or patch (CP) and underwent phlebotomy to measure surrogate biomarkers of thrombosis [sex hormone-binding globulin (SHBG), free protein S and activated protein C resistance (APC-r)] before switching, and during the fourth cycle of use of the new method. RESULTS: Of 142 reproductive age women enrolled, 120 sample pairs were available for analysis. SHBG increased significantly from baseline in CP users [mean change (95% CI), +29.9 nM (9.6-50)] but not in CR users [-1.6 (-16.6 to 13.5)]. Protein S decreased significantly from baseline in CP users [mean change -7.1% (-12.1 to -2.1)], but increased significantly in CR users [+5.3% (1.1-9.6)]. The APC-r ratio did not undergo a significant change from baseline in either group [CP +0.06 (-0.06 to 0.18), CR +0.02 (-0.10 to 0.14)]. Compared to CR users, subjects using the CP had significantly higher SHBG [187.5 (167.0-208), 146 (132.6-159.4), p=.012], significantly lower protein S [81.8 (76.8-86.8), 93.6 (89.1-98.1), p=.001] and similar APC-r ratios [2.99 (2.85-3.14), 3.09 (2.96, 3.22), p=.3] at the Cycle 4 visit. CONCLUSION: OC users who switch to the ring exhibit beneficial changes in biomarkers of thrombosis, while those switching to the patch display a shift favoring clot formation.

Different effects of oral contraceptives containing different progestogens on protein S and tissue factor pathway inhibitor.

BACKGROUND: Oral contraceptives (OC) containing different types of progestogens induce different sensitivities to activated protein C (APC) measured with the thrombin generation-based APC-resistance test. These differences in APC resistance may be the biological explanation for the differences in thrombotic risk of the various pills. The mechanistic basis of APC resistance observed in OC users is unknown. Our objective was to study the effect of OC on the two main determinants of the APC-resistance test, free protein S and free tissue factor pathway inhibitor (TFPI). PATIENTS/METHODS: We measured free protein S and free TFPI in 156 users of various types of OC. RESULTS: Users of desogestrel-containing OC, known to double the risk of thrombosis compared with levonorgestrel-containing OC, had lower free protein S (24 vs. 33 U dL(-1)) and TFPI free antigen (2.9 vs. 3.6 ng mL(-1)) levels than users of OC containing levonorgestrel. Women using cyproterone acetate-containing OC, known to confer a high thrombotic risk, had the lowest free protein S (19 U dL(-1)) and free TPFI antigen (2.5 ng mL(-1)) levels. Users of OC containing drospirenone had lower free protein S (23 U dL(-1)) and TFPI antigen levels (3.2 ng mL(-1)) than users of levonorgestrel-containing OC. Low free protein S and low free TFPI antigen levels were associated with an increased resistance to APC, an established risk factor for thrombosis. CONCLUSIONS: This study observed that the differences in APC resistance induced by OC containing different progestogens can at least in part be explained by different effects of OC on free protein S and TFPI.

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.

Differential impact of conventional and low-dose oral hormone therapy (HT), tibolone and raloxifene on functionality of the activated protein C system.

Recent studies have shown that hormone therapy (HT) is associated with an acquired resistance to activated protein C (APC). The aims of the present study were to evaluate a possible dose-response relationship and differential effects of different HT regimens on functionality of the APC system. Two hundred two healthy women were randomly assigned to receive treatment for 12 weeks with tablets containing either low-dose HT containing 1 mg 17ss-oestradiol + 0.5 mg norethisterone acetate (NETA) (n = 50), conventional-dose HT containing 2 mg 17ss-oestradiol and 1 mg NETA (n = 50), 2.5 mg tibolone (n = 51), or 60 mg raloxifene (n = 51). Normalized APC system sensitivity ratios (nAPCsr) were determined in plasma collected at baseline and after 12 weeks using a thrombin generation-based APC resistance test probed with either recombinant APC (rAPC) or thrombomodulin (rTM). NAPCsr increased in both the conventional- and low-dose HT groups, consistent with reduced sensitivity to APC. The increase was slightly more pronounced in the conventional-dose group, but the difference between the two HT groups was not statistically significant. The sensitivity to APC was only marginally altered in those allocated to tibolone. Consequently, tibolone showed a different phenotype as compared with the low-dose HT group. A small increase in nAPCsr with both rAPC and rTM was seen in the raloxifene-group, but the increase was less than in the low-dose HT group. Our findings indicate that oestrogen-progestin therapy induces an APC resistant phenotype, which may be related to dose, whereas tibolone and raloxifene only marginally alter the sensitivity to APC.

Decreased plasma sensitivity to activated protein C by oral contraceptives is associated with decreases in plasma glucosylceramide.

Oral contraceptive (OC) use increases venous thrombosis (VTE) risk and causes activated protein C (APC) resistance. Plasma glucosylceramide (GlcCer) deficiency is associated with VTE and GlcCer functions as an APC anticoagulant cofactor. Because estradiol decreases GlcCer in cultured cells, we hypothesized OC use would decrease plasma GlcCer and contribute to APC resistance. In a pilot study, seven female adults alternatively took second and third generation OCs and plasma samples were analyzed for GlcCer using high performance liquid chromatography and for APC sensitivity using modified prothrombin time assays. Second and third generation OC usage decreased the APC sensitivity ratio by 8.1% +/- 4.7% (P = 0.004) and 11.7% +/- 8.2% (P = 0.013) and plasma GlcCer levels by 10.1% +/- 6.8% (P = 0.008) and 11.0% +/- 5.1% (P = 0.002), respectively. The plasma GlcCer level correlated with the sensitivity of plasma to APC (P = 0.017, r = 0.51, n = 21 plasma samples). Thus, both second and third generation OC usage decreased plasma GlcCer which could cause a reduction in the plasma sensitivity to APC/protein S, thereby potentially increasing VTE risk.

Effect of raloxifene on activated protein C (APC) resistance in postmenopausal women and on APC resistance and homocysteine levels in elderly men: two randomized placebo-controlled studies.

Raloxifene, a selective estrogen receptor modulator, like hormonal replacement therapy increases the risk of venous thromboembolism in postmenopausal women. A possible explanation for the increased thrombotic risk could be an increase in acquired resistance to activated protein C (APC). In two randomized, placebo-controlled, double-blind studies we determined the effect of raloxifene on the normalized APC sensitivity ratios (nAPCsr). The nAPCsr were determined with the thrombin generation-based APC resistance test. In the first study 83 postmenopausal women (age, 51.1 +/- 2.7 years) randomly received daily 0.625 mg conjugated equine estrogen and 2.5 mg medroxyprogesterone acetate (n=17), 60 mg raloxifene (n=23), 150 mg raloxifene (n=20) or placebo (n=23) for 24 months. At baseline and after 6, 12 and 24 months the nAPCsr were measured. In the second study 30 elderly men (age, 64.4 +/- 2.4 years) randomly received 120 mg raloxifene (n=15) or placebo (n=15) for 3 months. At baseline and after 3 months the nAPCsr and fasting homocysteine levels were measured. In postmenopausal women conjugated equine estrogen/medroxyprogesterone acetate significantly increased the nAPCsr from 1.26 +/- 0.82 to 2.87 +/- 0.86 at 24 months (P <0.0005 compared with placebo). Raloxifene had no significant effect on nAPCsr compared with placebo in both women and men. The results did not change after excluding carriers of factor V Leiden. Also fasting homocysteine levels were not affected by raloxifene in the aging men. It is concluded that raloxifene, in contrast to combined hormonal replacement therapy, does not increase APC resistance.

Impact of progestagens on activated protein C (APC) resistance among users of oral contraceptives.

Oral contraceptive (OC) use is associated with an increased risk of venous thromboembolism. Previous data reported higher thrombotic risk in women using third-generation combined OC than in those using second generation OC. The difference could be explained by differential effects of progestagens on plasma sensitivity to activated protein C (APC). The main purpose of this cross-sectional study was to assess the influence of a progestagen-only OC (chlormadinone acetate) as well as the effect of several combined OC with different progestagen components on APC resistance. The effect of APC on endogenous thrombin potential (ETP) was investigated in the plasma of healthy women using either combined OC (n=82) or progestagen-only OC (n=28), and in non-users (n=64). Carriers of factor V Leiden were excluded. Compared with non-users, there was no significant change in APC resistance in women using progestagen-only OC. Women who used combined OC were less sensitive to APC than non-users (P < 0.001) and the difference was significantly more pronounced in women using third-generation OC (n=41) than in those who used second-generation OC containing levonorgestrel (n=22) (P < 0.05). Compared with OC containing levonorgestrel, use of norethisterone-containing OC (n = 9) was associated with an increased resistance to APC (P < 0.05). Women who used cyproterone-containing OC (n = 10) were less sensitive to APC than those using third-generation OC (P < 0.05) or second-generation OC containing levonorgestrel (P < 0.05). Protein S, factor II and FVIII levels explained in part the OC-related changes in APC sensitivity variations. ETP-based APC resistance may contribute to explain why different brands of OC can be associated with different levels of thrombogenicity.

[Thrombophilic syndrome associated to phenotypic resistance to activated protein C in postmenopausal women]. / Sindrome trombofilica associata a resistenza fenotipica alla proteina C attivata in donne in postmenopausa.

AIM: Hormone replacement therapy (HRT) may reduce the risk of cardiovascular events in healthy postmenopausal women. However recent studies suggest a 2-4 fold increased risk of idiopathic venous thromboembolism (VTE) among users of HRT. Our aim was to evaluate the overall effect of HRT on hemostatic variables probably related to increased VTE risk reported in epidemiological studies. METHODS: Therefore, 100 healthy postmenopausal women aged 45-60 years divided into 50 HRT non-users and 50 HRT users were examined. The authors assayed on the automated coagulometer ACL7000 (Instrumentation Laboratory, Milan) the procoagulant proteins: factor VIII (VIII:C) and factor VII (VII:C); the natural anticoagulant proteins: antithrombin (ATIII), protein C (PC), protein S (PS) and the resistance to anticoagulant action of activated protein C (APC-Resistance). The free tissue factor pathway inhibitor (TFPI) was measured with an ELISA method (Diagnostica Stagò; France, Roche). The in vivo coagulation and fibrinolysis activation was evaluated by the assays of prothrombin fragment 1+2 (F1+2) and plasmin- antiplasmin complexes (PAP) using ELISA techniques. RESULTS: Increased levels of FVIII:C and FVII:C were observed in HRT users and HRT non-users women compared to controls (FVIII:C= 126+/-58%, 120+/-59% vs 85+/-15% p=0.0001; FVII: C 113+/-23%, 103+/-19% vs 90+/-16% p=0.0001). The activation peptides were significantly different compared to those found in control subjects; higher values were observed in HRT users compared to HRT non-users (F1+2=1.11+/-0.44 nM, 077+/-0.31 nM vs 0.45+/-0.35 p=0.00001; P-AP= 606+/-406 ng/ml, 514+/-205 ng/ml vs 235+/-59 p=0.0001). The ATIII and the PC were similar among the 3 different groups of subjects, but reduced levels of PS were observed in HRT users (PS 93+/-23%, 105+/-22% vs 109+/-12 p=0.0001). The mean normalized APC sensitivity ratio (APC-SR) was lower in the two populations of women as compared with that of controls (nAPC-SR 1.02+/-0.7, 1.02+/-0.8 vs 1.1+/-25 p=0.02). The values of free TFPI were reduced in HRT users compared to HRT non-users (9.1+/-1.9 ng/ml, 10.1+/-2.3 ng/ml vs 4.6+/-1.5 ng/ml p<0.0001). CONCLUSION: HRT appears to be associated to a shift in the procoagulant-anticoagulant balance towards a procoagulant state. The changes in hemostatic system could explain the increased risk of VTE in healthy postmenopausal women during HRT, nevertheless this risk could be higher in women known to have a congenital or acquired thrombophilic state.