High proportion of patients with bleeding of unknown cause in persons with a mild-to-moderate bleeding tendency: Results from the Vienna Bleeding Biobank (VIBB).

INTRODUCTION: Data on clinical characteristics and the prevalence of underlying coagulopathies in patients with mild-to-moderate bleeding disorders (MBDs) are scarce. AIM: We established the Vienna Bleeding Biobank (VIBB) to characterize and thoroughly investigate Austrian patients with MBDs. RESULTS: Four hundred eighteen patients (female = 345, 82.5%) were included. A platelet function defect (PFD) was diagnosed in 26 (6.2%) and a possible PFD in 30 (7.2%) patients. Eight patients (1.9%) were diagnosed with von Willebrand disease (VWD) (type 1 n = 6; type 2 n = 2), and 29 patients had low VWF (30-50 IU/dL). Deficiencies in factor VIII, IX, XI or XIII were found in 11 (2.6%), 3 (0.7%), 3 (0.7%) and 1 patient(s), 2 patients had dysfibrinogenaemia, and further 2 had possible PFD and FXI deficiency. Probable causal mutations were detected in 8 of 11 patients with FVIII deficiency, 2 of 3 patients with FIX deficiency and 2 of 8 patients with VWD. Three hundred three patients (72.5%) had normal results in the coagulation assays and were categorized as patients with bleeding of unknown cause (BUC). The bleeding score did not differ between patients with and without established diagnosis. A diagnosis of a bleeding disorder was more frequently made in men than in women (49.3% vs 22.9%). Male sex (OR 3.55, 95% CI: 2.02-6.22; P < .001) and blood group 0 (OR 1.86, 95% CI: 1.17-2.94; P = .008) were independently associated with diagnosis of a bleeding disorder. CONCLUSION: The high rate of patients with BUC despite in-depth haemostatic assessment underlines the incompleteness of available routine laboratory tests. Males with MBDs were more likely to be diagnosed with an established bleeding disorder than females.

Parameters influencing FVIII pharmacokinetics in patients with severe and moderate haemophilia A.

In haemophilia A patients factor VIII (FVIII) recovery and half-life can vary substantially. There are parameters known to modulate FVIII pharmacokinetics (PK), but they explain only about 34% of the variability. The aim of this study was to identify new parameters that influence FVIII PK and thus to expand the current knowledge. FVIII PK were determined in 42 haemophilia A patients (37 severe, 5 moderate) without inhibitor. Patients' characteristics and laboratory parameters were evaluated for an association with FVIII PK. We analysed plasma levels of low-density lipoprotein receptor-related protein 1 (LRP1) and protein C (PC) activity, which had been hypothesized to influence FVIII activity. Furthermore, four variations in intron 6 of the LRP1 gene, which had been shown to influence LRP1, were investigated. FVIII half-life differed widely from 6.2 to 20.7 h, with a median of 10.0 h. Patients with blood group O had shorter FVIII half-life compared to patients with non-O blood group (median FVIII half-life 9.0 h vs. 10.4 h, P = 0.018). Age was significantly associated with FVIII half-life (r = 0.32, P = 0.035). Besides age, also VWF antigen (r = 0.52, P < 0.001) and blood group (r = -0.37, P = 0.015) was associated with FVIII half-life. No correlation was found with FVIII- or LRP1-genotype, LRP1 or PC concentrations. Our data showed large differences in FVIII PK between individual patients and revealed age, blood group and VWF levels as important determining factors for FVIII half-life. FVIII genotype or levels of LRP1 or PC had no influence on FVIII PK.

Determinants of factor VIII plasma levels in carriers of haemophilia A and in control women.

Factor VIII (FVIII) levels show a considerable variability in female carriers of haemophilia A. Presently, the reasons for this are poorly understood. The aim of the study was to elucidate the influence of genetic and non-genetic parameters on FVIII plasma levels in carriers (n = 42). Results were compared with age-matched healthy women without carriership of haemophilia A (n = 42). Each carrier was tested for the family-specific mutation, ABO blood group, FVIII level, von Willebrand factor (VWF) antigen and activity and C-reactive protein (CRP). FVIII levels were lower in carriers compared to non-carriers [74% (51-103) vs. 142% (109-169), P < 0.001]. No statistically significant differences were observed between the two groups with respect to VWF activity, prothrombin-time, hs-CRP, fibrinogen, body mass index (BMI), age and smoking status as well as the distribution of ABO blood groups. In non-carriers, FVIII was statistically significantly correlated with BMI, activated partial thromboplastin time (APTT), VWF antigen, hs-CRP and fibrinogen. In carriers, significant correlations between FVIII and APTT, VWF antigen and activity were found, whereas BMI, hs-CRP or fibrinogen did not correlate with FVIII. In non-carriers, the association of FVIII with ABO blood groups was statistically significant (P = 0.006), but not in carriers of haemophilia A (P = 0.234). The type of FVIII gene mutation did not influence FVIII levels. Carrier status is the major determinant of a carrier;s FVIII plasma level. Factors known to influence FVIII levels in the general population do not significantly affect FVIII activity in carriers, neither does the type of mutation influence FVIII levels.

High factor VIII levels independently predict venous thromboembolism in cancer patients: the cancer and thrombosis study.

OBJECTIVE: Patients with cancer are at an increased risk for venous thromboembolism (VTE). Clotting factor VIII activity (FVIII) has been established as risk factor of primary and recurrent VTE. We investigated FVIII as predictive parameter of VTE in cancer patients. METHODS AND RESULTS: The prospective observational Cancer and Thrombosis Study (CATS) includes patients with newly diagnosed cancer or disease progression, study end point is symptomatic VTE. FVIII was measured on a Sysmex CA 7000 analyzer. Data on 840 patients (median age: 62 years, 25th to 75th percentile 53 to 68, 378 women) were available for analyses, of these 111 patients had hematologic malignancies and 729 solid cancer. During a median observation time of 495 days 62 events occurred. Cumulative probability of VTE after 6 months was 14% in patients with elevated FVIII-levels and 4% in those with normal levels (P=0.001). The association was strongest in younger patients: whereas in 40-year-old patients a 2-fold VTE risk per factor VIII increase of 20% was observed (HR=2.0 [95% CI: 1.5 to 2.7], P<0.0001), this association was still present but attenuated in older patients. CONCLUSIONS: FVIII is independently associated with an increased risk of VTE in cancer patients. The association between FVIII and VTE risk declines with increasing age.

Thrombin generation in haemophilia A patients with factor VIII inhibitors after infusion of recombinant factor VIIa.

BACKGROUND: Development of factor VIII inhibitors is a serious complication in haemophilia A patients. Recombinant factor VIIa (rVIIa) is clinically effective, but its effects on haemostatic system need still to be fully elucidated. MATERIAL AND METHODS: In an open controlled study, we measured thrombin generation (peak thrombin) in venous blood and prothrombin fragment F1 + 2 (F1 + 2) and D-dimer in venous and in shed blood in five haemophilia A patients with inhibitors before and after rVIIa infusion. A total of five healthy individuals who did not receive rVIIa served as controls. RESULTS: At baseline, patients had lower mean (min-max) peak thrombin levels than controls [0.12 (0.0-0.6) vs. 186.9 (116.0-254.4) nM, P = 0.001]. After infusion, peak thrombin levels increased in average to 40.7 (28.3-51.6) nM, which translates into 80.2% (95% CI 65.4-88.6%) lower levels compared to that of controls. Mean (min-max) F1 + 2 levels in venous blood did not differ significantly between patients and controls [160.7 (89.8-331.3) vs. 160.8 (104.4-242.3) pmol L(-1)], but increased in average (min-max) by 39.4% (14.1-58.5%) after infusion. In blood emerging from incisions made to determine the bleeding (shed blood), F1 + 2 levels were lower in patients than controls [1383.3 (906.4-2044.6) vs. 2981.7 (1610.0-4539.6) pmol L(-1); P = 0.04], but were not affected by rVIIa; D-dimer levels were significantly higher in haemophiliacs than in controls and remained unchanged after infusion. CONCLUSIONS: Haemophilia A patients with factor VIII inhibitors have low thrombin generation. After rVIIa, the extent of coagulation activation as measured by levels of F1 + 2 is increased, but thrombin generation is restored to only 20%. Peak thrombin levels could reflect the effects of rVIIa on coagulation mechanisms, and their relevance with regard to the clinical coagulation defect of haemophilia A patients with factor VIII inhibitors might be evaluated.

Mechanisms underlying acquired von Willebrand syndrome associated with an IgM paraprotein.

Acquired von Willebrand (vW) syndrome is a rare bleeding disorder which is frequently associated with immunological, malignant or cardiovascular disorders. The underlying pathomechanisms, particularly in patients with IgM monoclonal gammopathies, often remain unknown. We report a patient with indolent small B-cell lymphoma (immunocytoma) and plasmacytic differentiation with an IgM kappa paraprotein who was admitted with retroperitoneal haematoma. Medical history and coagulation testing were consistent with acquired vW syndrome. vW immunohistochemistry showed normal cytoplasmic labelling of endothelial cells and megakaryocytes, whereas the lymphomatous infiltrate was negative. Acquired vW syndrome due to adsorption of vW factor on malignant cells was thus excluded. In the multimeric analysis, all multimers were present similar to that in type 1 vW syndrome, but the triplet structures were blurred. The bands on serum immunofixation electrophoresis were also atypically broadened, which suggested complex formation between the IgM and vW factor. Immunoprecipitation studies showed that the 176-kDa proteolytic fragment of vW factor co-precipitated with the IgM paraprotein in the patient but not in the controls, suggesting a specific interaction between vW factor and the paraprotein in the patient. The patient required surgery and was successfully managed by chemotherapy consisting of rituximab and fludarabin as well as plasma exchange.

Persistent arterial stiffness and endothelial dysfunction following successful pancreas-kidney transplantation in Type 1 diabetes.

OBJECTIVE: Successful simultaneous pancreas-kidney transplantation (SPK) in Type 1 diabetic (T1DM) patients results in improved cardiovascular outcome and survival. However, it is doubtful whether the impairment of cardiovascular and endothelial function in T1DM can be completely reversed. METHODS: Pulse-wave velocity, stroke volume, heart rate, serological markers of endothelial dysfunction (soluble intercellular, vascular cell-adhesion molecules, E-selectin, and plasminogen-activator-inhibitor-1) were measured in 10 T1DM patients after SPK with non-diabetic glucose levels, 10 T1DM patients with poor [T1DM>8; glycated haemoglobin (HbA1c)>8%], and 10 with good glucose control (T1DM<7, HbA1c<7%), in 6 non-diabetic patients after kidney transplantation (KT) and 9 non-diabetic control subjects (CON), matching for major anthropometric characteristics. RESULTS: Pulse-wave velocity was increased in SPK (P < 0.02 vs. CON, KT, T1DM<7) and in T1DM>8 (P < 0.02 vs. T1DM<7). Systolic blood pressure was increased in SPK (P < 0.05 vs. CON). Stroke volume was reduced in SPK, T1DM>8 and T1DM<7 and KT (P < 0.01 vs. CON). Heart rate was elevated in SPK and in T1DM>8 (P < 0.0003 vs. CON and T1DM<7). In SPK, soluble intercellular and vascular cell-adhesion molecules were 100% and 44% higher (P < 0.03 vs. CON), respectively, while plasminogen-activator-inhibitor-1 was decreased in SPK (P < 0.02 vs. CON). CONCLUSION: T1DM patients after SPK experience arterial stiffness, a higher heart-rate and blood pressure, reduced stroke volume and serological signs of endothelial dysfunction. Thus, functional and structural cardiovascular alterations as a result of glucotoxicity, uraemia and hypertension in T1DM might not be completely resolved by SPK.

Von Willebrand factor indicates bacterial translocation, inflammation, and procoagulant imbalance and predicts complications independently of portal hypertension severity.

BACKGROUND: Elevated plasma von Willebrand factor antigen (vWF) has been shown to indicate the presence of clinically significant portal hypertension, and thus, predicts the development of clinical events in patients with cirrhosis. AIM: To investigate the impact of bacterial translocation and inflammation on vWF, as well as the association between vWF and procoagulant imbalance. Moreover, we assessed whether vWF predicts complications of cirrhosis, independent of the severity of portal hypertension. METHODS: Our study population comprised 225 patients with hepatic venous pressure gradient (HVPG) ≥ 10 mm Hg without active bacterial infections or hepatocellular carcinoma. RESULTS: vWF correlated with markers of bacterial translocation (lipopolysaccharide-binding protein [LBP; ρ = 0.201; P = 0.021]), inflammation (interleukin 6 [IL-6; ρ = 0.426; P < 0.001] and C-reactive protein [CRP; ρ = 0.249; P < 0.001]), and procoagulant imbalance (factor VIII/protein C ratio; ρ = 0.507; P < 0.001). Importantly, the associations between vWF and these parameters were independent of HVPG. Moreover, vWF (per 10%) independently predicted variceal bleeding (hazard ratio [HR]: 1.08 [95% confidence interval (95% CI): 1.01-1.16]; P = 0.023), requirement of paracentesis (HR: 1.05 [95% CI: 1.01-1.1]; P = 0.023) and bacterial infections (HR: 1.04 [95% CI: 1-1.09]; P = 0.04) including spontaneous bacterial peritonitis (HR: 1.09 [95% CI: 0.999-1.18]; P = 0.053) on a trend-wise level. After backward elimination, vWF (HR: 1.05 [95% CI: 1.02-1.08]; P = 0.003) and CRP (per 10 mg/L; HR: 1.53 [95% CI: 1.14-2.05]; P = 0.005) remained in the final model for transplant-free mortality. Finally, the independent prognostic value of vWF/CRP groups for mortality was confirmed by competing risk analysis. CONCLUSION: Our results demonstrate that vWF is not only a marker of portal hypertension but also independently linked to bacterial translocation, inflammation and procoagulant imbalance, which might explain its HVPG-independent association with most clinical events. Prognostic groups based on vWF/CRP efficiently discriminate between patients with a poor 5-year survival and patients with a favourable prognosis.

Evidence of a U-shaped association between factor XII activity and overall survival.

INTRODUCTION: The clinical relevance of decreased coagulation factor XII (FXII) plasma activity as a risk factor for both venous and arterial thrombosis is still discussed controversially. The current study evaluated the predictive value of FXII levels for all-cause mortality in a large Viennese patient cohort. PATIENTS AND METHODS: Individuals, whose FXII activity levels were determined for suspected coagulation disorders or thrombophilia screening between 1991-2003 were included in this study (n = 8936, 51% male, 49% female, median age 43 years). Death/survival was determined by record linkage with the Austrian Death Registry. The median observation period was 5 years covering a total of 46 400 person years; the death rate was 17.1%. For Cox regression analysis, FXII plasma activity was divided into 11 categories of 10% steps with the category of > 100% FXII serving as a reference category. RESULTS: With decreasing FXII plasma activity, hazard ratios for all-cause mortality gradually increased linearly from 1.0 in the > 100% category to 1.5 (95% CI: 1.2-1.9) in the 80-90% category to 4.7 (95% CI: 3.4-6.5) in the 10-20% category. Similar results were obtained, when only vascular mortality or death as a result of ischemic heart disease was considered. No significant increase in all-cause mortality (HR: 1.4, 95%CI 0.7-2.8) was observed in the small group of FXII-deficient subjects [0-10% category (n = 58)]. CONCLUSIONS: This study first demonstrates a strong and almost linear association of FXII plasma activity between 90% and 10% with all-cause mortality in a large Viennese patient cohort. Interestingly, mortality rates are not increased when FXII activity is below 10%, resulting in a U-shaped survival curve.

Low-density lipoprotein receptor-related protein 1 polymorphism 663 C > T affects clotting factor VIII activity and increases the risk of venous thromboembolism.

BACKGROUND: Clotting factor (F) VIII is an independent risk factor for primary and recurrent venous thromboembolism (VTE). The causes for high plasma FVIII levels are not fully understood, but an involvement of genetic factors has been demonstrated. A multifunctional endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP1), mediates cellular uptake and subsequent degradation of FVIII and may contribute to variations in FVIII levels. OBJECTIVE: We assessed the association of a genetic variation of LRP1 (663C > T) with basal FVIII levels and the risk of venous thrombosis in a group of high-risk patients and in healthy controls. PATIENTS AND METHODS: One hundred and fifty-two patients with a history of recurrent VTE (median age 56 years, 47% women) were compared with 198 age- and sex-matched controls (median age 53 years, 50% women). The LRP1 663C > T genotype was analyzed by mutagenic separated polymerase chain reaction assay and heterozygosity was confirmed by sequence analysis. RESULTS: LRP1 663C > T genotype distribution differed significantly between patients (663CC n = 138, 663CT n = 14) and controls (663CC n = 190, 663CT n = 8; P = 0.048). In multivariable linear regression analysis including LRP1 663C > T, ABO blood group, von Willebrand factor antigen, C-reactive protein and age, LRP1 663CT was independently associated with FVIII activity (P = 0.02). LRP1 663CT was also associated with increased odds for VTE following adjustment for blood group O, FV Leiden and the prothrombin variation 20210G > A in multivariate analysis (odds ratio 3.3, 95% CI 1.3-8.5). CONCLUSIONS: According to our data the LRP1 663C > T polymorphism influences plasma FVIII levels independently of blood group, C-reactive protein and von Willebrand factor and is significantly associated with the risk of VTE.

Ristocetin-induced platelet aggregation for monitoring of bleeding tendency in CLL treated with ibrutinib.

Bleeding because of impaired platelet function is a major side effect of the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib. We quantitatively assessed ristocetin-induced platelet aggregation (RIPA) in 64 patients with chronic lymphocytic leukemia (CLL) under ibrutinib at 287 time points. Eighty-seven bleeding episodes in 39 patients were registered (85 Common Toxicity Criteria (CTC) grade 1 or 2, 2 CTC grade 3) during a median observation period of 10.9 months. At times of bleeding, RIPA values were significantly lower (14 vs 28 U; P<0.0001). RIPA was impaired in patients receiving concomitant antiplatelet therapy or anticoagulation (14 vs 25 U, P=0.005). A gradual decline of median RIPA values was observed with increasing bleeding severity. Importantly, no CTC grade 2 or 3 bleeding were observed with RIPA values of >36 U. Sequential monitoring indicated a decrease of RIPA values from a median of 17 to 9 U within 2 weeks after initiation of treatment as well as an increase above the critical threshold of 36 U within 7 days when ibrutinib was paused. Low RIPA values were similar during treatment with another BTK inhibitor, CC292. Quantitative assessment of platelet function is a practical tool to monitor bleeding tendency under BTK-inhibitor therapy.

Validation of rotation thrombelastography in a model of systemic activation of fibrinolysis and coagulation in humans.

BACKGROUND: Thrombelastography (TEG) is a whole blood assay to evaluate the viscoelastic properties during blood clot formation and clot lysis. Rotation thrombelastography (e.g. ROTEM) has overcome some of the limitations of classical TEG and is used as a point-of-care device in several clinical settings of coagulation disorders. Endotoxemia leads to systemic activation of the coagulation system and fibrinolysis in humans. OBJECTIVES: We validated whether ROTEM is sensitive to endotoxin induced, tissue factor-triggered coagulation and fibrinolysis and if its measures correlate with biohumoral markers of coagulation and fibrinolysis. PATIENTS AND METHODS: Twenty healthy male volunteers participated in this randomized placebo-controlled trial. Volunteers received either 2 ng kg(-1) National Reference Endotoxin or saline. RESULTS: Endotoxemia significantly shortened ROTEM clotting time (CT) by 36% (CI 0.26-0.46; P < 0.05) with a strong inverse correlation with the peak plasma levels of prothrombin fragments (F(1 + 2)) (r = -0.83, P < 0.05). Additionally, endotoxin infusion enhanced maximal lysis (ML) 3.9-fold (CI: 2.5-5.2) compared with placebo or baseline after 2 h (P < 0.05). Peak ML and peak tissue plasminogen activator (t-PA) values correlated excellently (r = 0.82, P < 0.05). ROTEM parameters clot formation time and maximal clot firmness were not affected by LPS infusion, whereas platelet function analyzer (PFA-100) closure times decreased. CONCLUSIONS: Rotation thrombelastography (ROTEM) detects systemic changes of in vivo coagulation activation, and importantly it is a point of care device, which is sensitive to changes in fibrinolysis in humans. The ex vivo measures CT and ML correlate very well with established in vivo markers of coagulation activation (F(1 + 2)) and fibrinolysis (t-PA), respectively.

Prediction of recurrent venous thromboembolism by the activated partial thromboplastin time.

BACKGROUND: Venous thromboembolism (VTE) is a multi-factorial disease. Extensive thrombophilia screening is costly and often inconclusive. Simple laboratory methods are required to predict the risk of recurrence. OBJECTIVE: To assess if measurement of activated partial thromboplastin time (APTT) allows stratification of patients with VTE into high- and low-risk categories with regard to recurrence. PATIENTS AND METHODS: We prospectively followed 918 patients with a first unprovoked VTE and studied the relationship between recurrence and an APTT after discontinuation of anticoagulation. APTT was expressed as a ratio of test to reference coagulation times. Study endpoint was symptomatic recurrent VTE. RESULTS: Venous thromboembolism recurred in 101 (11%) patients. Patients without recurrence had a greater APTT ratio than those with recurrence (0.97 +/- 0.09 vs. 0.93 +/- 0.09, P = 0.001). After 4 years, probability of recurrent VTE was 8.5% (95% CI: 5.5-11.5%) among patients with a ratio equal to or > 0.95 and 15.6% (95% CI: 11.4-19.9%) among patients with a lower ratio (P = 0.005). Compared with patients with an APTT ratio < 0.95, the relative risk (RR) of recurrence among patients with a ratio equal to or > 0.95 was 0.56 (95% CI: 0.38-0.84, P = 0.005) before and 0.58 (95% CI: 0.39-0.87, P = 0.009) after adjustment for sex, age, factor V Leiden, and factor II G20210A. CONCLUSIONS: Measurement of APTT allows stratification of patients with VTE into high- and low-risk categories with regard to recurrence.

Longitudinal analysis of hemostasis biomarkers in cancer patients during antitumor treatment.

UNLABELLED: ESSENTIALS: Hemostasis biomarkers impact thrombosis occurrence and survival in cancer patients. We performed a longitudinal analysis of hemostatic parameters in 112 cancer patients. Hemostatic parameters are associated with disease state, patients' prognosis, and the risk of VTE. The procoagulant state exists not only at diagnosis, but also during the course of disease. BACKGROUND: Hemostasis biomarkers are known to have an impact on venous thromboembolism (VTE) occurrence and survival in cancer patients. OBJECTIVES: As there are almost no data on longitudinal changes, we aimed to evaluate those in the present prospective observational study during chemotherapy and the course of disease. PATIENTS/METHODS: Patients with cancer of the brain (n = 39), lung (n = 41), colon (n = 15) or pancreas (n = 17) were included before initiation of antitumor therapy. Blood samples for determination of factor VIII, thrombin peak height, D-dimer, F1 + 2 , fibrinogen and soluble P-selectin (sP-selectin) were drawn on a monthly basis. The study endpoints were death, VTE occurrence, or completion of the study period. RESULTS: Overall, 546 blood samples of 112 patients were analyzed. D-dimer and sP-selectin levels were significantly higher in patients with distant metastasis than in those without. Patients with complete remission had significantly lower levels of F1 + 2 , D-dimer and fibrinogen. Peak height thrombin levels showed a decrease over time in all tumor types. Levels of biomarkers behaved differently in the various tumor types. Patients who developed VTE (n = 14) showed increasing levels of FVIII, sP-selectin, and D-dimer. At the last blood sampling time-point before VTE occurrence, in 13 patients the D-dimer level was above the median, and in seven of these patients it was even above the 75th percentile; however, the individual course was highly variable. Regarding survival, steadily increased FVIII, sP-selectin and D-dimer levels were associated with higher mortality. CONCLUSIONS: Hemostatic parameters show an association with disease state, prognosis, and the risk of VTE, not only at diagnosis, but also during the course of antineoplastic treatment.

Comparison between the impact of morning and evening doses of rivaroxaban on the circadian endogenous coagulation rhythm in healthy subjects.

UNLABELLED: ESSENTIALS: It is unknown whether single rivaroxaban doses should best be administered in the morning or evening. Circadian rhythm of coagulation/fibrinolysis was measured after morning or evening intake of rivaroxaban. Evening intake of rivaroxaban leads to prolonged exposure to rivaroxaban concentrations. Evening intake of rivaroxaban better matches the morning hypofibrinolysis. BACKGROUND: A circadian variation of the endogenous coagulation system exists with hypercoagulability and hypofibrinolysis and a corresponding peak of cardiovascular thromboembolic events in the morning. So far, no information is given as to whether single daily doses of the new oral anticoagulant drug rivaroxaban should best be administered in the morning or the evening. MATERIALS AND METHODS: Sixteen healthy male or female volunteers with a mean age of 26 ± 7 years were included in this randomized, controlled, analyst-blinded cross-over clinical trial. All subjects were given three morning and three evening single doses of 10 mg rivaroxaban. Circadian rhythms of prothrombin fragment 1 + 2, plasminogen activator inhibitor, and plasmin-antiplasmin complex were measured before any medication intake, as well as after morning or evening medication intake. Rivaroxaban concentrations were determined by an anti-activated factor X assay and liquid chromatography-mass spectrometry. MAIN RESULTS: Concentrations of rivaroxaban were higher 12 h after evening intake of rivaroxaban than 12 h after morning intake (53.3 ng mL(-1) [95% confidence interval 46.0-67.8] vs. 23.3 ng mL(-1) [19.4-29.1, respectively]). Rivaroxaban intake in the evening reduced morning F1+2 concentrations better at 8:00 AM than did administration on awakening (85 ± 25 nmol L(-1) vs. 106 ± 34 nmol L(-1) , CI: 9.4-32.1). In addition, this suppression effect was longer lasting after evening intake. CONCLUSIONS: Evening intake of rivaroxaban leads to prolonged exposure to rivaroxaban concentrations and better matches the morning hypofibrinolysis. These results might help to further improve the efficacy and safety of rivaroxaban treatment.

Endothelin 1 transcription is controlled by nuclear factor-kappaB in AGE-stimulated cultured endothelial cells.

Incubation of bovine aortic endothelial cells (BAECs) with erythrocytes from patients with type 2 diabetes induced an increase in endothelin 1 (ET-1) production. The effect of erythrocytes on ET-1 synthesis was dependent on glycemic control. ET-1 levels after incubation with erythrocytes derived from patients with HbA(1c) levels <6% were just half the levels observed after incubation with erythrocytes from patients with HbA(1c) levels >8%. Nepsilon-(carboxymethyl)lysine (CML)-containing protein isolated from patients' erythrocytes induced ET-1, and CML-containing protein-dependent ET-1 induction was blocked by the recombinant decoy peptide soluble receptor for advanced glycation end products (AGEs), which comprises the NH2-terminal Ig domain of the receptor for AGEs. In vitro-generated AGEs induced ET-1 mRNA transcription (nuclear run-on assay and Northern blot) in a time- and dose-dependent manner. Transient transfection of BAECs with a chimeric construct containing the 5' promoter region of the ET-1 gene linked to a reporter gene confirmed that AGE induced ET-1 promoter activity. Electrophoretic mobility shift assay confirmed AGE-inducible binding of members of the nuclear factor-kappab (NF-kappaB) family to a potential binding site at -2,090 bp. Binding was functionally significant because overexpression of the cytoplasmic inhibitor of NF-kappaB or deletion of the NF-kappaB binding site reduced ET-1 induction, whereas overexpression of NF-kappaB p65 induced ET-1 even in the absence of AGEs. Thus, ET-1 transcription is controlled by the AGE-inducible redox-sensitive transcription factor NF-kappaB.

Advanced glycation end product-induced activation of NF-kappaB is suppressed by alpha-lipoic acid in cultured endothelial cells.

Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxidative stress led to the activation of the transcription factor NF-kappaB and thus promoted the upregulation of various NF-kappaB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antioxidative defense with the natural occurring antioxidant alpha-lipoic acid before AGE albumin induction completely prevented the AGE albumin-dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-kappaB activation was also reduced in a time- and dose-dependent manner as long as alpha-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since alpha-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-kappaB. Western blots further demonstrated that alpha-lipoic acid inhibited the release and translocation of NF-kappaB from the cytoplasm into the nucleus. As a consequence, alpha-lipoic acid reduced AGE albumin-induced NF-kappaB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered alpha-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.

Risk of pregnancy-associated recurrent venous thromboembolism in women with a history of venous thrombosis.

BACKGROUND: Limited data exist on the risk of pregnancy-associated venous thromboembolism (VTE) in women with a history of VTE. OBJECTIVE: To evaluate the risk of recurrent pregnancy-associated thrombosis in women with previous VTE in a large retrospective cohort study. PATIENTS AND METHODS: One hundred and fifty-nine women with at least one pregnancy (293 pregnancies in total) after a VTE were included into the study. The patients underwent a standardized interview on their history of thrombosis and pregnancy-associated complications. RESULTS: Eight recurrent events occurred during 197 pregnancies without thrombosis prophylaxis. The probability of VTE during pregnancy without thrombosis prophylaxis was 6.2% (95% confidence interval 1.6-10.9%). The risk was constant over the whole period of pregnancy. Of the eight women with VTE during pregnancy four had heterozygous FV:R506Q, two in combination with hyperhomocysteinemia. No VTE occurred during 87 pregnancies with thrombosis prophylaxis. In the postpartum period 15 VTEs occurred, two of 83 (2.4%) after pregnancy termination, one of 53 (1.9%) after miscarriage, three of 10 (30%) after stillbirth and nine of 138 (6.5%) after live birth. CONCLUSIONS: Without thrombosis prophylaxis the risk for recurrent symptomatic VTE is substantial during the whole period of pregnancy in women with previous VTE. The majority of events occurred after delivery, reflecting the very high risk during the postpartum period. Prospective and comparative trials to ascertain efficacy and safety of prophylactic heparin are urgently needed.

The presence of IgG antibodies against beta2-glycoprotein I predicts the risk of thrombosis in patients with the lupus anticoagulant.

BACKGROUND: Lupus anticoagulant (LA) is a strong risk factor of thrombosis. However, a subgroup of patients positive for LA is unaffected by thrombosis and currently no predictive markers are available to identify patients positive for LA at increased risk for thrombosis. OBJECTIVE: The aim of the study was to investigate whether anti-beta-2-glycoprotein I (anti-beta2GPI) or anticardiolipin antibodies (ACA) are associated with an increased risk of thrombosis in patients persistently positive for LA. PATIENTS AND METHODS: A cohort of 87 consecutive patients persistently positive for LA was investigated, 55 with and 32 without a history of thrombosis. Immunoglobulin G (IgG) and M (IgM) antibodies against beta2GPI and cardiolipin were determined by enzyme-linked immunoassay. RESULTS: Patients positive for LA with thrombosis had significantly higher levels of anti-beta2GPI IgG (median 16.7 standard units, interquartile range 3.0-75.2, P = 0.002) and of ACA IgG (41.1 IgG phospholipid units per mL, 8.9-109.0, P = 0.002) than those without thrombosis (2.6, 1.4-7.9 and 9.7, 4.6-22.1, respectively). Levels of anti-beta2GPI IgM and ACA IgM did not differ significantly between LA patients with and without thrombosis (P = 0.25 and 0.12, respectively). Elevated anti-beta2GPI IgG was associated with an increased risk for thrombosis (OR = 4.0, 95% CI 1.2-13.1), especially for venous thromboembolism (OR = 5.2, 95% CI 1.5-18.0). CONCLUSIONS: Increased levels of anti-beta2GPI IgG were associated with thrombosis. We conclude that anti-beta2GPI IgG levels above normal predict an increased risk of thrombosis in patients persistently positive for LA.

Preeclampsia and fetal loss in women with a history of venous thromboembolism.

A higher prevalence of risk factors for venous thromboembolism (VTE) has been found in women with preeclampsia and fetal loss. We investigated whether women with a history of VTE have a higher prevalence of pregnancy-associated complications compared with control subjects. In 395 patients with a history of VTE and in 313 control women, the prevalence of complications during pregnancy and the mean birth weight of viable infants were evaluated. The prevalence of pregnancy-induced hypertension and preeclampsia was higher in patients (5.1% and 3.0%, respectively) compared with control subjects (1.3% each). The odds ratio was 4.13 for pregnancy-induced hypertension (95% CI 1.4 to 12.22, P=0.0058) and 2.43 for preeclampsia (95% CI 0.78 to 7.6, P=0.133). Stillbirth was slightly more frequent in patients (4.3%) than in control subjects (3.2%); the difference was not statistically significant. Miscarriage was equally frequent in patients (21.8%) and control subjects (21.3%). The birth weight of viable infants born to patients was, on average, 109 g lower than that of the infants born to the control subjects (P=0.014) after adjustment for the mother's body mass index. Our study demonstrates that women with a predisposition to VTE have, overall, a good chance for a successful pregnancy outcome. However, the findings from our study support the assumption that a predisposition to venous thrombosis is associated with a higher risk for complications during pregnancy and lower infant birth weight.