Activated Protein C Sensitivity Ratio Research Articles

Gene Dosage of F5 c.3481C>T Stop-Codon (p.R1161Ter) Switches the Clinical Phenotype from Severe Thrombosis to Recurrent Haemorrhage: Novel Hypotheses for Readthrough Strategy.

Inherited defects in the genes of blood coagulation essentially express the severity of the clinical phenotype that is directly correlated to the number of mutated alleles of the candidate leader gene (e.g., heterozygote vs. homozygote) and of possible additional coinherited traits. The F5 gene, which codes for coagulation factor V (FV), plays a two-faced role in the coagulation cascade, exhibiting both procoagulant and anticoagulant functions. Thus, defects in this gene can be predisposed to either bleeding or thrombosis. A Sanger sequence analysis detected a premature stop-codon in exon 13 of the F5 gene (c.3481C>T; p.R1161Ter) in several members of a family characterised by low circulating FV levels and contrasting clinical phenotypes. The propositus, a 29 y.o. male affected by recurrent haemorrhages, was homozygous for the F5 stop-codon and for the F5 c.1691G>A (p.R506Q; FV-Leiden) inherited from the heterozygous parents, which is suggestive of combined cis-segregation. The homozygous condition of the stop-codon completely abolished the F5 gene expression in the propositus (FV:Ag < 1%; FV:C < 1%; assessed by ELISA and PT-based one-stage clotting assay respectively), removing, in turn, any chance for FV-Leiden to act as a prothrombotic molecule. His father (57 y.o.), characterised by severe recurrent venous thromboses, underwent a complete molecular thrombophilic screening, revealing a heterozygous F2 G20210A defect, while his mother (56 y.o.), who was negative for further common coagulation defects, reported fully asymptomatic anamnesis. To dissect these conflicting phenotypes, we performed the ProC®Global (Siemens Helthineers) coagulation test aimed at assessing the global pro- and anticoagulant balance of each family member, investigating the responses to the activated protein C (APC) by means of an APC-sensitivity ratio (APC-sr). The propositus had an unexpectedly poor response to APC (APC-sr: 1.09; n.v. > 2.25), and his father and mother had an APC-sr of 1.5 and 2.0, respectively. Although ProC®Global prevalently detects the anticoagulant side of FV, the exceptionally low APC-sr of the propositus and his discordant severe-moderate haemorrhagic phenotype could suggest a residual expression of mutated FV p.506QQ through a natural readthrough or possible alternative splicing mechanisms. The coagulation pathway may be physiologically rebalanced through natural and induced strategies, and the described insights might be able to track the design of novel treatment approaches and rebalancing molecules.

Analytical performance of the endogenous thrombin potential–based activated protein C resistance assay on the automated ST Genesia system

BackgroundThe evaluation of activated protein C (APC) resistance based on the endogenous thrombin potential (ETP) is recommended during the development of steroid contraceptives in women. In 2019, this assay was validated on the calibrated automated thrombogram (CAT) device. However, in view of its screening potential, its automation is essential. ObjectivesTo transfer the ETP‐based APC resistance assay on the ST Genesia system using reagent STG‐ThromboScreen with exogenous APC added. MethodDose‐response curves were performed to define APC concentration leading to 90% ETP inhibition on healthy donors. Intra‐ and interrun reproducibility was assessed. The normal range was defined on the basis of 56 samples from healthy individuals. The sensitivity was assessed on 40 samples from women using combined oral contraceptives (COCs). A method comparison with the validated ETP‐based APC resistance on the CAT system was performed. Results were expressed in normalized APC sensitivity ratio (nAPCsr). ResultsThe APC concentration leading to 90% ETP inhibition was 652 mU/mL. Intra‐ and interrun reproducibility showed standard deviation <4%. The nAPCsr normal range stood between 0.00 and 2.20. Analyses of 40 samples from women using COCs confirmed the good sensitivity of the assay. Compared to the CAT system, nAPCsr values were slightly higher on the automated system. ConclusionThis study is the first reporting the analytical performances of the ETP‐based APC resistance assay on an automated platform. Results support the concept that this test, when incorporated into clinical routine, could become a promising regulatory and clinical tool to document on the thrombogenicity of female hormonal therapies.

Estetrol (E4) is a native fetal estrogen that does not modify coagulation markers in postmenopausal women and maintains sensitivity to activated protein C (APC)

Objective: Compared to non-users, the risk of venous thromboembolism (VTE) has been reported to be increased in women using hormonal replacement therapy (HRT). This risk increase is explained, at least in part, by an acquired resistance towards activated protein C (APCr) which is induced by the estrogenic component. The APCr is assessed by measuring the amount of thrombin generated in the absence and presence of exogenous APC. Results are reported as normalized APC sensitivity ratio (nAPCsr), a scale which has been found to correlate with the VTE risk observed in COC and HRT users.

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

IntroductionHormonal 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 methodsPlasma 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. ResultsWomen 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. DiscussionWe 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.

Inhibitory effects of amniotic fluid on the activated protein C anticoagulation system in maternal plasma.

Pulmonary thromboembolism (PTE) is one of the leading causes of maternal mortality. We previously reported that possible contamination of amniotic fluid (AF) into maternal circulation accelerated thrombin production and activated platelet function in maternal blood through the extrinsic pathway, which may be associated with the high incidence of PTE in early puerperium. However, it remains unclear whether the maternal anticoagulation system, e.g., the activated protein C (APC) pathway, contributes to the hypercoagulable condition induced by AF. Our previous study using an endogenous thrombin potential (ETP)-based assay revealed that sensitivity to APC was reduced during the postpartum first day, i.e., immediately after delivery, when parturients were supposed to be exposed to AF. Our aim is to investigate the susceptibility of maternal plasma to APC when mixed with AF. We collected plasma from 51 pregnant females and mixed with AF as well as APC. APC-sensitivity ratio (APC-sr) was calculated using the ETP-based assay. Addition of AF to maternal plasma showed a significant increase of ETP in the presence of APC. APC-sr was significantly increased, indicating decreased sensitivity to APC, after AF mixture to maternal plasma. The present APC-sr difference with AF contamination was smaller than that we reported previously in venous thromboembolism cases. The inhibitory effects of AF on the APC anticoagulation pathway may contribute, at least partly, to further promotion of thrombin production induced by AF. Combined with other classical thrombophilic risk factors, the present findings support possible involvements of AF exposure in the high incidence of PTE in early puerperium.

Anti–Domain I β2-Glycoprotein I Antibodies and Activated Protein C Resistance Predict Thrombosis in Antiphospholipid Syndrome: TAC(I)T Study

Antibodies binding to domain I of β2-glycoprotein I (aDI) and activated protein C (APC) resistance are associated with an increased risk of thrombosis in cross-sectional studies. The objective of this study was to assess their predictive value for future thromboembolic events in patients with antiphospholipid antibodies (aPL) or antiphospholipid syndrome. This prospective multicenter cohort study included consecutive patients with aPL or systemic lupus erythematosus. We followed 137 patients (43.5 ± 15.4 year old; 107 women) for a mean duration of 43.1 ± 20.7 months. We detected aDI IgG antibodies by ELISA in 21 patients. An APC sensitivity ratio (APCsr) was determined using a thrombin generation-based test. The APCsr was higher in patients with anti-domain I antibodies demonstrating APC resistance (0.75 ± 0.13 vs 0.48 ± 0.20, P < 0.0001). In univariate analysis, the hazard ratio (HR) for thrombosis over time was higher in patients with aDI IgG (3.31 [95% CI, 1.15-9.52]; P = 0.03) and patients with higher APC resistance (APCsr >95th percentile; HR, 6.07 [95% CI, 1.69-21.87]; P = 0.006). A sensitivity analysis showed an increased risk of higher aDI IgG levels up to HR 5.61 (95% CI, 1.93-16.31; P = 0.01). In multivariate analysis, aDI IgG (HR, 3.90 [95% CI, 1.33-11.46]; P = 0.01) and APC resistance (HR, 4.98 [95% CI, 1.36-18.28]; P = 0.02) remained significant predictors of thrombosis over time. Our study shows that novel tests for antibodies recognizing domain I of β2-glycoprotein I and functional tests identifying APC resistance are significant predictors of thrombosis over time and may be useful for risk stratification.

Proof of concept of a new scale for the harmonization and the standardization of the ETP‐based APC resistance

BackgroundThe evaluation of the activated protein C resistance (APCr) based on the endogenous thrombin potential (ETP) is recommended during the development of steroid contraceptives. Results are usually expressed as “normalized APC sensitivity ratio” (nAPCsr) using a reference plasma that should achieve an ETP ratio of 0.1 in presence of exogenous APC. Because of the interassay variability, achieving exactly an ETP ratio of 0.1 in each run is almost impossible, which significantly affects the theoretical 0‐10 scale of nAPCsr. ObjectivesTo compare the nAPCsr to the nAPCsr10, a newly proposed method to express the degree of APC resistance. MethodsIndividual plasma samples (n = 854) were analyzed to compare nAPCsr and nAPCsr10. These values were obtained using the validated ETP‐based APCr assay. ResultsThe Spearman correlation between nAPCsr and nAPCsr10 had a coefficient of 0.99. Linear regression showed the following equation y = 0.9315*x + 0.03942 (r2 = .97). When differences (nAPCsr10 – nAPCsr) were plotted against nAPCsr10, the mean difference equaled 0.16% or 4.95%. The correction obtained with the use of the nAPCsr10 showed that the results of the nAPCsr were statistically different (P < .0001). ConclusionsThis new scale provides a harmonization and normalization of the nAPCsr. Results show a better reproducibility with the nAPCsr10. It avoids the additional variability and the unharmonized scale introduced by the use of a reference plasma. This adapted method for the calculation of the APC resistance could provide the regulatory and scientific bodies with more reproducible and harmonized evaluations.

ACL/β2GPI and aPS/PT show synergic thrombogenic effects in suppressing anticoagulant activity of APC and stimulating tissue factor expression and TNF-α secretion by mononuclear cells

IntroductionPatients with systemic lupus erythematosus (SLE) possessing anti-phospholipid antibodies (aPLs) are often complicated by thrombotic vascular events. aPLs commonly associated with the complications are anti-cardiolipin/β2-glycoprotein I antibodies (aCL/β2GPI) and anti-phosphatidylserine/prothrombin antibodies (aPS/PT). However, the pathological mechanisms leading to thrombosis remain unclear. We explored clinical features of SLE patients with aCL/β2GPI and aPS/PT and investigated thrombogenic effects of their IgG fractions. Materials and methodsWe enrolled 97 SLE patients and 38 healthy control volunteers and performed activated protein C (APC) resistance screening test using their plasma samples. To detect the direct effect of aPLs IgG on APC, we developed an APC sensitivity ratio assay. Effects of aPLs IgG on monocytes were studied by measuring the surface expression of tissue factor (TF) and excretion of TNF-α from peripheral blood mononuclear cell culture. Results and conclusionThrombotic complications among SLE patients were closely associated with aCL/β2GPI or aPS/PT, with higher prevalence in patients with both antibodies. Addition of aPLs(+)-IgG to the APC sensitivity ratio assay led to significant suppression of the anticoagulant activity of APC. The suppression was more pronounced in double-positive cases. TF expression on monocytes and concentration of TNF-α in culture medium were increased by aPLs, again more pronounced in double-positive cases. These results indicate that the effects of aCL/β2GPI and aPS/PT are synergic both for APC anticoagulant activity and for production of TF and TNF-α from mononuclear cells. These modes of thrombogenic action of aPLs could be an important target for developing specific measures to prevent complications of SLE.

Endogenous thrombin potential changes during the first cycle of oral contraceptive use

ObjectivesVenous thromboembolism (VTE) risk increases within months of combination oral contraceptive (COC) initiation. Because elevated endogenous thrombin potential (ETP) has been found in several studies to be a VTE risk factor, we evaluated the extent of ETP changes during the initial cycle of an ethinyl estradiol (EE) and levonorgestrel (LNG) COC. We also assessed the relationship between ETP changes and systemic EE and LNG concentrations. Study designParticipants provided multiple blood samples during a first 21-day cycle of a 30-mcg EE/150-mcg LNG COC and after a further 7 days without an active COC. Thrombin generation measured with and without addition of activated protein C (APC) yielded ETP+APC and ETP-APC and the normalized APC sensitivity ratio (nAPCsr). EE and LNG pharmacokinetic analyses were conducted over 24 h after the first COC tablet and again at steady state. ResultsThrombin generation was determined in 16 of the 17 women who completed the study. Mean ETP-APC increased steadily to 21% above baseline at 24 h after the 6th COC tablet (COC624; p<.001) and to 28% above baseline at steady state (COC21; p<.001). The percentage increase in mean ETP+APC was considerably more — 54% at COC624 and 79% at steady state. Mean nAPCsr increased by 28% at COC624 and by 41% at steady state. Higher concentrations of EE or LNG were not correlated with greater increases in ETP. ConclusionsETP increases during the first COC cycle were substantial. ImplicationsThe early increases in ETP may provide biological support for the rapid increase in VTE risk during initial COC use. The lack of association between this clotting system perturbation and the systemic EE concentration is surprising and deserves further study.

Identification and functional characterization of a novel F5 mutation (Ala512Val, FVBonn) associated with activated protein C resistance

Essentials Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. A novel missense mutation (Ala512Val - FVBonn ) was characterized in vitro and in silico. FVBonn is a new cause of APC resistance and venous thrombosis. FVBonn expresses additionally enhanced procoagulant activity in the absence of APC. Background Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. This phenotype is most commonly associated with the factor V Arg506Gln mutation (FV Leiden), which impairs the APC-mediated inactivation of both activated FV (FVa) and activated FVIII (FVIIIa). Objectives Here, we report the identification and characterization of a novel FV mutation (Ala512Val, FVBonn ) in six patients with APC resistance and venous thrombosis or recurrent abortions. Methods FVBonn was expressed in a recombinant system and compared with recombinant wild-type (WT) FV and FV Leiden in several functional assays. Results FVBonn conferred APC resistance to FV-depleted plasma, both in the activated partial thromboplastin time (APTT)-based test (APC sensitivity ratio [APCsr] of 1.98 for FVBonn versus 4.31 for WT FV and 1.59 for FV Leiden) and in the thrombin generation-based test (normalized APCsr of 5.41 for FVBonn versus 1.00 for WT FV and 8.99 for FV Leiden). The APC-mediated inactivation of FVaBonn was slower than that of WT FVa (mainly because of delayed cleavage at Arg506), but was greatly stimulated by protein S. The APC cofactor activity of FVBonn in FVIIIa inactivation was ~ 24% lower than that of WT FV. In line with these findings, an in silico analysis showed that the Ala512Val mutation is located in the same loop as the Arg506 APC cleavage site and might hamper its interaction with APC. Moreover, FVBonn was more procoagulant than WT FV and FV Leiden in the absence of APC, because of an increased activation rate and, possibly, an enhanced interaction with activated FX. Conclusions FVBonn induces hypercoagulability via a combination of increased activation/procoagulant activity, decreased susceptibility to APC-mediated inactivation, and slightly reduced APC cofactor activity.

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

IntroductionThe 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 MethodsBlood 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.ResultsAll 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.ConclusionsIn 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.

Superficial vein thrombosis, thrombin generation and activated protein C resistance as predictors of thromboembolic events in lupus and antiphospholipid patients. A prospective cohort study

IntroductionPredicting thrombosis in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid antibodies (aPL) is still challenging. Our objective was to determine risk factors for thrombotic events including activated protein C (APC) resistance proven by a thrombin generation (TG) assay in patients with SLE and/or aPL. Materials and methodsWe performed a prospective cohort study in a French University Hospital and tertiary care center. Ninety-two consecutive patients with SLE and/or aPL without ongoing anticoagulant treatment were enrolled. The outcome was time to thrombotic event. We evaluated clinical and laboratory variables including APC sensitivity ratio (APCsr) determined by TG. An APCsr>90th percentile of a control population indicated APC resistance. ResultsPatients were followed-up for a median duration of 35months (inter-quartile range: 26 to 62; 320 patient-years). Thrombosis during follow-up occurred in 18 patients. In univariate analysis, together with history of hypertension, superficial vein thrombosis (SVT) and arterial thrombosis, patients with both aPL and APC resistance had an increased risk for incident thromboembolic events (HR, 3.67[95% confidence interval, 1.31 to 10.31]). In multivariate analysis, only history of hypertension (HR, 10.77 [95% confidence interval, 3.15 to 36.83]), SVT (HR, 7.45 [95% confidence interval, 2.25 to 24.66]) and arterial thrombosis (HR, 3.31 [95% confidence interval, 1.14 to 9.55]) remained independent risk factors. ConclusionsHistory of thrombosis including seemingly benign SVT have a higher predictive value for incident thrombotic events in SLE or aPL patients than APC resistance proven by TG.

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.

Coagulation Functions with Clinical Phenotype and Inhibitory Mechanisms in Acquired Factor V Inhibitors.

Abstract 2232Development of acquired factor V (aFV) inhibitors rarely occurs, but its clinical phenotype varies from asymptomatic to life-threatening bleeding. A recent systematic report describes that little bleeding symptom is present in 20% at the diagnosis for patients with acquired FV (aFV) inhibitors. However, the coagulation function and its mechanism(s) on the different clinical phenotype are poorly understood. In this study, we examined the coagulation function on aFV inhibitors by using comprehensive coagulation assays, thrombin generation test (TGT) and clot waveform analysis (CWA). TGT was performed using tissue factor (0.5 pM), phospholipid (PL; 4 μM) and ellagic acid (0.3 μM). CWA, that evaluates the parameters of min1 as maximum coagulation velocity and min2 as maximum coagulation acceleration, was performed on MDA-II® system. We tested 7 cases with aFV inhibitors. Four cases were asymptomatic (FV:C; 3.6±3.4 IU/dl, inhibitor 5.8±3.3 BU/ml: non-B group), and 3 cases had severe bleeding tendency (2.9 ± 4.5 IU/dl, 66 ±51 BU/ml; B group). In TGT, all cases in both groups little showed the thrombin generation within 60 min, independently of FV:C level and clinical phenotype, showing little informative in functional evaluation for aFV inhibitors. However, in a PT-based CWA, the clotting time observed in non-B group was markedly shorted compared to that in B group (62.2±17.0/112±15 sec; p=0.006). In addition, both parameters in non-B group were significantly greater than those in B group ( min1 ; 2.89±1.10/0.98±0.29 dT/dt; p=0.014) and ( min2 ; 0.75±0.40/0.15±0.07 d2T/dt2; p=0.028), suggesting that CWA was useful for the prediction and monitoring of hemorrhagic symptoms in patients with aFV inhibitors. To confirm the distinct mechanism(s) on both groups, the IgGs from aFV plasmas were immune-purified using protein G-Sepharose. In the reactant mixtures with normal plasma and aFV IgGs, all parameters obtained in CWA were similar to those obtained in patients’ plasmas. SDS-PAGE and western blotting revealed that 2 cases in B group reacted the light chain of FV(a). However, 2 cases in non-B group reacted the heavy chain, and other 2 cases were reacted with both chains (heavy>light), indicative of the distinct epitopes of IgGs in both groups. Since the light chain contains the PL-binding site(s), the effects of aFV IgGs were examined on the FV-PL binding in an ELISA. All IgGs in B group inhibited this binding (by 40–90%) dose-dependently, whilst little affected in non-B group. Since FV acts as a cofactor of activated protein C (APC) on inactivation of FVIIIa, the effects of aFV IgGs on the ability of APC on FVIIIa inactivation were examined using intrinsic FXa generation assay. The APC sensitivity ratio (APCsr) was expressed as ratio of amounts of generated FXa in the absence of APC relative to its presence. A low level of APCsr indicates the reduction in FVIIIa inactivation, consequently APC resistance. APCsr values in B group were >2.0 within normal range, whilst those in non-B group were decreased to 1.5, supportive of APCR in non-B group. Based on these findings, we propose that severe bleeding tendency in B group would be appeared through negligible prothrombinase activity, since aFV IgGs blocked the FV(a)-PL binding. While, clinical phenotype in non-B group would be asymptomatic, since aFV IgGs unaffect the FV(a)-PL binding and further cause the APC resistance. In addition, various clinical phenotypes in aFV inhibitors appear to be dependent on the recognizing epitope of these IgGs. Disclosures:No relevant conflicts of interest to declare.

Sex hormone‐binding globulin levels are not causally related to venous thrombosis risk in women not using hormonal contraceptives

Oral contraceptive use increases the risk of venous thrombosis as well as sex hormone-binding globulin (SHBG) levels. Furthermore, increased SHBG levels are positively associated with activated protein C (APC) resistance and thrombotic risk in oral contraceptive users. To determine whether increased SHBG levels are causally related to venous thrombosis in women not using hormonal contraceptives. Premenopausal women were selected from a case-control study on venous thrombosis, the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA) study (23 patients; 258 controls). Women using hormonal contraceptives were excluded. First, the risk of venous thrombosis with SHBG levels above the normal reference range (70 nm) was determined. Second, because multiple regulatory factors affect SHBG levels and residual confounding may remain, we determined six single-nucleotide polymorphisms (SNPs) in the SHBG gene and assessed the risk of venous thrombosis in a different case-control study, the Leiden Thrombophilia Study (LETS) (20 patients; 74 controls), and in the MEGA study. Finally, the association between SHBG levels and the normalized activated partial thromboplastin time-based APC resistance (an intermediate endpoint for venous thrombosis) was determined. Elevated SHBG levels (> 70.0 nm) were associated with venous thrombosis (odds ratio 1.92; 95% confidence interval [CI] 0.74-5.00). However, this finding can be explained by residual confounding. Two SNPs in the SHBG gene affected SHBG levels, but not venous thrombosis risk. Furthermore, SHBG levels in controls were not associated with APC resistance (SHBG level, > 70.0 vs. ≤ 70.0 nm: mean difference in normalized APC sensitivity ratio, 0.03; 95% CI -0.05 to 0.10). Exclusion of women with FV Leiden did not materially change these results. Increased SHBG levels are not causally related to the risk of venous thrombosis.

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

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. 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. We conducted an observational study in 262 users of different contraceptives, and measured nAPCsr and SHBG levels. 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. SHBG is a useful marker with which to estimate the thrombotic safety of a preparation.

Genetic modulation of the FVLeiden/normal FV ratio and risk of venous thrombosis in factor V Leiden heterozygotes

The factor (F)V Leiden mutation causes activated protein C (APC) resistance by decreasing the susceptibility of FVa to APC-mediated inactivation and by impairing the APC-cofactor activity of FV in FVIIIa inactivation. However, APC resistance and the risk of venous thromboembolism (VTE) vary widely among FV Leiden heterozygotes. Common F5 genetic variation probably contributes to this variability. APC resistance was determined in 250 FV Leiden heterozygotes and 133 normal relatives using the prothrombinase-based assay, which specifically measures the susceptibility of plasma FVa to APC. The effects of 12 F5 single-nucleotide polymorphisms (SNPs) on the normalized APC sensitivity ratio (nAPCsr) and on FV levels were determined by multiple regression analysis. In FV Leiden heterozygotes,VTE risk increased with increasing nAPCsr, reaching an odds ratio (OR) of 9.9 (95% confidence interval [CI] 1.2–80.5) in the highest nAPCsr quartile. The minor alleles of several F5 SNPs, including 327 A/G (Q51Q), 409 G/C (D79H), 2663 A/G(K830R, T2 haplotype), 6533 T/C (M2120T) and 6755 A/G (D2194G, R2 haplotype), increased the nAPCsr in FV Leiden heterozygotes, but not in their normal relatives. Most of these effects could be attributed to a shift in the FV(Leiden)/normal FV ratio. Four FV Leiden heterozygotes with extremely high nAPCsr turned out to be pseudo-homozygotes, i.e. they carried a deleterious mutation on the non-Leiden allele. In FV Leiden heterozygotes, the prothrombinase-based nAPCsr is a marker of VTE risk and is modulated by common F5 SNPs that affect the FV(Leiden)/normal FV ratio in plasma.

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

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. To investigate the influence of the TFPI/protein S anticoagulant system on the risk of myocardial infarction (MI) in young women. 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. 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)] 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.

Resistance to activated protein C is a risk factor for pregnancy‐related venous thrombosis in the absence of the F5 rs6025 (factor V Leiden) polymorphism

Resistance to activated protein C (aPC) is most commonly due to the F5 rs6025 (factor V Leiden) polymorphism, which increases the risk of venous thrombosis. In the present population-based study of 313 cases and 353 controls, we investigated whether reduced sensitivity to aPC was associated with a history of pregnancy-related venous thrombosis. Calibrated automated thrombography was used to determine the sensitivity to aPC, and normalized aPC sensitivity ratio (n-aPC-sr) was calculated. Pregnant women and women using oral contraceptives and/or anticoagulants were excluded due to the effect on the n-aPC-sr. In women without the F5 rs6025 polymorphism, free tissue factor pathway inhibitor (TFPI), free protein S and protein C activity were associated with n-aPC-sr. Unadjusted odds ratio for venous thrombosis for women with n-aPC-sr in the 4th quartile as compared with n-aPC-sr below the 4th quartile was 2·4 (95% confidence interval 1·7-3·6). Adjusting for free protein S, free TFPI and age did not influence the odds ratios. Also in carriers of the F5 rs6025 polymorphism the risk for venous thrombosis was increased for women with higher n-aPC-sr. Our findings substantiate the importance of the aPC resistant phenotype as a risk factor for pregnancy-related venous thrombosis.

A Rapid Activated Protein C Sensitivity Test as a Diagnostic Marker for a Suspected Venous Thromboembolism in Pregnancy and Puerperium

Objective: The purpose of the study was to examine whether changes in response to activated protein C (APC) can be a diagnostic marker of venous thromboembolism (VTE) during pregnancy and puerperium. Methods: The normalized APC sensitivity ratio (sr) was examined in arbitrarily selected healthy Japanese pregnant females and compared with those in non-pregnant females and patients with VTE at the onset before anticoagulation in pregnancy and puerperium using an endogenous thrombin potential-based assay with a computer-assisted calibrated automated thrombography. Results:Sensitivity to APC in patients with VTE at onset was reduced in comparison to that in late pregnancy period and puerperium (p < 0.01, Student’s t test). The odds ratio for VTE was 31.9 with statistical significance in pregnant females with suspected clinical symptoms and APC-sr (≧5), although the odds ratio for VTE was not significant with D-dimer (≧5). Conclusion: These data suggest that an APC sensitivity test can be a possible surrogate diagnostic marker of suspected VTE during pregnancy and puerperium.