Stella Thomassen

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

Summary.  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.

Direct inhibition of factor VIIa by TFPI and TFPI constructs

Tissue factor pathway inhibitor (TFPI) is a multi‐Kunitz domain protease inhibitor that down‐regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa.

Rapid activation of haemostasis after hormonal emergency contraception

Hormonal emergency contraception (EC) is a well established contraceptive method, recommended to all women, although the effects on haemostais are not fully evaluated. The aim of this study was to ...

Inhibition of tissue factor:factor VIIa–catalyzed factor IX and factor X activation by TFPI and TFPI constructs

TFPI is a Kunitz‐type protease inhibitor that downregulates the extrinsic coagulation pathway by inhibiting factor Xa (FXa) and FVIIa. All three Kunitz domains (KD1, KD2, and KD3) and protein S are required for optimal inhibition of FXa and FVIIa. There is limited information on Kunitz domain requirements of the inhibition of TF:FVIIa–catalyzed FIX and FX activation by TFPI.

Role of exosite binding modulators in the inhibition of Fxa by TFPI.

Tissue factor pathway inhibitor (TFPI) down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. Both TFPI and FXa interact with several plasma proteins (e. g. prothrombin, FV/FVa, protein S) and non-proteinaceous compounds (e. g. phospholipids, heparin). It was our aim to investigate effects of ligands that bind to FXa and TFPI on FXa inhibition by full-length TFPI (designated TFPI) and truncated TFPI (TFPI1-150). Inhibition of FXa by TFPI and TFPI1-150 and effects of phospholipids, heparin, prothrombin, FV, FVa, and protein S thereon was quantified from progress curves of conversion of the FXa-specific chromogenic substrate CS11-(65). Low concentrations negatively charged phospholipids (~10 µM) already maximally stimulated (up to 5- to 6-fold) FXa inhibition by TFPI. Unfractionated heparin at concentrations (0.2-1 U/ml) enhanced FXa inhibition by TFPI ~8-fold, but impaired inhibition at concentrations > 1 U/ml. Physiological protein S and FV concentrations both enhanced FXa inhibition by TFPI 2- to 3-fold. In contrast, thrombin-activated FV (FVa) impaired the ability of TFPI to inhibit FXa. FXa inhibition by TFPI1-150 was not affected by FV, FVa, protein S, phospholipids and heparin. TFPI potently inhibited FXa-catalysed prothrombin activation in the absence of FVa, but hardly inhibited prothrombin activation in the presence of thrombin-activated FVa. In conclusion, physiological concentrations TFPI (0.25-0.5 nM TFPI) inhibit FXa with a t1/2 between 3-15 minutes. Direct FXa inhibition by TFPI is modulated by physiological concentrations prothrombin, FV, FVa, protein S, phospholipids and heparin indicating the importance of these modulators for the in vivo anticoagulant activity of TFPI.

APC resistance during the normal menstrual cycle

Increased serum levels of endogenous as well as exogenous estrogen are regarded to be responsible for acquired activated protein C (APC) resistance. It was the objective of this study to evaluate whether the physiological increase in serum estradiol concentration during the normal menstrual cycle affects the individuals sensitivity to APC. Seventy-two women with normal menstrual cycles were included in the study. Blood samples for analysis of estradiol (E2), progesterone (P4) and APC resistance were drawn at two time points of the menstrual cycle (day 3-5 and day 22-25). Two methods of measuring APC resistance were used: the activated partial thromboplastin time (aPTT)-based assay and the endogenous thrombin potential (ETP)-based APC resistance test. Independent of the method used, no changes in APC resistance were found, even though the E2 concentration increased significantly between the two menstrual phases. No correlations between E2 levels and APC resistance, P4 levels and APC resistance or changes in E2 concentrations and changes in APC resistance were detected. Ten women were carriers of the factor V(Leiden) mutation. Their baseline APC resistance was increased, but their response to elevated E2 during the menstrual cycle did not differ from that of non-carriers. In conclusion, our observations suggest that physiological differences in serum levels of estradiol and progesterone between the early follicular and the luteal phase in a normal menstrual cycle do not have any significant impact on the individuals sensitivity to APC.

Resistance to APC and SHBG levels during use of a four-phasic oral contraceptive containing dienogest and estradiol valerate: a randomized controlled trial

The use of combined oral contraceptives is associated with a 3‐ to 6‐fold increased risk of venous thrombosis. This increased risk depends on the estrogen dose as well as the progestogen type of combined oral contraceptives. Thrombin generation‐based activated protein C resistance (APC resistance) and sex hormone‐binding globulin (SHBG) levels predict the thrombotic risk of a combined hormonal contraceptive. Recently, a four‐phasic oral contraceptive containing dienogest (DNG) and estradiol valerate (E2V) has been marketed. The aim of this study was to evaluate the thrombotic risk of the DNG/E2V oral contraceptive by comparing APC resistance by measuring normalized APC sensitivity ratios (nAPCsr) and SHBG levels in users of oral contraceptives containing dienogest and estradiol valerate (DNG/E2V) and oral contraceptives containing levonorgestrel and ethinyl estradiol (LNG/EE).

Platelet full length TFPI-α in healthy volunteers is not affected by sex or hormonal use

Background Only 10% of plasma TFPIα (TFPI) exists in the full length form, the rest circulates as a C-terminally truncated form. However, blood platelets exclusively contain full length TFPI, which is released at the site of injury upon platelet activation, and which could play an important local regulatory role in thrombin generation and prevention of thrombosis. Methods The anticoagulant activities of full length and truncated TFPI were investigated using thrombin generation assays. Blood samples were obtained from 30 healthy volunteers (10 male subjects, 10 female subjects, and 10 females using oral contraceptives). Platelet TFPI was released in platelet rich plasma and in platelet isolates using convulxin or thrombin, and measured by free TFPI ELISA and thrombin generation assays. Results Full length TFPI and platelet TFPI were much more potent inhibitors of thrombin generation than truncated TFPI, which was virtually inactive. Although mean plasma TFPI antigen levels decreased from men (0.30 nM) to women (0.20 nM) to women using oral contraceptives (0.11 nM), no relevant differences were found in platelet TFPI among those subgroups. Conclusions Platelets release similar amounts of TFPI regardless of plasma TFPI concentrations and is unaffected by sex or oral contraceptive use. We speculate that platelet TFPI is important to prevent systemic coagulation and thrombosis and restrict thrombus formation to the site of the growing platelet plug. The stable contribution of platelet TFPI to the anticoagulant potential in plasma is likely to become particularly relevant under conditions of low plasma TFPI levels in combination of oral contraceptives use.

Sex hormone-binding globulin as a marker for the thrombotic risk of hormonal contraceptives: reply to a rebuttal: Letters to the Editor

M. RAPS ,* F . M. HELMERHORST ,*† K . FLE I SCHER ,‡ V. A . VAN HYLCKAMA,* B . H . STEGEMAN,§¶ S . THOMASSEN ,** F . R . ROSENDAAL ,* J . ROS ING ,** B . E . P . B . BALL I EUX†† and H . A . A . M. VAN VL IET†‡‡ *Department of Clinical Epidemiology, Leiden University Medical Center; †Department of Reproductive Medicine, Leiden University Medical Center, Leiden; ‡Department of Obstetrics and Gynecology, University Medical Center St Radboud, Nijmegen; §Department of Thrombosis and Haemostasis, Leiden University Medical Center; ¶Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center; **Department of Biochemistry, Maastricht University, Maastricht; ††Department of Chemistry, Leiden University Medical Center, Leiden; and ‡‡Department of Gynecology, Catharina Hospital, Eindhoven, the Netherlands

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

OBJECTIVES Venous 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 DESIGN Participants 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. RESULTS Thrombin 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. CONCLUSIONS ETP increases during the first COC cycle were substantial. IMPLICATIONS The 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.