The common VWF variant p.Y1584C: detailed pathogenic examination of an enigmatic sequence change.

BACKGROUND: As knowledge of the human genome has advanced, so too has the recognition that interpretation of the pathogenic nature of sequence variants can be challenging. The von Willebrand factor (VWF) gene exhibits a significant degree of sequence variability, and the first VWF variant associated with type 1 von Willebrand disease (VWD), c.4751 A>G, p.Y1584C, was described in 2003. However, since that time, the pathogenic nature of this variant has remained unclear, being assigned properties ranging from a risk factor to a pathogenic variant. OBJECTIVES: To provide additional evaluation on the interpretation of pathogenicity for this common VWF variant. METHODS: Fifty-eight subjects with only the p.Y1584C variant were recruited from 2 cohort studies (the Zimmerman Program and the Canadian type 1 VWD study). Clinical and laboratory phenotypes were assessed. RESULTS: The prevalence of the p.Y1584C variant in our cohorts was 23- to 27-fold higher than that in large normal population databases. Significantly more p.Y1584C subjects had an abnormal bleeding score when compared to Y1584 individuals. In comparison with a group of 35 subjects without the p.Y1584C variant, subjects with the variant had lower mean VWF:antigen and VWF:ristocetin cofactor values and significantly higher VWF propeptide/VWF:antigen ratios suggestive of enhanced clearance. CONCLUSION: Collectively, the results of this analysis suggest that p.Y1584C is likely pathogenic, however, due to influences such as incomplete penetrance, variable expressivity, and other genetic modifiers like ABO blood group, the straightforward assignment of pathogenicity to this variant is inevitably challenging.

Von Willebrand Factor (VWF) multiplex activity assay differentiation of type 1 von Willebrand Disease (VWD) and variant VWD.

INTRODUCTION: VWD diagnosis is challenging requiring multiple VWF activity tests using many individual assays. We have developed an ELISA-based VWF Multiplex Activity Assay (VWF-MAA) to address this concern; however, the ability of the VWF-MAA to discriminate between type 1 VWD, variant VWD, and normal subjects has not been evaluated. AIM: To evaluate the VWF-MAA and its ability to differentiate between type 1 VWD, variant VWD and normal subjects in individuals undergoing an initial laboratory evaluation for bleeding. METHODS: A total of 177 plasma samples from the Zimmerman Program: Comparative Effectiveness in the Diagnosis of VWD were evaluated from 11 centres across the US and Canada. The VWF-MAA was compared to Versiti Blood Research Institute (VBRI) and Local Center (LC) assigned VWD diagnosis. RESULTS: Overall, 129/177 (72.9%) were correctly assigned as normal (non-VWD), type 1, or variant VWD compared to the VBRI assigned diagnosis. VWF-MAA assigned non-VWD accurately in 29/57 (50.9%) samples, and type 1 VWD accurately in 93/110 (84.6%) samples. Considering LC diagnosis where there was agreement with VWF-MAA and not VBRI diagnosis, type 1 VWD was accurate in 105/110 (95.5%) samples. Bland-Altman analysis demonstrated good correlation between laboratory methods. VWD, types 2A, 2B, 1C VWD were also assigned by the VWF-MAA. CONCLUSIONS: We demonstrate that the VWF-MAA has utility in differentiating type 1 VWD, variant VWD and normal subjects in individuals undergoing an initial laboratory evaluation for bleeding.

Characterization of copy-number variants in a large cohort of patients with von Willebrand disease reveals a relationship between disrupted regions and disease type.

Background: Genetic analysis for von Willebrand disease (VWD) commonly utilizes DNA sequencing to identify variants in the von Willebrand factor (VWF) gene; however, this technique cannot always detect copy-number variants (CNVs). Additional mapping of CNVs in patients with VWD is needed. Objectives: This study aimed to characterize CNVs in a large sample of VWF mutation-negative VWD patients. Methods: To determine the role of CNVs in VWD, a VWF high-resolution comparative genomic hybridization array was custom-designed to avoid multiple sequence variations, repeated sequences, and the VWF pseudogene. This was performed on 204 mutation-negative subjects for whom clinical variables were also available. Results: Among the 204 patients, 7 unique CNVs were found, with a total of 24 CNVs (12%). Of the 7 unique CNVs, 1 was novel, 1 was found in a VWF database, and 5 were previously reported. All patients with type 1C VWD and a CNV had the same exon 33 and 34 in-frame deletion. Certain clinical variables were also significantly different between those with and without CNVs. Conclusion: The in-frame deletion in patients with type 1C VWD exactly matches the D4N module of the D4 domain, a region where mutations and deletions are known to affect clearance. We observed significantly higher VWF-to-ristocetin cofactor levels in patients with type 1C VWD and a CNV than in patients without a CNV, suggesting a relationship between CNVs and the increased clearance observed in patients with type 1C VWD. Glycoprotein IbM activity was significantly lower in patients with type 1 VWD and a CNV than in patients without a CNV, suggesting that platelet binding is more affected by CNVs than single base pair mutations. This work elucidates some of the underlying genetic mechanisms of CNVs in these patients.

Variability in International Society on Thrombosis and Haemostasis-Scientific and Standardization Committee endorsed Bleeding Assessment Tool (ISTH-BAT) score with normal aging in healthy females: contributory factors and clinical significance.

BACKGROUND: Bleeding assessment tools are key screening tests used in the evaluation of patients with suspected inherited bleeding disorders. The International Society on Thrombosis and Haemostasis-Scientific and Standardization Committee endorsed Bleeding Assessment Tool (ISTH-BAT) has differing reference ranges for adult males (0-3), adult females (0-5), and children (0-2), reflecting differing bleeding symptoms and exposure to hemostatic challenges in these healthy population subgroups. Age is known to markedly impact bleeding score in individuals with von Willebrand disease. However, the influence of age on bleeding score in healthy adult controls is poorly understood. OBJECTIVES: We aimed to assess variability in ISTH-BAT score with age among healthy control females. METHODS: We used the legacy "Merging Project" dataset of normal healthy controls upon which current ISTH-BAT normal ranges are based. We included women, totaling 646 individuals. The normal range (middle 95th percentile) of total ISTH-BAT and grouped subdomain scores between age quartiles was assessed. RESULTS: The normal range of ISTH-BAT scores increased with age, ranging from 0 to 4 in the youngest quartile (age range, 18-30) to 0 to 6 in the oldest (age range, 52-88). This increased variability with aging was related both to high menorrhagia domain scores in older women and an increase in postprocedural bleeding with accumulated exposure to hemostatic challenges. CONCLUSIONS: Cumulatively, our data highlight that normal aging leads to increased variability in bleeding scores in healthy adult females. Further refinement of the ISTH-BAT with age-adjusted reference ranges may improve the sensitivity and specificity of the tool among females.

Ristocetin dependent cofactor activity in von Willebrand disease diagnosis: Limitations of relying on a single measure.

Background: Von Willebrand disease (VWD) is a common inherited bleeding disorder, however the diagnosis can be complicated by a subjective bleeding history and issues with some current von Willebrand factor (VWF) laboratory assays. Objectives: In the Zimmerman Program, we sought to determine how often a type 1 diagnosis was based on a single low VWF ristocetin cofactor (VWF:RCo) level resulting from the common genetic variant p.D1472H or an isolated assay issue, if that low value was corroborated by the VWF glycoprotein-IbM (VWF:GPIbM) assay, and if retesting confirmed original levels. Methods: New patients being evaluated for bleeding were consented. Analysis included VWF sequencing, bleeding scores, and comparisons of local VWF antigen (VWF:Ag) and VWF:RCo to central VWF:Ag and VWF:GPIbM. Results: A total of 18% of VWD subjects had a low local VWF:RCo, but normal VWF:Ag and normal central testing including VWF:GPIbM. Seventy percent of the low VWF:RCo cohort had no pathogenic VWF variants; however, 33% carried p.D1472H. Low VWF:RCo subjects with follow-up local testing within 2 years showed those with p.D1472H continued to have low VWF:RCo and VWF:RCo/VWF:Ag ratio with normal VWF:GPIbM. Subjects without p.D1472H had an increase mean VWF:RCo, resulting in 59% with normal levels on repeat testing. Conclusions: The diagnosis of VWD based on a single low VWF:RCo but normal VWF:Ag, was often attributed to p.D1472H or variability in VWF:RCo that was eliminated with VWF:GPIbM. Our study suggests that using VWF:RCo alone for diagnostic purposes may be insufficient while repeat VWF:RCo or VWF:GPIbM testing can be valuable in establishing a VWD diagnosis.

Longitudinal bleeding assessment in von Willebrand disease utilizing an interim bleeding score.

BACKGROUND: Assessment of bleeding phenotype is critically important in the diagnosis of von Willebrand disease (VWD). Despite advances in bleeding assessment tools (BATs), standardized tools to evaluate bleeding following diagnosis (interim bleeding) are lacking. OBJECTIVES: We assessed the clinical utility of an interim bleeding protocol in a multicenter, international study involving patients with VWD. METHODS: The enrolment ISTH BAT formed the original bleeding score (0 BS). At follow-up, the International Society on Thrombosis and Haemostasis BAT was repeated but included only interval bleeding (Interim BS, 1 BS). Both scores were annualized (0 BS/yr, 1 BS/yr). BS were analyzed by VWD subtype, plasma VWF level, sex, and age. RESULTS: Interim BS discriminated by subtype, with significantly increased 0 BS and 1 BS in patients with type 3 VWD. In patients with type 1 VWD, a positive or negative 0 BS did not predict future bleeding, with similar 1 BS/yr (median 1.0 vs. 0.7, p = .2). Despite significantly higher 0 BS in females with type 1 VWD than males (median 7 vs. 5, p = .0012), 1 BS were not significantly different (median 4 vs. 4, p = .16). While 0 BS were lower in children than adults with type 1 VWD, interim BS were similar (median 5 vs. 3, p = .5; 1BS/yr, median 1 vs. 0.8, p = .7). Interestingly, in those with plasma von Willebrand factor:ristocetin cofactor levels >50 IU/dl, interim BS rates were similar to those 30-50 IU/dl (1 BS/yr 0.8 vs. 1.3, p = .5). CONCLUSION: This study provides both a new approach to longitudinal bleeding assessment and insights into the evolution of bleeding in VWD.

Molecular pathogenesis and heterogeneity in type 3 VWD families in U.S. Zimmerman program.

BACKGROUND: Type 3 von Willebrand Disease (VWD) is a rare and severe form of VWD characterized by the absence of von Willebrand factor (VWF). OBJECTIVES: As part of the Zimmerman Program, we sought to explore the molecular pathogenesis, correlate bleeding phenotype and severity, and determine the inheritance pattern found in type 3 VWD families. PATIENTS/METHODS: 62 index cases with a pre-existing diagnosis of type 3 VWD were analyzed. Central testing included FVIII, VWF:Ag, VWF:RCo, and VWFpp. Bleeding symptoms were quantified using the ISTH bleeding score. Genetic analysis included VWF sequencing, comparative genomic hybridization and predictive computational programs. RESULTS: 75% of subjects (46) had central testing confirming type 3, while 25% were re-classified as type 1-Severe or type 1C. Candidate VWF variants were found in all subjects with 93% of expected alleles identified. The majority were null alleles including frameshift, nonsense, splice site, and large deletions, while 13% were missense variants. Additional studies on 119 family members, including 69 obligate carriers, revealed a wide range of heterogeneity in VWF levels and bleeding scores, even amongst those with the same variant. Co-dominant inheritance was present in 51% of families and recessive in 21%, however 28% were ambiguous. CONCLUSION: This report represents a large cohort of VWD families in the U.S. with extensive phenotypic and genotypic data. While co-dominant inheritance was seen in approximately 50% of families, this study highlights the complexity of VWF genetics due to the heterogeneity found in both VWF levels and bleeding tendencies amongst families with type 3 VWD.

The impact of aberrant von Willebrand factor-GPIbα interaction on megakaryopoiesis and platelets in humanized type 2B von Willebrand disease model mouse.

Type 2B von Willebrand disease (VWD) is caused by gain-of-function mutations in von Willebrand factor (VWF). Increased VWF affinity for GPIba results in loss of high molecular weight multimers and enhanced platelet clearance, both contributing to the bleeding phenotype. Severity of the symptoms vary among type 2B VWD patients, with some developing thrombocytopenia only under stress conditions. Efforts have been made to study underlying pathophysiology for platelet abnormalities, but animal studies have been limited because of species specificity in the VWF-GPIba interaction. Here, we generated a severe form of type 2B VWD (p.V1316M) knockin mice in the context of human VWF exon 28 (encoding A1 and A2 domains) and crossed them with human GPIba transgenic strain. Heterozygous mutant mice recapitulated the phenotype of type 2B VWD in autosomal dominant manner and presented severe macrothrombocytopenia. Of note, platelets remaining in the circulation had extracytoplasmic GPIba shed-off from the cell surface. Reciprocal bone marrow transplantation determined mutant VWF produced from endothelial cells as the major cause of the platelet phenotype in type 2B VWD mice. Moreover, altered megakaryocyte maturation in the bone marrow and enhanced extramedullary megakaryopoiesis in the spleen were observed. Interestingly, injection of anti-VWF A1 blocking antibody (NMC-4) not only ameliorated platelet count and GPIba expression, but also reversed MK ploidy shift. In conclusion, we present a type 2B VWD mouse model with humanized VWF-GPIba interaction which demonstrated direct influence of aberrant VWF-GPIba binding on megakaryocytes.

A novel mouse model of type 2N VWD was developed by CRISPR/Cas9 gene editing and recapitulates human type 2N VWD.

Type 2N von Willebrand disease is caused by mutations in the factor VIII (FVIII) binding site of von Willebrand factor (VWF), resulting in dysfunctional VWF with defective binding capacity for FVIII. We developed a novel type 2N mouse model using CRISPR/Cas9 technology. In homozygous VWF2N/2N mice, plasma VWF levels were normal (1167 ± 257 mU/mL), but the VWF was completely incapable of binding FVIII, resulting in 53 ± 23 mU/mL of plasma FVIII levels that were similar to those in VWF-deficient (VWF-/-) mice. When wild-type human or mouse VWF was infused into VWF2N/2N mice, endogenous plasma FVIII was restored, peaking at 4 to 6 hours post-infusion, demonstrating that FVIII expressed in VWF2N mice is viable but short-lived unprotected in plasma due to dysfunctional 2N VWF. The whole blood clotting time and thrombin generation were impaired in VWF2N/2N but not in VWF-/- mice. Bleeding time and blood loss in VWF2N/2N mice were similar to wild-type mice in the lateral tail vein or ventral artery injury model. However, VWF2N/2N mice, but not VWF-/- mice, lost a significant amount of blood during the primary bleeding phase after a tail tip amputation injury model, indicating that alternative pathways can at least partially restore hemostasis when VWF is absent. In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in severe type 2N patients. This unique model can be used to investigate the biological properties of VWF/FVIII association in hemostasis and beyond.

von Willebrand factor levels in the diagnosis of von Willebrand disease: a systematic review and meta-analysis.

von Willebrand disease (VWD) is associated with significant morbidity as a result of excessive mucocutaneous bleeding. Early diagnosis and treatment are important to prevent and treat these symptoms. We systematically reviewed the accuracy of diagnostic tests using different cutoff values of von Willebrand factor antigen (VWF:Ag) and platelet-dependent von Willebrand factor (VWF) activity assays in the diagnosis of VWD. We searched Cochrane Central Register for Controlled Trials, MEDLINE, and Embase databases for eligible studies. We pooled estimates of sensitivity and specificity and reported patient-important outcomes when relevant. This review included 21 studies that evaluated VWD diagnosis. The results showed low certainty in the evidence for a net health benefit from reconsidering the diagnosis of VWD vs removing the disease diagnosis in patients with VWF levels that have normalized with age. For the diagnosis of type 1 VWD, VWF sequence variants were detected in 75% to 82% of patients with VWF:Ag < 0.30 IU/mL and in 44% to 60% of patients with VWF:Ag between 0.30 and 0.50 IU/mL. A sensitivity of 0.90 (95% confidence interval [CI], 0.83-0.94) and a specificity of 0.91 (95% CI, 0.76-0.97) were observed for a platelet-dependent VWF activity/VWF:Ag ratio < 0.7 in detecting type 2 VWD (moderate certainty in the test accuracy results). VWF:Ag and platelet-dependent activity are continuous variables that are associated with an increase in bleeding risk with decreasing levels. This systematic review shows that using a VWF activity/VWF:Ag ratio < 0.7 vs lower cutoff levels in patients with an abnormal initial VWD screen is more accurate for the diagnosis of type 2 VWD.

Screening for von Willebrand disease does not impact posttonsillectomy bleeding in a low-risk population.

BACKGROUND: Bleeding is an important complication in children following tonsillectomy. Screening with coagulation tests prior to procedure is common to assess bleeding risk in the perioperative period, although ASH/ASPHO Choosing Wisely guidelines recommend against routine PT/PTT testing. Our aim was to compare von Willebrand factor antigen (VWF:Ag) and activity levels among patients with postoperative bleeding following tonsillectomy to evaluate for potential risk for bleeding. PROCEDURE: Eligible subjects were aged 0-18 without significant personal or family history of major bleeding. Postoperative bleeding diaries were collected and symptoms measured using a postoperative bleeding score. Plasma VWF levels were drawn at time of anesthesia administration. RESULTS: Postoperative bleeding occurred in 248 cases out of 1399 total subjects. Median VWF:Ag was 86 in patients with postoperative bleeding scores of 1-2, 86 for scores 3-4, 84 for scores 5-6, and 83 for scores >6, with no significant difference among groups (p = .98). Additionally, no difference was observed for subjects with multiple days of postoperative bleeding as compared to those with only 1 day of postoperative bleeding. Finally, no difference in VWF:Ag was observed for subjects whose first reported bleed occurred early in the postoperative course compared to those whose first reported bleed occurred later. VWF:Ag does not correlate with severity of bleeding, time of onset of first bleeding event, or recurrence of bleeding in healthy children with no personal or family history of bleeding who have postoperative bleeding following tonsillectomy. CONCLUSIONS: This data does not support routine von Willebrand disease screening prior to tonsillectomy.

Thromboelastometry assessment of hemostatic properties in various murine models with coagulopathy and the effect of factor VIII therapeutics.

BACKGROUND: Rotational thromboelastometry (ROTEM) has been commonly used to assess the viscoelastic properties of the blood clotting process in the clinic for patients with a hemostatic or prothrombotic disorder. OBJECTIVE: To evaluate the capability of ROTEM in assessing hemostatic properties in whole blood from various mouse models with genetic bleeding or clotting disease and the effect of factor VIII (FVIII) therapeutics in FVIIInull mice. METHODS: Mice with a genetic deficiency in either a coagulation factor or a platelet glycoprotein were used in this study. The properties of platelet- or plasma-FVIII were also assessed. Citrated blood from mice was recalcified and used for ROTEM analysis. RESULTS: We found that blood collected from the vena cava could generate reliable results from ROTEM analysis, but not blood collected from the tail vein, retro-orbital plexus, or submandibular vein. Age and sex did not significantly affect the hemostatic properties determined by ROTEM analysis. Clotting time (CT) and clot formation time (CFT) were significantly prolonged in FVIIInull (5- and 9-fold, respectively) and FIXnull (4- and 5.7-fold, respectively) mice compared to wild-type (WT)-C57BL/6J mice. Platelet glycoprotein (GP)IIIanull mice had significantly prolonged CFT (8.4-fold) compared to WT-C57BL/6J mice. CT and CFT in factor V (FV) Leiden mice were significantly shortened with an increased α-angle compared to WT-C57BL/6J mice. Using ROTEM analysis, we showed that FVIII expressed in platelets or infused into whole blood restored hemostasis of FVIIInull mice in a dose-dependent manner. CONCLUSION: ROTEM is a reliable and sensitive assay for assessing therapeutics on hemostatic properties in mouse models with a bleeding or clotting disorder.

von Willebrand factor antigen levels are associated with burden of rare nonsynonymous variants in the VWF gene.

Approximately 35% of patients with type 1 von Willebrand disease (VWD) do not have a known pathogenic variant in the von Willebrand factor (VWF) gene. We aimed to understand the impact of VWF coding variants on VWD risk and VWF antigen (VWF:Ag) levels, studying 527 patients with low VWF and VWD and 210 healthy controls. VWF sequencing was performed and VWF:Ag levels assayed. A combined annotation-dependent depletion (CADD) score >20 was used as a predicted pathogenicity measure. The number of rare nonsynonymous VWF variants significantly predicted VWF:Ag levels (P = 1.62 × 10-21). There was an association between average number of rare nonsynonymous VWF variants with VWD type 1 (P = 2.4 × 10-13) and low VWF (P = 1.6 × 10-27) compared with healthy subjects: type 1 subjects possessed on average >2 times as many rare variants as those with low VWF and 8 times as many as healthy subjects. The number of rare nonsynonymous variants significantly predicts VWF:Ag levels even after controlling for presence of a variant with a CADD score >20 or a known pathogenic variant in VWF (P = 2.7 × 10-14). The number of rare nonsynonymous variants in VWF as well as the presence of a variant with CADD >20 are both significantly associated with VWF levels. The association with rare nonsynonymous variants holds even when controlling for known pathogenic variants, suggesting that additional variants, in VWF or elsewhere, are associated with VWF:Ag levels. Patients with higher VWF:Ag levels with fewer rare nonsynonymous VWF gene variants could benefit from next-generation sequencing to find the cause of their bleeding.

Laboratory variability in the diagnosis of type 2 VWD variants.

Essentials Patients with von Willebrand disease were enrolled in our study. Type 2 VWD diagnoses were based on original test results. Repeat evaluation resulted in many patients receiving a different type 2 diagnosis. Some genetic variants were particularly likely to move type 2 subcategories. ABSTRACT: Introduction Type 2 von Willebrand disease (VWD) refers to patients with a qualitative defect in von Willebrand factor. Accurate diagnosis of type 2 VWD subtypes can be challenging. Aim of the study To compare the historical diagnosis of type 2 VWD with current laboratory testing. Methods Subjects were enrolled in the Zimmerman Program either because of a preexisting diagnosis of VWD (retrospective cohort) or from evaluation for bleeding symptoms or suspected VWD (prospective cohort). Original diagnosis was assigned by the local center and central diagnosis was based on central laboratory testing. Results Two hundred and seventeen index cases in the retrospective cohort and 35 subjects in the prospective cohort carried a local diagnosis of type 2 VWD (29% and 6% of enrolled index cases, respectively). In the retrospective cohort, the diagnosis was confirmed in 66% of cases with a preexisting diagnosis of 2A, 77% 2B, 54% 2M, and 72% 2N. In the prospective cohort, 31% were confirmed 2A, 60% 2B, 23% 2M, and 100% 2N. Several genetic variants were repeatedly implicated in subjects with changed diagnosis: p.M1304R, p.R1315C, p.R1374C, and p.R1374H. Conclusions Both the prospective and retrospective cohorts demonstrated consistent variation in subjects whose diagnosis changed between 2A, 2B, and 2M. The importance of accurately diagnosing type 2 VWD may be most significant in the 2B subtype given potential concerns with the use of desmopressin in type 2B VWD. Some genetic variants appear in multiple types of VWD, making specific diagnoses challenging.

von Willebrand factor variant D1472H has no effect in mice with humanized VWF-platelet interactions.

The von Willebrand factor ristocetin cofactor activity assay (VWF:RCo) is used for diagnosis of von Willebrand disease (VWD) because of its ability to evaluate VWF binding to platelets. VWF sequence variant p.D1472H is associated with lower VWF:RCo levels in the absence of associated bleeding symptoms, indicating the VWF:RCo may not be accurate for characterizing VWF function in individuals with this variant. Thus, this study aimed to determine the implications of the variant on VWF functioning in vivo. Mice were engineered with humanized wild-type (WT*) VWF A1/A2 and VWF with the p.D1472H (1472H) variant along with humanized platelet GPIbα and bred to homozygosity. VWF antigen and VWF binding to GPIbα were measured using enzyme-linked immunosorbent assay. Gel electrophoresis was used for VWF multimer analysis. Tail bleeding assays were performed at a 3-mm defined length. Normal VWF multimers were preserved in both WT* and 1472H mice. VWF expression was normal in the WT* and 1472H mice, and VWF binding to GPIbα did not statistically differ between the groups. Additionally, tail bleeding times were similar for WT* and 1472H mice. These results show the p.D1472H variant does not impair hemostasis in mice, and support the conclusion that p.D1472H is a normal variant in humans.

A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9.

BACKGROUND: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder, caused by quantitative and qualitative changes in von Willebrand factor (VWF). The biology of VWD, studied in canine, porcine, and murine models, differ in species-specific biology of VWF and the amenability to experimental manipulations such as phlebotomy. The factor VIII (FVIII) levels in these models are higher than in humans with type 3 VWD, suggesting functional differences between FVIII and VWF.ObjectivesTo develop a VWF knock out (VWF-/-) rat by excision of all 52 exons of the VWF locus. METHODS: The entire VWF gene was eliminated in Sprague-Dawley (Crl:SD) rats via CRISPR/Cas9-mediated gene editing. VWF antigen (VWF:Ag), VWF propeptide, and VWF collagen IV binding (VWF:CB4) levels were determined by ELISA assays and FVIII chromogenic activity (FVIII:C) levels by chromogenic FVIII assays. Lateral tail veins were transected to measure bleeding time. VWF-/- rats were infused with FVIII-/- rat platelet poor plasma (PPP) to determine response of plasma FVIII. RESULTS: Breeding of VWF ± rats yielded VWF-/- offspring at normal Mendelian ratios. VWF:Ag, VWF propeptide, VWF:CB4, and FVIII:C plasma levels were undetectable in VWF-/- rats. VWF-/- rats bled longer and more than VWF+/- and VWF+/+ rats when challenged. Transfusion of FVIII-deficient platelet-poor plasma induced a rapid rise in endogenous FVIII:C in VWF-/- rats. CONCLUSION: This rat model of severe VWD due to elimination of the entire VWF gene recapitulates the severe secondary deficiency of FVIII seen in human type 3 VWD and facilitates the study of VWF and FVIII and their interactions.

Low VWF levels in children and lack of association with bleeding in children undergoing tonsillectomy.

von Willebrand disease is a common bleeding disorder, but diagnosis can be difficult in young children who have not had bleeding challenges. We sought to evaluate the correlation between bleeding and von Willebrand factor (VWF) levels in children undergoing surgical challenge with tonsillectomy. Children ages 0 to 18 undergoing tonsillectomy without a personal or family history of bleeding were enrolled prospectively following informed consent and institutional review board approval. VWF levels were obtained at the time of surgery. VWF antigen (VWF:Ag) and VWF activity (VWF:GPIbM) were tested via enzyme-linked immunosorbent assay. Bleeding score was calculated using the International Society of Hematology bleeding assessment tool (BAT). Surgical and postoperative bleeding were determined using questionnaires filled out by the surgeon and patient/family. A total of 1399 subjects were enrolled with evaluable data, with a median age of 5 years. The median VWF:Ag was 85 IU/dL and the median VWF:GPIbM was 100 U/dL. Median BAT for the entire population was 0, including those with postoperative bleeding. There was no difference in VWF level between those who experienced postoperative bleeding and those who did not, with median VWF:Ag 85 vs 85 (P = .89) and mean VWF:GPIbM 98 vs 100 (P = .5). Interestingly, there was a difference in VWF levels with age, with median VWF:Ag 81 for those younger than 3 years, 82 for those 3 to 6 years, 90 for those 7 to 10 years, and 100 for those 11 to 18 years. A similar trend was noted for VWF:GPIbM. Of the 2 to 6 year olds, 5% had VWF:Ag <50, which would meet criteria for low VWF, but only 1.8% had an abnormal BAT at study entry and only 2.5% bled after surgery. Only 1 subject with low VWF had an elevated postoperative BAT >2. These data suggest that low VWF levels do not correlate with bleeding in children undergoing tonsillectomy. In addition, VWF levels outside the adult normal range in young children may be more common than previously thought and do not necessarily predict surgical bleeding.

The severe spontaneous bleeding phenotype in a novel hemophilia A rat model is rescued by platelet FVIII expression.

Previous studies have shown that platelet-specific factor VIII (FVIII) expression (2bF8) restores hemostasis and induces immune tolerance in hemophilia A (HA) mice even with preexisting inhibitors. Here we investigated for the first time whether platelet FVIII expression can prevent severe spontaneous bleeding in rat HA, a model mimicking the frequent spontaneous bleeding in patients with severe HA. A novel FVIII-/- rat model in a Dahl inbred background (Dahl-FVIII-/-) with nearly the entire rat FVIII gene inverted was created by using a CRISPR/Cas9 strategy. There was no detectable FVIII in plasma. Spontaneous bleeding in the soft tissue, muscles, or joints occurred in 100% of FVIII-/- rats. Sixty-one percent developed anti-FVIII inhibitors after ≥2 doses of recombinant human FVIII infusion. However, when 2bF8 transgene was crossed into the FVIII-/- background, none of the resulting 2bF8tg+FVIII-/- rats (with platelet FVIII levels of 28.26 ± 7.69 mU/108 platelets and undetectable plasma FVIII) ever had spontaneous bleeding. When 2bF8tg bone marrow (BM) was transplanted into FVIII-/- rats, only 1 of 7 recipients had a bruise at the early stage of BM reconstitution, but no other spontaneous bleeding was observed during the study period. To confirm that the bleeding diathesis in FVIII-/- rats was ameliorated after platelet FVIII expression, rotational thromboelastometry and whole-blood thrombin generation assay were performed. All parameters in 2bF8tg BM transplantation recipients were significantly improved compared with FVIII-/- control rats. Of note, neither detectable levels of plasma FVIII nor anti-FVIII inhibitors were detected in 2bF8tg BM transplantation recipients. Thus, platelet-specific FVIII expression can efficiently prevent severe spontaneous bleeding in FVIII-/- rats with no anti-FVIII antibody development.

von Willebrand factor binding to myosin assists in coagulation.

von Willebrand factor (VWF) binds to platelets and collagen as a means of facilitating coagulation at sites of injury. Recent evidence has shown that myosin can serve as a surface for thrombin generation and binds to activated factor V and factor X. We studied whether VWF can also bind myosin as a means of bringing factor VIII (FVIII) to sites of clot formation. A myosin-binding assay was developed using skeletal muscle myosin to measure VWF binding, and plasma-derived and recombinant VWF containing molecular disruptions at key VWF sites were tested. Competition assays were performed using anti-VWF antibodies. FVIII binding to myosin was measured using a chromogenic FVIII substrate. Thrombin generation was measured using a fluorogenic substrate with and without myosin. Wild-type recombinant VWF and human plasma VWF from healthy controls bound myosin, whereas plasma lacking VWF exhibited no detectable myosin binding. Binding was multimer dependent and blocked by anti-VWF A1 domain antibodies or A1 domain VWF variants. The specific residues involved in myosin binding were similar, but not identical, to those required for collagen IV binding. FVIII did not bind myosin directly, but FVIII activity was detected when VWF and FVIII were bound to myosin. Myosin enhanced thrombin generation in platelet-poor plasma, although no difference was detected with the addition of myosin to platelet-rich plasma. Myosin may help to facilitate delivery of FVIII to sites of injury and indirectly accelerate thrombin generation by providing a surface for VWF binding in the setting of trauma and myosin exposure.

How we approach: Training pediatric coagulationists.

Unique expertise is required for the care of children, adolescents, and young adults with bleeding and clotting disorders. A number of Hemophilia Treatment Centers have developed pediatric hemostasis and thrombosis fellowship programs to facilitate subspecialty training and recruitment and retention in this field. This manuscript reviews an approach to training pediatric coagulationists including a description of current programs, sample curriculum, funding sources, and expected outcomes.