Idiopathic pulmonary arterial hypertension - a unrecognized cause of high-shear high-flow haemostatic defects (otherwise referred to as acquired von Willebrand syndrome) in children.

Acquired von Willebrand syndrome (AVWS) is reported in high-flow high-shear congenital cardiac disorders. We hypothesized that the narrowed pulmonary vasculature in idiopathic pulmonary arterial hypertension (IPAH) may induce AVWS. We conducted a cross-sectional evaluation of children with IPAH. Patients with bleeding symptoms and/or laboratory abnormalities (thrombocytopenia, anomalies in coagulation screening tests) were tested in-depth for haemostatic defects. Fourteen children were followed with IPAH of which 8 were eligible. Four children exhibited abnormal bleeding scores (International Society on Thrombosis and Haemostasis Bleeding Assessment Tool: 3-5). All 8 patients showed very prolonged platelet function analyser (PFA)-100 closure times. Six children demonstrated either mild thrombocytopenia or low-normal von Willebrand factor (VWF) antigen (VWF:Ag) or VWF activity [mean (range), in iu/dl: VWF:Ag: 70 (61-91); VWF activity: 57 (34-70)]. Average VWF collagen binding capacity (VWF:CB) was 64 iu/dl (range: 53-123 iu/dl), with low-normal VWF activity/VWF:Ag or VWF:CB/VWF:Ag ratios occurring in five patients. All children had normal multimers distribution patterns. One patient underwent a lung transplantation, with normalization of haemostatic abnormalities post-surgery. Overall, 8 out of 14 children with IPAH had mild to moderate bleeding symptoms and/or laboratory abnormalities in keeping with AVWS. Normalization of the haemostatic defects following lung transplantation and lack of family history of bleeding attests to the acquired nature of their defects.

Characterization of aberrant splicing of von Willebrand factor in von Willebrand disease: an underrecognized mechanism.

Approximately 10% of von Willebrand factor (VWF) gene mutations are thought to alter messenger RNA (mRNA) splicing through disruption of consensus splice sites. This mechanism is likely underrecognized and affected by mutations outside consensus splice sites. During VWF synthesis, splicing abnormalities lead to qualitative defects or quantitative deficiencies in VWF. This study investigated the pathologic mechanism acting in 3 von Willebrand disease (VWD) families with putative splicing mutations using patient-derived blood outgrowth endothelial cells (BOECs) and a heterologous human embryonic kidney (HEK 293(T)) cell model. The exonic mutation c.3538G>A causes 3 in-frame splicing variants (23del, 26del, and 23/26del) which cannot bind platelets, blood coagulation factor VIII, or collagen, causing VWD through dominant-negative intracellular retention of coexpressed wild-type (WT) VWF, and increased trafficking to lysosomes. Individuals heterozygous for the c.5842+1G>C mutation produce exon 33 skipping, exons 33-34 skipping, and WT VWF transcripts. Pathogenic intracellular retention of VWF lacking exons 33-34 causes their VWD. The branch site mutation c.6599-20A>T causes type 1 VWD through mRNA degradation of exon 38 skipping transcripts. Splicing ratios of aberrant transcripts and coexpressed WT were altered in the BOECs with exposure to shear stress. This study provides evidence of mutations outside consensus splice sites disrupting splicing and introduces the concept that VWF splicing is affected by shear stress on endothelial cells.

Bleeding Scores for the Diagnosis of von Willebrand Disease.

Obtaining a personal history of bleeding is a critical component to the diagnosis of von Willebrand disease (VWD). The collection of this information can be challenging for physicians, however, as the reporting and interpretation of bleeding symptoms is subjective. The need for more precise quantification of bleeding symptoms was recognized and the Vicenza Bleeding Questionnaire was developed in 2005. This questionnaire collects data regarding the presence and severity of bleeding symptoms and generates a bleeding score by summing the severity of all symptoms reported by a patient. Several subsequent bleeding assessment tools (BATs) have been developed based from this original questionnaire and there has been a surge in the use of BATs in various clinical settings for the diagnosis and evaluation of VWD. This review will discuss the evolution of BATs over the past decade, as well as their use and validation in various settings for the diagnosis and evaluation of VWD. Additionally, we will discuss the clinical utility of BATs, the limitations of these tools, and future directions.

Von Willebrand factor regulates complement on endothelial cells.

Atypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura have traditionally been considered separate entities. Defects in the regulation of the complement alternative pathway occur in atypical hemolytic uremic syndrome, and defects in the cleavage of von Willebrand factor (VWF)-multimers arise in thrombotic thrombocytopenic purpura. However, recent studies suggest that both entities are related as defects in the disease-causing pathways overlap or show functional interactions. Here we investigate the possible functional link of VWF-multimers and the complement system on endothelial cells. Blood outgrowth endothelial cells (BOECs) were obtained from 3 healthy individuals and 2 patients with Type 3 von Willebrand disease lacking VWF. Cells were exposed to a standardized complement challenge via the combination of classical and alternative pathway activation and 50% normal human serum resulting in complement fixation to the endothelial surface. Under these conditions we found the expected release of VWF-multimers causing platelet adhesion onto BOECs from healthy individuals. Importantly, in BOECs derived from patients with von Willebrand disease complement C3c deposition and cytotoxicity were more pronounced than on BOECs derived from normal individuals. This is of particular importance as primary glomerular endothelial cells display a heterogeneous expression pattern of VWF with overall reduced VWF abundance. Thus, our results support a mechanistic link between VWF-multimers and the complement system. However, our findings also identify VWF as a new complement regulator on vascular endothelial cells and suggest that VWF has a protective effect on endothelial cells and complement-mediated injury.

Vascular endothelial cells evade complement-mediated membrane injury via Weibel-Palade body mobilization.

BACKGROUND: Defective complement inhibition can lead to the formation of membrane attack complexes (MAC; C5b-9) on the plasma membranes of vascular endothelial cells, resulting in injury that drives the progression of thrombotic microangiopathy (TMA), a key pathology in kidney disease. OBJECTIVE/METHODS: We examined the response of human endothelial cells to complement-mediated damage using blood outgrowth endothelial cells (BOECs) derived from healthy donors. BOECs were sensitized to complement factors present in normal human serum to induce the formation of C5b-9 on their plasma membranes. RESULTS: This triggered an expected abrupt rise in intracellular Ca2+ reflecting membrane leakage. Remarkably, while intracellular Ca2+ remained elevated, membrane leakage ceased within 30 minutes, and cells did not show significant death. Extensive mobilization of Weibel-Palade bodies (WPBs) was observed along with secretion of von Willebrand factor (VWF). The potential role of WPBs and VWF in mitigating complement-mediated damage was examined by comparing the effects of C5b-9 on BOECs derived from von Willebrand disease (VWD) patients expressing reduced amounts of VWF, lacking expression of functional VWF, or lacking both VWF and WPBs. BOECs lacking WPBs were not resistant to complement-mediated damage, but became resistant when transfected to express VWF (and thus WPBs). CONCLUSION: We conclude that BOECs exposed to C5b-9 attack respond by mobilizing WPBs, which mitigate and repair damage by fusing with the plasma membrane. We propose that a similar cell-specific response may protect the vascular endothelium from complement-mediated damage in vivo.

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.