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Introduction Hormonal 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 methods Plasma 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. Results Women 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. Discussion We 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.

BACKGROUND The 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. OBJECTIVES To compare the nAPCsr to the nAPCsr10 , a newly proposed method to express the degree of APC resistance. METHODS Individual plasma samples (n = 854) were analyzed to compare nAPCsr and nAPCsr10 . These values were obtained using the validated ETP-based APCr assay. RESULTS The 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). CONCLUSIONS This 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.

Hypothesis/aims of study. The current analysis was undertaken to elucidate the role of Factor Va resistance to proteolytic cleavage by activated protein C in FVL(1691)GA female carriers in the development of acute and recurrent thromboses. Study design, materials and methods. A prospective clinical cohort study of 1100 women of reproductive age was conducted, with the course and outcomes of 2,707 pregnancies analyzed. Two cohorts were specified: the main group consisted of 500 patients with FV(1691)GA genotype, and the control group consisted of 600 patients with FVL(1691)GG genotype. Results. FVL(1691)GA genotype was significantly associated with the development of venous thromboembolic complications (VTEC) compared to FVL(1691)GG genotype (OR 9.3; p < 0.0001). Episodes of recurrent thrombosis during and outside of pregnancy were registered only in FVL(1691)GA patients (OR 5.7, p = 0.2). In all cases, at the time of the thrombotic event and during the period before the episode of acute or recurrent thrombosis, an APC resistance normalized ratio (NR) value was ≤ 0.49, with no episodes of VTEC registered with an APC resistance NR value ≥ 0.5. Conclusion. Venous thromboses occur under the condition of expressed APC resistance with underlying FVL(1691)GA carriage. The APC resistance index can serve as an objective biochemical marker to determine the feasibility of thromboprophylaxis within the framework of personalized medicine.

We present a case of the multiple venous intestinal infarction in patient with two inherited thrombophilias: Leiden factor V (LFV) and factor VIII elevation. The patient had a critical hypocoagulation caused by vitamin K antagonist (VKA) overdose. At laparotomy, several intestinal segments were necrotic and ischemic. Coagulopathy was corrected by the transfusion of the fresh frozen plasma. Because of the 4th duodenal segment infarction distal segmental duodenectomy with side-to-side duodenojejunostomy was done, which is a rarely performed procedure. On postoperative day 6 deep vein thrombosis developed, despite nadroparin profillaxes, early mobilisation and compressive stockings. Our case demonstrated that in patients with congenital thrombophilia, development of the mesenteric venous thrombosis is possible even with VKA induced severe hypocoagulation. Venous infarction of the small bowel can be associated with the hemoperitoneum and gastrointestinal bleeding. After resection of the fourth duodenal segment, side-to-side duodenojejunostomy is a feasible method of reconstruction.

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. SummaryBackground 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.

Summary. Background and Objectives: 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. Patients/methods: 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. Results: 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 FVLeiden/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. Conclusions: In FV Leiden heterozygotes, the prothrombinase-based nAPCsr is a marker of VTE risk and is modulated by common F5 SNPs that affect the FVLeiden/normal FV ratio in plasma.

Proteolytic inactivation of factors Va (FVa) and VIIIa (FVIIIa) by activated protein C (APC) and its cofactors protein S and factor V (FV) is a key process in the physiological down-regulation of blood coagulation. Functional abnormalities of this pathway, which manifest themselves in vitro as a poor anticoagulant response of plasma to added APC (APC resistance), are prevalent in the general population and are associated with an increased risk of venous thrombosis. APC resistance was originally discovered in thrombophilic families and later shown to be associated with the common FV Arg506Gln (FV(Leiden)) mutation, which abolishes one of the APC-cleavage sites in FV. Although FV(Leiden) is the major cause of hereditary APC resistance, it is becoming increasingly clear that several other genetic and acquired conditions contribute to APC resistance and thereby increase the risk of venous thrombosis. This paper reviews the multifactorial etiology of APC resistance and discusses its clinical implications.

Point（1）妊娠中の抗凝固療法は胎盤通過性のないヘパリン（もしくは類似物質）のみである．（2）ヘパリン誘発性血小板減少（HIT）発症時は代替薬剤としてのヒルジンアナログ（アルガトロバン）は胎盤通過性のため使用できない．（3）低用量アスピリンは動静脈血栓症を起こしうる抗リン脂質抗体症候群（APS）のみならず，胎盤内凝固の抑制を目的として妊娠中使用することがある．（4）胎盤内ならびに産褥子宮内での凝固線溶現象の結果，FDP-DD値は妊娠産褥期，DVTの診断の参考にならない．（5）妊婦では生理的に遊離型プロテインS活性低下がおこり，妊娠産褥期は活性化プロテインC（APC）に対する感受性が低下する．（6）妊娠中は凝固因子の増加により過凝固状態となる．

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.

The incidence of venous thrombosis in the general population is low, approximately 3 per 10 000 women per year among women in reproductive age. Despite the low incidence, use of hormonal contraceptives frequently causes venous thrombosis since millions of women worldwide use hormonal contraceptives. This use is associated with a two- to eight-fold increased risk of venous thrombosis, dependent on the type and dose of the contents of the hormonal contraceptive. The aim of this thesis was to study the risk of venous thrombosis during use of different hormonal contraceptives, focusing on the pathogenesis, the evaluation of a marker to estimate the risk of venous thrombosis, and the assessment of effectiveness, side effects including the risk of venous thrombosis and acceptability of a new hormonal contraceptive.

Acquired resistance to activated protein C has been reported during oral contraception and pregnancy. Its thrombogenic potential was studied in 41 neurosurgical patients who were enrolled in the placebo group of a thromboprophylaxis trial, Normalized activated protein C sensitivity ratio (nAPC-SR), clotting activity of factors V and VIII, and levels of protein C antigen were measured prior to and at days 3 and 7 after surgery. Bilateral venography was done in all patients at days 8-10 to demonstrate deep vein thrombosis. A lowered nAPC-SR was found in 76% (baseline), 80% (day 3), and 88% (day 7) of patients. It was inversely related to factor VIII clotting activity (p = .0003) and protein C antigen, (p = .02). Deep vein thrombosis was demonstrated in 30% of patients with a normal nAPC-SR and in 23% of patients with a lowered nAPC-SR. Pulmonary embolism was not observed. Multivariate analysis did not identify a lowered nAPC-SR as a thrombotic risk factor, in contrast with gender (women, p = .02) and Quetelet index (greater than or equal to 25 kg/m(2), p = .006). Our data provide no evidence that acquired activated protein C resistance, frequently found in neurosurgical patients, contributes to their high risk of postoperative deep vein thrombosis.

A successful pregnancy is dependent on the development of adequate placental circulation. The abnormalities of placental vasculature may result in a number of gestational pathologies, including fetal loss. The aim of our study was to determine whether women with f V Leiden are at an increased risk of pregnancy loss. For this purpose we assessed three groups of women. In a prospective group we examined 30 females with spontaneous abortions for f V Leiden. In a retrospective group we assessed the frequency of abortions in 80 women (l72 pregnancies) with f V Leiden (72 heterozygous, 8 homozygous) from 57 unrelated families. In a control group we evaluated the frequency of abortions in 45 women without f V Leiden. Factor V Leiden was found in 3% of women in the 1st group. Fetal loss occurred in 10% of women in the 2nd group and in 9% in the 3rd group. Factor V Leiden was not found to be a risk factor for fetal loss in our study group.

Objective: To raise awareness among health care providers caring for an active population to an uncommon genetic mutation that increases the risk for a potentially fatal venous thromboembolism. Background: A 19-year-old previously healthy female collegiate soccer athlete complained of coughing and progressively decreased exercise tolerance, which were attributed to a recent illness and lack of sleep. Later that evening, she complained of dyspnea and pleuritic pain and was referred to the emergency department. Bilateral pulmonary emboli were identified with computed tomography, and a hypercoagulable panel revealed that the patient was heterozygous for the factor V Leiden mutation. Differential Diagnosis: Pneumonia, pneumothorax, pericarditis, pleuritis, gastroesophageal reflux disease, pulmonary embolism. Treatment: Intravenous heparin therapy was initiated immediately in the emergency department. This was followed by inpatient anticoagulant therapy for 5 days and outpatient anticoagulant therapy for an additional 12 months. During this time, the patient was unable to participate in soccer drills or return to competition and was limited to conditioning activities due to the risk of increased bleeding time. Uniqueness: Documented cases of pulmonary embolism in a young athletic population are rare and are usually associated with genetic risk factors. Factor V Leiden is a relatively uncommon genetic mutation that dramatically increases the risk for venous thromboembolism. Although the fatality rate in this population is low, fatality is preventable if the condition is recognized early and managed properly. Conclusions: Athletes should be encouraged to communicate with their athletic trainers regarding any changes in health status or medication usage. When an athlete presents with nonspecific symptoms such as dyspnea and chest pain, athletic trainers should consider the possibility of pulmonary embolism. A high degree of suspicion results in early diagnosis and treatment and may prevent a fatal event.

Poor anticoagulant response to activated protein C (APC), present in 20–60% of thrombophilic patients, is most often caused by abnormal factor Va due to the mutation of Arg506 to Gln, and DNA sequencing confirms this finding. At Scripps Reference Laboratory (SRL), we have validated an in-house assay to detect APC resistance. A study of 80 normal subjects (40 males and 40 females, 21–60 years old) showed that adult males and females have statistically significant differences in their anticoagulant response to APC. Furthermore, APC response is increased in older individuals. APC responses of the same 80 normal samples determined using the SRL assay were compared to a commercial kit (Chromogenix®, Coatest). Although both procedures are similar, the SRL assay gave a greater difference between male and female normal ranges. Eight of 18 (44%) thrombophilic patients were identified as APC-resistant. Samples from individuals having the factor V Arg506Gln mutation yielded low responses to APC in both assays. Determination of APC resistance assists physicians in determining the cause of thrombotic disorders and may eventually help in predicting the risk of familial thrombosis.

BACKGROUND High body mass index (BMI) is associated with an increased risk of venous thrombosis (VT). Clotting factor VIII levels are increased in obese subjects, possibly because of a chronic inflammatory state, which increases activated protein C (APC) resistance. The APC resistance in FV Leiden carriers could be aggravated and further worsened by high FVIII levels in blood group non-O carriers. We hypothesized that an association exists between BMI and APC resistance, and that this is amplified by the presence of FV Leiden and/or blood group non-O. METHODS We used the Leiden Thrombophilia Study (LETS) to determine whether an association exists between BMI and APC resistance, and whether the combination of high BMI and APC resistance increases the risk of VT. In a pooled analysis of LETS and a Norwegian case-cohort study (TROL), we verified whether FV Leiden modified the risk of the occurrence of VT with increasing BMI, and whether this risk was further increased by blood group non-O. RESULTS APC resistance increased linearly with increasing BMI, partly because of a concurrent rise in FVIII. A BMI in the median or upper tertile was associated with a 1.9-fold (95% confidence interval [CI] 1.0-2.5) and 2.2-fold (95% CI 1.4-3.4) increased risk as compared with the lowest tertile. Both relative risks decreased slightly after FVIII and APC resistance adjustments. The effect of BMI on VT risk was enhanced two-fold to 10-fold in FV Leiden or blood group non-O carriers. CONCLUSIONS The increased risk of VT in individuals with high BMI is partly mediated by FVIII-related APC resistance. This risk is more pronounced when other causes of increased APC resistance are also present.

Summary. Background: 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. Objectives: To investigate the influence of the TFPI/protein S anticoagulant system on the risk of myocardial infarction (MI) in young women. Methods: 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. Results: 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)] Conclusion: 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.

In this issue of Blood, Nogami et al report on a novel factor V (FV) gene mutation (FV Trp1920?Arg, FVNara) associated with activated protein C (APC) resistance and a severe thrombotic phenotype in a young Japanese patient. Since the affected amino acid residue is located in the light chain of FV, far from the known APC-cleavage sites, this discovery may afford new insights into the molecular mechanisms of APC resistance.

Autistic disorder is a severe neurodevelopment disorder characterized by a triad of impairments in reciprocal social interaction, verbal and nonverbal communication, and a pattern of repetitive stereotyped activities, behaviours and interests. There are strong lines of evidence to suggest that the immune system plays an important role in the pathogenesis of autistic disorder. The aim of this study was to analyze quantitative plasma concentration of immunoglobulin classes, and subclasses in autistic patients and their families. The investigation was performed retrospectively in 50 persons with autistic disorder in the Republic of Macedonia. Infantile autistic disorder was diagnosed by DSM-IV and ICD-10 criteria. Plasma immunoglobulin classes (IgM, IgA, and IgG) and subclasses (IgG1, IgG2, IgG3, and IgG4) were determined using Nephelometer Analyzer BN-100. Multiple comparisons for the IgA variable have shown statistically significant differences between three pairs: male autistic from the fathers (p = 0,001), female autistic from the mothers (p = 0,008), as well as healthy sisters from the fathers (p = 0,011). Statistically significant differences found between three groups regarding autistic disorder (person with autistic disorder, father/mother of a person with autistic disorder, and brother/sister) independent of sex belongs to IgA, IgG2, and IgG3 variables. Multiple comparisons for the IgA variable have shown statistically significant differences between children with autistic disorder from the fathers and mothers (p < 0,001), and healthy brothers and sisters from the fathers and mothers (p < 0,001). Comparison between healthy children and children with autistic disorder from the same family should be tested for immunoglobulin classes and subclasses in order to avoid differences between generations.

To The Editor: Pseudo-homozygous APC resistance[1][1] is defined by finding the heterozygous factor V (FV) R506Q substitution (Leiden mutation,[2][2] 1691 G/A in the coagulation FV gene) in the presence of APC resistance ratios[3][3] similar to those of FV Leiden homozygotes. Although partial FV

Pentapharm Ltd. has a long tradition of producing and proceeding snake venom components as APIs and tools in pharmaceutical and diagnostic applications, beginning with the snake venom-derived product Reptilase® about 60 years ago. In recent years diagnostic test kits in the field of haemostasis have been developed, like Pefakit® APC-R Factor V Leiden and Pefakit® PiCT®, which make use of one or more snake venom compounds as key reactants. The venom-derived compounds are responsible for many outstanding properties of these tests and account for their top ranking performance qualities confirmed in non-clinical and clinical studies. Both tests are on the market in Europe. Pefakit® APC-R Factor V Leiden is also marketed in the US since January 2005. Research on snake venom-derived compounds for diagnostic and pharmaceutical use is ongoing.

Summary. Background: Functional defects of the protein C pathway, detectable in plasma as activated protein C (APC) resistance, are a prevalent risk factor for venous thrombosis. The factor V (FV) Leiden mutation causes APC resistance by interfering with the APC-mediated inactivation of both FVa and FVIIIa. Co-inheritance of FV Leiden and quantitative FV deficiency on different alleles, a rare condition known as pseudo-homozygous APC resistance, is associated with pronounced APC resistance and 50% reduced FV levels, because of non-expression of the non-Leiden FV allele. Objectives: The role of normal FV in modulating the APC resistance phenotype in carriers of FV Leiden was investigated in patients with pseudo-homozygous APC resistance and in model systems. Patients/methods: Four functional plasma assays probing both components of APC resistance (susceptibility of FVa to APC and cofactor activity of FV in FVIIIa inactivation) were employed to compare seven clinically and genetically characterized FV Leiden pseudo-homozygotes to 30 relatives with different FV genotypes (including 12 FV Leiden heterozygotes and seven carriers of FV deficiency) and to 32 unrelated FV Leiden homozygotes. Results and conclusions: All assays consistently indicated that FV Leiden pseudo-homozygotes are significantly more APC-resistant than heterozygotes and indistinguishable from homozygotes. Thrombin generation measurements in FV-deficient plasma reconstituted with purified normal FV and FV Leiden confirmed these observations and showed that the expression of the normal FV allele is an important modulator of APC resistance in FV Leiden heterozygotes. These findings provide an explanation for the higher thrombotic risk of FV Leiden pseudo-homozygotes when compared with heterozygotes.

BACKGROUND: Epidemiological studies have shown that both the estrogen dose and progestogen type of oral contraceptives contribute to the increased risk of thrombosis in oral contraceptive users. Thrombin generation-based activated protein C (APC) sensitivity is a global test for the net prothrombotic effect of oral contraceptives and predicts the thrombotic risk. Our objective was to test the usefulness of sex hormone-binding globulin (SHBG) as a marker for the thrombotic risk of an oral contraceptive. METHODS: We measured SHBG and APC resistance in 156 healthy users of various types of oral contraceptives. RESULTS: Users of oral contraceptives with a moderately increased risk of thrombosis (gestodene and desogestrel pills) had higher SHBG levels than users of low-risk oral contraceptives containing levonorgestrel. Similarly, for higher doses of estrogen in oral contraceptives we found higher SHBG levels. Women using oral contraceptives with the highest thrombotic risk (cyproterone acetate pills) rendered the highest SHBG levels. Users of oral contraceptives containing gestodene, desogestrel or cyproterone acetate were more resistant to APC than users of levonorgestrel pills. SHBG levels were positively associated with the increased APC resistance. CONCLUSIONS: Our findings support the hypothesis that the effect of an oral contraceptive on SHBG levels might be a marker for the thrombotic risk.

Objective—Functional polymorphisms contributing to coagulation factor levels are preferential markers for association studies aimed at identifying prothrombic genetic components.Methods and Results—Factor V (FV) microsatellite genotypes were found to be associated with FV levels (P=0.003). Single nucleotide polymorphisms analysis and sequencing of the promoter and of coding regions identified two polymorphisms (Met2120Thr, Asp2194Gly) present in 20% of the population (n=1013) that are responsible for genotype-phenotype associations. The effect of the Met2120Thr polymorphism, both in plasma (mean reduction of FV level in the heterozygous condition: 25%) and in recombinant FV studies (34% reduction), was comparable to that of the Asp2194Gly change (20% and 34%, respectively). The study of 10 subjects with a rare genotype indicated that the Asp2194Gly substitution is the functional determinant of the reduced FV levels associated with the FVHR2 haplotype. Among Leiden carriers, the doubly heterozygous condition for FV2120Thr was found to be associated with a significantly increased activated protein-C resistance (APCR) (PP=0.009) in symptomatic than in asymptomatic subjects.Conclusions—Extensive analysis of FV polymorphisms indicated that changes in the C2 domain modulate FV levels and might increase APCR and thrombotic risk in FV Leiden carriers through a pseudohomozygous mechanism.

Acquired resistance to activated protein C has been reported during oral contraception and pregnancy. Its thrombogenic potential was studied in 41 neurosurgical patients who were enrolled in the placebo group of a thromboprophylaxis trial, Normalized activated protein C sensitivity ratio (nAPC-SR), clotting activity of factors V and VIII, and levels of protein C antigen were measured prior to and at days 3 and 7 after surgery. Bilateral venography was done in all patients at days 8-10 to demonstrate deep vein thrombosis. A lowered nAPC-SR was found in 76% (baseline), 80% (day 3), and 88% (day 7) of patients. It was inversely related to factor VIII clotting activity (p = .0003) and protein C antigen, (p = .02). Deep vein thrombosis was demonstrated in 30% of patients with a normal nAPC-SR and in 23% of patients with a lowered nAPC-SR. Pulmonary embolism was not observed. Multivariate analysis did not identify a lowered nAPC-SR as a thrombotic risk factor, in contrast with gender (women, p = .02) and Quetelet index (greater than or equal to 25 kg/m(2), p = .006). Our data provide no evidence that acquired activated protein C resistance, frequently found in neurosurgical patients, contributes to their high risk of postoperative deep vein thrombosis.

Venous thromboembolism including deep vein thrombosis (DVT) and pulmonary embolism (PE) is the third most common cardiovascular disease after ischemic heart disease and stroke and is contributing to escalating health care costs in the industrialised part of the world. Most epidemiological data derives from hospital studies and knowledge of the incidence and risk factors of the disease in the general population is limited.Within the framework of a general population study, "The Study of Men Born in 1913", 855 men were followed prospectively from the age of 50 to the age of 80. Four men had suffered a venous thromboembolic event (VTE) before the age of 50 and another 65 men had their first VTE during the 30-year follow-up period. The cumulative incidence of venous thromboembolism was 10.7% by the age of 80 years. The incidence rate of a first DVT was 1.4 per 1,000 observation-years. The corresponding incidence rates were 0.7 for non-fatal PE, and 1.1 for fatal PE. Waist circumference (p=0.004) and smoking (p=0.022) at baseline predicted a venous thromboembolic event in multivariate survival analysis. Men in the highest decile of waist circumference (= 100 cm) had an adjusted relative risk of 3.92 (95% CI: 2.10-7.29, p

The aim of this thesis was to improve background knowledge for making a reliable medical evaluation at the first visit of a woman in her 13th gestational week, to the antenatal clinic. We have focused on the prediction and the risk estimation of preeclampsia, intra-uterine growth restriction (IUGR), and thrombosis. The thesis was based on five studies, in which we have evaluated biochemical analyzes, genetic tests, anamnestic information, and statistical information (based on the data from medical files and from the Swedish Medical Birth and Hospital Discharge Registers). High maternal urine human chorionic gonadotropin level in early pregnancy was associated with a 3-fold increased risk of preeclampsia, vis-à-vis low values, while low epidermal growth factor levels were associated with IUGR pregnancies. Maternal smoking was associated with an increased risk of thrombosis (Odds ratio (OR)=1.24; 95% Confidence interval (CI) 1.02-1.51), which was consumption dependent. Moderate smoking was associated with lower incidence of preeclampsia associated with preterm birth in both study series (OR=0.1; CI 0.01-0.7, and OR=0.6; CI 0.5-0.8, respectively). Apart from an 1.1% risk of thrombosis, APC resistance was not associated with preeclampsia, IUGR, or spontaneous abortion. However, the carriers of APC resistance had fewer profuse hemorrhages at delivery (3.7% vs. 7.9%), which might have given them an evolutionary advantage, explaining the high prevalence (10.7%). The incidence of pregnancy-associated thrombosis in Sweden was 13/10000, evenly distributed in the ante- and postpartum periods. APC resistance was associated with an 8-fold increased risk of thrombosis. Overweight, heredity of thrombosis, and cesarean delivery were all associated with a roughly 5-fold increased risk of thrombosis. Preeclampsia was associated with a 3-fold increased risk of thrombosis in the postpartum period. Inledning För att kunna sätta in resurser där de behövs bäst, finns det en önskan att kunna förutsäga vilka graviditeter som kommer att bli komplicerade. Vid preeklampsi (havandeskapsförgiftning), tillväxthämning hos fostret samt blodproppssjukdom finns det idag inget säkert sätt att tidigt i graviditeten veta vilka som kommer att drabbas. Denna oförmåga att kunna förutse vilka som kommer att drabbas har försvårat studier på bakomliggande mekanismer samt sökandet efter en behandling. Syftet med dessa studier är att öka kunskapen om risken för gravida kvinnor att drabbas av ovan nämnda komplikationer. Utgångspunkten har varit att förbättra underlaget till den bedömning som görs vid första besöket, när kvinnan är i 13:e graviditetsveckan. För att uppnå detta mål har vi i fem arbeten studerat och analyserat kemiska tester, genetiska tester, anamnestiska uppgifter samt relevanta statistiska uppgifter. Graviditetshormon och "epidermal growth factor" De kemiska tester som vi har studerat är epidermal growth factor (EGF), en tillväxtfaktor för bland annat huden och magtarmkanalen. Djurförsök har visat att brist på EGF gav tillväxthämmade foster. Hypotesen var att EGF var med och styrde moderkakans funktion och fostrets tillväxt samt att brist kunde ge tillväxthämning eller preeklampsi. Graviditetshormonet HCG finns normalt bara i blodet hos gravida och används därför som graviditetstest. Funktionen av HCG är ej klarlagd, men man har sett att halten HCG i blodet hos kvinnor som får preeklampsi är högre än hos dem som inte drabbas av detta tillstånd. APC-resistens Vi undersökte det nyupptäckta ärftliga tillståndet, APC-resistens, som ger blodet en ökad tendens att levra sig (koagulera) samt en livslång ökad risk att drabbas av venös blodpropp. APC-resistens uppstod för cirka 25 000 år sedan genom att en aminosyra i vår genetiska uppsättning förändrades till en annan, en punktmutation. Denna förändring finns nu hos cirka 1 miljon svenskar och cirka 50 miljoner Kaukasier, men den finns nästan inte alls i t.ex Afrika och Asien. Både vid preeklampsi och tillväxthämning utvecklas moderkakan på ett felaktigt sätt i den tidiga graviditeten, men tillstånden visar sig först under graviditetens andra hälft. Vid båda tillstånden finns det en aktiverad koagulation, vilket ofta leder till ökat antal blodproppar (infarkter) i moderkakan. Eftersom cirkulationen i moderkakan liknar den venösa, med lågt tryck och flöde, var det logiskt att anta att APC-resistens kunde predisponera till den bakomliggande sjukligheten i moderkakan vid ovan nämnda tillstånd. Om ett starkt samband hittades, skulle det kunna användas för att förutsäga vilka graviditeter som skulle kunna drabbas. Ett potentiellt farligt tillstånd som APC-resistens skall normalt vara sällsynt, det skall selekteras bort i människans evolution. Den höga förekomsten tyder på att APC-resistenta individer måste ha en evolutionär fördel, annars hade inte tillståndet varit så vanligt. Publikation I I det första arbetet följe vi 1009 kvinnor som lämnat urinprov i tidig graviditet för att hitta en analys som kunde förutse utvecklingen av preeklampsi och / eller tillväxthämning. Kvinnor som födde tillväxthämmade barn hade lägre EGF-halt i urinen i den tidiga graviditeten. Fyndet indikerar att EGF kan vara ett av de tillväxthormon som styr moderkakans funktion eller fostrets tillväxt. Kvinnor med höga värden av HCG i urinen hade en trefaldigt ökad risk att få preeklampsi än kvinnor med lägre halt. Detta skulle kunna användas för att identifiera den grupp gravida som har en ökad risk för preeklampsi. Även rökare hade lägre nivå av både EGF och HCG i urinen. Vi spekulerade i möjligheten att rökning kunde ge tillväxthämmning genom att påverka tillväxtfaktorer, såsom EGF. Detta är intressant, då vi sen länge vet att rökning orsakar tillväxthämning, men utan att känna till de exakta mekanismerna. Publikation II Det andra arbetet bestod av två delar, ett Malmö material bestående av alla preeklamptiker under 5 år och ett landsomfattande material. Båda dessa jämfördes med friska gravida kvinnor. Målet var att undersöka om rökare hade ett skydd mot att utveckla preeklampsi samt att diskutera den eventuella orsaken. Eftersom tillståndet framförallt påverkar sjuklighet och dödlighet hos spädbarn genom för tidig (preterm) födsel, var vi särskilt intresserade av denna delgrupp. Måttlighetsrökare (1-9 cigaretter dagligen) hade lägre förekomst av både preeklampsi och av den tidiga födsel som ofta hänger ihop med svår preeklampsi. Vi framförde flera tänkbara förklaringar och spekulerade i att detta "skydd" kunde vara en effekt av nikotin. Om detta visar sig vara sant, skulle man i framtiden kunna använda nikotin i annan form än cigaretter mot preeklampsi. Men det krävs ytterligare studier innan det kan bli aktuellt. Publikation III I det tredje arbetet har vi studerat om kvinnor med tidigare preeklampsi eller tillväxthämning (IUGR), hade en ökad förekomst av APC-resistens samt om de APC-resistenta kvinnorna blödde mindre vid förlossningen. Studiegruppen som helhet hade högre förekomst av APC-resistens (18% mot 10%). Denna skillnad var koncentrerad till den lilla grupp kvinnor som hade både IUGR och preeklampsi, och det fanns ingen skillnad hos de kvarvarande i studiegruppen (de med bara preeklampsi eller IUGR). Det fanns ingen skillnad i antalet missfall per kvinna mellan APC-resistenta och icke APC-resistenta kvinnor. Däremot hade de förra mindre blödningsmängd samt färre stora blödningar. Publikation IV I det fjärde arbetet studerades graviditetens naturalförlopp hos APC-resistenta kvinnor. Vi följde 2480 kvinnor, som lämnade blod och intervjuades i början av sin graviditet. Förekomsten av APC-resistens var 10.7% hos de gravida kvinnorna i Malmö. Förutom en 8-faldigt förhöjd risk att få blodpropp, fann vi ingen ökad risk för graviditetskomplikationer såsom; preeklampsi, tillväxthämning och sen- eller tidigt missfall hos de APC-resistenta kvinnorna. Däremot hade de mindre blödningsmängd och färre stora blödningar än andra kvinnor. Vid jämförelse av andra parametrar fann vi att överviktiga kvinnor samt kvinnor med blodpropp hos nära släktingar hade cirka 5 gånger ökad risk att få blodpropp, jämfört med övriga. Publikation V I det femte arbetet gjorde vi en statistisk studie från Medicinska Födelseregistret och Patientregistret, omfattande alla kvinnor med graviditetsrelaterad blodproppsdiagnos. Av de 479 422 kvinnor som födde barn under denna tid fann vi 608 kvinnor med 625 blodproppstillbud i samband med graviditet. Dessa 608 kvinnor jämfördes med alla kvinnor som var förlösta 1993 (utan blodpropp). Vi fann att förekomsten av blodpropp var 13/10 000 graviditeter, jämnt fördelade före och efter förlossningen. Kejsarsnitt var förknippat med en 5-faldigt ökad risk och preeklampsi med en 3-faldigt ökad risk för blodpropp i efterbördsskedet. Men det finns inget samband mellan preeklampsi och blodpropp före förlossningen. Det var förvånande att ett tillstånd som preeklampsi, som har aktiverad koagulation, inte ledde till en ökad risk för blodpropp under graviditeten. Hos rökare fann vi en ökad risk för blodpropp, en risk som steg med antalet cigarretter per dag. Tidigare har man ej funnit något samband mellan venös blodpropp och rökning. Evolutionär fördel Fyndet att APC-resistens (en genetisk förändring som ökar blodets förmåga att koagulera) medförde färre stora blödningar vid förlossningen verkar kanske inte så intressant. Men det har uppskattats att var tionde kvinna avslutade sitt liv i graviditetskomplikationer för två hundra år sedan, samt att uppemot hälften av dessa dödsfall hade ett samband med blodbrist eller stora blödningar. Det minskade antalet stora blödningar i samband med förlossningen hos APC- resistenta, kan ha medfört en historisk minskad risk att dö vid förlossning. Detta kan ha gett kvinnorna med APC-resistens en evolutionär fördel gentemot de icke APC-resistenta och att familjer med APC-resistens därigenom blivit större än andra, vilket skulle kunna förklara den höga förekomsten av APC- resistens idag. Detta är inte den första gången en ärftlig avvikelse har visat sig ha en evolutionär fördel. Tidigare har individer med sickelcellsanemi (en typ av ärftlig blodbrist) visats ha en fördel i områden där malaria är vanligt. Malaria har en hög dödlighet, men dessa bärarne får malaria mindre ofta och mildare och överlever på så vis. Slutsats Vi har försökt förutse vilka patienter som kommer att utveckla preeklampsi och tillväxthämning och vi har funnit att ett HCG-prov av urinen skulle kunna användas för att identifiera kvinnor med högre risk att utveckla dessa symptom. Att kvinnor som fick tillväxthämmade barn hade lägre EGF-nivå i urinen kan indikera att EGF deltar i styrningen av moderkakan och fostrets tillväxt. Fyndet att måttlighetsrökarna med preeklampsi mer sällan födde för tidigt kan vara en effekt av nikotin. Våra studier har förbättrat kännedomen om hur vissa faktorer (APC-resistens, rökning, kejsarsnitt, preeklampsi, paritet, ålder, övervikt, samt blodpropps förekomst i nära släkten) påverkar risken att få blodpropp. APC- resistens förekom hos cirka 11% av gravida kvinnor och förutom en ökad risk för blodpropp fann vi ingen ökad risk för graviditetskomplikationer. Det lägre antalet stora blödningar vid förlossningen kan ha gett APC-resistenta kvinnor en evolutionär fördel i form av färre dödsfall vid förlossningen. Detta kan vara orsaken till den höga förekomsten av APC-resistens.