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Antithrombin deficiency, the most severe congenital thrombophilia, might be underestimated, as some pathogenic variants are not detected by routine functional methods. We have identified 2 new SERPINC1 variants, p.Glu227Lys and p.Asn224His, in 4 unrelated thrombophilic patients with early and recurrent thrombosis that had normal antithrombin activity. In one case, the mutation was identified by whole genome sequencing, while in the 3 remaining cases, the mutation was identified by sequencing SERPINC1 based on a single functional positive finding supporting deficiency. The 2 variants shared a common functional defect, an impaired or null N-glycosylation of Asn224 according to a eukaryotic expression model. Carriers had normal anti-FXa or anti-FIIa activities but impaired anti-FVIIa activity and a detectable loss of inhibitory function when incubating the plasma for 1 hour at 41°C. Moreover, the b glycoform of the variants, lacking 2 N-glycans, had reduced secretion, increased heparin affinity, no inhibitory activity, and a potential dominant–negative effect. These results explain the increased thrombin generation observed in carriers. Mutation experiments reflected the role that Lysine residues close to the N-glycosylation sequon have in impairing the efficacy of N-glycosylation. Our study shows new elements involved in the regulation of N-glycosylation, a key posttranslational modification that, according to our results, affects folding, secretion, and function, providing new evidence of the pathogenic consequence of an incorrect N-glycosylation of antithrombin. This study supports that antithrombin deficiency is underestimated and encourages the development of new functional and genetic tests to diagnose this severe thrombophilia.

Background There are no data on the effect of low-dose anticoagulation as secondary prophylaxis for venous thromboembolism (VTE) in cancer patients. We assessed the efficacy and safety of low-dose apixaban for 30 months, after initial 6 months of full-dose treatment. Methods We included 298 patients with cancer and any type of VTE in a single arm interventional clinical trial. All patients were treated with full-dose apixaban (5 mg twice daily) for 6 months. Total 196 patients with active cancer after 6 months treatment continued with apixaban 2.5 mg twice daily for another 30 months. The main endpoints were recurrent VTE, major bleeding and clinically relevant non-major bleeding. Results During the 30 months of treatment with low-dose apixaban 14 (7.6%; 95% confidence interval (CI) 4.0%–11.7%) patients experienced recurrent VTE, six (3.1%; 95% CI 1.1%–6.5%) experienced major bleeding and 16 (8.1%, 95% CI: 4.7%–12.8%) experienced clinically relevant non-major bleeding. The incidence rate per person month of recurrent VTE was 0.8% (95% CI 0.41–1.6) at 2–6 months with full-dose apixaban, and 1.0% (95% CI 0.5–1.9) at 7–12 months with low-dose apixaban. The incidence rate of major bleeding was 1.1% (95% CI 0.6–2.0) at 2–6 months, and 0.3% (95% CI 0.1–1.0) at 7–12 months. Between 12 and 36 months the incidence rate of recurrent VTE and major bleedings remained low. Conclusion Dose reduction of apixaban to 2.5 mg twice daily seems safe after 6 months of full-dose treatment. After 12 months the incidence rate of recurrent VTE and major bleeding remained low.

Introduction: Physical activity may reduce the development of breast cancer. Whereas hypercoagulability has been linked to adverse outcomes in breast cancer patients, the effects of physical activity on their hemostatic factors are unknown. The study aimed to assess whether long-term (1 year) physical activity can affect hemostatic factors in breast cancer patients. Methods: Fifty-five women (35–75 years) with invasive breast cancer stage I/II were randomized to a physical activity intervention (n = 29) lasting 1 year or to a control group (n = 26), and analyzed as intention to treat. Fibrinogen, factor VII antigen, tissue factor pathway inhibitor, and von Willebrand factor (VWF) antigen as well as prothrombin fragment 1 + 2, the endogenous thrombin potential and D-dimer, were measured in plasma before intervention (baseline), and then after 6 and 12 months. Results: Maximal oxygen uptake (measure of cardiorespiratory fitness) decreased the first 6 months among the controls, but remained stable in the intervention group. We found no significant differences between the two study groups regarding any of the hemostatic factors, except a significantly higher increase in factor VII antigen in the intervention group. The effect of the intervention on VWF was, however, significantly affected by menopausal stage, and a significant effect of the intervention was found on VWF among postmenopausal women, even after adjustment for dietary intake. Conclusion: Long-term physical activity had no effect on the majority of the hemostatic factors measured, but led to increased plasma concentrations of factor VII antigen and prevented an increase in VWF concentration after breast cancer treatment in postmenopausal women. The clinical impact of these findings for risk of vascular thrombosis warrants further studies.

The randomized “Testicular cancer and Aerobic and Strength Training trial” (TAST‐trial) aimed to evaluate the effect of high‐intensity interval training (HIIT) on cardiorespiratory fitness during cisplatin‐based chemotherapy (CBCT) for testicular cancer (TC). Here, we report on an unexpected high number of thromboembolic (TE) events among patients randomized to the intervention arm, and on a review of the literature on TE events in TC patients undergoing CBCT. Patients aged 18 to 60 years with a diagnosis of metastatic germ cell TC, planned for 3 to 4 CBCT cycles, were randomized to a 9 to 12 weeks exercise intervention, or to a single lifestyle counseling session. The exercise intervention included two weekly HIIT sessions, each with 2 to 4 intervals of 2 to 4 minutes at 85% to 95% of peak heart rate. The study was prematurely discontinued after inclusion of 19 of the planned 94 patients, with nine patients randomized to the intervention arm and 10 to the control arm. Three patients in the intervention arm developed TE complications; two with pulmonary embolism and one with myocardial infarction. All three patients had clinical stage IIA TC. No TE complications were observed among patients in the control arm. Our observations indicate that high‐intensity aerobic training during CBCT might increase the risk of TE events in TC patients, leading to premature closure of the TAST‐trial.

Background and Purpose— In randomized stroke trials, central adjudication of a trial’s primary outcome is regularly implemented. However, recent evidence questions the importance of central adjudication in randomized trials. The aim of this review was to compare outcomes assessed by central adjudicators with outcomes assessed by site investigators. Methods— We included randomized stroke trials where the primary outcome had undergone an assessment by site investigators and central adjudicators. We searched MEDLINE, EMBASE, CENTRAL (Cochrane Central Register of Controlled Trials), Web of Science, PsycINFO, and Google Scholar for eligible studies. We extracted information about the adjudication process as well as the treatment effect for the primary outcome, assessed both by central adjudicators and by site investigators. We calculated the ratio of these treatment effects so that a ratio of these treatment effects >1 indicated that central adjudication resulted in a more beneficial treatment effect than assessment by the site investigator. A random-effects meta-analysis model was fitted to estimate a pooled effect. Results— Fifteen trials, comprising 69 560 participants, were included. The primary outcomes included were stroke (8/15, 53%), a composite event including stroke (6/15, 40%) and functional outcome after stroke measured on the modified Rankin Scale (1/15, 7%). The majority of site investigators were blind to treatment allocation (9/15, 60%). On average, there was no difference in treatment effect estimates based on data from central adjudicators and site investigators (pooled ratio of these treatment effects=1.02; 95% CI, [0.95–1.09]). Conclusions— We found no evidence that central adjudication of the primary outcome in stroke trials had any impact on trial conclusions. This suggests that potential advantages of central adjudication may not outweigh cost and time disadvantages in stroke studies if the primary purpose of adjudication is to ensure validity of trial findings.

Endoplasmic reticulum (ER) stress has been shown to play a key role during the initiation and clinical progression of the cardiovascular diseases, such as atherosclerosis. We have recently shown that expression of tissue factor pathway inhibitor (TFPI) in human monocyte-derived macrophages (MDMs) was induced by cholesterol crystals (CC). In the present study we aimed to determine the role of TFPI under ER stress conditions using human MDMs. qRT-PCR and immunohistochemistry analysis were performed to determine the presence of the ER stress marker CCAAT/enhancer binding protein homologous protein (CHOP) and TFPI in human carotid plaque material and also in human MDMs polarized into pro-inflammatory M1 or anti-inflammatory M2 populations. CHOP mRNA levels were upregulated in the plaques compared to healthy vessels, and CHOP protein was localized in the same area as TFPI in the plaques. Both CHOP and TFPI mRNA levels were upregulated after CC treatment, especially in the M2 phenotype, and the ER stress inhibitor 4-phenylbutyric acid (PBA) reversed this effect. Furthermore, CC treatment increased the levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-8, which for TNF-α and IL-8 was inhibited by PBA, and reduced the levels of the anti-inflammatory cytokine IL-10 in M2-polarized macrophages. Knockdown of TFPI prior to CC treatment exacerbated TNF-α and IL-6 levels, but reduced IL-8 and IL-10 levels. Our results show that CC induce TFPI and cytokine expression in M2-polarized macrophages through activation of the ER stress pathway and that TFPI has a protective effect against TNF-α and IL-6 mediated inflammation. These mechanisms may have implications for the pathogenesis of atherosclerosis.

INTRODUCTION: Tissue factor (TF) pathway inhibitor (TFPI) is the physiological inhibitor of TF induced blood coagulation and two isoforms exists, TFPIα and TFPIβ. In atherosclerotic plaques, TFPI may inhibit TF activity and thrombus formation, which is the main cause of ischemic stroke in carotid artery disease. We aimed to identify the isoforms of TFPI present in human carotid plaques and potential sources of TFPI. MATERIALS AND METHODS: Human atherosclerotic plaques from carotid endarterectomies were used for mRNA and immunohistochemistry analyses. hPBMCs isolated from buffy coats and THP-1 cells were differentiated and polarized into M1 or M2 macrophages, and subsequently cultured with or without cholesterol crystals (CC). mRNA and protein expression were measured with qRT-PCR and ELISA, respectively, and procoagulant activity was assessed using a two-stage chromogenic assay. RESULTS: TFPIα and TFPIβ mRNA levels were significantly increased in carotid plaques, whereas TF levels were unchanged as compared to healthy arteries. Antibodies against total TFPI showed elevated levels compared to antibodies against free TFPIα, both by immunohistochemical and ELISA detection in plaques. The antibody against total TFPI also co-localized with CD68 and the M1 and M2 markers CD80 and CD163, respectively. The TFPI mRNA expression was elevated and the procoagulant activity was decreased in M2 compared to M1 polarized human macrophages. TFPI was present in early foam cell formation and CC treatment increased the TFPI mRNA expression even further in M2 macrophages. CONCLUSIONS: Our data indicate that both isoforms of TFPI are present in advanced plaques and that anti-inflammatory M2 macrophages may be a potential source of TFPI.

Neovascularization and hemorrhaging are evident in advanced atherosclerotic plaques due to hypoxic conditions, and mediate the accumulation of metabolic substrates, inflammatory cells, lipids, and other blood born factors inside the plaque. Tissue factor (TF) pathway inhibitor (TFPI) is mainly expressed by endothelial cells and is the endogenous inhibitor of the coagulation activator TF, which together with the downstream product thrombin can drive plaque progression and atherogenesis. We aimed to investigate the effect of hypoxic conditions on endothelial cell expression and activity of TFPI and TF that are important in coagulation initiation. Hypoxia was induced in primary human umbilical vein endothelial cells using chemicals or 1% oxygen tension, and mRNA and protein expressions were measured using qRT-PCR, ELISA, and Western blot analysis. Microscopy of fluorescence-labeled cells was used to visualize cell-associated TFPI. Cell-surface factor Xa (FXa) activity was measured using a two-stage chromogenic substrate method. We found that hypoxia reduced the TFPI mRNA and protein levels and increased the TF mRNA expression in a dose-dependent manner. The effect on TFPI was apparent on all the protein pools of TFPI, i.e., secreted TFPI, cell-surface associated TFPI, and intracellular TFPI, and seemed to be dependent upon hypoxia inducible factor-2α (HIF-2α). An increase in FXa activity was also observed on the endothelial cell surface, reflecting an increase in pro-thrombotic potential of the cells. Our findings indicate that hypoxic conditions may enhance the pro-coagulant activity of endothelial cells, which may promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders.

Post-thrombotic syndrome (PTS) is a long-term complication of deep-vein thrombosis (DVT). The Villalta scale is the recommended tool for diagnosing PTS, but requires a clinician’s assessment in addition to patient self-assessment. In the present study, we validated a self-administered tool for patient reporting of leg symptoms and signs as a mean to assess PTS. We first validated a form for patient self-reported Villalta (PRV1), then developed and validated a visually assisted form (PRV2). The validity of PRV1 and PRV2 was assessed in patients diagnosed with DVT between 2004 and 2012. Median time from DVT to inclusion was 5.1 and 3.5 years for PRV1 (n=162) and PRV2 (n=94), respectively. Patients were requested to complete the PRV form before a scheduled visit. PTS diagnosed by the original Villalta scale during the visit served as the reference method. PRV1 showed only moderate agreement for diagnosing PTS compared with the original Villalta scale (kappa agreement 0.60, 95 % CI 0.48–0.72), whereas PRV2 showed very good agreement (0.82, 95 % CI 0.71–0.94). In the validation of PRV2, PTS was diagnosed in 54 (57 %) patients according to the original Villalta scale and in 60 (64 %) by PRV2. The sensitivity of PRV2 to detect PTS was 98 % and the specificity was 83 %. We conclude that the visually assisted form for PRV is a valid and sensitive tool for diagnosing PTS. Such a tool could be applied in further clinical studies of PTS, making studies less resource demanding by reducing the need for in-person clinic visits.

OBJECTIVE: We conducted a systematic review and meta-analysis to address benefits and harms of using elastic compression stockings after lower-extremity deep vein thrombosis. METHODS: We searched 7 electronic databases through January 15, 2015, including randomized controlled trials (RCTs)/quasi-randomized trials reporting on elastic compression stocking efficacy on postthrombotic syndrome incidence, recurrent venous thromboembolism, mortality, and acute pain after deep vein thrombosis. Two reviewers independently screened records, extracted data, assessed risk of bias, and assessed confidence in effect estimates using Grading of Recommendations Assessment, Development, and Evaluation methodology. We applied random-effects meta-analysis models. RESULTS: We included 5 RCTs (n = 1418) reporting on postthrombotic syndrome. The hazard ratio (HR) for postthrombotic syndrome with elastic compression stockings was 0.69 (95% confidence interval [CI], 0.47-1.02). We have very low confidence in this estimate due to heterogeneity and inclusion of unblinded studies at high risk of bias. Excluding high risk of bias studies, a single large RCT at low risk of bias provided moderate-quality evidence of no effect on postthrombotic syndrome (HR 1.00; 95% CI, 0.81-1.24). Moderate-quality evidence including all 5 studies suggests no effect of elastic compression stockings on recurrent venous thromboembolism (relative risk [RR] 0.88; 95% CI, 0.63-1.24) or mortality (RR 1.00; 95% CI, 0.73-1.37, 5 studies). Moderate-quality evidence from one large RCT does not suggest effect on acute pain after deep vein thrombosis. CONCLUSIONS: The highest-quality evidence available suggests no effect of elastic compression stockings on postthrombotic syndrome or pain relief, from a single large RCT. However, results for preventing postthrombotic syndrome differ substantially across studies, and future guideline updates should reflect uncertainty about treatment effects. Elastic compression stockings are unlikely to prevent death or recurrent venous thromboembolism.

BACKGROUND: Adaptation of guidelines for use at the national or local level can facilitate their implementation. We developed and evaluated an adaptation process in adherence with standards for trustworthy guidelines and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology, aiming for efficiency and transparency. This article is the first in a series describing our adaptation of Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines for a Norwegian setting. METHODS: Informed by the ADAPTE framework, we developed a five-step adaptation process customized to guidelines developed using GRADE: (1) planning, (2) initial assessment of the recommendations, (3) modification, (4) publication, and (5) evaluation. We developed a taxonomy for describing how and why recommendations from the parent guideline were modified and applied a mixed-methods case study design for evaluation of the process. RESULTS: We published the adapted guideline in November 2013 in a novel multilayered format. The taxonomy for adaptation facilitated transparency of the modification process for both the guideline developers and the end users. We excluded 30 and modified 131 of the 333 original recommendations according to the taxonomy and developed eight new recommendations. Unforeseen obstacles related to acquiring a licensing agreement and procuring a publisher resulted in a 9-month delay. We propose modifications of the adaptation process to overcome these obstacles in the future. CONCLUSIONS: This case study demonstrates the feasibility of a novel guideline adaptation process. Replication is needed to further validate the usefulness of the process in increasing the organizational and methodologic efficiency of guideline adaptation.

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.

Objective. To estimate incidence and risk factors for intrauterine fetal death (IUFD) in a Norwegian study-population applying two different control groups. Design. Case-control study. Setting. Two university hospitals in ̽����ѡ, Norway, January 1990 through December 2003. Sample. The cases: 377 women with IUFD. Controls: 1) all women delivering at the study-hospitals in the period (facility-based), and 2) 1 215 women with live births at one study-hospital in the period (selected). Methods. Information from cases and selected controls was collected from medical records. Data on facility-based controls were provided by the Medical Birth Registry of Norway. Data were analyzed using chi-squared test and logistic regression. Main outcome measures. Incidence of IUFD and adjusted odds ratios of risk factors. Results. The incidence was 4.1/1 000 deliveries. Small-for-gestational age (SGA) and placental abruption were the strongest risk factors for IUFD. Hypertensive disorders were of low risk if not associated with SGA. Low to moderate risk factors were pre-pregnancy diabetes mellitus, thyroid disease, placenta previa, gestational diabetes, smoking and twin pregnancy. Advanced maternal age was significant when compared with facility-based controls. Risk estimates pointed in the same direction independent of control-group. Hypertension appeared overestimated when using facility-based controls, whereas advanced age was underestimated in the analysis among selected controls. Conclusion. SGA has a strong association with IUFD, and the risk of hypertensive disorders is mediated through SGA. The other risk factors, except placental abruption, are of low prevalence and of limited importance in the prevention of a relatively low incidence, although dramatic, event like IUFD.

We previously found an association between the circadian variation of free tissue factor pathway inhibitor (TFPI) and melatonin in able-bodied males and in men with complete cervical spinal cord injuries. We therefore examined whether melatonin modifies production and/or secretion of TFPI in endothelial cells. We sampled supernatants from cultures of primary human umbilical vein endothelial cells (HUVECs) and of human coronary artery endothelial cells (HCAECs), that had been exposed to varying doses (0-300 pg/ml) of melatonin for 0.5-24 h. We then measured the protein concentrations of free TFPI, tissue factor and plasminogen activator inhibitor type 1 (PAI-1). We also measured endothelial TFPI, tissue factor and PAI-1 transcripts using quantitative real-time PCR. Melatonin dose dependently increased free TFPI levels about 25-30-fold in supernatants of both HUVEC and HCAEC, and independent of incubation duration. In contrast, TF and PAI-1 remained unaltered upon increasing doses of melatonin. Neither TFPI mRNAs nor tissue factor mRNAs nor PAI-1-mRNAs were changed in cell cultures added melatonin. The ratio of free TFPI in cell supernatants to free TFPI in cell lysates about doubled upon addition of melatonin, indicating that melatonin increased release from intracellular storages of free TFPI or from membrane-bound free TFPI. Our data indicate that melatonin stimulates vascular endothelial cells to secrete TFPI without altering transcription of the TFPI gene. If melatonin increases TFPI release in a similar fashion in vivo as in vitro, this could have potential clinical implications in both prophylaxis and treatment of thromboembolic events.

Heparin-induced thrombocytopenia (HIT, type II) is an immune-mediated disorder due to antibodies formed against heparin-platelet factor 4 complexes, usually appearing at days 5 to 14 after initiation of heparin. It is important to recognize HIT because heparin prophylaxis or treatment paradoxically associates with new venous and/or arterial thrombosis. Early clinical suspicion and diagnosis together with proper pharmacotherapy and close laboratory monitoring are the cornerstones for successful management. This includes monitoring of Thrombocytopenia, its Timing to heparin administration, appearance of new Thrombosis or resistance to treatment, and differential diagnosis by exclusion of o Ther causes (the 4T's). Specific attention should be paid to the absence or presence of thrombosis and to tailoring thromboprophylaxis or anticoagulant therapy with a nonheparin alternative. Even in the absence of HIT-associated thrombosis, an active policy for prolonged thromboprophylaxis is demanded. Rapid and reliable assays should be developed for diagnosis and anticoagulation monitoring to secure safe management with nonheparins. Semiquantitative testing for on-call hours should be available and later confirmed as clinically needed. Alternative therapeutic options are available, but because their use is infrequent, experienced coagulation treatment centers should provide guidance in the treatment and in laboratory monitoring. Most of the evidence in HIT is grade IC, and thus the best evidence is provided by clinical experience. New anticoagulants and platelet inhibitors may offer future alternatives in the management of HIT, but the current treatment options provide the best experience and benefit. The joint clinical and laboratory guidelines provided in this article along with two practical case scenarios were prepared by a Nordic expert panel. They will be valuable for hematologists and colleagues who do not routinely encounter HIT.

Background –  Patients with acute ischemic stroke and atrial fibrillation are at increased risk of stroke progression and recurrence. We sought to assess whether D-dimer and other markers of hemostatic activation could predict these adverse events in such patients. Method –  Blood samples were obtained from patients included in the Heparin in Acute Embolic Stroke Trial. Stroke progression was defined as a ≥3-point worsening on the Scandinavian Stroke Scale during the first 48 h after randomization. Blood samples were analyzed for D-dimer, prothrombin fragment 1 + 2, soluble fibrin monomer, and C-reactive protein. Results –  A total of 382 patients were included in the analyses. Levels of D-dimer and other markers of hemostatic activation were not significantly higher in patients with stroke progression than in other patients (D-dimer median values: 1025 ng/ml vs 970 ng/ml, P = 0.73). The same was true for recurrent stroke (D-dimer: 720 ng/ml vs 973 ng/ml, P = 0.96), and the combined endpoint of stroke progression, recurrent stroke, and death (D-dimer: 991 ng/ml vs 970 ng/ml, P = 0.91). Multivariable analyses did not alter the results. Conclusion –  D-dimer and other markers of hemostatic activation were not associated with stroke progression, recurrent stroke, or death in patients with acute ischemic stroke and atrial fibrillation.

Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening adverse drug reaction that may develop in certain patients exposed to heparin and is caused by antibodies with specificity for chemokine CXCL4 (formerly known as platelet factor 4)/heparin complexes. Rapid diagnosis and intervention is key to prevent severe thrombotic complications. The immunobiology of HIT is atypical as the immune reaction most often involves rapid generation of immunoglobulin class G within 5-14 days after heparin exposure, and apparently lacks memory as patients may be reexposed to heparin. This report reviews clinical presentation, diagnostic issues, and immunobiology of HIT.

INTRODUCTION: Protein C (PC) is a key anticoagulant that regulates hemostasis, and inherited deficiency of PC is an established risk factor for venous thrombosis (VT). The factor V Leiden mutation causing activated PC (APC) resistance is an additional risk factor for VT. Reduced PC levels in the circulation and/or APC resistance do not necessarily lead to thrombotic disease. In the present study we describe and characterize an ethnic Lebanese family in which individuals with reduced PC levels and APC resistance have various clinical symptoms. METHODS: PC activity and antigen levels and APC resistance in the family members were quantified with commercial kits. Sequencing of PC DNA and mRNA was performed with BigDye Terminator Cycle Sequencing kit on the ABI 3730 Genetic Analyzer. RESULTS: PC antigen and anticoagulant activity in the plasma of the proband and family members ranged from 9% to 69% and 3% to 63%, respectively, compared to levels measured in pooled normal plasma. Sequencing analysis of the PC gene of family members revealed that they were either homozygous or heterozygous for the Ala267Thr mutation. In addition, three of them exhibited APC resistance. None of the family members, except the proband, have had a history of VT despite that two of them have two genetic risk factors for thrombosis.

Background Pregnancy is associated with a ten-fold increased risk of venous thrombosis (VT) with different risk profiles for the ante- and postnatal periods. The purpose of this study was to assess the risk of pregnancy-related VT associated with the factor V Leiden and the prothrombin gene G20210A polymorphisms. Materials and Methods The study comprised 377155 women with 613232 pregnancies at 18 Norwegian hospitals from 1 January 1990 to 31 December 2003. Of total 559 cases with a validated first lifetime diagnosis of VT in pregnancy or within 14 weeks post-partum, and 1229 controls naive for VT, 313 cases and 353 controls donated biological material. Results The odds ratios for venous thrombosis during pregnancy or puerperium were 5.0 (95% CI 3.1 to 8.3) and 9.4 (2.1-42.4) for heterozygous carriers of the factor V Leiden and the prothrombin gene polymorphisms, respectively. All homozygous carriers of the factor V Leiden (n=8) and the prothrombin polymorphism (n=1) developed VT indicating a very high risk of VT. We estimated that pregnancy-related VT occurred in 1.1/1000 non-carriers, in 5.4/1000 heterozygous carriers of factor V Leiden, and in 9.4/1000 heterozygous carriers of the prothrombin polymorphism. To avoid one VT, the number of pregnant women needed to screen for these two polymorphisms and the number needed to be given thromboprophylaxis were 2015 and 157, respectively. Conclusions Although the relative risk for VT during pregnancy and after delivery was increased among carriers of the factor V Leiden and the prothrombin polymorphisms, the overall probability for pregnancy-related VT was low. Trial registration www.clinicaltrials.gov identifier number NCT00856076.

Background Activated protein C (PC) is a serine protease that regulates blood coagulation by inactivating coagulation factors Va and VIIIa. PC deficiency is an autosomally inherited disorder associated with a high risk of recurrent venous thrombosis. The aim of the study was to explore the mechanisms responsible for severe PC deficiency in a patient with the protein C A267T mutation by in-vitro expression studies. Results Huh7 and CHO-K1 cells were transiently transfected with expression vectors containing wild-type (WT PC) and mutated PC (A267T PC) cDNAs. PC mRNA levels were assessed by qRT-PCR and the PC protein levels were measured by ELISA. The mRNA levels of WT PC and A267T PC were similar, while the intracellular protein level of A267T PC was moderately decreased compared to WT PC. The secretion of A267T PC into the medium was severely impaired. No differences in molecular weights were observed between WT and A267T PC before and after treatment with endo-beta-N-acetylglucosaminidase. Proteasomal and lysosomal degradations were examined using lactacystin and bafilomycin, respectively, and revealed that A267T PC was slightly more susceptible for proteasomal degradation than WT PC. Intracellular co-localization analysis indicated that A267T PC was mainly located in the endoplasmic reticulum (ER), whereas WT PC was observed in both ER and Golgi. Conclusions In contrast to what has been reported for other PC mutants, intracellular degradation of A267T PC was not the main/dominant mechanism underlying the reduced intracellular and secretion levels of PC. Our results indicate that the A267T mutation most likely caused misfolding of PC, which might lead to increased retention of the mutated PC in ER.

Purpose: To assess image quality, vessel visualization, preliminary diagnostic properties, and interobserver variability of a novel balanced turbo field echo (b-TFE) sequence and contrast-enhanced T1 fast field echo (CE-FFE) sequence with blood pool agent (BPA). Materials and Methods: A total of 15 healthy volunteers and six patients with ultrasound-verified proximal deep vein thrombosis (DVT) were examined from the inferior vena cava (IVC) to the proximal calf veins. Results: The great majority of deep veins were completely visualized on both sequences. In healthy volunteers the IVC was completely visualized in five b-TFE and 11 CE-FFE scans, and partially in seven b-TFE and four CE-FFE scans (P = 0.008). Poorest image quality was in the pelvis. Contrast-to-noise ratio (CNR) was higher on b-TFE compared to CE-FFE, with significant difference in calf images (P = 0.036). Sensitivity was 100% for proximal DVT with both methods. Specificity was 70% (CE-FFE) and 80% (b-TFE) for proximal femoral DVT; 100% in distal femoral. Interobserver reliability was kappa 1.0 (b-TFE), 0.9 (CE-FFE) for proximal, and overall poor for distal DVT. Conclusion: Contrast-enhancement did not add valuable information in visualizing deep veins of the lower limbs compared to b-TFE, though the IVC was slightly better visualized. Diagnostic properties and interobserver reliability of both sequences were good for proximal DVT and poor for distal DVT.

In this population-based case-control study we explored the association of antiphospholipid antibodies with pregnancy-related venous thrombosis. From 1990 through 2003 615 pregnant women were identified at 18 hospitals in Norway with a diagnosis of first time VT. In 2006, 531 of 559 eligible cases and 1092 of 1229 eligible controls were invited for further investigations. The final study population comprised 313 cases and 353 controls, who completed a comprehensive questionnaire and donated a single blood sample, 3-16years after index pregnancy. We report the results on lupus anticoagulant, anticardiolipin antibodies, and anti-ss(2) glycoprotein-1 antibodies alone, in combination, and with the contribution of the factor V Leiden and the prothrombin gene G20210A polymorphisms. Cut-off values for APAs were chosen according to current international consensus. 29 (9.3%) of the cases and 24 (6.8%) of the controls had at least one positive test for APAs (OR 1.4; 95% CI 0.8-2.5). Nine cases (2.8%) and no controls had more than one positive test (multi-positivity) for APAs. After excluding women with factor V Leiden or prothrombin polymorphisms, still 6 cases were multi-positive for APAs. We conclude that multi-positivity, but not single-positivity, for APAs was weakly associated with a history of ante- and postnatal VT.

Tissue factor pathway inhibitor (TFPI) is the primary physiological inhibitor of tissue factor (TF) induced coagulation. Low plasma TFPI levels have been shown to be associated with increased risk of arterial and venous thrombosis. Several clinical studies have reported that single nucleotide polymorphisms (SNPs) in the regulatory regions of the gene, such as the -287T/C, the -399C/T, and the -33T/C SNPs, may affect plasma TFPI levels. However, molecular studies investigating the functionality of the polymorphisms are lacking. In this study, we found that the -287C and -399T alleles affected the activity of the promoter using a reporter gene system. This was also the case for the -33T/C polymorphism. An association regarding the transcriptional activity of the reporter gene was detected between the -287C allele and the -33T/C polymorphism. Analysis of the polymorphic sites with electrophoretic mobility shift assay (EMSA) showed that all three polymorphisms potentially alter DNA protein interactions. Based on these findings, we speculate that the -287C and the -33C alleles can be associated with lowered risk of thrombosis.

Thrombosis is a major complication and an important cause of death in cancer patients. Tumor cells may trigger coagulation and induce a prothrombotic phenotype, which in return may enhance angiogenesis, tumor growth, and metastasis. Tissue factor pathway inhibitor (TFPI) has been reported to reduce tumor growth and metastasis in vivo and to induce apoptosis and inhibit proliferation in normal cells in vitro. However, no effect has so far been observed in cancer cells. We therefore aimed to characterize the functional effects of ectopic overexpression and endogenous downregulation of TFPI in cancer cells, and to elucidate possible mechanisms involved. The tumor derived breast cancer cells SK-BR-3 and Sum102 were used to construct stable cell lines overexpressing TFPIa and TFPIb, and with TFPI knocked down, respectively. Effects of altered TFPI expression were evaluated by measuring apoptosis and proliferation of the cells, and gene expressions were analyzed using PCR arrays. Increased DNA fragmentation and Caspase 3 activity was observed in SK-BR-3 cells overexpressing TFPIa and TFPIb, while a decrease in apoptosis was seen in Sum102 cells with TFPI expression knocked down. An increase and reduction in expression of pro- and antiapoptotic genes, respectively, were seen in TFPI overexpressing cells, and the majority of the upregulated genes encoded proteins involved in the death receptor pathway, among them the death receptor ligand TNF-a. In conclusion, TFPIa and TFPIb induced apoptosis in breast cancer cells and increased expression of apoptotic genes indicating a possible involvement of the death receptor pathway.

Combined oral contraceptives and combined oral postmenopausal hormone therapy are associated with a weak, but clinically significant risk of arterial and venous thrombosis (VT). The effects are related to dose of estrogen and type of progestin. The main effects are increase in markers of activated coagulation, reduction in coagulation inhibitors, and acquired activated protein C resistance. Reduction in tissue factor pathway inhibitor (TFPI) is probably an important mechanism, which predicts activation of coagulation and acquired resistance to activated protein C. Coagulation markers should be used as intermediate or surrogate markers in early pharmacodynamic studies to evaluate the risk associated with new formulations.

Bone marrow angiogenesis is increased in non-Hodgkin lymphomas (NHL). Compounds affecting extracellular matrix (ECM) may modify angiogenesis. Here we investigated ECM regulators in 48 unselected NHL patients compared with 35 controls. Untreated patients had elevated (P < 0.05) serum matrix metalloproteinase (MMP) 9 and tissue inhibitor of metalloproteinase (TIMP) 1, while MMP-2, TIMP-2 and syndecan-1 were not significantly different from controls. MMP-9 mRNA was significantly up-regulated in blood mononuclear cells, while mRNA expressions of the other ECM regulators were unaltered. We found strong correlations between mRNA expressions of both vascular endothelial growth factor and fibroblast growth factor 2, and MMP-9, TIMP-1 and TIMP-2. After therapy, serum MMP-2 increased while MMP-9 decreased (P < 0.05), the others being unchanged. Several compounds affecting ECM may be involved in angiogenic activity in NHL.

Background: Approximately one in four patients with acute proximal deep vein thrombosis (DVT) given anticoagulation and compression therapy develop post-thrombotic syndrome (PTS). Accelerated removal of thrombus by thrombolytic agents may increase patency and prevent PTS. Objectives: To assess short-term efficacy of additional catheterdirected thrombolysis (CDT) compared with standard treatment alone. Patients and methods: Open, multicenter, randomized, controlled trial. Patients (18–75 years) with iliofemoral DVT and symptoms < 21 days were randomized to receive additional CDT or standard treatment alone. After 6 months, iliofemoral patency was investigated using duplex ultrasound and air-plethysmography assessed by an investigator blinded to previous treatment. Results: One hundred and three patients (64 men, mean age 52 years) were allocated additional CDT (n = 50) or standard treatment alone (n=53). After CDT, grade III (complete) lysis was achieved in 24 and grade II (50%–90%) lysis in 20 patients. One patient suffered major bleeding and two had clinically relevant bleeding related to the CDT procedure. After 6 months, iliofemoral patency was found in 32 (64.0%) in the CDT group vs. 19 (35.8%) controls, corresponding to an absolute risk reduction (RR) of 28.2% (95% CI: 9.7%–46.7%; P = 0.004). Venous obstruction was found in 10 (20.0%) in the CDT group vs. 26 (49.1%) controls; absoluteRR29.1%(95%CI: 20.0%–38.0%; P = 0.004). Femoral venous insufficiency did not differ between the two groups. Conclusions: After 6 months, additional CDT increased iliofemoral patency from 36% to 64%. The ongoing long-term follow-up of this study will document whether patency is related to improved functional outcome.

Antiangiogenic drugs are currently tested in haematological malignancies. As these drugs target different angiogenic regulators, and as cancers are inherently heterogeneous, a detailed characterization of angiogenesis in individual cancers is needed. Hence, we measured bone marrow microvessel density (MVD), plasma concentrations of eight angiogenesis-related parameters and the expression in blood mononuclear cells of 40 angiogenesis-related mRNAs in 93 patients with haematological neoplasias (acute myeloid leukaemia; chronic lymphatic leukaemia; multiple myeloma (MM); or non-Hodgkin's lymphoma (NHL)) before start and after completion of cancer therapy. Compared with healthy individuals, the patients had significantly increased bone marrow MVD, especially patients with advanced stage disease. A novel finding was that patients with NHL also had increased bone marrow MVD. The plasma levels of vascular endothelial growth factor (VEGF), interleukin (IL)-6 and IL-8 were significantly increased. VEGF levels were highest in those who did not achieve complete remission after cancer therapy. The mRNA expression of IL-8 was upregulated 15-fold. Our data show that patients with haematological malignancies have increased bone marrow MVD; hence, supporting the notion that bone marrow angiogenesis plays a role in the pathogenesis and progression of these cancers. VEGF, IL-6 and IL-8 seem to contribute to the malignant phenotype.

Introduction: Tissue factor (TF) pathway inhibitor (TFPI) is the endogenous inhibitor regulating TF-induced blood coagulation. Several polymorphisms have been identified in the TFPI gene and some of them correlated with variations in plasma TFPI levels. The aim of the present study was to characterize the TFPIV264M mutant in comparison with the wild type protein (TFPIWT). Materials and methods: We have overexpressed the TFPIV264M mutant and TFPIWT in human coronary artery endothelial cells and compared the expression and activity levels of the mutated protein relative to the TFPIWT. The protein levels were determined by ELISA, the inhibitory activity of the proteins was assessed with a chromogenic substrate assay. The mRNA level of the two TFPI variants was determined using real time RT-PCR. MFOLD was used to predict mRNA secondary structure. Results and conclusions: TFPIV264M displayed increased protein levels and activity compared to TFPIWT accompanied by an increase in mRNA levels of TFPIV264M due to prolonged stability of TFPIV264M mRNA. The specific activity of the TFPIV264M was similar to TFPIWT, indicating that the mutation does not affect the enzymatic function of the protein.

Objective: To estimate the incidence of venous thromboembolism (VTE) in pregnancy and puerperium and to identify risk factors for pregnancy-related VTE. Study Design: A register-based case-control study with 613232 pregnancies from 1990 to 2003 in eleven Norwegian counties. Medical records for eligible cases were revisited and relevant medical data were transferred to a specific case-report-form. The diagnosis of VTE was based on strict criteria. Data were analyzed by chi-square test and forward stepwise logistic regression. Results: In total 615 cases were detected. The incidence of VTE was 1/1000 pregnancies. The ante- and postnatal incidences were quite similar. Antenatal risk factors were assisted reproduction, gestational diabetes, age over 35 years, multiple pregnancies, and primi-parity. Postnatal risk factors were Cesarean section, preeclampsia, assisted reproduction, abruptio placenta, and placenta previa. Conclusion: We found different ante- and postnatal risk patterns. Assisted reproduction and gestational diabetes were significant antenatal risk factors while Cesarean section and preeclampsia were strong postnatal risk factors.

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

Objective To evaluate impact of different postmenopausal hormone therapy (HT) regimens and raloxifene on mammographic breast density. Design Open, randomised, comparative clinical trial. Setting Women were recruited through local newspapers and posters. They were examined at the departments of haematology, gynaecology, and radiology in a University Hospital. Population 202 healthy postmenopausal women, between the age of 45 and 65 years. Methods Subjects were randomly assigned to receive daily treatment for 12 weeks with tablets containing either low-dose HT containing 1 mg 17 -estradiol + 0.5 mg norethisterone acetate (NETA)(n=50), conventional-dose HT containing 2 mg 17 -estradiol and 1 mg NETA (n=50), 2.5 mg tibolone (n=51), or 60 mg raloxifene (n=51). Mammographic density was determined at baseline and after 12 weeks by an automated technique in full-field digital mammograms (FFDM). Main outcome measures Mammographic density was expressed as Volumetric Breast Density Estimations. Results Mammographic breast density increased significantly and to a similar degree in both the conventional- and low-dose HT group. A small reduction in mammographic breast density was seen in the raloxifene group, whereas those allocated to tibolone treatment only showed minor changes. Conclusions Our findings demonstrated a significant difference in impact on mammographic breast density between the regimens. Although these results indicate a differential effect of these regimens on breast tissue the relation to breast cancer risk remains unresolved.

Objective: To study ante- and postnatal risk factors of venous thrombosis (VT) in pregnancy. Methods: A hospital-based case-control study. Cases were women with objectively verified VT during pregnancy or postpartum. Two controls were selected for each case. Validated risk factors were analyzed by chi-square test and logistic regression. Results: In total 559 cases with no prior VT, 268 ante- and 291 postnatal cases, were identified together with 1229 controls. Risk factor for antenatal VT were assisted reproduction technique (ART), antepartum immobilization, cigarette smoking, and slight weight gain (<7 kg). Conception after ART and multiple pregnancy showed additive effect, whereas antepartum immobilization and high body mass index (BMI) showed multiplicative effect on the risk for antepartum VT. No other interaction was found between risk factors for antepartum VT. Risk factors for postnatal VT were antepartum immobilization, cigarette smoking, IUGR, preeclampsia, emergency caesarean section, postpartum hemorrhage, infection, and surgery, and age and parity. Antepartum immobilization and high BMI and reoperation on the indication bleeding showed multiplicative effects on the risk of postnatal VT. Conclusions: Ante- and postpartum risk factors differed markedly. More attention should be paid to pregnant women of high BMI who are at risk for immobilization.

Background Postmenopausal hormone therapy (HT) is associated with an increased risk for arterial and venous thrombosis. Objectives To compare the impact of HT, tibolone, and raloxifene on C-reactive protein (CRP) and other inflammatory markers, and to investigate possible underlying mechanisms for changes in CRP and D-dimer. Methods 202 healthy women were randomly assigned to treatment for 12 weeks with either low-dose HT containing 1 mg 17 -estradiol + 0.5 mg norethisterone acetate (NETA)(n=50), conventional-dose HT containing 2 mg 17 -estradiol and 1 mg NETA (n=50), 2.5 mg tibolone (n=51), or 60 mg raloxifene (n=51). Results CRP increased in the conventional and low-dose HT-group. These changes were significantly more pronounced in the conventional-dose group (RMANOVA p=0.02). Also tibolone was associated with an increase in CRP, in contrast to raloxifene which reduced CRP. Reduction in levels of Lp (a), ICAM, P-selectin, E-selectin, MCP-1 and IL-6 were observed in all treatment groups. The changes were most pronounced for the conventional dose HT group, least pronounced for the raloxifene, whereas the changes in those allocated to tibolone and low-dose HT were intermediary. Increased levels of TNF- and vWf ag were seen in the raloxifene group. We observed positive associations between changes in IL-6, vWf ag, MCP-1 and CRP. Conclusions The regimens had markedly different impact on markers of inflammation. The average increase in CRP was not accompanied by an increase in average IL-6 or TNF- or other markers, but women with large reductions in IL-6 had reduced increase in CRP.

Patients with haematological malignancies carry increased risk of venous thrombosis (VT). However, the mechanisms that link these malignancies to activated coagulation have not been fully identified. Since anti-haemostatic agents are studied in clinical trials for their potential to prolong survival in cancer patients, a detailed characterisation of haemostatic markers in cancer subtypes is needed. Hence, in this study, we measured the plasma concentrations and mRNA expression in blood mononuclear cells of haemostatic parameters in 93 patients with haematological neoplasias (acute myeloid leukaemia, chronic lymphatic leukaemia, multiple myeloma, and non-Hodgkin’s lymphoma) before start and after completion of cancer therapy. At diagnosis we found activation of coagulation and fibrinolysis, especially in patients with acute myeloid leukaemia. This hypercoagulation was not associated with increased levels of tissue factor (TF) or factor VII (fVII) antigen or mRNA, or levels of activated fVII. In conclusion we found a hypercoagulable state in patients with haematological malignancy that did not seem to be initiated by TF.

Background: We have previously found that activation of coagulation in patients with various hematological malignancies was apparently not initiated by tissue factor (TF). Acquired activated protein C (APC) resistance may be another mechanism responsible for such hypercoagulation, and has been demonstrated in patients with solid tumors, but not in patients with hematological malignancy. Objective: To investigate acquired APC resistance in a hypercoagulable cohort of patients with hematological malignancies. Patients/methods: Blood samples from 93 patients with acute myeloid leukemia (AML), chronic lymphatic leukemia, multiple myeloma, or non-Hodgkin's lymphoma, were analyzed before start and after completion of cancer therapy. APC resistance was measured using calibrated automated thrombography. The APC sensitivity ratio (APC-SR) was calculated as the ratio of the endogenous thrombin potential (ETP) determined in plasma probed with either APC or buffer. Results: Untreated patients were found to have higher APC-SR than healthy controls, and AML patients had higher APC-SR as compared to the other diagnoses, both findings being consistent with acquired APC resistance. The acquired APC resistance was partly ameliorated with cancer treatment. Decreased levels of protein S and tissue factor pathway inhibitor were inversely correlated to APC resistance. Conclusions: APC resistance may contribute to the hypercoagulable state in hematological malignancies.

The aim of the study was to investigate the association between the proximal level of the clot and the severity of pulmonary embolism (PE). The cohort consisted of 99 consecutive patients with PE diagnosed by multi-detector CT. A new score was constructed by calculating the mean value of the largest affected vessel (sub-segmental=1, segmental=2, lobar=3, main pulmonary artery (MPA)=4) in each lung. A significant association was found between the most proximal level of PE and pulmonary artery obstruction index (PAOI) (p<0.0001), right ventricular (RV)/left ventricular (LV) ratio (p<0.0001), and PaO2 (p=0.004). No significant association was found between systolic blood pressure and the level of PE. Troponin-T was elevated in none of the sub-segmental, 5% of segmental, 20% of lobar, and in 56% of PEs in the MPA (p=0.001). Significant association was found between the proposed score and PAOI (p<0.0001), RV/LV ratio (p<0.0001), PaO2 (p<0.008). Troponin-T was elevated in 10% of level 1, 0% of level 2, 43% level of 3, 66% of level 4 PE (p<0.0001). Cut-off level score 4 yielded a sensitivity of 84% and a specificity of 74% for the detection of elevated Troponin-T. In conclusion the study indicates that both the most proximal level of PE and the proposed score are related to the severity of PE as determined by blood oxygenation, biochemical and radiological parameters and could therefore be of value for rapid risk stratification of PE. However, the prognostic value of these classifications and their clinical significance needs to be evaluated in properly designed studies.

Introduction. We have recently reported that different hormone regimens given to healthy post-menopausal women had markedly different effects on activation of coagulation. Low-dose hormone therapy (HT) and raloxifene, as opposed to conventional-dose HT and tibolone, were associated with no or minor activation of coagulation. The aim of this study was to elucidate the mechanism(s) for differences in coagulation activation by analysing clotting and fibrinolytic factors and coagulation inhibitors. Materials and Methods. 202 healthy women were randomly assigned to receive treatment for 12 weeks with either low dose HT containing 1 mg 17 -estradiol + 0.5 mg norethisterone acetate (NETA) (n=50), conventional dose HT containing 2 mg 17 -estradiol and 1 mg NETA (n=50), 2.5 mg tibolone (n=51), or 60 mg raloxifene (n=51) in an open-label design. Results. The conventional- and low-dose HT groups generally showed similar effects, i.e., reductions in both clotting factors and inhibitors, but the effects were markedly more pronounced in the conventional-dose HT group. Compared with the low-dose HT group those treated with tibolone showed more pronounced decreases in factor VII, less reduction of antithrombin and protein C and even increased levels in protein S and tissue factor pathway inhibitor. As opposed to the low-dose HT group the reductions in inhibitors in the raloxifene group were smaller. Moreover in those allocated to raloxifene reduced levels of fibrinogen were seen. Conclusions. Our study demonstrates that the different HT regimens and raloxifene exert differential effects on coagulation factors, inhibitors and fibrinolytic factors.

BACKGROUND AND PURPOSE: We wanted to investigate whether common prothrombotic mutations are more prevalent in patients with atrial fibrillation who have had a stroke than in healthy controls. We also wanted to assess whether early recurrent ischemic cerebrovascular events were more frequent among carriers of the factor V Leiden or the prothrombin gene mutations than among others. METHODS: We used a case-control design with 367 patients with acute ischemic stroke and atrial fibrillation (cases) and 482 healthy blood donors (controls). All mutations were detected with conventional polymerase-chain reaction protocols. RESULTS: The odds ratios for carriers of the factor V Leiden, prothrombin gene 20210GA, methylenetetrahydrofolate reductase 677CT, or platelet glycoprotein IIIa 1565TC (Pl(A2)) mutation were 0.91, (95% CI, 0.51 to 1.59), 2.25 (95% CI, 0.61 to 8.90), 0.83 (0.61 to 1.13), and 0.79 (0.57 to 1.10), respectively. Early recurrent ischemic stroke and total recurrent ischemic cerebrovascular events were slightly more frequent among carriers of the factor V Leiden mutation than among noncarriers: odds ratio 1.45 (95% CI, 0.41 to 5.1), and 1.59 (0.61 to 4.1), respectively. None of the patients with recurrent ischemic cerebrovascular events had the prothrombin gene mutation. CONCLUSIONS: These mutations are not important risk factors for thromboembolic stroke associated with atrial fibrillation. Carriers of the factor V Leiden mutation had a small, nonsignificantly higher risk of early recurrent ischemic cerebrovascular events.

Background The Coagulation Inhibitor Potential (CIP) assay is a global assay that may detect major thrombophilia at a sensitivity of 100% and a specificity of 70-80%. Subnormal CIP may be associated with increased risk of venous thromboembolism (VTE). Aims Compare the effect on CIP in plasma samples from postmenopausal women treated with four different therapeutic regimens. Methods Fibrin aggregation in plasma was monitored after activation of coagulation with tissue factor, and the effect of potentiated inhibition of coagulation was measured. The assay was performed on plasma samples from 202 healthy women who were randomly assigned to receive treatment for twelve weeks with conventional-dose hormone therapy (HT), low-dose HT, raloxifene or tibolone. Major thrombophilias were excluded. Twelve women were heterozygous carriers of the factor V Leiden mutation. Results Compared to baseline the median level in CIP was reduced by 64% in the conventional-dose HT group, by 38% in the low-dose HT group, 31% in the raloxifene group, whereas for those treated with tibolone median CIP increased by 9%. The median changes in CIP were significant for both the HT groups (P  0.0001) and for those allocated to raloxifene (P = 0.003), but not for the tibolone group (P = 0.653). Women with heterozygous factor V Leiden mutation had significantly reduced median CIP level (P  0.0001) at baseline. Conclusions: HT and raloxifene, associated with VTE, reduce CIP. Tibolone does not reduce CIP.

Fibrinogen in plasma includes three main fractions; high-molecular-weight (HMW)-fibrinogen, low-molecular-weight (LMW)-fibrinogen, and very-low-molecular-weight (LMW')-fibrinogen. During acute-phase conditions, plasma fibrinogen levels and the HMW-/LMW-fibrinogen ratio increase rapidly due to increased synthesis of HMW-fibrinogen. The consequences of elevated plasma fibrinogen levels and local deposition of fibrin in inflammatory tissues observed during acute-phase conditions are not clear. We wanted to investigate proinflammatory effects of fibrinogen and fibrin on peripheral blood mononuclear cells (PBMC) as reflected by altered mRNA expression and synthesis of the proinflammatory cytokines IL-6, TNF-alpha and IL-1beta, and to explore the significance of altered HMW-/LMW-fibrinogen ratio. PBMC were isolated from whole blood using Lymphoprep((R)). HMW-fibrinogen was separated from unfractioned fibrinogen by ammonium sulphate precipitation. Cells were incubated with unfractioned fibrinogen, HMW-fibrinogen or fibrin. Cytokine levels in cell lysates were determined using ELISA assays. Real-time PCR was used for mRNA quantification. We found that fibrinogen significantly increased mRNA levels, and induced synthesis of the proinflammatory cytokines IL-6 and TNF-alpha in PBMC in a dose dependent manner. Median (25, 75 percentile) IL-6 and TNF-alpha concentrations were 12 (5, 40) pg/ml and 16 (0,61) pg/ml, respectively. Median mRNA quantity was increased 12.3- (6.6, 48.6) and 1.7- (1.5, 6.5) fold for IL-6 and TNF-alpha compared to controls. The stimulatory effect of unfractioned fibrinogen was not significantly different from HMW-fibrinogen. Fibrinogen and fibrin were equally effective in promoting cytokine synthesis from PBMC. The results support that fibrin and fibrinogen may actively modulate the inflammatory process by inducing synthesis of proinflammatory cytokines from PBMC.

Purpose: To assess the interobserver variability of radiologists with varied levels of experience in the interpretation of multidetector computed tomography (MDCT) pulmonary angiographies. Material and Methods: Review of CT pulmonary angiographies performed on patients included in a diagnostic study evaluating a decision-based algorithm for diagnosing pulmonary embolism (PE). Five radiologists, three board-certified general radiologists and two radiology trainees with 2 years’ experience, participated in the study. Results: According to the consensus reading, PE was present in 91 (31%) and absent in 194 (67%) patients, while in five patients (1.7%) the interpretations were regarded as equivocal. The per-patient agreement on the diagnosis of PE achieved by each of the four readers compared to the consensus reading was very good (k range 0.85–0.92), but peripheral emboli were missed in four to six patients by three of four observers. The agreement on the most proximal level of PE (per-proximal level) assessed by mean k value was 0.83 (k range 0.68–0.91) for the detection of proximal emboli, 0.61 for segmental emboli (k range 0.40–0.80), and 0.38 for emboli in the subsegmental vessels (k range 0.0–0.89). Conclusion: The overall agreement on the diagnosis of PE by MDCT for general radiologists and radiology trainees is very good, and we therefore believe that the initial management of patients with suspected PE could be based on the preliminary assessment performed by on-call radiologists with 2 years of experience. Key words: Alternative diagnosis; interobserver agreement; multidetector CT; pulmonary embolism

Atherosclerotic disease is denoted by low density lipoprotein (LDL) influx and retention in the artery intima, triggering the recruitment of monocytes causing a low grade chronic inflammation. Tissue factor (TF) pathway inhibitor (TFPI) regulates TF induced blood coagulation. Two isoforms exist, TFPIα and TFPIβ. In addition, TFPIα is also found complexed with LDL with unknown function. Moreover, ApoE-/- TFPI-/+ double KO mice display an atherogenic phenotype. We aimed to identify the cells expressing TFPI isoforms in human atherosclerotic plaques and the function of TFPI in these cells.

Endoplasmic reticulum (ER) stress is an important event during the initiation and clinical progression of several cardiovascular diseases, including atherosclerosis. In the present study we determined the role of tissue factor pathway inhibitor (TFPI) under ER stress conditions using human monocyte-derived macrophages (PBMC) and human carotid endarterectomies. TFPI was expressed in carotid plaques from patients with internal carotid stenosis, especially the TFPIα isoform. Moreover, TFPI colocalized with the ER stress marker CHOP in the plaques. Cholesterol crystals (CC) were used in human M1 and M2-polarized macrophages in vitro as an inducer of ER stress. CHOP and TFPI mRNA levels were upregulated after CC treatment, especially in the M2 phenotype. The ER stress inhibitor 4-Phenylbutyric acid (PBA) reversed the CC-mediated upregulation of CHOP and TFPI. Furthermore, M2-polarized macrophages incubated with CC had higher levels of Bcl-2:Bax mRNA ratios and Bcl-2 protein expression, and these effects were reversed by TFPI knockdown and PBA treatment. Our results indicate that TFPI could promote survival of the M2-polarized macrophages under ER stress conditions. These findings may have implications for the pathogenesis of atherosclerosis.

Nye retningslinjer med 250 anbefalinger for antitrombotisk behandling og tromboseprofylakse er nå tilgjengelig på nett. Retningslinjene fra Norsk selskap for trombose og hemostase imøtekommer nye standarder for troverdige retningslinjer og benytter seg av innovative teknologiske løsninger.