von Willebrand factor propeptide variants lead to impaired storage and ER retention in patient-derived endothelial colony-forming cells.

BACKGROUND: von Willebrand factor (VWF) is synthesized by vascular endothelial cells and megakaryocytes. The VWF propeptide is critical for multimerization and acts as an intra-molecular chaperone for mature VWF in sorting to its storage organelles, Weibel-Palade bodies (WPBs). In the Canadian Type 3 VWD study, almost half of the identified variants were in the VWF propeptide and these were associated with an increased bleeding phenotype. OBJECTIVE: To investigate VWF propeptide variants that cause quantitative von Willebrand disease (VWD) by utilizing patient-derived endothelial colony-forming cells (ECFCs). PATIENTS/METHODS: Endothelial colony-forming cells were isolated from five Type 3 VWD patients from four families with the following variants: (1) homozygous p.Asp75_Gly178del (deletion of exons 4 and 5 deletion; Ex4-5del), (2) homozygous p.Cys633Arg, (3) homozygous p.Arg273Trp, and (4) p.Pro293Glnfs*164 and p.Gln419* inherited in the compound heterozygous state. Additionally, ECFCs were isolated from six family members (two Type 1 VWD, four unaffected). RESULTS: Endothelial colony-forming cells from the Type 3 patient with the compound heterozygous genotype exhibited a true null VWF cellular phenotype, with negligible VWF detected. In contrast, the other three propeptide variants presented a similar expression pattern in homozygous ECFCs where VWF was synthesized but not packaged in WPBs, and variant VWF had an increased association with the endoplasmic reticulum (ER) marker, protein disulfide-isomerase (PDI), indicating an ER-retention phenotype. The biosynthetic phenotype was similar but to a lesser degree in heterozygous ECFCs expressing the non-null variants. CONCLUSION: This study further elucidates the importance of the VWF propeptide in the VWD phenotype using patient-derived cells.

A two-pronged approach to reduce noise levels in the neonatal intensive care unit.

OBJECTIVES: The aim of this study was to assess the efficacy of a visual noise feedback system and "quiet time" in reducing noise levels in the neonatal intensive care unit (NICU). DESIGN: A prospective cross-sectional study was performed in a combined level II/III NICU at a Canadian tertiary care hospital. Noise levels were recorded continuously for three weeks without and then three weeks with visual noise feedback system. Noise levels were compared after one year of using visual feedback, and subsequently with the addition of two "quiet times." RESULTS: Visual feedback reduced noise levels from 54.2 dB (95% CI 53.8-54.7 dB) to 49.4 dB (95% CI 48.9-49.8 dB; P < 0.0001) and increased the amount of time spent under 45 dB from 0 to 25% (P < 0.0001) after three weeks of use. However, this effect was not sustained at one year of visual feedback, with noise levels at 54.7 dB (95% CI 54.5-55.0 dB, P = 0.55). Quiet Time did not further reduce daily noise in the NICU (average noise levels 54.7, 95% CI 54.4-55.0 dB, P = 0.836). CONCLUSIONS: While visual noise feedback system reduced noise levels in the short term, these effects were not sustainable at one year and could not be remediated with the addition of a Quiet Time initiative. Continuing education regarding the detrimental effects of noise is paramount to ensure persistent noise reduction in the NICU.

Patients with aortic stenosis have von Willebrand factor abnormalities and increased proliferation of endothelial colony forming cells.

BACKGROUND: Patients with aortic stenosis (AS) can experience bleeding complications including gastrointestinal bleeding from angiodysplastic lesions due to acquired von Willebrand syndrome. Studies have pointed to a role for von Willebrand factor (VWF) in angiogenesis. OBJECTIVE: The objective of this study was to assess VWF defects in AS patients over time and the impact on angiogenesis using patient-derived endothelial colony-forming cells (ECFCs). PATIENTS/METHODS: Plasma sample collection and ECFC isolations were performed before valve replacement surgery, 3 to 5 days after, and 6 months after surgery. Plasma VWF antigen, activity, propeptide, collagen binding, multimers, factor VIII coagulant activity, and ADAMTS13 activity (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs 13) were determined. ECFCs were assessed for VWF and angiopoietin-2 (Ang-2) storage and secretion, cell proliferation, and tubule formation in Matrigel. RESULTS AND CONCLUSIONS: Aortic stenosis patients exhibited quantitative and qualitative abnormalities of VWF including significantly increased VWF antigen, activity, and propeptide levels following surgery (P < .01). Increased high molecular weight VWF multimers were observed at all time points and in particular 3 to 5 days after surgery (mean = 14% ± 6%) relative to before (mean = 10% ± 4%), suggesting increased proteolysis by ADAMTS13 pre-operatively in a shear-dependent manner. ECFCs from patients with aortic stenosis were more proliferative than controls (P < .05) and had increased retention of Ang-2 (P < .05) suggesting epigenetic modification of the cells. Overall, there are hemostatic changes in AS patients that are present before valve replacement surgery and these persist long after surgery has occurred. These findings have implications for the current clinical management of AS patients.

Determinants of Successful Direct Breastfeeding at Hospital Discharge in High-Risk Premature Infants.

BACKGROUND: Despite a mother's intention to breastfeed, there are many barriers to feeding preterm infants that decrease breastfeeding rates. OBJECTIVE: The objective of this research was to determine factors associated with successful direct breastfeeding (DBF) of the preterm infant at hospital discharge. MATERIALS AND METHODS: A retrospective chart review of 69 preterm (<34 weeks' gestational age) infants in the neonatal intensive care unit, whose mothers intended to breastfeed, was conducted. Infant-, mother-, and feeding-related factors were examined by chi-square or t test for their relationship with breastfeeding success, and by multiple logistic regression to identify predictive factors. RESULTS: Successful DBF at discharge occurred in 64%. Mothers of infants who were breastfed were older (p < 0.0001); had less psychiatric illness (p = 0.03); and were less likely to smoke (p < 0.0001) and use recreational drugs (p = 0.04). The infants had higher birth weights (p = 0.03) and lower incidence of bronchopulmonary dysplasia (p = 0.04). A higher proportion of infants received DBF at their first oral feed (p < 0.001), and were discharged earlier (p = 0.03). Reduced milk supply was cited for breastfeeding failure in 36%. Older maternal age (odds ratio [OR] = 1.24, 95% confidence interval [CI] 1.02-1.51) and DBF at the first oral feed (OR = 7.72, 95% CI 1.37-43.6) were associated with successful DBF at discharge. CONCLUSION: Maternal age and method of first oral feed are critical predictors of breastfeeding success in preterm infants. Mothers should be encouraged to breastfeed at the infant's first oral attempt and strategic breastfeeding support should be provided before initiation of oral feeding.

Generation and optimization of the self-administered pediatric bleeding questionnaire and its validation as a screening tool for von Willebrand disease.

OBJECTIVE: Our objective was to generate, optimize, and validate a self-administered pediatric bleeding questionnaire (Self-PBQ) as a screening tool for von Willebrand disease (VWD) in children referred to the hematology clinic for the first time. STUDY DESIGN: The Self-PBQ was generated by combining the validated expert-administered PBQ and the International Society on Thrombosis and Hemostasis (ISTH) bleeding assessment tool (BAT). Medical terminology was translated into lay language requiring a grade 4 reading level. In Phase 1, the Self-PBQ was optimized and the level of agreement between the Self-PBQ and the expert-administered PBQ was determined. Phase 2 established the normal range of bleeding scores (BSs) of the Self-PBQ. Phase 3 examined the Self-PBQ as a screening tool for first-time referrals to the hematology clinic. RESULTS: The Self-PBQ is a reliable surrogate for the expert-administered PBQ with an excellent intraclass correlation (ICC) of 0.917. The Self-PBQ was scored with the PBQ and the ISTH-BAT scoring systems, for which its normal BS ranges are -1 to 2 or 0 to 2, respectively. A positive Self-PBQ BS (≥3) had a sensitivity of 78%, a specificity of 37%, a positive predictive value of 0.18, and a negative predictive value of 0.91 for identifying VWD in children being investigated by a hematologist for a bleeding disorder. CONCLUSION: The Self-PBQ generates comparable BSs to the expert-administered PBQ and is a reliable, reasonably sensitive screening tool to incorporate into the assessment of children presenting to a hematologist for the investigation of an inherited bleeding disorder.

Abnormal angiogenesis in blood outgrowth endothelial cells derived from von Willebrand disease patients.

: Bleeding associated with angiodysplasia is a common, often intractable complication in patients with von Willebrand disease (VWD). von Willebrand factor (VWF), the protein deficient or defective in VWD, is a negative regulator of angiogenesis, which may explain the pathologic blood vessel growth in VWD. This study explores the normal range of angiogenesis in blood outgrowth endothelial cells (BOECs) derived from healthy donors and compares this to angiogenesis in BOECs from VWD patients of all types and subtypes. BOECs were assessed for VWF and angiopoietin-2 (Ang-2) gene expression, secretion, and storage. To explore angiogenic potential, we characterized cellular proliferation, matrix protein adhesion, migration, and tubule formation. We found great angiogenic variability in VWD BOECs with respect to each of the angiogenesis parameters. However, type 1 and 3 VWD BOECs had higher Ang-2 secretion associated with impaired endothelial cell migration velocity and enhanced directionality. Type 2A and 2B BOECs were the most proliferative and multiple VWD BOECs had impaired tubule formation in Matrigel. This study highlights the angiogenic variability in BOECs derived from VWD patients. Abnormal cell proliferation, migration, and increased Ang-2 secretion are common features of VWD BOECs. Despite the many abnormalities of VWD BOECs, significant heterogeneity among individual VWD phenotypes precludes a simple description of relationship between VWD type and in vitro surrogates for angiodysplasia.