Sensitive Measurement of Clinically Relevant Factor VIII Levels in Thrombin Generation Assays Requires Presence of Factor XIa.

BACKGROUND: Hemophilia A (HA) is characterized by decreased or absent factor VIII (FVIII) activity. Current FVIII assays are based on clotting time and thus only provide information about the initiation of coagulation. In contrast, thrombin generation assays (TGAs) can be used to measure the full coagulation spectrum of initiation, propagation, and termination that provide information on the whole course of thrombin generation and inhibition. However, the commercially available TG kits lack sensitivity for measurements of hemophilia plasma within lower FVIII ranges, which is essential for explaining differences in bleeding phenotypes in hemophiliacs at clinically low levels of FVIII. AIMS: Optimization of the TGA for measurements of low FVIII levels in severe HA patients. METHODS: TGA measurements were performed in severe HA pooled plasma (n = 10). Investigations of several preanalytical and analytical variables of the assay were performed in a stepwise process and adjusted based on sensitivity toward intrinsic coagulation activation. RESULTS: TGA initiated by tissue factor (TF) alone at varying concentrations was unable to significantly differentiate between FVIII levels below 20%. In contrast, TGA activation with low concentrations of TF in presence of FXIa appeared to be highly sensitive for FVIII changes both in high and low ranges. In addition, a representative TGA curve at trough levels could only be produced using the dual TF/FXIa TGA. CONCLUSION: We propose a critical optimization for the setup of the TGA for measurements in severe HA plasma. The dual TF/FXIa TGA shows increased sensitivity, especially in lower FVIII ranges, which allows for better individual characterization at baseline, prediction of interventions, and follow-up.

Protein arginine deiminase 4 inactivates tissue factor pathway inhibitor-alpha by enzymatic modification of functional arginine residues.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an important regulator of coagulation and a link between inflammation and thrombosis. During thrombotic events, TFPI is proteolytically inactivated by neutrophil elastase while bound to neutrophil extracellular traps (NETs). Protein arginine deiminase 4 (PAD4) catalyzes the conversion of arginine to citrulline and is crucial for NET formation. OBJECTIVES: Here, we show that PAD4 inactivates full-length TFPIα by citrullination of its functional arginines. METHODS: Citrullination of TFPIα and of TFPI-constructs by PAD4 was studied using western blotting and mass spectrometry. Binding of TFPIα to PAD4 was investigated using a solid-phase assay. Functional consequences were investigated by factor Xa inhibition and thrombin generation assays. RESULTS: Nanomolar PAD4 amounts eliminated factor Xa inhibition by TFPIα. A citrullinated mutant Kunitz 2 domain did not inhibit factor Xa. Citrullination of TFPIα was found to be time- and concentration-dependent. Immunoprecipitation of citrullinated proteins from whole blood after neutrophil activation suggested the presence of TFPIα. Negatively charged phospholipids inhibited citrullination and truncated variants K1K2 and TFPI 1-161, and the isolated K2 domain were less efficiently citrullinated by PAD4. TFPIα bound to PAD4 with nanomolar affinity and involved the basic C-terminus. Thrombin generation in TFPI-deficient plasma demonstrated reduced anticoagulant activity of citrullinated TFPI. Mass spectrometry demonstrated citrullination of surface-exposed arginine residues in TFPIα after incubation with PAD4. CONCLUSION: Full-length TFPIα is sensitive to citrullination by PAD4, which causes loss of factor Xa inhibition. This process may play a role in the increased thrombosis risk associated with inflammation.

Resistance to activated protein C and impaired TFPI activity in women with previous hormone-induced venous thromboembolism.

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.

Effect of blood loss during caesarean section on coagulation parameters.

INTRODUCTION: Venous thrombosis is the leading cause of pregnancy-related maternal morbidity and mortality. The thrombosis risk is increased by caesarean section and blood loss, though underlying mechanisms of these prothrombotic changes remain unknown. MATERIALS AND METHODS: This prospective study recruited 50 pregnant women at term undergoing elective caesarean section at University Hospital Magdeburg, Germany. Blood loss during surgery was correlated with the changes in total protein S, full length TFPI (TFPIfl), prothrombin, the endogenous thrombin potential (ETP) and resistance to activated protein C (APCsr) determined via calibrated automated thrombography. RESULTS: Mean blood loss was 506 ml (95%CI: 456 to 557 ml). Total protein S was 0.63 (95%CI: 0.60 to 0.67) U/ml preoperatively, decreased by 14.8% after caesarean section and almost normalised five days later. TFPIfl was 0.47 (95%CI: 0.41 to 0.53) U/ml before, remained unchanged immediately after and increased by 11.5% five days after surgery. Prothormbin was 1.10 (95%CI: 1.03 to 1.16) U/ml preoperatively, reduced by 10.4% immediately after and increased again five days after caesarean section, exceeding the preoperative values by 4.4% (-0.7 to 9.6). The ETP decreased by 3.9%, whereas the APCsr increased by 37.0% immediately after caesarean section. The changes in total protein S, prothrombin, thrombin generation and APC resistance showed a trend to be more pronounced in the subgroups with higher blood loss. DISCUSSION: Moderate blood loss during caesarean section hardly reduces thrombin generation but aggravates pregnancy-induced APC resistance and combined deficiency of TFPI and protein S, which can account for the increased thrombosis risk in early puerperium.

Severe thrombophilia in a factor V-deficient patient homozygous for the Ala2086Asp mutation (FV Besançon).

BACKGROUND: Coagulation factor V (FV), present in plasma and platelets, has both pro- and anticoagulant functions. OBJECTIVE: We investigated an FV-deficient patient (FV:C 3%, FV:Ag 4%) paradoxically presenting with recurrent venous thrombosis (11 events) instead of bleeding. METHODS/RESULTS: Thrombophilia screening revealed only heterozygosity for the F2 20210G>A mutation. Although thrombin generation in the patient's platelet-poor plasma was suggestive of a hypocoagulable state, thrombin generation in the patient's platelet-rich plasma (PRP) was higher than in control PRP and extremely resistant to activated protein C (APC). This was partially attributable to the complete abolition of the APC-cofactor activity of FV and a marked reduction of plasma tissue factor pathway inhibitor antigen and activity. The patient was homozygous for a novel missense mutation (Ala2086Asp, FVBesançon ) that favors a "closed conformation" of the C2 domain, predicting impaired binding of FV(a) to phospholipids. Recombinant FVBesançon was hardly secreted, indicating that this mutation is responsible for the patient's FV deficiency. Model system experiments performed using highly diluted plasma as a source of FV showed that, compared with normal FVa, FVaBesançon has slightly (≤1.5-fold) unfavorable kinetic parameters (Km , Vmax ) of prothrombin activation, but also a lower rate of APC-catalyzed inactivation in the presence of protein S. CONCLUSIONS: FVBesançon induces a hypercoagulable state via quantitative (markedly decreased FV level) and qualitative (phospholipid-binding defect) effects that affect anticoagulant pathways (anticoagulant activities of FV, FVa inactivation, tissue factor pathway inhibitor α level) more strongly than the prothrombinase activity of FVa. A possible specific role of platelet FV cannot be excluded.

Double-dose cefuroxime concentrations in bone, synovial fluid of the knee joint and subcutaneous adipose tissue-A randomised porcine microdialysis study.

This study evaluated target tissue concentrations of double dose cefuroxime administered intravenously as either one 15 min infusion of 3000 mg (Group 1) or two single 15 min infusions of 1500 mg administered 4 h apart (Group 2). Sixteen pigs were randomised into two groups of eight. Cortical and cancellous bone, synovial fluid of the knee joint and subcutaneous adipose tissue concentrations were measured based on sampling via microdialysis. Plasma samples were collected as a reference. Comparison of the groups was based on time with concentrations above relevant minimal inhibitory concentrations (fT>MIC) of 4 µg/mL. The mean time fT>MIC (4 µg/mL) across compartments was longer for Group 2 (280-394 min) than for Group 1 (207-253 min) (p<0.01). Cortical bone showed a tendency towards longer fT>MIC (4 µg/mL) in Group 2 (280 min) than in Group 1 (207 min) (p = 0.053). Within 50 min after administration, the mean concentration of 4 µg/mL was reached in all compartments for both groups. The mean concentrations decreased below 4 µg/mL after approximately 4 h (Group 1) and 3 h (Group 2) from initiation of administration (time zero). During an 8 h interval, double-dose cefuroxime administered as 2 × 1500 mg with a 4 h interval provides longer time above MIC breakpoint for Staphylococcus aureus (4 µg/mL) than a single bolus of 3000 mg cefuroxime. To maintain sufficient tissue concentrations during longer surgeries, re-administration of cefuroxime (1500 mg) should be considered 3 h after the first administration.

Targeted next-generation sequencing of adult gliomas for retrospective prognostic evaluation and up-front diagnostics.

AIMS: We aimed to reclassify a population-based cohort of 529 adult glioma patients to evaluate the prognostic impact of the 2016 World Health Organization (WHO) central nervous system tumour classification. Moreover, we evaluated the feasibility of gene panel next-generation sequencing (NGS) in daily diagnostics of 225 prospective glioma patients. METHODS: The retrospective cohort was reclassified according to WHO 2016 criteria by immunohistochemistry for IDH-R132H, fluorescence in situ hybridization for 1p/19q-codeletion and gene panel NGS. All tumours of the prospective cohort were subjected to NGS analysis up-front. RESULTS: The entire population-based cohort was successfully reclassified according to WHO 2016 criteria. NGS results were obtained for 98% of the prospective patients. Survival analyses in the population-based cohort confirmed three major prognostic subgroups, that is, isocitrate dehydrogenase (IDH)-mutant and 1p/19q-codeleted oligodendrogliomas, IDH-mutant astrocytomas and IDH-wildtype glioblastomas. The distinction between WHO grade II and III was prognostic in patients with IDH-mutant astrocytoma. The survival of patients with IDH-wildtype diffuse astrocytomas carrying TERT promoter mutation and/or EGFR amplification overlapped with the poor survival of IDH-wildtype glioblastoma patients. CONCLUSIONS: Gene panel NGS proved feasible in daily diagnostics. In addition, our study confirms the prognostic role of glioma classification according to WHO 2016 in a large population-based cohort. Molecular features of glioblastoma in IDH-wildtype diffuse glioma were linked to poor survival corresponding to IDH-wildtype glioblastoma patients. The distinction between WHO grade II and III retained prognostic significance in patients with IDH-mutant diffuse astrocytic gliomas.

Evaluation of tumor-infiltrating lymphocytes and association with prognosis in BRCA-mutated breast cancer.

BACKGROUND: Patients with a BRCA1 or BRCA2 mutation (BRCA-mutated breast cancer) are frequently diagnosed with low differentiated and highly proliferating breast cancer characterized by high amounts of tumor-infiltrating lymphocytes (Tils). Stromal Tils (sTils) are highly prognostic in sporadic triple-negative and HER2 positive breast cancer however, their prognostic importance in BRCA-mutated breast cancers is unknown. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded primary tumor tissue from 411 patients with a germline BRCA1 or BRCA2 mutation and diagnosed with early breast cancer was included. The percentage of sTils was quantified on full HE sections according to guidelines proposed by the Immuno-Oncology Biomarker in Breast Cancer Working Group. Distribution of sTils and associates with patient and tumor characteristics were assessed according to categorical sTils groups defined as low (<10%), intermediate (10-59%) and high (≥60%). Prognostic associations of sTils were evaluated as a continuous variable in univariate and multivariate models. Only follow-up time beyond date of BRCA mutation test was included. RESULTS: A large proportion had high sTils (27% in the full cohort, 36% in BRCA1-mutated, and 44% in ER negative breast cancers). Higher sTils were associated with BRCA1, ER negative breast cancer, high histological grade and medullary histology. In combined analysis for BRCA1 and BRCA2-mutated breast cancers, increasing sTils in 10% intervals were significantly associated with OS (HR 0.92, 95% CI 0.84-1.00, p = .05). For each 10% increment of sTils in BRCA1 breast cancers, a 10% reduction of mortality (adjusted HR 0.90 95% CI 0.81-0.99, p = .03) and a 13% reduction in risk of DFS-event (HR 0.87 95% CI 0.76-1.00, p = .05) was observed even after adjustment for ER status. No significant association with survival was of observed in the BRCA2 subgroup. Test for interaction of sTils and BRCA status was not statistically significant (p = .3). CONCLUSIONS: Breast cancer patients with a germline BRCA mutation had higher sTils than previously reported in sporadic breast cancers, and sTils were associated with favorable survival among BRCA carriers.

Clinical and molecular characterization of BRCA-associated breast cancer: results from the DBCG.

BACKGROUND: In breast cancer (BC) patients a cancer predisposing BRCA1/2 mutation is associated with adverse tumor characteristics, risk assessment and treatment allocation. We aimed to estimate overall- (OS) and disease-free survival (DFS) according to tumor characteristics and treatment among women who within two years of definitive surgery for primary BC were shown to carry a mutation in BRCA1/2 . MATERIAL AND METHODS: From the clinical database of the Danish Breast Cancer Group we included 141 BRCA1 and 96 BRCA2 BC patients. Estrogen receptor and HER2 status were centrally reviewed on paraffin-embedded tumor tissue. Information on risk reducing surgery was obtained from the Danish Pathology and Patient Registries and included as time-dependent variables in Cox proportional hazard models. RESULTS: Ten-year OS and DFS for BRCA1 BC patients were 78% (95% CI 69-85) and 74% (95% CI 64-81). Ten-year OS and DFS for BRCA2 BC were 88% (95% CI 78-94) and 84% (95% CI 74-91). BRCA1 BC patients as compared to BRCA2 BC patients had a higher risk of BC relapse or non-breast cancer within ten years of follow-up, independent of ER status (adjusted HR 2.78 95% CI 1.28-6.05, p = .01), but BRCA mutation was not associated with OS (adjusted HR 1.98, 95% CI 0.87-4.52, p = .10). In multivariate analysis, including both BRCA1 and BRCA2 carriers, no chemotherapy was associated with a higher risk of death (adjusted OS HR 3.58, 95% CI 1.29-9.97, p = .01) and risk reducing contralateral mastectomy (RRCM) was associated with a significantly reduced risk of death (adjusted OS HR 0.42, 95% CI =0.21-0.84, p = .01). CONCLUSION: Difference in OS between BRCA1 and BRCA2 BC patients could be ascribed to tumor-biology. BRCA1 BC patients may have a shorter ten-year DFS than BRCA2 BC patients. Chemotherapy and risk reducing contralateral mastectomy reduce mortality for both BRCA1 and BRCA2 BC patients.

Lecocytes mutation load declines with age in carriers of the m.3243A>G mutation: A 10-year Prospective Cohort.

Carriers of the mitochondrial mutation m.3243A>G presents highly variable phenotypes including mitochondrial encephalomyopathy, lactoacidosis and stroke-like episodes (MELAS). We conducted a follow-up study to evaluate changes in leucocyte heteroplasmy and the clinical phenotypes in m.3243A>G carriers. Leucocyte heteroplasmy was determined by next generation sequencing covered by 100 000X reads in 32 individuals with a median follow-up of 10.2 years. Ten-year clinical follow-up is reported in 46 individuals. The annual leucocyte mutation level declined by -0.7 (±0.4) percentage points/year (P < .0001), and correlated with the level of the initial sample (ρ = -0.92, P < .0001). Eleven of 46 m.3243A>G carriers died and clinical symptoms progressed. This longitudinal study shows the decline in leucocyte m.3243A>G heteroplasmy associates with the level of the initial sample. Further, there was a high mortality among carriers.

New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma.

Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation. SUMMARY: Background Protein S plays an important role in the down-regulation of coagulation as cofactor for activated protein C (APC) and tissue factor pathway inhibitor (TFPI). Aim To develop functional assays to quantify the APC- and TFPI-cofactor activities of protein S in plasma. Methods APC- and TFPI-cofactor activities of protein S in plasma were measured using calibrated automated thrombography in protein S-depleted plasma supplemented with a small amount of sample plasma either in the presence of anti-TFPI antibodies and APC (APC-cofactor activity) or at excess full-length TFPI without APC (TFPI-cofactor activity). Total and free protein S levels in plasma were measured by ELISAs. Results Average APC-cofactor activities of protein S were 113%, 108% and 89% in plasma from normal individuals (n = 15), FV Leiden heterozygotes (n = 14) and FV Leiden homozygotes (n = 7), respectively, whereas the average APC-cofactor activity of protein S in plasma from heterozygous protein S-deficient individuals (n = 21) was significantly lower (55%). Similar trends were observed for the TFPI-cofactor activity of protein S, with averages of 109%, 115% and 124% in plasma from individuals with normal protein S levels and different FV Leiden genotypes, and 64% in plasma from protein S-deficient patients. APC-cofactor activities of protein S correlated significantly with free and total protein S antigen levels, whereas TFPI-cofactor activities correlated less with protein S antigen levels. Conclusion We have developed functional protein S assays that measure both the APC- and TFPI-cofactor activities of protein S in plasma, which are hardly if at all affected by the FV Leiden mutation.

Differences in fat accumulation between immature male and female Atlantic salmon Salmo salar after dietary administration of tetradecylthioacetic acid.

This study provoked sex-specific differences in fat metabolism in Atlantic salmon Salmo salar, by dietary administration of tetradecylthioacetic acid (TTA) during their first spring and winter in the sea. The effects of TTA were evaluated in June of the first spring and May of the second spring in the sea, by analysing white muscle-fat content. Muscle fat in males and females differed significantly as a result of TTA in their diet and diet interacted with the sex of the fish. The fat content during the first spring after dietary TTA was lowered by a greater amount in females than in males, 3·1-4·3%, respectively (P < 0·05). In contrast, during the second spring, fat content was lowered by a greater amount in males than in females, 15·8-16·7%, respectively (P < 0·01). Condition factor followed a similar pattern to the muscle fat. The results indicate that the difference in male and female fat accumulation dynamics is related to sex-specific reproduction biology of S. salar. In addition, the findings show that it is important to consider the sex of the fish and the season of the year when studying fat dynamics and reproductive biology of S. salar.

Acute hypoxia induces upregulation of microRNA-210 expression in glioblastoma spheroids.

AIM: Tumor hypoxia and presence of tumor stem cells are related to therapeutic resistance and tumorigenicity in glioblastomas. The aim of the present study was therefore to identify microRNAs deregulated in acute hypoxia and to identify possible associated changes in stem cell markers. MATERIALS & METHODS: Glioblastoma spheroid cultures were grown in either 2 or 21% oxygen. Subsequently, miRNA profiling was performed and expression of ten stem cell markers was examined. RESULTS: MiRNA-210 was significantly upregulated in hypoxia in patient-derived spheroids. The stem cell markers displayed a complex regulatory pattern. CONCLUSION: MiRNA-210 appears to be upregulated in hypoxia in immature glioblastoma cells. This miRNA may represent a therapeutic target although it is not clear from the results whether this miRNA may be related to specific cancer stem cell functions.

Tissue factor-independent inhibition of thrombin generation by tissue factor pathway inhibitor-α.

BACKGROUND: Tissue factor pathway inhibitor-α (TFPIα) inhibits factor Xa by forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. TF-FVIIa forms a quaternary complex with TFPIα and FXa, which shuts off the initiation of coagulation via the extrinsic pathway. AIM: To investigate whether direct inhibition of FXa by TFPIα independently of TF plays a role in downregulating coagulation. METHODS: Inhibition of FXa by TFPIα in plasma was determined by measuring thrombin generation triggered with FXa, the FX activator from Russell's viper venom (RVV-X), FXIa, or FIXa. TF-independent anticoagulant activities of TFPIα and its cofactor, protein S, were quantified: (i) after neutralization of TFPIα and protein S with anti-TFPI or anti-protein S antibodies; and (ii) in TFPI-depleted or protein S-depleted plasmas supplemented with varying amounts of TFPIα or protein S. RESULTS: Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation in plasma triggered with RVV-X, FXa, FIXa, or FXIa. Anti-TFPI and anti-protein S antibodies decreased the lag time and increased the peak height of thrombin generation to the same extent, indicating that inhibition of FXa by TFPIα requires the presence of protein S. TFPIα and protein S titrations in TFPI-depleted or protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF also revealed TF-independent anticoagulant activity of TFPIα, which was completely dependent on the presence of protein S. CONCLUSION: Direct inhibition of FXa by TFPIα contributes to the downregulation of coagulation.

Changes in haemostatic parameters during the menstrual cycle and subsequent use of drospirenone-containing oral contraceptives.

INTRODUCTION: Oral contraceptives (OC) increase the risk of venous thromboembolism that depends on the OC formulation and could at least partially be explained by impaired function of the protein C-system (APC resistance) and the tissue factor pathway inhibitor (TFPI)-system. There is limited information available on the effects of OC, containing a newer progestogen- drospirenone (DRSP-OC) on these two major anticoagulant pathways, thrombin generation, reflecting the overall state of coagulation, and other coagulation parameters. METHODS: In a study population consisting of 14 healthy women (age 21-33 years) we investigated the effect of the menstrual cycle and subsequent use of DRSP-OC on APC resistance, the function of the TFPI-system, thrombin generation and on their major determinants, i.e. prothrombin, antithrombin, FV, FX, FVIII, protein C, protein S(total and free) and TFPI(full-length and free). RESULTS: All studied parameters remained unchanged during the menstrual cycle. During DRSP-OC use we observed a significant increase in APC resistance (~2.4-fold), thrombin generation measured at low (~2.2-fold) and high tissue factor concentrations (~1.4-fold), plasma concentrations of prothrombin (19%), FX (31%), FVIII (17%) and protein C (43%). DRSP-OC use impaired the function of the TFPI-system and decreased plasma levels of antithrombin (-6%), FV (-22%), protein Stotal (-21%), protein Sfree (-20%), TFPIfull-length (-36%) and TFPIfree (-46%). CONCLUSIONS: DRSP-OC caused procoagulant changes in all studied haemostatic parameters. The impairment of the protein C- and TFPI-systems was more pronounced than the impairment of the coagulation pathways and can at least partially account for the increased risk of venous thromboembolism in users of DRSP-OC.

A preliminary study: effects of football training on glucose control, body composition, and performance in men with type 2 diabetes.

The effects of regular football training on glycemic control, body composition, and peak oxygen uptake (VO2 peak) were investigated in men with type 2 diabetes mellitus (T2DM). Twenty-one middle-aged men (49.8 ± 1.7 years ± SEM) with T2DM were divided into a football training group (FG; n = 12) and an inactive control group (CG; n = 9) during a 24-week intervention period (IP). During a 1-h football training session, the distance covered was 4.7 ± 0.2 km, mean heart rate (HR) was 83 ± 2% of HRmax, and blood lactate levels increased (P < 0.001) from 2.1 ± 0.3 to 8.2 ± 1.3 mmol/L. In FG, VO2 peak was 11% higher (P < 0.01), and total fat mass and android fat mass were 1.7 kg and 12.8% lower (P < 0.001), respectively, after IP. After IP, the reduction in plasma glucose was greater (P = 0.02) in FG than the increase in CG, and in FG, GLUT-4 tended to be higher (P = 0.072) after IP. For glycosylated hemoglobin (HbA1), an overall time effect (P < 0.01) was detected after 24 weeks. After IP, the number of capillaries around type I fibers was 7% higher (P < 0.05) in FG and 5% lower (P < 0.05) in CG. Thus, in men with T2DM, regular football training improves VO2 peak, reduces fat mass, and may positively influence glycemic control.

Small peptides blocking inhibition of factor Xa and tissue factor-factor VIIa by tissue factor pathway inhibitor (TFPI).

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nM). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded ß-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nM) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nM). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.

Incorporation of disulfide containing protein modules into multivalent antigenic conjugates: generation of antibodies against the thrombin-sensitive region of murine protein S.

Antigenic peptide conjugates can be used as vaccines and for the production of antibodies for clinical and research use. A method is presented here for the construction of conjugates incorporating oxidatively folded protein domains in their native conformation. This method was used to prepare multiple antigenic peptide constructs of the thrombin-sensitive loop region of murine anticoagulant protein S.

Association of PHB 1630 C>T and MTHFR 677 C>T polymorphisms with breast and ovarian cancer risk in BRCA1/2 mutation carriers: results from a multicenter study.

BACKGROUND: The variable penetrance of breast cancer in BRCA1/2 mutation carriers suggests that other genetic or environmental factors modify breast cancer risk. Two genes of special interest are prohibitin (PHB) and methylene-tetrahydrofolate reductase (MTHFR), both of which are important either directly or indirectly in maintaining genomic integrity. METHODS: To evaluate the potential role of genetic variants within PHB and MTHFR in breast and ovarian cancer risk, 4102 BRCA1 and 2093 BRCA2 mutation carriers, and 6211 BRCA1 and 2902 BRCA2 carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2 (CIMBA) were genotyped for the PHB 1630 C>T (rs6917) polymorphism and the MTHFR 677 C>T (rs1801133) polymorphism, respectively. RESULTS: There was no evidence of association between the PHB 1630 C>T and MTHFR 677 C>T polymorphisms with either disease for BRCA1 or BRCA2 mutation carriers when breast and ovarian cancer associations were evaluated separately. Analysis that evaluated associations for breast and ovarian cancer simultaneously showed some evidence that BRCA1 mutation carriers who had the rare homozygote genotype (TT) of the PHB 1630 C>T polymorphism were at increased risk of both breast and ovarian cancer (HR 1.50, 95%CI 1.10-2.04 and HR 2.16, 95%CI 1.24-3.76, respectively). However, there was no evidence of association under a multiplicative model for the effect of each minor allele. CONCLUSION: The PHB 1630TT genotype may modify breast and ovarian cancer risks in BRCA1 mutation carriers. This association need to be evaluated in larger series of BRCA1 mutation carriers.

Changes in texture, colour and fatty acid composition of male and female pig shoulder fat due to different dietary fat sources.

Two experiments with 72 slaughter pigs in each were conducted. Entire males and females were individually fed restricted. Palm kernel-, soybean- and fish-oil were used in varying combinations, giving different dietary fat levels (29-80g/kg) and iodine values ranging from 50 to 131. Shoulder fat was analysed for fatty acid composition (inner and outer layer), firmness and colour. A clear dose-response relationship was seen between fatty acids in diets and in shoulder fat. Interestingly, the very long chain n-3 fatty acids seemed to be deposited more efficiently when additional fat was included in the diet. Both high and low dietary iodine values changed towards less extreme iodine values in fat. Low-fat diets enhanced de novo synthesis of fatty acids. Males revealed a higher percentage of PUFA and a lower percentage of C18:1 and MUFA. Fat firmness, but not colour, was influenced by sex and dietary fat source.