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.

Blood group genotyping in a multitrauma patient: a case report.

Currently DNA-based analysis of blood groups is mainly used to improve transfusion safety by reducing alloantibody formation in multiply transfused patients and by monitoring pregnancies at risk for hemolytic disease of the fetus and newborn. We present a case in which genotyping was performed after massive transfusion with unmatched group O, D- blood in a trauma setting. Our patient was genotyped as O1A1 and predicted to be D-, and we therefore transfused group A, D- red blood cell concentrates. This case demonstrates how the use of blood group genotyping in an acute setting can lead to a decrease in the unnecessary use of group O, D- blood products.

In vitro comparison of platelet storage in plasma and in four platelet additive solutions, and the effect of pathogen reduction: a proposal for an in vitro rating system.

BACKGROUND: The introduction of platelet (PLT) additive solutions (PASs) and pathogen reduction (PR) technologies possibly allow extension of PLT shelf life. It was our aim to compare in vitro quality of leucocyte-reduced PLT concentrates (PCs) stored in various PASs, including PR, with those in plasma during 8 days of storage. The study was performed in four blood centres where each tested four conditions. STUDY DESIGN AND METHODS: In paired experiments (n = 12), buffy coat pools were made to which various storage media were added. Plasma served as reference; two centres used InterSol followed by PR (InterSol+PR) and InterSol without PR; T-sol, SSP+ and Composol were also studied. RESULTS: All PCs fulfilled release criteria (pH(37 degrees C)>6.6; swirl present) until Day 8. Marked differences were seen for other parameters, including CD62P expression: 28 +/- 5; 31 +/- 7; and 39 +/- 9% for T-sol, Intersol+PR and without PR, respectively, which were higher as found for Composol (12 +/- 3%), SSP+ (15 +/- 5%) and plasma (15 +/- 6%). Three parameters (CD62P, Annexin A5, and lactate concentration) were collapsed into one rating value (6 = good quality, 0 = poor quality); PLTs in plasma had a rating of 2.8 +/- 1.0, which was higher as for T-Sol (1.5 +/- 0.5), InterSol+PR (1.3 +/- 0.6) and without PR (1.7 +/- 0.5). PLTs in potassium- and magnesium-containing PASs showed higher ratings as plasma, 4.3 +/- 0.5 for Composol and 3.8 +/- 0.8 for SSP+. CONCLUSION: PLT concentrates in plasma, SSP+ and Composol scored better using an arbitrary rating system as PLTs stored in T-Sol or InterSol; PR further impaired rating parameters. The applicability of these differences in rating for clinical effects needs a clinical study.

Increased thrombin generation in women with a history of preeclampsia.

INTRODUCTION: Women with a history of preeclampsia have an increased risk for cardiovascular disease in later life. We evaluated thrombogenic characteristics of women with a previous history of preeclampsia, expressed in levels of thrombin generation, number of microparticles and related to menstrual cycle and endothelial function, measured as flow-mediated dilatation. MATERIALS AND METHODS: We included 18 primipara women with a history of preeclampsia and 17 healthy primipara controls, 15 (+/-3) months after the index pregnancy. Thrombin generation was measured by tissue factor triggered assay, microparticle levels were measured by flow cytometry and the endothelial function was previously examined by measuring flow-mediated dilatation by high-resolution ultrasound, during follicular and luteal phases of the menstrual cycle. RESULTS: Women with previous preeclampsia produced more total amount of thrombin as calculated from thrombin max, thrombin potential and max slope levels p<0.05, 0.01 and 0.01 respectively. Platelet derived microparticle levels were higher in women with a history of preeclampsia, p=0.07. Flow-mediated dilatation was significantly decreased in comparison to healthy controls (p<0.0001). There were no variation in levels of thrombin, microparticles and flow-mediated dilatation during the menstrual phases. CONCLUSION: Women with a history of preeclampsia show signs of hypercoagulability as indicated by higher thrombin generation and higher platelet derived microparticle levels. Since these women were investigated more than one year after delivery, these results may be indicative of an increased risk of cardiovascular events later in life.

Microparticles adhere to collagen type I, fibrinogen, von Willebrand factor and surface immobilised platelets at physiological shear rates.

Microparticles (MPs), shed during the storage of platelets, support blood coagulation and could be helpful in restoring the haemostatic system in thrombocytopenic patients. The mechanisms by which MPs support haemostasis under flow conditions were investigated. Fluorescent-labelled MPs were perfused at shear rates of 100 and 1000/s over surfaces coated with collagen, fibrinogen, von Willebrand factor (VWF) or surface-adherent platelets. Adhesion was monitored in real-time by fluorescence microscopy. In addition, thrombin-antithrombin (TAT) complex formation was measured in flowing thrombocytopenic blood. MPs attained the capacity to firmly adhere to collagen, VWF, fibrinogen and surface-adherent platelets at high and low shear rate. Antibodies against glycoprotein Ibalpha and alpha(IIb)beta(3) were used to demonstrate the specificities of these interactions. The addition of MPs to thrombocytopenic blood did not affect platelet adhesion. TAT complex formation was increased in the presence of MPs in capillaries coated with fibrinogen, but not on collagen fibres. We confirmed that MPs adhere to a damaged vascular bed in vivo after infusion in denuded arteries in a mouse model. MPs have platelet-like adhering properties and accelerate thrombin generation. These properties strongly support the notion that MPs can be beneficial in maintaining normal haemostasis when platelet function is impaired or reduced, as in thrombocytopenic patients.

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, 17beta-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 (Delta E2 = 6.6 nmol/l) and the APCsr (Delta APCsr = 0.30). Six women who participated in this study became pregnant. Compared with baseline, the APCsr was increased 1.9-fold (Delta APCsr = 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.

Oral contraceptives, thrombosis and haemostasis.

The use of oral contraceptives is a well-established acquired risk factor for venous thrombosis. In 1995, a number of epidemiological studies were published which suggested that women who use third generation oral contraceptives that contain desogestrel or gestodene as progestagen are exposed to a two- to threefold higher risk for venous thrombosis than women using second generation oral contraceptives which contain levonorgestrel. In this paper, the effects of oral contraceptives on the haemostatic system are discussed. It appears that plasma from oral contraceptive users is resistant to the anticoagulant action of activated protein C (APC). This phenomenon, called acquired APC resistance, is more pronounced in users of desogestrel or gestodene-containing oral contraceptives than in women who use oral contraceptive pills with levonorgestrel. On the basis of these observations, it was proposed that acquired APC resistance may be the mechanistic basis of the increased risk for venous thrombosis during oral contraceptive use and for the further increased thrombotic risk of third generation oral contraceptive users. Furthermore, the results of a recent cross-over study are discussed. This study indicated that a large number of other haemostatic parameters were changed during oral contraceptive use. Some of these changes were more pronounced on desogestrel-containing oral contraceptives. The cross-over study also showed that the increased fibrinolytic activity during OC use is counterbalanced by an enhanced activity of thrombin-activatable fibrinolysis inhibitor (TAFI), a protein that participates in the inhibition of fibrinolysis.

Activated protein C resistance during in vitro fertilization treatment.

Acquired resistance to the anticoagulant action of activated protein C (APC) has been proposed to explain the increased risk of venous thrombosis associated with pregnancy, hormone replacement therapy and the use of oral contraceptives. In this study, we have investigated whether the hormonal changes induced during in vitro fertilization (IVF) treatment are also associated with acquired APC resistance. Twenty-nine women, who were planned for an IVF cycle, donated blood at four time points during treatment, i.e. at baseline, down-regulation, hyperstimulation and luteal support. In the plasma samples, APC sensitivity ratios (APCsr) and the levels of progesterone and estradiol were measured. The changes in plasma concentrations of hormones were in accordance with literature. The APCsr increased significantly during hyperstimulation and remained high during luteal support. The extent of APC resistance occurring during IVF treatment was comparable to that observed during the use of second generation OC and was less pronounced than that occurring during pregnancy. The change in estradiol between baseline and hyperstimulation correlated with the change in APCsr. Although this suggests that plasma estrogen levels are an important determinant for acquired APC resistance, it remains to be established which plasma proteins are responsible for estrogen-induced APC resistance.

A randomized cross-over study on the effects of levonorgestrel- and desogestrel-containing oral contraceptives on the anticoagulant pathways.

The use of oral contraceptives (OC) causes disturbances of the procoagulant, anticoagulant and fibrinolytic pathways of blood coagulation which may contribute to the increased risk of venous thrombosis associated with OC therapy. Here we report the results of a cycle-controlled randomized cross-over study, in which we determined the effects of so-called second and third generation OC's on a number of anticoagulant parameters. In this study, 28 non-OC using women were randomly prescribed either a second generation (150 microg levonorgestrel/30 microg ethinylestradiol) or a third generation OC (150 microg desogestrel/30 microg ethinylestradiol) and who switched to the other OC after a two month wash out period. The anticoagulant parameters determined were: antithrombin (AT), alpha2-macroglobulin (alpha2-M), alpha1-antitrypsin, protein C inhibitor (PCI), protein C, total and free protein S and activated protein C sensitivity ratios (APC-sr) measured with two functional APC resistance tests which quantify the effect of APC on either the activated partial thromboplastin time (aPTT) or on the endogenous thrombin potential (ETP). During the use of desogestrel-containing OC the plasma levels of alpha2-M, alpha1-antitrypsin, PCI and protein C significantly increased, whereas AT and protein S significantly decreased. Similar trends were observed with levonorgestrel-containing OC, although on this kind of OC the changes in AT, PCI and protein S (which was even slightly increased) did not reach significance. Compared with levonorgestrel, desogestrel-containing OC caused a significant decrease of total (p <0.005) as well as free protein S (p <0.0001) and more pronounced APC resistance in both the aPTT (p = 0.02) and ETP-based (p <0.0001) APC resistance tests. These observations indicate that the activity of the anticoagulant pathways in plasma from users of desogestrel-containing OC is more extensively impaired than in plasma from users of levonorgestrel-containing OC.

Time course of alterations in phospholipid fatty acids and number of beta-adrenoceptors in the rat heart during adrenergic stimulation in vivo.

The purpose of this study was to test the hypothesis that catecholamine induced down-regulation of beta-adrenoceptors in cardiac muscle is facilitated by modifications of the lipid milieu in cellular membranes. The time course of down-regulation of beta -adrenoceptors and changes in the fatty acid composition of phospholipids was examined in the rat heart during adaptation to repeated epinephrine administration. By this we studied a possible relationship between modulation of the membrane phospholipids and the properties of beta-adrenoceptors during 7 days of epinephrine administration. The fatty acid composition of cardiac membrane phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and Bmax and Kd of [3H]dihydroalprenolol binding to beta -adrenoceptors were measured in rats after 1, 2, 3, 5 and 7 days of epinephrine administration. The in vivo adrenergic stimulation led to a significant response of phospholipid fatty acyl chains. In both PC and PE the linoleic acid (18:2n-6) level decreased markedly. The docosahexanoic acid (22:6n-3) level increased in PE and the arachidonic acid (20:4n-6) level increased in PC. These fatty acid changes were all significant after 3-5 days of epinephrine administration. During the 7 day epinephrine administration, the polyunsaturated fatty acid levels in the phospholipids of purified cardiac sarcolemma changed in the same way as in the phospholipids of whole ventricular muscle. The number of binding sites of beta -adrenoceptors (Bmax) decreased as expected. The decrease in Bmax occurred later than the changes in their lipid environment and was only significant after 7 days of epinephrine administration. The conclusion is that during adaptation to epinephrine administration, the down-regulation of beta-adrenoceptors is preceded by alterations in the polyunsaturated fatty acid composition of phospholipids in heart muscle. This supports the concept of a regulatory role of membrane lipids in the response of beta-adrenoceptors to prolonged stimulation.

Acquired APC resistance and oral contraceptives: differences between two functional tests.

Resistance to activated protein C (APC) is often associated with a mutation in factor V (factor VLeiden). Individuals without factor VLeiden who exhibit a response in functional APC-resistance tests similar to that of carriers of factor VLeiden are considered to be acquired APC resistant. This phenomenon is particularly observed in women using oral contraceptives (OC). In the present study we compared the response to APC in plasma from normal individuals, carriers of factor VLeiden and women who use OC using functional tests that either quantify the effect of APC on the endogenous thrombin potential (ETP) or on the activated partial thromboplastin time (aPTT). Both tests discriminated equally well between individuals with and without factor VLeiden who were not using OC. In contrast to the aPTT-based test, the ETP-based assay yielded significant differences in sensitivity to APC between non-OC users and OC users and between users of second and third generation OC. Since there was no correlation between APC-sensitivity determined with both assays in non-carriers of factor VLeiden and in women who use OC and a poor correlation in carriers of factor VLeiden, we propose that other plasma components differentially modulate the response to APC in the aPTT- and ETP-based APC-resistance tests and that OC change the level of plasma protein(s) that modulate the effect of APC on thrombin formation initiated via the extrinsic coagulation pathway.

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.

PIP: This cycle-controlled randomized cross-over study examined the effects of a second-generation oral contraceptive (OC) containing levonorgestrel and a third-generation OC containing desogestrel on the anticoagulant action of activated protein C (APC) in the plasma. The response to APC in plasma was assessed in 28 women who received two consecutive cycles of a second-generation OC (150 mcg levonorgestrel and 30 mcg ethinyl estradiol) or a third-generation OC (150 mcg desogestrel and 30 mcg ethinyl estradiol), and who switched preparations after two pill-free cycles. Normalized APC sensitivity ratio was also taken from these women. Results showed that in the 28 women the normalized APC sensitivity ratio increased during treatment with both preparations. Compared with levonorgestrel, desogestrel-containing OC treatment caused a highly significant (p 0.0001) additional increase in normalized APC sensitivity ratio (0.51; 95% CI, 0.37-0.66). In conclusion, OC treatment diminishes the efficacy with which APC down-regulates in-vitro thrombin formation.