Relationship between sex hormone binding globulin and blood coagulation in women on postmenopausal hormone treatment.

: Postmenopausal hormone therapy increases the risk of venous thrombosis. Sex hormone binding globulin (SHBG) is a suggested marker of 'total estrogenicity'. The study objective was to evaluate the impact of hormone therapy on SHBG and the association with coagulation variables. The study populations comprised 202 healthy postmenopausal women randomized to treatment with low-dose or conventional-dose hormone therapy, tibolone or raloxifene (RET-study) and 140 women with a history of venous thrombosis randomized to conventional-dose hormone therapy or placebo (EVTET-study). SHBG was determined in serum collected at baseline and after 12 weeks. In healthy women, conventional-dose hormone therapy increased SHBG with mean 9.7 (95% confidence interval 4.8-14.5) nmol/l, low-dose hormone therapy by mean 5.9 (0.4-11.5) nmol/l, raloxifene by mean 7.2 (3.9-10.4) nmol/l, while tibolone reduced SHBG with mean -25.1 (-29.9 to -20.4) nmol/l. SHBG correlated with protein S, tissue factor pathway inhibitor (TFPI) and protein C at baseline, and with protein S and TFPI after 12 weeks, but the change in SHBG from baseline to 12 weeks was only associated with the change in activated protein C (APC) resistance. In women with a history of venous thrombosis, the mean increase in SHBG was 13.6 (8.4-18.9) nmol/l in the conventional-dose hormone therapy group, with no change in the placebo group. Baseline SHBG was higher among women who developed recurrent venous thrombosis on conventional-dose hormone therapy. SHBG correlated with several coagulation inhibitors, but the change in SHBG induced by postmenopausal hormone therapy was only associated with the change in APC resistance.

The reversal effect of prothrombin complex concentrate (PCC), activated PCC and recombinant activated factor VII in apixaban-treated patients in vitro.

BACKGROUND: The number of patients under treatment with FXa inhibitors is increasing, but there is no concensus on how to reverse their anticoagulant effect in case of a life-threatening bleeding. A specific antidote is not yet commercially available. Prothrombin complex concentrate (PCC), activated PCC (aPCC) and recombinant factor VIIa (rFVIIa) are suggested available reversal agents. OBJECTIVES: To find the most effective reversal agent to apixaban and to determine the optimal dose. PATIENTS/METHODS: PCC, aPCC, and rFVIIa at concentrations imitating 80%, 100%, and 125% of suggested therapeutic doses were added to blood drawn from apixaban-treated patients (n=30). aPCC was also tested in a 50% dose. Samples from healthy subjects (n=40) were used as controls. Thromboelastometry in whole blood (WB) and thrombin generation in platelet-poor plasma (PPP) were measured to assess the reversal effect. RESULTS: aPCC shortened clotting time (CT) in WB, and increased the peak thrombin concentration and velocity index in PPP to a greater extent than PCC and rFVIIa. No significant differences were seen between rFVIIa and aPCC on thrombin generation lag time, or between PCC and aPCC on endogenous thrombin potential (ETP). The 50% dose of aPCC had a slightly inferior effect, but was comparable to the other reversal agents. CONCLUSIONS: In this in vitro study the 80% dose of aPCC (40 IU/kg) reversed the anticoagulant effect of apixaban more effectively than the corresponding dose of rFVIIa and PCC both in WB (CT) and PPP (peak, ETP).

Reduced Levels of D-dimer and Changes in Gut Microbiota Composition After Probiotic Intervention in HIV-Infected Individuals on Stable ART.

BACKGROUND: Microbial translocation and chronic inflammation may contribute to non-AIDS morbidity in patients with HIV. This study assessed the impact of probiotic intervention on microbial translocation and inflammation in patients on antiretroviral therapy with viral suppression and subnormal CD4 count. METHODS: Thirty-two patients receiving antiretroviral therapy (CD4 <500 cells/µL) were randomized in a double-blind fashion to multistrain daily probiotics (n = 15), placebo (n = 9), or controls (n = 8) for 8 weeks. Soluble inflammation markers, D-dimer, lipopolysaccharide (LPS), sCD14, T-cell activation, tryptophan metabolites, and gut microbiota composition were analyzed at baseline and end of study. Nonparametric statistics were applied. RESULTS: Twenty-four participants completed the study and were included in as-treated analyses. In patients receiving probiotics, there was a significant reduction in D-dimer levels (median change 33%, P = 0.03) and a tendency to reduced levels of C-reactive protein (CRP) (P = 0.05) and interleukin (IL)-6 (P = 0.06). The changes in CRP and IL-6 were highly correlated (r = 0.95, P < 0.01), whereas changes in D-dimer did not correlate with changes in CRP or IL-6. Increases in Bifidobacteria (P = 0.04) and Lactobacilli (P = 0.06) were observed in the probiotic group, whereas the relative abundance of Bacteroides decreased (P ≤ 0.01). No significant changes were seen in markers of microbial translocation or T-cell activation. However, the expansion of Bifidobacteria correlated negatively with differences in LPS (r = -0.77, P = 0.01), whereas the reduction in Bacteroides correlated positively with changes in LPS during the study period (r = 0.72, P = 0.02). CONCLUSIONS: Probiotic intervention seemed to reduce markers of coagulation and inflammation without overt changes in microbial translocation. These findings warrant further studies in larger cohorts with long-term follow-up.

Results of a consensus meeting on the use of argatroban in patients with heparin-induced thrombocytopenia requiring antithrombotic therapy - a European Perspective.

Argatroban has been introduced as an alternative parenteral anticoagulant for HIT-patients in several European countries in 2005. In 2009 a panel of experts discussed their clinical experience with argatroban balancing risks and benefits of argatroban treatment in managing the highly procoagulant status of HIT-patients. This article summarizes the main conclusions of this round table discussion. An ongoing issue is the appropriate dosing of argatroban in special patient groups. Therefore, dosing recommendations for different HIT-patient groups (ICU patients; non-ICU patients, paediatric patients, and for patients undergoing renal replacement therapies) are summarized in this consensus statement. Because of the strong correlation between argatroban dosing requirements and scores used to characterize the severity of illness (APACHE; SAPS, SOFA) suitable dosing nomograms are given. This consensus statement contributes to clinically relevant information on the appropriate use and monitoring of argatroban based on the current literature, and provides additional information from clinical experience. As the two other approved drugs for HIT, danaparoid and lepirudin are either currently not available due to manufacturing problems (danaparoid) or will be withdrawn from the market in 2012 (lepirudin), this report should guide physicians who have limited experience with argatroban how to use this drug safely in patients with HIT.

The association of inherited thrombophilia and intrauterine fetal death: a case-control study.

Inherited thrombophilias are probably associated with placenta-mediated pregnancy complications, but the strength of the association between inherited thrombophilias and intrauterine fetal death after 22 gestational weeks varies due to small sample size and different methodologies used across studies. The objective of the present study was to investigate the association of inherited thrombophilia and intrauterine fetal death in a case-control design. We studied 105 women with a history of intrauterine fetal death after 22 gestational weeks and 262 controls with live births. We investigated the prevalence of the factor V Leiden (F5 rs6025) and prothrombin gene G20210A (F2 rs1799963) polymorphisms, and antithrombin, protein C and protein S deficiencies, and their association with intrauterine fetal death. Results were presented as percentages and odds ratios (ORs) with 95% confidence intervals (CIs). A total of 18.4% of cases and 11.8% of controls were positive for at least one inherited thrombophilia (OR 1.7; 95% CI 0.9-3.1). The prothrombin gene G20210A polymorphism (OR 4.0; 95% CI 1.1-14.4), but not the factor V Leiden polymorphism, or antithrombin, protein C or protein S deficiencies, was associated with intrauterine fetal death after 22 weeks of gestation. Compared with women with live births only, women with a history of intrauterine fetal death after 22 gestational weeks were significantly more often carriers of the prothrombin gene G20210A polymorphism.

An exploratory trial of cyclooxygenase type 2 inhibitor in HIV-1 infection: downregulated immune activation and improved T cell-dependent vaccine responses.

Chronic HIV infection is characterized by chronic immune activation and dysfunctional T cells with elevated intracellular cyclic AMP (cAMP), which inhibits the T cell activation capability. cAMP may be induced by prostaglandin E(2) following lipopolysaccharide (LPS)-induced upregulation of cyclooxygenase type 2 (COX-2) in monocytes due to the elevated LPS levels in patients with chronic HIV infection. This hypothesis was tested using celecoxib, a COX-2 inhibitor, for 12 weeks in HIV-infected patients without antiretroviral treatment in a prospective, open, randomized exploratory trial. Thirty-one patients were randomized in the trial; 27 completed the study, including 13 patients on celecoxib. Celecoxib reduced chronic immune activation in terms of CD38 density on CD8(+) T cells (-24%; P = 0.04), IgA levels (P = 0.04), and a combined score for inflammatory markers (P < 0.05). Celecoxib further reduced the inhibitory surface receptor programmed death 1 (PD-1) on CD8(+) T cells (P = 0.01), including PD-1 on the HIV Gag-specific subset (P = 0.02), enhanced the number of CD3(+) CD4(+) CD25(+) CD127(lo/-) Treg or activated cells (P = 0.02), and improved humoral memory recall responses to a T cell-dependent vaccine (P = 0.04). HIV RNA (P = 0.06) and D dimers (P = 0.07) tended to increase in the controls, whereas interleukin-6 (IL-6) possibly decreased in the treatment arm (P = 0.10). In conclusion, celecoxib downmodulated the immune activation related to clinical progression of chronic HIV infection and improved T cell-dependent functions in vivo.

Fibrinogen and fibrin induce synthesis of proinflammatory cytokines from isolated peripheral blood mononuclear cells.

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-1 beta, and to explore the significance of altered HMW-/LMW-fibrinogen ratio. PBMC were isolated from whole blood using Lymphoprep. 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.

Altered hemostatic balance and endothelial activation in pregnant women with familial hypercholesterolemia.

INTRODUCTION: Patients with familial hypercholesterolemia (FH) are prone to premature cardiovascular disease. During pregnancy plasma lipids reach higher absolute values in FH than in healthy women. Pregnancy is associated with activation of coagulation and possibly also of vascular endothelium, which might further increase the risk of cardiovascular disease in FH. However, whether hemostatic and endothelial activation markers are increased in pregnant FH women compared with non-FH pregnancies, is unknown. MATERIALS AND METHODS: Activation markers of hemostasis and endothelium were analyzed in blood samples collected prospectively from 22 heterozygous FH women during pregnancy and compared with those of a reference group of 149 healthy, pregnant women. RESULTS: A procoagulant pattern was detected in both groups, but was more evident among FH women at least partly due to their enhanced thrombin generation, and because tissue factor pathway inhibitor type 1 increased in the reference group only. Furthermore, plasminogen activator inhibitor type 2 antigen increased more in FH than in the reference group. Whereas C-reactive protein, intercellular adhesion marker-1 and E-selectin did not change appreciably, vascular endothelial cell adhesion molecule 1 rose markedly in FH. CONCLUSION: Increased lipid levels as well as a net procoagulant activity and an enhanced endothelial activation possibly confer additional risks of cardiovascular disease among pregnant FH women.

A daily glass of red wine induces a prolonged reduction in plasma viscosity: a randomized controlled trial.

Moderate red wine consumption has been associated with decreased risk of coronary heart disease. Reduced plasma viscosity and fibrinogen levels have been launched as possible contributors to this risk reduction. The effect of moderate red wine consumption on plasma viscosity, however, has not been investigated in a prospective, randomized trial. We wanted to evaluate the effect of moderate red wine consumption on plasma viscosity, fibrinogen concentration and fibrinogen subfractions. Healthy, nonsmoking volunteers were assigned to consume one glass of red wine daily for 3 weeks in a prospective, randomized cross-over study. In the second 3-week period the volunteers abstained from alcohol use. The plasma viscosity, fibrinogen concentration and the distribution of the main fibrinogen subfractions were determined at inclusion, after wine drinking and after abstention. Plasma viscosity was reduced by 0.026 and 0.024 mPa.s in the two groups following wine intake (95% confidence interval, 0.009-0.043, P = 0.004; 95% confidence interval, 0.0083-0.039, P = 0.003). The decrease in plasma viscosity was maintained following 3 weeks of abstention. The fibrinogen concentration was reduced by 0.17 g/l following wine drinking in the group starting with abstention (95% confidence interval, 0.04-0.29, P = 0.01). The distribution of the fibrinogen subfractions remained unaltered. We conclude that a daily glass of red wine for 3 weeks significantly reduces plasma viscosity. Fibrinogen concentrations are also significantly reduced, when preceded by an abstention period. The decreased viscosity levels are maintained after 3 weeks of abstention, suggesting a sustained viscosity lowering effect of red wine.

Tissue factor pathway inhibitor anticoagulant activity: risk for venous thrombosis and effect of hormonal state.

Full-length tissue factor pathway inhibitor (TFPI) is assumed to be biologically more important than truncated TFPI because of its stronger anticoagulant effect in the diluted prothrombin time (dPT) assay. We have developed a dPT-based assay for TFPI anticoagulant activity. Here, we report the effect of hormonal state on TFPI anticoagulant activity and whether TFPI anticoagulant activity assesses the risk for deep-vein thrombosis (DVT) better than conventional TFPI assays. We undertook a case-control study of 474 patients with DVT and 474 controls and compared the odds ratio (OR) for DVT for those with low TFPI anticoagulant activity with the ORs obtained for TFPI free and total antigen, and TFPI chromogenic substrate activity. Hormonal state affected clot time in dPT, but this effect was ameliorated by anti-TFPI antibodies. Low TFPI anticoagulant activity gave an OR of 1.5 (0.97-2.1) for DVT, similar to the ORs obtained with conventional TFPI assays, ranging from 1.2 to 1.4. Individuals low in both the activity assays obtained an OR of 5.9 (1.7-20). We concluded that the effect of hormonal state on dPT was mediated through TFPI, and a dPT-based assay of TFPI anticoagulant activity did not assess the risk for DVT better than conventional TFPI assays.

INR calibration of Owren-type prothrombin time based on the relationship between PT% and INR utilizing normal plasma samples.

Prothrombin time (PT) is clinically important and is used to monitor oral anticoagulant therapy. To obtain PT results in international normalized ratio (INR), the current standardization procedure is complex and involves reference reagents. The PT of diluted plasma samples can be determined with a combined thromboplastin (the Owren-type procedure), but not necessarily with a plain thromboplastin (the Quick-type procedure). Owren-type PT procedures can therefore, as an alternative to the INR calibration, be calibrated with diluted normal plasma to give PT results in percent of normal PT activity (PT%). The present study explored if a plasma-based calibration of an Owren-type PT procedure can be used to obtain results in INR. The approach was to establish a relationship between PT% and INR by multi-center analysis of 365 samples from healthy individuals and patients on warfarin treatment. INR values were obtained by manual Quick-type reference procedure and PT% values by various automated Owren-type procedures. A relationship INR = (1/PT% + 0.018)/0.028 was found. A calibration procedure, based on the relationship, was investigated. Calibrators were the median PT of 21 normal plasma at dilutions representing 100%, 50%, 25%, 12.5% and 6.25% of normal PT activity. These were assigned INR values of 1.00, 1.36, 2.07, 3.05 and 6.36. Calibration of various Owren-type assays was repeatedly performed by 5 expert laboratories during 3 consecutive years. The INR values of certain lyophilised or frozen control plasmas were determined. The frozen control plasmas had externally assigned INR values according to WHO guide-lines. Within the laboratory, CV was typically below 3%. No appreciable difference among the results of the different laboratories or the three assay occasions was found. Externally assigned and INR values were essentially identical to those found. These and other results indicated that the calibration procedure was reproducible, precise and accurate. Thus, an Owren-type PT assay can be calibrated with normal plasma samples to give results in INR and the investigated calibration procedure can be proposed for this purpose.

The viscosity of fibrinogen subfractions and of EDTA denatured fibrinogen do not differ from that of native fibrinogen.

INTRODUCTION: Fibrinogen is a major determinant of plasma viscosity. The increased risk of atherothrombotic disease associated with a high fibrinogen concentration may partly be attributed to its effect on viscosity. Since the ratio between the three main fibrinogen subfractions high molecular weight (HMW)-, low molecular weight (LMW)-, and very low molecular weight (LMW')-fibrinogen is altered during acute phase conditions, and an increased HMW/LMW-fibrinogen ratio is associated with increased thromboembolic risk, we have examined how these subfractions affect viscosity. The viscosity of plasma is usually determined in ethylenediaminetetra-acetic acid (EDTA) plasma at 37 degrees C. Under such conditions the clotting properties of fibrinogen is affected due to denaturation. Denaturation of plasma proteins may affect their viscosity. Therefore, we have also investigated the effects of EDTA on the viscosity of fibrinogen. MATERIALS AND METHODS: Purified fibrinogen was obtained by beta-alanine precipitation of plasma from healthy donors. Separation of the fibrinogen fractions was performed by gradual precipitation of purified fibrinogen by ammonium sulphate. The viscosity was determined using a Haake Microvisco 2 viscometer. RESULTS: There was no statistically significant difference between the viscosity of native fibrinogen and the three fibrinogen subfractions. A substantial prolongation of the thrombin clotting time was observed in the fibrinogen solution containing EDTA at 37 degrees C compared to 20 degrees C. However, the viscosity of EDTA anticoagulated purified fibrinogen and plasma samples did not differ from that of heparin anticoagulated samples. CONCLUSION: The viscosity of the main fibrinogen subfractions HMW-, LMW- and LMW-fibrinogen did not differ from that of native fibrinogen, and the use of EDTA as anticoagulant did not significantly affect the viscosity of fibrinogen at 37 degrees C.