von Willebrand disease.

von Willebrand disease (VWD) is the most common inherited bleeding disorder. The disorder is characterized by excessive mucocutaneous bleeding. The most common bleeding manifestations of this condition include nosebleeds, bruising, bleeding from minor wounds, menorrhagia or postpartum bleeding in women as well as bleeding after surgery. Other less frequent symptoms include gastrointestinal bleeding, haematomas or haemarthroses. VWD pathophysiology is complex and results from defects in von Willebrand factor (VWF) glycoprotein. Quantitative deficiencies are responsible for type 1 VWD with a partial decrease of VWF and type 3 with the complete absence of VWF. Qualitative abnormalities cause type 2 VWD, being further divided into types 2A, 2B, 2M and 2N. Although common, VWD is at risk of misdiagnosis, overdiagnosis and underdiagnosis owing to several factors, including complex diagnosis, variability of bleeding symptoms, presence of external variables (blood groups and other physiological modifiers such as exercise, thyroid hormones, oestrogens, and ageing), and lack of disease awareness among non-specialist health-care providers. Establishing the correct VWD diagnosis requires an array of specialized phenotypic assays and/or molecular genetic testing of the VWF gene. The management of bleeding includes increasing endogenous VWF levels with desmopressin or infusion of exogenous VWF concentrates (plasma-derived or recombinant). Fibrinolytic inhibitors, topical haemostatic agents and hormonal therapies are used as effective adjunctive measures.

Risk Factors for Antiphospholipid Antibodies and Antiphospholipid Syndrome.

Persistence of serum antiphospholipid antibodies (aPL) is associated with a high thrombotic risk, both arterial and venous, and with pregnancy complications. Due to the potential morbidity and mortality associated with the presence of aPL, identifying and recognizing risk factors for the development of aPL and thrombosis in aPL carriers may help to prevent and reduce the burden of disease. Multiple elements are involved in the pathomechanism of aPL development and aPL-related thrombosis such as genetics, malignancy, and infections. This review will address the role of both well-known risk factors and their evolution, and of emerging risk factors, including COVID-19, in the development of aPL and thrombosis in aPL carriers.

Colorectal cancer screening in patients with inherited bleeding disorders: high cancer detection rate in hemophilia patients.

BACKGROUND: The population-based colorectal cancer (CRC) screening program in individuals aged 55 to 75 years in the Netherlands uses fecal immunochemical testing (FIT), to detect hemoglobin in feces, followed by colonoscopy in individuals with a positive FIT. OBJECTIVES: The objectives of this study are to assess the false-positive rate, detection rate, and positive predictive value of FIT for CRC and advanced adenoma (AA) in patients with Von Willebrand disease (VWD) or hemophilia. METHODS: We performed a multicenter, nationwide cross-sectional study embedded in 2 nationwide studies on VWD and hemophilia in the Netherlands. RESULTS: In total, 493 patients with hemophilia (n = 329) or VWD (n = 164) were included, of whom 351 patients participated in the CRC screening program (71.2%). FIT positivity and false-positive rate in patients with hemophilia and VWD were significantly higher than those in the general population (14.8% vs. 4.3%, p < .001 and 10.3% vs. 2.3%, p <.001, respectively). In patients with hemophilia, the detection rate of CRC/AA was significantly higher than that in the general male population (4.5% vs. 1.8%, p = .02), and the positive predictive value of FIT for CRC/AA was comparable (32.3% vs. 39.7%, n.s.). In patients with VWD, the detection rate was similar to that of the general population (0.8% vs. 1.4%, n.s.), whereas the positive predictive value was significantly lower than that in the general population (6.3% vs. 36.8%, p = .02). CONCLUSION: This study indicates that despite a high false-positive rate of FIT in patients with inherited bleeding disorders, the detection rate of CRC and/or AA in hemophilia patients is high. FIT performs different in patients with hemophilia or VWD compared with the general population.

Accuracy of point-of-care coagulation testing during cardiopulmonary bypass in a patient post COVID-19 infection.

INTRODUCTION: Extracorporeal circulation (ECC) in cardiac surgery is performed under systemic heparinization. Adequacy of heparin therapy and anticoagulation during ECC is assessed by activated clotting time (ACT), although there are concerns regarding the reliability of this measure. The ACT can be affected by factors other than heparin anticoagulation. A novel factor that should be considered is the influence of a COVID-19 infection. More than half of the hospitalized COVID-19 patients develop coagulation abnormalities with dysregulated coagulation test results. Patients recently recovered from COVID-19 may still demonstrate some forms of coagulation disorder affecting the ACT. This case describes an inaccurate point-of-care ACT testing in a patient with previous COVID-19 infection undergoing cardiac surgery with ECC and the alternative coagulation testing performed. CASE PRESENTATION: A 77-years-old Caucasian male presented with symptomatic severe mitral valve regurgitation for which he underwent surgery. Medical history revealed a COVID-19 infection one month before surgery. Pre-operative hematological lab results were normal and baseline ACT during surgery was 100 s. To achieve an adequate ACT of > 400 s, multiple doses of heparin were needed and after administration of a triple dose (75,000 IE heparin in total) this adequate ACT was achieved. In the meanwhile we measured anti-Xa level and APTT, which were at adequate levels when ACT was still < 400 s. DISCUSSION: This case emphasizes the need of alternative methods for monitoring heparin therapy in case ACT does not respond adequately. Another point to highlight in this case is the poorly correlated relation between ACT and APTT and anti-Xa in light of the recent COVID-19 infection. Although studies have shown that COVID-19 infection can cause coagulopathy and altered hemostatic parameters, ACT has never been investigated in COVID-19 patient. Understanding the correlation between ACT, APTT and anti-Xa in COVID-19 patients is mandatory.

Clinical value of early assessment of hyperfibrinolysis by rotational thromboelastometry during postpartum hemorrhage for the prediction of severity of bleeding: A multicenter prospective cohort study in the Netherlands.

INTRODUCTION: Coagulopathy may be the result of hyperfibrinolysis and could exacerbate bleeding following childbirth. Timely recognition of hyperfibrinolysis during the earliest stages of postpartum hemorrhage could identify women at risk of more severe blood loss who may benefit from targeted anti-fibrinolytic therapy. Rotational thromboelastometry (ROTEM® ) is a point-of-care test that could detect hyperfibrinolysis. The aim of this study was to evaluate whether early assessment of hyperfibrinolysis by ROTEM during postpartum hemorrhage could predict progression to severe postpartum hemorrhage. MATERIAL AND METHODS: During a prospective cohort study in the Netherlands among women with postpartum hemorrhage (total blood loss at least 1000 ml within 24 h after childbirth) ROTEM measurements were performed following 800-1500 ml of blood loss. Hyperfibrinolysis was defined as an enzymatic fibrinolysis index (ROTEM EXTEM maximum clot lysis [ML] minus the ROTEM APTEM ML) above 15%. Severe postpartum hemorrhage was defined as a composite end point of total blood loss greater than 2000 ml, transfusion of four or more units of packed cells, and/or need for an invasive intervention. The predictive value of hyperfibrinolysis for progression to severe postpartum hemorrhage was assessed by area under the receiver operating curve (AUC) and positive and negative predictive values. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02149472). RESULTS: Of 390 women included, 82 (21%) had severe postpartum hemorrhage. Four (1%) women had thromboelastometric evidence of hyperfibrinolysis, of whom two developed severe postpartum hemorrhage. The AUC for enzymatic fibrinolysis index more than 15% for progression to severe postpartum hemorrhage was 0.47 (95% CI 0.40-0.54). Positive and negative predictive values for this index were 50.0% (95% CI 6.8-93.2) and 79.3% (95% CI 74.9-83.2), respectively. CONCLUSIONS: Thromboelastometric evidence of hyperfibrinolysis was rare in women with postpartum hemorrhage when assessed between 800 and 1500 ml of blood loss. The clinical predictive value of viscoelastometric point-of-care testing for hyperfibrinolysis for progression to severe postpartum hemorrhage during early postpartum hemorrhage is limited.

Bleeding assessment tools in the diagnosis of VWD in adults and children: a systematic review and meta-analysis of test accuracy.

Von Willebrand disease (VWD) can be associated with significant morbidity. Patients with VWD can experience bruising, mucocutaneous bleeding, and bleeding after dental and surgical procedures. Early diagnosis and treatment are important to minimize the risk of these complications. Several bleeding assessment tools (BATs) have been used to quantify bleeding symptoms as a screening tool for VWD. We systematically reviewed diagnostic test accuracy results of BATs to screen patients for VWD. We searched Cochrane Central, MEDLINE, and EMBASE for eligible studies, reference lists of relevant reviews, registered trials, and relevant conference proceedings. Two investigators screened and abstracted data. Risk of bias was assessed using the revised tool for the quality assessment of diagnostic accuracy studies and certainty of evidence using the Grading of Recommendations Assessment, Development and Evaluation framework. We pooled estimates of sensitivity and specificity. The review included 7 cohort studies that evaluated the use of BATs to screen adult and pediatric patients for VWD. The pooled estimates for sensitivity and specificity were 75% (95% confidence interval, 66-83) and 54% (29-77), respectively. Certainty of evidence varied from moderate to high. This systematic review provides accuracy estimates for validated BATs as a screening modality for VWD. A BAT is a useful initial screening test to determine who needs specific blood testing. The pretest probability of VWD (often determined by the clinical setting/patient population), along with sensitivity and specificity estimates, will influence patient management.

Clinical value of early viscoelastometric point-of-care testing during postpartum hemorrhage for the prediction of severity of bleeding: A multicenter prospective cohort study in the Netherlands.

INTRODUCTION: To evaluate rotational fibrin-based thromboelastometry (ROTEM® FIBTEM) with amplitude of clot firmness at 5 min (A5) as an early point-of-care parameter for predicting progression to severe postpartum hemorrhage, and compare its predictive value with that of fibrinogen. MATERIAL AND METHODS: Prospective cohort study in the Netherlands including women with 800-1500 ml of blood loss within 24 h following birth. Blood loss was quantitatively measured by weighing blood-soaked items and using a fluid collector bag in the operating room. Both FIBTEM A5 values and fibrinogen concentrations (Clauss method) were measured between 800 and 1500  ml of blood loss. Predictive accuracy of both biomarkers for the progression to severe postpartum hemorrhage was measured by area under the receiver operating curves (AUC). Severe postpartum hemorrhage was defined as a composite endpoint of (1) total blood loss >2000 ml, (2) transfusion of ≥4 packed cells, and/or (3) need for an invasive intervention to cease bleeding. RESULTS: Of the 391 women included, 72 (18%) developed severe postpartum hemorrhage. Median (IQR) volume of blood loss at blood sampling was 1100 ml (1000-1300) with a median (interquartile range [IQR]) fibrinogen concentration of 3.9 g/L (3.4-4.6) and FIBTEM A5 value of 17 mm (13-20). The AUC for progression to severe postpartum hemorrhage was 0.53 (95% confidence interval [CI] 0.46-0.61) for FIBTEM A5 and 0.58 (95% CI 0.50-0.65) for fibrinogen. Positive predictive values for progression to severe postpartum hemorrhage for FIBTEM A5 ≤12 mm was 22.5% (95% CI 14-33) and 50% (95% CI 25-75) for fibrinogen ≤2 g/L. CONCLUSIONS: The predictive value of FIBTEM A5 compared to fibrinogen concentrations measured between 800 and 1500 ml of blood loss following childbirth was poor to discriminate between women with and without progression towards severe postpartum hemorrhage.

Treatment of acquired hemophilia A, a balancing act: results from a 27-year Dutch cohort study.

Acquired hemophilia A (AHA) is a severe auto-immune bleeding disorder. Treatment of AHA is burdensome and optimal management is still unresolved. Therefore a retrospective nationwide multi-center cohort study (1992-2018) was performed to evaluate clinical presentation and treatment efficacy and safety of AHA in the Netherlands. Multivariate logistic and Cox regression analysis was used to study independent associations between patient characteristics and clinical outcomes. A total of 143 patients (median age 73 years; 52.4% male) were included with a median follow-up of 16.8 months (IQR 3.6-41.5 months). First-line immunosuppressive treatment was mostly steroid monotherapy (67.6%), steroids/cyclophosphamide (11.9%) and steroids/rituximab (11.9%), with success rates of 35.2%, 80.0% and 66.7% respectively, P < .05. Eventually 75% of patients achieved complete remission (CR). A high anti-FVIII antibody titer, severe bleeding and steroid monotherapy were associated with lower CR rates. Infections, the most important adverse event, occurred significantly more often with steroid combination therapy compared to steroids alone (38.7% vs 10.6%; P = .001). Overall mortality was 38.2%, mostly due to infections (19.2%) compared to 7.7% fatal bleeds. Advanced age, underlying malignancy and ICU admission were predictors for mortality. This study showed that AHA is characterized by significant disease-related and treatment-related morbidity and mortality. A high anti-FVIII titer, severe bleeding and steroid monotherapy were associated with a lower CR rate. The efficacy of steroid combination therapies however, was overshadowed by higher infection rates and infections represented the most important cause of death. The challenging and delicate balance between treatment effectivity and safety requires ongoing monitoring of AHA and further identification of prognostic markers.

Mortality, life expectancy, and causes of death of persons with hemophilia in the Netherlands 2001-2018.

BACKGROUND: Treatment of patients with hemophilia has advanced over the past decades, but it is unknown whether this has resulted in a normal life expectancy in the Netherlands. OBJECTIVE: This observational cohort study aimed to assess all-cause and cause-specific mortality in patients with hemophilia in the Netherlands between 2001 and 2018 and to compare mortality and life expectancy with previous survival assessments from 1973 onward. PATIENTS/METHODS: All 1066 patients with hemophilia who participated in a nationwide survey in 2001 were followed until July 2018. RESULTS: Information on 1031 individuals (97%) was available, of whom 142 (14%) deceased during follow-up. Compared with the general Dutch male population, mortality of patients with hemophilia was still increased (standardized mortality ratio: 1.4, 95% confidence interval: 1.2-1.7). Intracranial bleeding and malignancies were the most common causes of death. Estimated median life expectancy of patients with hemophilia was 77 years, 6 years lower than the median life expectancy of the general Dutch male population (83 years). Over the past 45 years, death rates of patients with hemophilia have consistently decreased, approaching the survival experience of the general population. Over the past decades, mortality due to human immunodeficiency virus and hepatitis C virus infections has decreased, death due to intracranial hemorrhages has increased, and death due to ischemic heart disease has remained consistently low over time. CONCLUSIONS: Survival in patients with hemophilia in the Netherlands has improved over time but is still lower than that of the general population.

Rise of levels of von Willebrand factor and factor VIII with age: Role of genetic and acquired risk factors.

BACKGROUND: Von Willebrand factor (VWF) levels are regulated by genetic and acquired factors. The acquired factors are mostly related to age and could be mediators of the age effect on VWF levels. OBJECTIVES: To disentangle the role of genetic (sex, blood group) and acquired factors (comorbidities, body mass index, reduced kidney function, hormone use, and inflammation) in regulating von Willebrand factor antigen (VWF:Ag) and factor VIII activity (FVIII:C) levels in the normal population. METHODS: Analysis were performed in a large population sample (2923 individuals) from the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA study), after exclusion of individuals with active cancer and women who were pregnant or within nine months postpartum. The increase of VWF:Ag and FVIII:C with age was evaluated by linear regression after the age of 40 years. Analyses were adjusted for acquired factors and stratified for sex and blood group. RESULTS: VWF:Ag and FVIII:C increased with age: increase per decade of age for VWF:Ag 18 IU/dL (95%CI 15-20) and for FVIII:C 12 IU/dL (95%CI 10-14). After adjustment for acquired factors, the increase per decade was 13 IU/dL (95%CI 10-16) for VWF:Ag and 9 IU/dL (95%CI 6-11) for FVIII:C. The stratified analysis for blood group showed higher increase in the non-O group, but these differences were annulled after adjustment for acquired factors. CONCLUSIONS: VWF:Ag and FVIII:C increase with age. Carriers of blood group non-O present a steeper increase of VWF:Ag and FVIII:C with age, that is mediated by acquired factors.

Endothelial characteristics in healthy endothelial colony forming cells; generating a robust and valid ex vivo model for vascular disease.

BACKGROUND: Endothelial colony forming cells (ECFCs) derived from peripheral blood can be used to analyze the pathophysiology of vascular diseases ex vivo. However, heterogeneity is observed between ECFC clones and this variability needs to be understood and standardized for ECFCs to be used as a cell model for applications in vascular studies. OBJECTIVE: Determine reference characteristics of healthy control ECFCs to generate a valid ex vivo model for vascular disease. METHODS: Putative ECFCs (n = 47) derived from 21 individual healthy subjects were studied for cell morphology and specific cell characteristics. Clones were analyzed for the production and secretion of von Willebrand factor (VWF), cell proliferation, and the expression of endothelial cell markers. RESULTS: Based on morphology, clones were categorized into three groups. Group 1 consisted of clones with classic endothelial cell morphology, whereas groups 2 and 3 contained less condensed cells with increasing cell sizes. All clones had comparable endothelial cell surface expression profiles, with low levels of non-endothelial markers. However, a decrease in CD31 and a group-related increase in CD309 and CD45 expression, combined with a decrease in cell proliferation and VWF production and secretion, was observed in clones in group 3 and to a lesser extent in group 2. CONCLUSIONS: We observed group-related variations in endothelial cell characteristics when clones lacked the classic endothelial cell morphology. Despite this variation, clones in all groups expressed endothelial cell surface markers. Provided that clones with similar characteristics are compared, we believe ECFCs are a valid ex vivo model to study vascular disease.

von Willebrand Factor and Factor VIII Clearance in Perioperative Hemophilia A Patients.

BACKGROUND: von Willebrand factor (VWF) is crucial for optimal dosing of factor VIII (FVIII) concentrate in hemophilia A patients as it protects FVIII from premature clearance. To date, it is unknown how VWF behaves and what its impact is on FVIII clearance in the perioperative setting. AIM: To investigate VWF kinetics (VWF antigen [VWF:Ag]), VWF glycoprotein Ib binding (VWF:GPIbM), and VWF propeptide (VWFpp) in severe and moderate perioperative hemophilia A patients included in the randomized controlled perioperative OPTI-CLOT trial. METHODS: Linear mixed effects modeling was applied to analyze VWF kinetics. One-way and two-way analyses of variance were used to investigate perioperative VWFpp/VWF:Ag ratios and associations with surgical bleeding. RESULTS: Fifty-nine patients with median age of 48.8 years (interquartile range: 34.8-60.0) were included. VWF:Ag and VWF:GPIbM increased significantly postoperatively. Blood type non-O or medium risk surgery were associated with higher VWF:Ag and VWF:GPIbM levels compared with blood type O and low risk surgery. VWFpp/VWF:Ag was significantly higher immediately after surgery than 32 to 57 hours after surgery (p < 0.001). Lowest VWF:Ag quartile (0.43-0.92 IU/mL) was associated with an increase of FVIII concentrate clearance of 26 mL/h (95% confidence interval: 2-50 mL/h) compared with highest VWF antigen quartile (1.70-3.84 IU/mL). VWF levels were not associated with perioperative bleeding F(4,227) = 0.54, p = 0.710. CONCLUSION: VWF:Ag and VWF:GPIbM levels increase postoperatively, most significantly in patients with blood type non-O or medium risk surgery. Lower VWF antigen levels did not lead to clinically relevant higher FVIII clearance. VWF:Ag or VWF:GPIbM levels were not associated with perioperative hemorrhage.

Bleeding symptoms in patients diagnosed as type 3 von Willebrand disease: Results from 3WINTERS-IPS, an international and collaborative cross-sectional study.

BACKGROUND: Type 3 von Willebrand's disease (VWD) patients present markedly reduced levels of von Willebrand factor and factor VIII. Because of its rarity, the bleeding phenotype of type 3 VWD is poorly described, as compared to type 1 VWD. AIMS: To evaluate the frequency and the severity of bleeding symptoms across age and sex groups in type 3 patients and to compare these with those observed in type 1 VWD patients to investigate any possible clustering of bleeding symptoms within type 3 patients. METHODS: We compared the bleeding phenotype and computed the bleeding score (BS) using the MCMDM-1VWD bleeding questionnaire in patients enrolled in the 3WINTERS-IPS and MCMDM-1VWD studies. RESULTS: In 223 unrelated type 3 VWD patients, both the BS and the number of clinically relevant bleeding symptoms were increased in type 3 as compared to type 1 VWD patients (15 versus 6 and 5 versus 3). Intracranial bleeding, oral cavity, hemarthroses, and deep hematomas were at least five-fold over-represented in type 3 VWD. A more severe bleeding phenotype was evident in patients having von Willebrand factor antigen levels < 20 IU/dL at diagnosis in the two merged cohorts. In type 3 patients, there was an apparent clustering of hemarthrosis with gastrointestinal bleeding and epistaxis, whereas bleeding after surgery or tooth extraction clusters with oral bleeding and menorrhagia. CONCLUSIONS: In the largest cohort of type 3 VWD patients, we were able to describe a distinct clinical phenotype that is associated with the presence of a more severe hemostatic defect.

One piece of the puzzle: Population pharmacokinetics of FVIII during perioperative Haemate P® /Humate P® treatment in von Willebrand disease patients.

INTRODUCTION: Many patients with von Willebrand disease (VWD) are treated on demand with von Willebrand factor and factor VIII (FVIII) containing concentrates present with VWF and/or FVIII plasma levels outside set target levels. This carries a risk for bleeding and potentially for thrombosis. Development of a population pharmacokinetic (PK) model based on FVIII levels is a first step to more accurate on-demand perioperative dosing of this concentrate. METHODS: Patients with VWD undergoing surgery in Academic Haemophilia Treatment Centers in the Netherlands between 2000 and 2018 treated with a FVIII/VWF plasma-derived concentrate (Haemate® P/Humate P®) were included in this study. Population PK modeling was based on measured FVIII levels using nonlinear mixed-effects modeling (NONMEM). RESULTS: The population PK model was developed using 684 plasma FVIII measurements of 97 VWD patients undergoing 141 surgeries. Subsequently, the model was externally validated and reestimated with independent clinical data from 20 additional patients undergoing 31 surgeries and 208 plasma measurements of FVIII. The observed PK profiles were best described using a one-compartment model. Typical values for volume of distribution and clearance were 3.28 L/70 kg and 0.037 L/h/70 kg. Increased VWF activity, decreased physical status according to American Society of Anesthesiologists (ASA) classification (ASA class >2), and increased duration of surgery were associated with decreased FVIII clearance. CONCLUSION: This population PK model derived from real world data adequately describes FVIII levels following perioperative administration of the FVIII/VWF plasma-derived concentrate (Haemate® P/Humate P® ) and will help to facilitate future dosing in VWD patients.

Home treatment of patients with cancer-associated venous thromboembolism - An evaluation of daily practice.

BACKGROUND: Home treatment of cancer-associated venous thromboembolism (VTE) is challenging due to the high risk of adverse events. While home treatment is quite agreeable to cancer patients, studies evaluating the safety of VTE home treatment in this setting are largely unavailable. METHODS: This was an observational study in patients with cancer-associated VTE. The main outcomes were the proportion of patients treated at home (hospital discharge <24 h after diagnosis) and the 3-month incidence of VTE-related adverse events (major bleeding, recurrent VTE and/or suspected VTE-related mortality) in patients managed in hospital versus at home. RESULTS: A total of 183 outpatients were diagnosed with cancer-associated VTE: 69 had deep vein thrombosis (DVT) and 114 had pulmonary embolism (PE ±â€¯DVT). Of those, 120 (66%) were treated at home; this was 83% for patients with DVT and 55% for patients with PE (±DVT). The 3-month incidence of any VTE-related adverse event was 13% in those treated at home versus 19% in the hospitalized patients (HR 0.48; 95%CI 0.22-1.1), independent of initial presentation as PE or DVT. All-cause 3-month mortality occurred in 33 patients treated as inpatient (54%) compared to 29 patients treated at home (24%; crude HR 3.1 95%CI 1.9-5.0). CONCLUSIONS: Two-third of patients with cancer-associated VTE - including PE - were selected to start anticoagulant treatment at home. Cancer-associated VTE is associated with high rates of VTE-related adverse events independent of initial in hospital or home treatment. However, home treatment may be a good option for selected patients with cancer-associated DVT or PE.

Variability of von Willebrand factor-related parameters in endothelial colony forming cells.

Essentials Endothelial colony forming cells (ECFCs) are a powerful tool to study vascular diseases ex vivo. Separate ECFC lines show variations in morphology and von Willebrand factor-related parameters. Maximum cell density is correlated with von Willebrand factor expression in ECFCs. Variations in ECFC lines are dependent on the age and mesenchymal state of the cells. ABSTRACT: Background Endothelial colony forming cells (ECFCs) are cultured endothelial cells derived from peripheral blood. ECFCs are a powerful tool to study pathophysiological mechanisms underlying vascular diseases, including von Willebrand disease. In prior research, however, large variations between ECFC lines were observed in, among others, von Willebrand factor (VWF) expression. Objective Understand the relation between phenotypic characteristics and VWF-related parameters of healthy control ECFCs. Methods ECFC lines (n = 16) derived from six donors were studied at maximum cell density. Secreted and intracellular VWF antigen were measured by ELISA. The angiogenic capacity of ECFCs was investigated by the Matrigel tube formation assay. Differences in expression of genes involved in angiogenesis, aging, and endothelial to mesenchymal transition (EndoMT) were measured by quantitative PCR. Results Different ECFC lines show variable morphologies and cell density at maximum confluency and cell lines with a low maximum cell density show a mixed and more mesenchymal phenotype. We identified a significant positive correlation between maximum cell density and VWF production, both at protein and mRNA level. Also, significant correlations were observed between maximum cell density and several angiogenic, aging and EndoMT parameters. Conclusions We observed variations in morphology, maximum cell density, VWF production, and angiogenic potential between healthy control ECFCs. These variations seem to be attributable to differences in aging and EndoMT. Because variations correlate with cell density, we believe that ECFCs maintain a powerful tool to study vascular diseases. It is however important to compare cell lines with the same characteristics and perform experiments at maximum cell density.

Desmopressin treatment combined with clotting factor VIII concentrates in patients with non-severe haemophilia A: protocol for a multicentre single-armed trial, the DAVID study.

INTRODUCTION: Haemophilia A is an inherited bleeding disorder characterised by factor VIII (FVIII) deficiency. In patients with non-severe haemophilia A, surgery and bleeding are the main indications for treatment with FVIII concentrate. A recent study reported that standard dosing frequently results in FVIII levels (FVIII:C) below or above FVIII target ranges, leading to respectively a bleeding risk or excessive costs. In addition, FVIII concentrate treatment carries a risk of development of neutralising antibodies. An alternative is desmopressin, which releases endogenous FVIII and von Willebrand factor. In most patients with non-severe haemophilia A, desmopressin alone is not enough to achieve FVIII target levels during surgery or bleeding. We hypothesise that combined pharmacokinetic (PK)-guided administration of desmopressin and FVIII concentrate may improve dosing accuracy and reduces FVIII concentrate consumption. METHODS AND ANALYSIS: In the DAVID study, 50 patients with non-severe haemophilia A (FVIII:C ≥0.01 IU/mL) with a bleeding episode or undergoing surgery will receive desmopressin and FVIII concentrate combination treatment. The necessary dose of FVIII concentrate to reach FVIII target levels after desmopressin administration will be calculated with a population PK model. The primary endpoint is the proportion of patients reaching FVIII target levels during the first 72 hours after start of the combination treatment. This approach was successfully tested in one pilot patient who received perioperative combination treatment. ETHICS AND DISSEMINATION: The DAVID study was approved by the medical ethics committee of the Erasmus MC. Results of the study will be communicated trough publication in international scientific journals and presentation at (inter)national conferences. TRIAL REGISTRATION NUMBER: NTR5383; Pre-results.

Predictive value of a bleeding score for postpartum hemorrhage.

BACKGROUND: A reliable screening tool that could contribute to the identification of women with an increased risk of postpartum hemorrhage would be of great clinical significance. OBJECTIVES: The aim of this study was to examine the added predictive value of a bleeding assessment tool for postpartum hemorrhage exceeding 1000 mL. PATIENTS/METHODS: Prospective two-center cohort study among 1147 pregnant women visiting the outpatient clinic or the maternity ward who completed a bleeding assessment tool prior to birth. The condensed MCMDM-1VWD bleeding assessment tool was adjusted to a questionnaire that could be used as a self-assessment bleeding tool. A score of ≥4 was considered to be abnormal. RESULTS: In the 1147 pregnant women in our cohort, bleeding scores ranged from -3 to 13, with a median of 1 (IQR -1 to 3); 197 (17%) women developed postpartum hemorrhage. Among women with a history of postpartum hemorrhage 29% developed postpartum hemorrhage. Among 147 women with an abnormal bleeding score (≥4), 27 (18%) developed postpartum hemorrhage, whereas the remaining 170 cases of postpartum hemorrhage had a normal bleeding score. Despite the high incidence of postpartum hemorrhage, the ability of the bleeding score to predict postpartum hemorrhage was poor: area under receiver operating curve 0.53 (95% CI 0.49-0.58) for postpartum hemorrhage (PPH) ≥1000 mL. CONCLUSIONS: A history of significant postpartum hemorrhage was associated with an increased risk of subsequent postpartum hemorrhage. However, screening with a bleeding assessment tool did not help to discriminate women who will develop postpartum hemorrhage from women who will not.

Von Willebrand Disease: From In Vivo to In Vitro Disease Models.

Von Willebrand factor (VWF) plays an essential role in primary hemostasis and is exclusively synthesized and stored in endothelial cells and megakaryocytes. Upon vascular injury, VWF is released into the circulation where this multimeric protein is required for platelet adhesion. Defects of VWF lead to the most common inherited bleeding disorder von Willebrand disease (VWD). Three different types of VWD exist, presenting with varying degrees of bleeding tendencies. The pathophysiology of VWD can be investigated by examining the synthesis, storage and secretion in VWF producing cells. These cells can either be primary VWF producing cells or transfected heterologous cell models. For many years transfected heterologous cells have been used successfully to elucidate many aspects of VWF synthesis. However, those cells do not fully reflect the characteristics of primary cells. Obtaining primary endothelial cells or megakaryocytes with a VWD phenotype, requires invasive procedures, such as vessel collection or a bone marrow biopsy. A more recent and promising development is the isolation of endothelial colony forming cells (ECFCs) from peripheral blood as a true-to-nature cell model. Alternatively, various animal models are available but limiting, therefore, new approaches are needed to study VWD and other bleeding disorders. A potential versatile source of endothelial cells and megakaryocytes could be induced pluripotent stem cells (iPSCs). This review gives an overview of models that are available to study VWD and VWF and will discuss novel approaches that can be considered to improve the understanding of the structural and functional mechanisms underlying this disease.

Comorbidities associated with higher von Willebrand factor (VWF) levels may explain the age-related increase of VWF in von Willebrand disease.

Some comorbidities, such as hypertension, are associated with higher von Willebrand factor (VWF) levels in the general population. No studies have been conducted to assess this association in patients with von Willebrand disease (VWD). Therefore, we studied this association in patients with type 1 (n = 333) and type 2 (n = 203) VWD from the 'WiN" study. VWF antigen (VWF:Ag) was higher in type 1 VWD patients with hypertension [difference: 0·23 iu/ml, 95% confidence interval (CI): 0·11-0·35], diabetes mellitus (0·11 iu/ml, 95% CI: -0·02 to 0·23), cancer (0·14 iu/ml, 95% CI: 0·03-0·25) and thyroid dysfunction (0·14 iu/ml, 95% CI: 0·03-0·26) than in patients without these comorbidities (all corrected for age, sex and blood group). Similar results were observed for VWF collagen binding capacity (VWF:CB), VWF activity as measured by the VWF monoclonal antibody assay (VWF:Ab) and factor VIII (FVIII) coagulant activity (FVIII:C). In type 1 VWD, age was associated with higher VWF:Ag (0·03 iu/ml; 95% CI: 0·01-0·04), VWF:CB (0·02 iu/ml; 95% CI: 0·00-0·04), VWF:Ab (0·04 iu/ml; 95% CI: 0·02-0·06) and FVIII:C (0·03 iu/ml; 95% CI: 0·01-0·06) per decade increase. After adjustment for relevant comorbidities, these associations were no longer significant. Despite the higher VWF and FVIII levels, type 1 VWD patients with comorbidities had more bleeding episodes, particularly during surgery. There was no association between comorbidities and VWF/FVIII levels or bleeding phenotype in type 2 VWD patients. In conclusion, comorbidities are associated with higher VWF and FVIII levels in type 1 VWD and may explain the age-related increase of VWF and FVIII levels.