The von Willebrand factor-binding aptamer rondaptivon pegol as a treatment for severe and nonsevere hemophilia A.

Factor VIII (FVIII) circulates in a noncovalent complex with von Willebrand Factor (VWF), the latter determining FVIII half-life. The VWF-binding aptamer rondaptivon pegol (BT200) increases plasma levels of VWF/FVIII in healthy volunteers. This trial assessed its safety, pharmacokinetics, and pharmacodynamics in hemophilia A. Nineteen adult patients (ages 20-62 years, 4 women) with hemophilia A (8 mild, 2 moderate, and 9 severe) received subcutaneous injections of rondaptivon pegol. After an initial fixed dose of 3 mg on days 0 and 4, patients received weekly doses of 2 to 9 mg until day 28. Severe hemophilia A patients underwent sparse-sampling population pharmacokinetics individual profiling after the final dose of rondaptivon pegol. Adverse events, pharmacokinetics, and pharmacodynamics were assessed. FVIII activity and VWF levels were measured. All patients tolerated rondaptivon pegol well. The geometric mean half-life of rondaptivon pegol was 5.4 days and rondaptivon pegol significantly increased VWF levels. In severe hemophilia A, 6 doses of rondaptivon pegol increased the half-lives of 5 different FVIII products from a median of 10.4 hours to 31.1 hours (range, 20.8-56.0 hours). Median FVIII increased from 22% to 48% in mild hemophilia A and from 3% to 7.5% in moderate hemophilia A. Rondaptivon pegol is a first-in-class prohemostatic molecule that extended the half-life of substituted FVIII approximately 3-fold and increased endogenous FVIII levels approximately 2-fold in hemophilia patients. This trial was registered at www.clinicaltrials.gov as #NCT04677803.

The plasma miRNome and venous thromboembolism in high-grade glioma: miRNA Sequencing of a nested case-control cohort.

Patients with high-grade gliomas are at high risk of venous thromboembolism (VTE). MicroRNAs (miRNAs) are small non-coding RNAs with multiple roles in tumour biology, haemostasis and platelet function. Their association with VTE risk in high-grade glioma has not been comprehensively mapped so far. We thus conducted a nested case-control study within 152 patients with WHO grade IV glioma that had been part of a prospective cohort study on VTE risk factors. At inclusion a single blood draw was taken, and patients were thereafter followed for a maximum of 2 years. During that time, 24 patients (16%) developed VTE. Of the other 128 patients, we randomly selected 24 age- and sex-matched controls. After quality control, the final group size was 21 patients with VTE during follow-up and 23 without VTE. Small RNA next-generation sequencing of plasma was performed. We observed that hsa-miR-451a was globally the most abundant miRNA. Notably, 51% of all miRNAs showed a correlation with platelet count. The analysis of miRNAs differentially regulated in VTE patients-with and without platelet adjustment-identified potential VTE biomarker candidates such as has-miR-221-3p. Therewith, we here provide one of the largest and deepest peripheral blood miRNA datasets of high-grade glioma patients so far, in which we identified first VTE biomarker candidates that can serve as the starting point for future research.

Multicenter assessment and longitudinal study of the prevalence of antibodies and related adaptive immune responses to AAV in adult males with hemophilia.

Adeno-associated virus (AAV) based gene therapy has demonstrated effective disease control in hemophilia. However, pre-existing immunity from wild-type AAV exposure impacts gene therapy eligibility. The aim of this multicenter epidemiologic study was to determine the prevalence and persistence of preexisting immunity against AAV2, AAV5, and AAV8, in adult participants with hemophilia A or B. Blood samples were collected at baseline and annually for ≤3 years at trial sites in Austria, France, Germany, Italy, Spain, and the United States. At baseline, AAV8, AAV2, and AAV5 neutralizing antibodies (NAbs) were present in 46.9%, 53.1%, and 53.4% of participants, respectively; these values remained stable at Years 1 and 2. Co-prevalence of NAbs to at least two serotypes and all three serotypes was present at baseline for ~40% and 38.2% of participants, respectively. For each serotype, ~10% of participants who tested negative for NAbs at baseline were seropositive at Year 1. At baseline, 38.3% of participants had detectable cell mediated immunity by ELISpot, although no correlations were observed with the humoral response. In conclusion, participants with hemophilia may have significant preexisting immunity to AAV capsids. Insights from this study may assist in understanding capsid-based immunity trends in participants considering AAV vector-based gene therapy.

Tissue factor pathway inhibitor is associated with risk of venous thromboembolism and all-cause mortality in patients with cancer.

Venous thromboembolism (VTE) is a common complication in patients with cancer. Data on the role of natural inhibitors of coagulation for occurrence of cancer-associated VTE are limited, thus, we investigated the association of tissue factor pathway inhibitor (TFPI) with risk of VTE and all-cause mortality in patients with cancer. Total TFPI antigen levels were measured with a commercially available enzyme-linked immunosorbant assay in patients included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study with the primary outcome VTE. Competing risk analysis and Cox regression analysis were performed to explore the association of TFPI levels with VTE and all-cause mortality. TFPI was analyzed in 898 patients (median age 62 years; interquartile range [IQR], 53-68; 407 (45%) women). Sixty-seven patients developed VTE and 387 died (24-month cumulative risk 7.5% and 42.1%, respectively). Patients had median TFPI levels at study inclusion of 56.4 ng/mL (IQR, 45.7-70.0), with highest levels in tumor types known to have a high risk of VTE (gastroesophageal, pancreatic and brain cancer: 62.0 ng/mL; IQR, 52.0-75.0). In multivariable analysis adjusting for age, sex, cancer type and stage, TFPI levels were associated with VTE risk (subdistribution hazard ratio per doubling =1.63, 95% confidence interval [CI]: 1.03-2.57). When patients with high and intermediate/low VTE risk were analyzed separately, the association remained independently associated in the high risk group only (subdistribution hazard ratio =2.63, 95% CI: 1.40-4.94). TFPI levels were independently associated with all-cause mortality (hazard ratio =2.36, 95% CI: 1.85-3.00). In cancer patients increased TFPI levels are associated with VTE risk, specifically in patients with high-risk tumor types, and with all-cause mortality.

Natural IgM antibodies inhibit microvesicle-driven coagulation and thrombosis.

Thrombosis and its associated complications are a major cause of morbidity and mortality worldwide. Microvesicles (MVs), a class of extracellular vesicles, are increasingly recognized as mediators of coagulation and biomarkers of thrombotic risk. Thus, identifying factors targeting MV-driven coagulation may help in the development of novel antithrombotic treatments. We have previously identified a subset of circulating MVs that is characterized by the presence of oxidation-specific epitopes and bound by natural immunoglobulin M (IgM) antibodies targeting these structures. This study investigated whether natural IgM antibodies, which are known to have important anti-inflammatory housekeeping functions, inhibit the procoagulatory properties of MVs. We found that the extent of plasma coagulation is inversely associated with the levels of both free and MV-bound endogenous IgM. Moreover, the oxidation epitope-specific natural IgM antibody LR04, which recognizes malondialdehyde adducts, reduced MV-dependent plasmatic coagulation and whole blood clotting without affecting thrombocyte aggregation. Intravenous injection of LR04 protected mice from MV-induced pulmonary thrombosis. Of note, LR04 competed the binding of coagulation factor X/Xa to MVs, providing a mechanistic explanation for its anticoagulatory effect. Thus, our data identify natural IgM antibodies as hitherto unknown modulators of MV-induced coagulation in vitro and in vivo and their prognostic and therapeutic potential in the management of thrombosis.

Incidence, risk factors, and outcomes of venous and arterial thromboembolism in immune checkpoint inhibitor therapy.

The risk of venous thromboembolism (VTE) and arterial thromboembolism (ATE) associated with immune checkpoint inhibitors is currently unclear. Our aim was to quantify the risk of VTE/ATE in patients with cancer treated with immune checkpoint inhibitors, explore clinical impact, and investigate potential clinical risk factors. Patients treated with immune checkpoint inhibitors at the Medical University of Vienna from 2015 to 2018 were identified using in-house pharmacy records (n = 672; most frequent entities: 30.4% melanoma, 24.1% non-small cell lung cancer; 86% stage IV disease). A retrospective chart review was performed to screen for VTE and/or ATE. Cumulative incidences and between-group differences were estimated in competing-risk analysis. The impact of VTE/ATE on mortality was studied by multistate modelling. Over a median follow-up of 8.5 months, 47 VTEs and 9 ATEs were observed. Cumulative incidences of VTE and ATE were 12.9% (95% confidence interval [CI], 8.2-18.5) and 1.8% (95% CI, 0.7-3.6). Occurrence of VTE was associated with increased mortality (transition hazard ratio, 3.09; 95% CI, 2.07-4.60). History of VTE predicted VTE occurrence (subdistribution hazard ratio [SHR], 3.69; 95% CI, 2.00-6.81), and distant metastasis was nonsignificantly associated with VTE risk (SHR, 1.71; 95% CI, 0.62-4.73). No association of VTE with Eastern Cooperative Oncology Group performance status, Charlson comorbidity index, or Khorana score was observed, and rates of VTE were comparable between tumor types and checkpoint-inhibitory agents. In conclusion, patients with cancer under immune checkpoint inhibitor therapy are at high risk of thromboembolism, especially VTE. Furthermore, VTE occurrence was associated with increased mortality.

Interleukin-4 receptor alpha signaling regulates monocyte homeostasis.

Interleukin-4 (IL-4) and its receptors (IL-4R) promote the proliferation and polarization of macrophages. However, it is unknown if IL-4R also influences monocyte homeostasis and if steady state IL-4 levels are sufficient to affect monocytes. Employing full IL-4 receptor alpha knockout mice (IL-4Rα-/- ) and mice with a myeloid-specific deletion of IL-4Rα (IL-4Rαf/f LysMcre ), we show that IL-4 acts as a homeostatic factor regulating circulating monocyte numbers. In the absence of IL-4Rα, murine monocytes in blood were reduced by 50% without altering monocytopoiesis in the bone marrow. This reduction was accompanied by a decrease in monocyte-derived inflammatory cytokines in the plasma. RNA sequencing analysis and immunohistochemical staining of splenic monocytes revealed changes in mRNA and protein levels of anti-apoptotic factors including BIRC6 in IL-4Rα-/- knockout animals. Furthermore, assessment of monocyte lifespan in vivo measuring BrdU+ cells revealed that the lifespan of circulating monocytes was reduced by 55% in IL-4Rα-/- mice, whereas subcutaneously applied IL-4 prolonged it by 75%. Treatment of human monocytes with IL-4 reduced the amount of dying monocytes in vitro. Furthermore, IL-4 stimulation reduced the phosphorylation of proteins involved in the apoptosis pathway, including the phosphorylation of the NFκBp65 protein. In a cohort of human patients, serum IL-4 levels were significantly associated with monocyte counts. In a sterile peritonitis model, reduced monocyte counts resulted in an attenuated recruitment of monocytes upon inflammatory stimulation in IL-4Rαf/f LysMcre mice without changes in overall migratory function. Thus, we identified a homeostatic role of IL-4Rα in regulating the lifespan of monocytes in vivo.

Applicability of the European Society of Cardiology Guidelines on the management of acute coronary syndromes to older people with haemophilia A - A modified Delphi consensus by the ADVANCE Working Group.

INTRODUCTION: As people with haemophilia (PWH) receive better treatment and live longer they are more likely to encounter cardiovascular disease (CVD) and other comorbidities. ESC guidelines for the acute management of patients presenting with acute coronary syndrome (ACS) are based on the non-haemophilia population. AIM: To review the guidelines and propose relevant adaptations for PWHA without inhibitors who are treated with prophylaxis and present with ACS. METHODS: As part of the ADVANCE Group, 20 European haemophilia experts used a modified Delphi approach to develop and gain consensus on proposed adaptations of the ESC guidelines for PWHA without inhibitors. RESULTS: Of the 32 Class I recommendations across both guidelines, adaptions were considered necessary and proposed for 15. The adaptions highlight the need to provide sufficient FVIII trough levels at the time of antithrombotic treatment in people with haemophilia A (HA) without inhibitors. Patients receiving emicizumab prophylaxis and requiring oral anticoagulation therapy or combined single antiplatelet plus oral anticoagulation therapy will require additional FVIII replacement therapy. CONCLUSION: In the absence of high-quality clinical evidence, the combined expert opinion used to develop these adaptions to the current ESC guidelines may help to guide clinicians in their treatment decisions when a PWHA presents with ACS.

Growth differentiation factor-15 predicts major bleeding, major adverse cardiac events and mortality in patients with end-stage kidney disease on haemodialysis: findings from the VIVALDI study.

BACKGROUND: Patients with end-stage kidney disease (ESKD) are at high risk of cardiovascular events and bleeding. Optimizing risk assessment of ESKD patients regarding the risk of thromboembolism and bleeding complications in comorbid conditions, including atrial fibrillation and coronary heart disease, is challenging. To improve risk prediction we investigated growth differentiation factor-15 (GDF-15), a promising cardiovascular biomarker, and its relation to adverse outcomes. METHODS: In this prospective, multicentre, population-based cohort study, GDF-15 was measured in 594 ESKD patients on haemodialysis (median age 66 years, 38% female), who were followed up for a median of 3.5 years. The association of GDF-15 with major bleeding, arterial thromboembolism, major adverse cardiac events (MACE) and death was analysed within a competing risk framework. Further, we evaluated the additive predictive value of GDF-15 to cardiovascular and death risk assessment. RESULTS: GDF-15 levels were in median 5475 ng/l (25th-75th percentile 3964-7533) and independently associated with major bleeding {subdistribution hazard ratio [SHR] 1.31 per double increase [95% confidence interval (CI) 1.00-1.71]}, MACE [SHR 1.47 (95% CI 1.11-1.94)] and all-cause mortality [SHR 1.58 (95% CI 1.28-1.95)] but not arterial thromboembolism [SHR 0.91 (95% CI 0.61-1.36)]. The addition of GDF-15 to the HAS-BLED score significantly improved discrimination and calibration for predicting major bleeding [C-statistics increased from 0.61 (95% CI 0.52-0.70) to 0.68 (95% CI 0.61-0.78)]. Furthermore, we established an additive predictive value of GDF-15 beyond current risk models for predicting MACE and death. CONCLUSION: GDF-15 predicts the risk of major bleeding, cardiovascular events and death in ESKD patients on haemodialysis and might be a valuable marker to guide treatment decisions in this challenging patient population.

Circulatory miR-411-5p as a Novel Prognostic Biomarker for Major Adverse Cardiovascular Events in Patients with Atrial Fibrillation.

The risk stratification of patients with atrial fibrillation (AF) for subsequent cardiovascular events could help in guiding prevention strategies. In this study, we aimed at investigating circulating microRNAs as prognostic biomarkers for major adverse cardiovascular events (MACE) in AF patients. We conducted a three-stage nested case-control study within the framework of a prospective registry, including 347 AF patients. First, total small RNA-sequencing was performed in 26 patients (13 cases with MACE) and the differential expression of microRNAs was analyzed. Seven candidate microRNAs with promising results in a subgroup analysis on cardiovascular death were selected and measured via using RT-qPCR in 97 patients (42 cases with cardiovascular death). To further validate our findings and investigate broader clinical applicability, we analyzed the same microRNAs in a subsequent nested case-control study of 102 patients (37 cases with early MACE) by using Cox regression. In the microRNA discovery cohort (n = 26), we detected 184 well-expressed microRNAs in circulation without overt differential expression between the cases and controls. A subgroup analysis on cardiovascular death revealed 26 microRNAs that were differentially expressed at a significance level < 0.05 (three of which with an FDR-adjusted p-value <0.05). We, therefore, proceeded with a nested case-control approach (n = 97) focusing on patients with cardiovascular death and selected, in total, seven microRNAs for further RT-qPCR analysis. One microRNA, miR-411-5p, was significantly associated with cardiovascular death (adjusted HR (95% CI): 1.95 (1.04-3.67)). Further validation (n = 102) in patients who developed early MACE showed similar results (adjusted HR (95% CI) 2.35 (1.17-4.73)). In conclusion, circulating miR-411-5p could be a valuable prognostic biomarker for MACE in AF patients.

2022 international clinical practice guidelines for the treatment and prophylaxis of venous thromboembolism in patients with cancer, including patients with COVID-19.

The International Initiative on Thrombosis and Cancer is an independent academic working group of experts aimed at establishing global consensus for the treatment and prophylaxis of cancer-associated thrombosis. The 2013, 2016, and 2019 International Initiative on Thrombosis and Cancer clinical practice guidelines have been made available through a free, web-based mobile phone application. The 2022 clinical practice guidelines, which are based on a literature review up to Jan 1, 2022, include guidance for patients with cancer and with COVID-19. Key recommendations (grade 1A or 1B) include: (1) low-molecular-weight heparins (LMWHs) for the initial (first 10 days) treatment and maintenance treatment of cancer-associated thrombosis; (2) direct oral anticoagulants for the initial treatment and maintenance treatment of cancer-associated thrombosis in patients who are not at high risk of gastrointestinal or genitourinary bleeding, in the absence of strong drug-drug interactions or of gastrointestinal absorption impairment; (3) LMWHs or direct oral anticoagulants for a minimum of 6 months to treat cancer-associated thrombosis; (4) extended prophylaxis (4 weeks) with LMWHs to prevent postoperative venous thromboembolism after major abdominopelvic surgery in patients not at high risk of bleeding; and (5) primary prophylaxis of venous thromboembolism with LMWHs or direct oral anticoagulants (rivaroxaban or apixaban) in ambulatory patients with locally advanced or metastatic pancreatic cancer who are treated with anticancer therapy and have a low risk of bleeding.

Hemostatic Biomarkers and Venous Thromboembolism Are Associated With Mortality and Response to Chemotherapy in Patients With Pancreatic Cancer.

Objective: Pancreatic cancer activates coagulation and increases risk of venous thromboembolism (VTE). We aimed at characterizing the association of hemostatic biomarkers and VTE with mortality and chemotherapy response. Approach and Results: Pancreatic cancer patients (N=145) were included in a prospective, observational cohort study (CATS [Vienna Cancer and Thrombosis Study]). Hemostatic biomarkers (D-dimer, extracellular vesicle-tissue factor activity, prothrombin fragment 1+2, fibrinogen, factor VIII, PAI-1 [plasminogen activator inhibitor 1], sP-selectin [soluble P-selectin], thrombin generation assay) were measured at inclusion. The impact of VTE on overall survival/progression-free survival (OS/PFS) was evaluated by multistate modeling. The association of biomarkers with OS was analyzed by Cox-regression and with PFS and disease control rate in patients initiating palliative chemotherapy (n=95) by Cox-regression and logistic regression. Multivariable analysis included stage, grade, sex, age, performance status, VTE (time-dependent), vascular infiltration/compression, and tumor marker levels (carbohydrate-antigen 19-9, carcinoembryonic antigen). VTE occurrence was associated with shorter OS (transition hazard ratio, 3.40 [95% CI, 2.05-5.64]) and shorter PFS (transition hazard ratio, 2.10 [1.16-3.79]). Median post-VTE OS/PFS in months was 5.5 [2.2-6.5] and 3.0 [1.5-3.9], compared with 13.4 [9.7-16.6] and 7.5 [5.9-9.8] in patients without VTE (both P<0.001). D-dimer, extracellular vesicle-tissue factor activity, PAI-1, and sP-selectin were associated with increased mortality (hazard ratio per doubling, 1.27 [1.00-1.61]; 1.63 [1.14-2.36]; 1.25 [1.06-1.47]; 1.52 [1.05-2.20]). In patients initiating palliative chemotherapy, higher D-dimer predicted shorter PFS (hazard ratio per doubling, 1.27 [1.01-1.60]) and lower disease control rate (odds ratio per doubling, 0.59 [0.36-0.98]). Conclusions: VTE diagnosis is associated with shorter OS and PFS. Higher baseline levels of D-dimer, extracellular vesicle-tissue factor activity, PAI-1, and sP-selectin were independently prognostic for increased mortality, and D-dimer predicted response to palliative chemotherapy.

Atrial fibrillation and anticoagulation are associated with hospitalisations in patients with end-stage kidney disease on haemodialysis: a prospective population-based cohort study.

BACKGROUND: Patients with end-stage kidney disease on haemodialysis suffer from frequent complications requiring hospitalisation. Atrial fibrillation is a burdensome comorbidity amongst patients on haemodialysis. We aimed to assess frequency, reasons, and duration of hospitalisations in haemodialysis patients and their association with atrial fibrillation and anticoagulation. METHODS: Prevalent patients with end-stage kidney disease on haemodialysis were recruited into a prospective cohort study and observed for a median observation time of 3.4 years. Hospitalisations were recorded from discharge letters, medical records, and patient interviews. The association of atrial fibrillation, anticoagulation, and time-in-therapeutic range of vitamin K antagonist treatment with hospitalisations was analysed using negative binomial regression. RESULTS: Out of 625 patients, 238 (38.1%) had atrial fibrillation. Median number of hospitalisations per patient was 3.0 (1.0-5.0). Incidence rate of hospitalisation was 1.7 per patient-year in all and 1.9 in atrial fibrillation patients, median duration per hospitalisation was 7.9 (4.8-12.9) and 8.8 (5.7-13.3) days, respectively. Most frequent reasons for hospitalisation were vascular access complication/intervention (11.7%) and infection/fever (11.4%), while bleeding events comprised 6.0% of all hospitalisations. Atrial fibrillation patients had 27% higher risk of hospitalisation than patients without atrial fibrillation (incidence rate ratio [IRR] 1.27, 95% confidence interval [CI] 1.10-1.47). In atrial fibrillation patients, anticoagulation (enoxaparin or phenprocoumon, 41.6% of AF patients) was associated with increased risk of all-cause (IRR 1.38, 95%CI 1.14-1.69) and bleeding-related hospitalisation (IRR 1.96, 95%CI 1.06-3.63). There was no association between anticoagulation and stroke-related hospitalisation. In atrial fibrillation patients on phenprocoumon, increasing time-in-therapeutic range was associated with decreased risk of all-cause (IRR 0.35, 95%CI 0.14-0.87), but not bleeding-related hospitalisation (IRR 0.13, 95%CI 0.01-1.38). CONCLUSION: In haemodialysis patients, presence of atrial fibrillation and, among those with atrial fibrillation, anticoagulation were associated with higher risk of all-cause hospitalisation, including bleeding-related hospitalisation in the latter. Increasing time-in-therapeutic range in patients on vitamin K antagonist treatment was associated with decreased risk of all-cause, but not bleeding-related hospitalisation.

Relative risk of arterial and venous thromboembolism in persons with cancer vs. persons without cancer-a nationwide analysis.

AIMS: An interrelation between cancer and thrombosis is known, but population-based studies on the risk of both arterial thromboembolism (ATE) and venous thromboembolism (VTE) have not been performed. METHODS AND RESULTS: International Classification of Disease 10th Revision (ICD-10) diagnosis codes of all publicly insured persons in Austria (0-90 years) were extracted from the Austrian Association of Social Security Providers dataset covering the years 2006-07 (n = 8 306 244). Patients with a history of cancer or active cancer were defined as having at least one ICD-10 'C' diagnosis code, and patients with ATE and/or VTE as having at least one of I21/I24 (myocardial infarction), I63/I64 (stroke), I74 (arterial embolism), and I26/I80/I82 (venous thromboembolism) diagnosis code. Among 158 675 people with cancer, 8559 (5.4%) had an ATE diagnosis code and 7244 (4.6%) a VTE diagnosis code. In contrast, among 8 147 569 people without cancer, 69 381 (0.9%) had an ATE diagnosis code and 29 307 (0.4%) a VTE diagnosis code. This corresponds to age-stratified random-effects relative risks (RR) of 6.88 [95% confidence interval (CI) 4.81-9.84] for ATE and 14.91 (95% CI 8.90-24.95) for VTE. ATE proportion was highest in patients with urinary tract malignancies (RR: 7.16 [6.74-7.61]) and lowest in patients with endocrine cancer (RR: 2.49 [2.00-3.10]). The corresponding VTE proportion was highest in cancer of the mesothelium/soft tissue (RR: 19.35 [17.44-21.47]) and lowest in oropharyngeal cancer (RR: 6.62 [5.61-7.81]). CONCLUSION: The RR of both ATE and VTE are significantly higher in persons with cancer. Our population-level meta-data indicate a strong association between cancer, ATE and VTE, and support the concept of shared risk factors and pathobiology between these diseases.Relative risk of ATE and VTE in persons with a cancer diagnosis code versus persons without a cancer diagnosis code.

Antithrombin Activity and Association with Risk of Thrombosis and Mortality in Patients with Cancer.

Venous and arterial thromboembolism (VTE/ATE) are common complications in cancer patients. Antithrombin deficiency is a risk factor for thrombosis in the general population, but its connection to risk of cancer-associated thrombosis is unclear. We investigated the association of antithrombin activity levels with risk of cancer-associated VTE/ATE and all-cause mortality in an observational cohort study including patients with cancer, the Vienna Cancer and Thrombosis Study. In total, 1127 patients were included (45% female, median age: 62 years). Amongst these subjects, 110 (9.7%) patients were diagnosed with VTE, 32 (2.8%) with ATE, and 563 (49.9%) died. Antithrombin was not associated with a risk of VTE (subdistribution hazard ratio (SHR): 1.00 per 1% increase in antithrombin level; 95% CI: 0.99-1.01) or ATE (SHR: 1.00; 95% CI: 0.98-1.03). However, antithrombin showed a u-shaped association with the risk of all-cause death, i.e., patients with very low but also very high levels had poorer overall survival. In the subgroup of patients with brain tumors, higher antithrombin levels were associated with ATE risk (SHR: 1.02 per 1% increase; 95% CI: 1.00-1.04) and mortality (HR: 1.01 per 1% increase; 95% CI: 1.00-1.02). Both high and low antithrombin activity was associated with the risk of death. However, no association with cancer-associated VTE and ATE across all cancer types was found, with the exception of in brain tumors.

Neutrophil subpopulations and their activation potential in patients with antiphospholipid syndrome and healthy individuals.

OBJECTIVES: Patients with APS are at increased risk of thromboembolism. Neutrophils have been shown to play a role in inducing thrombosis. We aimed to investigate differences in neutrophil subpopulations, their potential of activation and neutrophil extracellular trap (NET) formation comparing high and low-density neutrophils (HDNs/LDNs) as well as subpopulations in patients with APS and controls to gain deeper insight into their potential role in thrombotic manifestations in patients with APS. METHODS: HDNs and LDNs of 20 patients with APS and 20 healthy donors were isolated by density gradient centrifugation and stimulated. Neutrophil subpopulations, their activation and NET release were assessed by flow cytometry. RESULTS: LDNs of both groups showed higher baseline activation, lower response to stimulation (regulation of activation markers CD11b/CD66b), but higher NET formation compared with HDNs. In patients with APS, the absolute number of LDNs was higher compared with controls. HDNs of APS patients showed higher spontaneous activation [%CD11b high: median (interquartile range): 2.78% (0.58-10.24) vs 0.56% (0.19-1.37)] and response to stimulation with ionomycin compared with HDNs of healthy donors [%CD11b high: 98.20 (61.08-99.13) vs 35.50% (13.50-93.85)], whereas no difference was found in LDNs. NET formation was increased in patients' HDNs upon stimulation. CONCLUSION: HDNs and LDNs act differently, unstimulated and upon various stimulations in both healthy controls and APS patients. Differences in HDNs and LDNs between patients with APS and healthy controls indicate that neutrophils may enhance the risk of thrombosis in these patients and could thus be a target for prevention of thrombosis in APS.

How I treat patients with hereditary antithrombin deficiency.

Genetic predispositions to venous thromboembolism (VTE) are relatively frequent in the general population and comprise a heterogeneous group of disorders. Whereas the most frequent congenital risk factors for thrombosis only moderately increase the risk, a deficiency in antithrombin (AT), one of the most important natural inhibitors of blood coagulation, carries a higher risk. Congenital AT deficiency is an infrequently encountered genetic risk factor for VTE, and different subtypes vary with regard to their thrombotic risk. Patients with congenital AT deficiency, especially those with quantitative deficiency (type 1), may develop thrombosis early in life and often have a conspicuous family history of first- and second-degree relatives with VTE. Women are particularly affected because of the risk potentiation by combined estrogen/progestogen oral contraceptive use or pregnancy. The lack of controlled trials or even observational studies of large cohorts does not allow therapeutic decisions to be based on scientific evidence. In this review, we will discuss cases with thrombotic manifestations and the tailored management of patients with this congenital thrombosis risk factor.

Diagnostic high-throughput sequencing of 2396 patients with bleeding, thrombotic, and platelet disorders.

A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multidisciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbor variants associated with rare bleeding, thrombotic, or platelet disorders (BTPDs). The molecular diagnostic rate was determined by the clinical phenotype, with an overall rate of 49.2% for all thrombotic, coagulation, platelet count, and function disorder patients and a rate of 3.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 745 unique variants, including copy number variants (CNVs) and intronic variants, as pathogenic, likely pathogenic, or variants of uncertain significance. Half of these variants (50.9%) are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPDs. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 894 index patients providing evidence that introducing an HTS genetic test is a valuable addition to laboratory diagnostics in patients with a high likelihood of having an inherited BTPD.

Alternative activation of human macrophages enhances tissue factor expression and production of extracellular vesicles.

Macrophages are versatile cells that can be polarized by the tissue environment to fulfill required needs. Proinflammatory polarization is associated with increased tissue degradation and propagation of inflammation whereas alternative polarization within a Th2 cytokine environment is associated with wound healing and angiogenesis. To understand if polarization of macrophages can lead to a procoagulant macrophage subset we polarized human monocyte derived macrophages to a proinflammatory and an alternative activation state. Alternative polarization with interleukin-4 and IL-13 led to a macrophage phenotype characterized by increased tissue factor (TF) production and release and by an increase in extracellular vesicle production. In addition, also TF activity was enhanced in extracellular vesicles of alternatively polarized macrophages. This TF induction was dependent on signal transducer and activator of transcription-6 signaling and poly ADP ribose polymerase activity. In contrast to monocytes, human macrophages did not show increased tissue factor expression upon stimulation with lipopolysaccharide and interferon-γ. Previous polarization to either a proinflammatory or an alternative activation subset does not change the subsequent stimulation of TF. The inability of proinflammatory activated macrophages to respond to lipopolysaccharide and interferon-γ with an increase in TF production seems to be due to an increase in TF promoter methylation and was reversible when treating these macrophages with a demethylation agent. In conclusion, we provide evidence that proinflammatory polarization of macrophages does not lead to enhanced procoagulatory function, whereas alternative polarization of macrophages leads to an increased expression of TF and increased production of TF bearing extracellular vesicles by these cells suggesting a procoagulatory phenotype of alternatively polarized macrophages.

Natural anticoagulants: A missing link in mild to moderate bleeding tendencies.

INTRODUCTION: There is a growing interest in natural anticoagulants as a cause of mild to moderate bleeding disorders (MBDs), particularly in patients with bleeding of unknown cause (BUC), which is defined as having a mild to moderate bleeding phenotype without a definite diagnosis despite exhaustive and repeated laboratory investigations. Recently, abnormalities in two natural anticoagulant pathways, thrombomodulin (TM), and tissue factor pathway inhibitor (TFPI), were identified in single patients or families as the underlying cause for a bleeding tendency. AIM: The objective of this review is to discuss the current understanding of the role of natural anticoagulants in MBDs using available clinical and translational data. METHODS: A Cochrane Library and PubMed (MEDLINE) search focusing on selected natural anticoagulants and their role in MBDs was conducted. RESULTS: Data on the influence of natural anticoagulants including protein C, protein S, antithrombin, TM, and TFPI or factors with anticoagulant properties like fibrinogen gamma prime (γ') on MBDs are scarce. Observations from sepsis treatment and from translational research highlight their importance as regulators of the haemostatic balance, especially via the activated protein C-related pathway, and suggest a role in some MBDs. CONCLUSION: Similar to the distinct genetic variants of natural anticoagulants linked to thrombosis, we hypothesize that novel variants may be associated with a bleeding tendency and could be identified using next generation sequencing.