Von Willebrand Factor Ristocetin Cofactor Activity Research Articles

Von Willebrand factor Ristocetin co-factor activity to von Willebrand factor antigen level ratio for diagnosis of acquired von Willebrand syndrome caused by aortic stenosis

BackgroundSevere aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress–dependent cleavage of high molecular weight multimers of von Willebrand factor (VWF). While the current standard diagnostic method is so-called VWF multimer analysis that is western blotting under nonreducing conditions, it remains unclear whether a ratio of VWF Ristocetin co-factor activity (VWF:RCo) to VWF antigen levels (VWF:Ag) of <0.7, which can be measured with an automated coagulation analyzer in clinical laboratories and is used for the diagnosis of hereditary von Willebrand disease. ObjectivesTo evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome. MethodsVWF:RCo and VWF:Ag were evaluated with the VWF large multimer index as a reference, which represents the percentage of a patient’s VWF high molecular weight multimer ratio to that of standard plasma in the VWF multimer analysis. ResultsWe analyzed 382 patients with AS having transaortic valve maximal pressure gradients of >30 mmHg, 27 patients with peripheral artery disease, and 46 control patients free of cardiovascular disease with osteoarthritis, diabetes, and so on. We assumed a large multimer index of <80% as loss of VWF large multimers since 59.0% of patients with severe AS had the indices of <80%, while no control patients or patients with peripheral artery disease, except for 2 patients, exhibited the indices of <80%. The VWF:RCo/VWF:Ag ratios, measured using an automated blood coagulation analyzer, were correlated with the indices (rs = 0.470, P < .001). When the ratio of <0.7 was used as a cut-off point, the sensitivity and specificity to VWF large multimer indices of <80% were 0.437 and 0.826, respectively. ConclusionVWF:RCo/VWF:Ag ratios of <0.7 may indicate loss of VWF large multimers with high specificity, but low sensitivity. VWF:RCo/VWF:Ag ratios in patients with AS having a ratio of <0.7 may be useful for monitoring the loss of VWF large multimers during their clinical courses.

Impact of Blood Group O and Von Willebrand Factor on Bleeding Severity in Patients with Von Willebrand Disease and Bleeding Disorder of Unknown Cause

Background: In patients with bleeding, von Willebrand disease (VWD) is diagnosed at von Willebrand factor (VWF) levels of ≤50 IU/dL ( James et al. Blood Adv. 1:280-300. 2021). Nevertheless, the majority of patients with a mild to moderate bleeding disorder (MBD) is diagnosed with bleeding disorder of unknown cause (BDUC), upon exclusion of VWD, platelet function defects (PFD), and coagulation factor deficiencies (CFD). Bleeding severity in patients with VWD does not correlate well with VWF levels in type 1 VWD, suggesting other modifiers of the bleeding tendency, when patients with severe VWD are excluded. Blood group O (BG O) is associated with lower VWF levels and has been found to be overrepresented in VWD. In BDUC patients, BGO is also overrepresented and recognized as an independent risk factor for bleeding severity and recurrent bleeding ( Mehic et al. Blood Adv.20: 5157-5164. 2020; Mehic et al. JTH. 7: 1757-1768. 2023). Aim: This study aimed to comprehensively investigate the clinical phenotype of patients with VWD or BDUC from the Vienna Bleeding Biobank (VIBB) and evaluate the direct effect of ABO BG on bleeding severity beyond its effect on VWF levels. Methods: Patients with VWD or BDUC from the VIBB, a single-center cohort study (EC 603/2009), were categorized by lowest VWF, as measured by VWF:antigen (VWF:Ag) or VWF ristocetin co-factor activity (VWF:RCo), into four groups: &amp;lt;30 IU/dL (very low VWF), 30-50 IU/dL (low VWF), 51-80 IU/dL, and &amp;gt;80 IU/dL. Patients diagnosed with PFD (n=221), CFD (n=28), or other diagnoses (n=4) were excluded. To study the association of BG O with bleeding severity irrespective of VWF levels, we fitted linear regression models adjusting for age, sex, and lowest VWF. We allowed for non-linearity by modelling age and sex with restricted cubic splines. Results: A total of 637 patients, 18 (2.8%) with very low VWF, 55 (8.6%) with low VWF, 237 (37.2%) with VWF levels ranging from 51 to 80 IU/dL and 327 (51.3%) with VWF levels &amp;gt;80 IU/dl, were included in this analysis, as summarized in Table 1. BG O was more prevalent in patients with very low VWF levels (61%), low VWF levels (71%), and VWF levels 51-80 IU/dL (66%) than in patients with VWF levels &amp;gt;80 IU/dL (30%). Patients with low VWF were youngest, followed by very low VWF and those with VWF 51-80 IU/dL. Age was highest in the patient group with VWF levels &amp;gt;80 IU/dL at clinical presentation and might influence VWF levels and bleeding scores. With regard to bleeding severity, patients with very low VWF levels had highest median bleeding scores or number of bleeding manifestations, while the bleeding severity was similar amongst the other three categories (Table 1). VWF levels did not correlate with bleeding severity scores (Vicenza score: Spearman rho=-0.03, p=0.53; ISTH BAT: rho=-0.02, p=0.75; number of bleeding manifestations: rho=-0.02, p=0.66). In linear regression, holding age, sex, and lowest VWF levels constant, predicted mean bleeding scores and number of bleeding manifestations were higher with BG O (Vicenza score mean difference [MD], 0.64; 95% confidence interval [95%CI], 0.12 to 1.25; p=0.015; ISTH BAT MD 0.52; 95%CI, -0.25 to 1.28; p=0.18; number of bleeding manifestations MD, 0.24; 95%CI, -0.06 to 0.54; p=0.12) (Figure 1). Conclusion: Our findings demonstrate an overlap between VWD and BDUC with a similar clinical phenotype and bleeding phenotype, but increased severity in patients with VWF levels &amp;lt;30 IU/dL. Patients with BG O had increased bleeding scores compared to those with BG non-O. BG O appears to be an independent factor in modifying bleeding severity in both patients with VWD or BDUC across all VWF ranges.

Trend in Von Willebrand Factor Profile in Women With Von Willebrand Disease During Pregnancy and Postpartum

Introduction: Pregnancy is considered a hypercoagulable state, with levels of multiple coagulation factors including Factor VIII (FVIII) and von Willebrand Factor (VWF), progressively increasing throughout, reaching their peak levels during the third trimester. However, in women with von Willebrand disease (VWD), these changes are variable and could be blunted or absent due to the wide heterogeneity of phenotypes and pathophysiological mechanisms linked to this disorder, posing a significant clinical challenge. This study aims to describe the trend in the VWF profile in women with VWD during each trimester of pregnancy and in the postpartum period. Methods: This is a retrospective single-center study that included adult pregnant women diagnosed with VWD, who were followed at Henry Ford Health between 03/2012 and 11/2022. Patients with other bleeding disorders were excluded from the study. Data on baseline characteristics, including age, race, age at childbirth, and parity were collected. Additional data collected included VWF antigen (VWF:Ag) level, FVIII activity level, and VWF ristocetin cofactor (VWF:Rco) activity during the first, second, and third trimesters, as well as in the postpartum period. Results: A total of 33 cases were included in our study. At baseline, the mean VWF:Ag level in our patients was 50.5% (reference range 50%-150%). Compared to baseline, the VWF:Ag level increased by 48.71% in the first trimester, 80.59% in the second trimester, and 168.71% in the third trimester. Postpartum, the VWF:Ag level decreased by 51.29% compared to the third trimester; however, it remained 30.89% higher than baseline levels. While the FVIII level (reference range 50%-150%) was an average of 64.8% at baseline, it increased by 21.91% in the first trimester, 74.23% in the second trimester, and 143.67% in the third trimester, compared to baseline. Postpartum, the FVIII level decreased by 48.45% from the third trimester but remained 25.62% higher than baseline. Additionally, the VWF:Rco level was an average of 44.3% (reference range 51%-215%) at baseline. Compared to baseline, the VWF:Rco level increased by 61.85% in the first trimester, 90.52% in the second trimester, and 142.66% in the third trimester. It decreased by 49.12% postpartum compared to the third trimester but remained 23.48% higher than baseline. Conclusion: In women with VWD, VWF:Ag level, FVIII level, and VWF:Rco levels steadily increase during pregnancy, reaching their maximum levels during the third trimester. All three levels decreased in the postpartum period; however, they remain higher compared to baseline at a median follow up of 105 days after delivery.

Constructing and evaluating the effect of micron-scale structures on von Willebrand factor damage

The incidence of clinical complication gastrointestinal bleeding has been proved as consequence of von Willebrand factor (VWF) damage after mechanical circulatory support in clinic. Many studies have been conducted to evaluate VWF damage, of which the most studied influencing factors are mechanical factors such as shear stress. However, in addition to mechanical factors, VWF damage may also be affected by interface factors. To address this issue, a roller pump circulation platform was established to investigate the effect of material surface micron-scale structures distribution on VWF damage in flow state. A composite micro-structure combining microngrating and micronpost was designed and constructed on the surface of Si wafer by lithography and reactive ion etching, and detailed characterization of material surfaces was also performed. Then the changes of VWF antigen, VWF ristocetin cofactor activity, and the degradation of high molecular weight VWF on these surfaces were investigated and compared. The results showed that, with the encryption of surface micro-structures arrangement, the material surface tends to be more hydrophobic, which is beneficial to reduce VWF damage. Therefore, in the design of material surface inside the mechanical circulatory support devices, it can be considered to add some surface micro-structures with a certain distribution density to change the hydrophilicity and hydrophobicity, so as to minimize the VWF damage. These results can provide important references for the evaluation of VWF damage caused by interface factors, and aid in designing material surface inside the mechanical circulatory support devices.

Impact of ABO Blood Group on Thromboembolic and Bleeding Complications in Patients with Left Ventricular Assist Devices.

The ABO blood group system is linked to hemostasis via its relationship with von Willebrand factor (VWF) and factor VIII (FVIII). In the current study, we investigated the association of the ABO system with clinical outcomes as well as VWF and platelet function in patients with left ventricular assist devices (LVADs). Bleeding and thromboembolic complications were assessed in 111 patients during 1 year after LVAD implantation. In 67 LVAD patients, VWF antigen, VWF activity, VWF ristocetin cofactor, VWF collagen-binding, and FVIII activity were assessed. Platelet surface P-selectin and activated glycoprotein IIb/IIIa were determined by flow cytometry, and soluble P-selectin was measured with an enzyme-linked immunoassay. Platelet aggregation was assessed by light transmission and impedance aggregometry. Thirty-six patients (32.4%) experienced a bleeding and 22 patients (19.8%) a thromboembolic event. In univariate analyses, patients with blood group O had numerically more bleeding complications and less thromboembolic events as compared to patients with blood group non-O (both p ≥ 0.05). After multivariable adjustment, blood group O was significantly associated with a higher risk of bleeding (hazard ratio 2.42 [95% confidence interval 1.03-5.70], p = 0.044) but not linked to thromboembolic complications. Patients with blood group O had significantly lower levels of VWF and FVIII (all p < 0.05), whereas P-selectin expression in response to thrombin-receptor activating peptide and soluble P-selectin were higher as compared to patients with blood group non-O (both p < 0.05). LVAD patients with blood group O are at an increased bleeding risk, potentially due to lower VWF and FVIII levels.

Type 2M VWD Laboratory Defects More Consistent Than Bleeding Symptoms across Family Members

Background Von Willebrand disease (VWD) is an inherited bleeding disorder characterized by a defect or decrease of von Willebrand factor (VWF). Type 2 VWD results from qualitative defects in VWF. Type 2M VWD is defined by defective platelet binding to GPIb and/or collagen. While the inheritance of type 2M VWD is autosomal dominant, the extent of symptoms across family members has not been well characterized. Methods Subjects included index cases with type 2M VWD and all enrolled family members from a large US study of VWD (Zimmerman Program). Index cases were enrolled due to a prior diagnosis of VWD. All available family members were enrolled if willing. VWF testing performed included VWF antigen (VWF:Ag), VWF platelet binding by either VWF ristocetin cofactor activity (VWF:RCo) or VWF:GPIbM, collagen binding with types I, III or IV collagen (VWF:CB1, VWF:CB3, VWF:CB4) and VWF multimer distribution. Bleeding scores were calculated using the ISTH bleeding assessment tool (BAT). DNA sequencing was performed for the full length VWF gene. Statistics were calculated using GraphPad Prism with a p value of <0.05 considered significant. Family members were defined as affected if they had VWF activity levels consistent with type 2M VWD and presence of genetic variant if identified. Results 24 index cases, 51 affected family members and 40 unaffected family members were available for analysis. The mean VWF:RCo/VWF:Ag ratio for index cases was 0.5 and the mean VWF:CB/VWF:Ag ratio was 1.05. The VWF:RCo/VWF:Ag ratio was similar in affected family members (0.44, p=NS) but higher as expected in unaffected members (0.89, p<0.001 for index case vs. unaffected, p<0.001 for affected vs. unaffected). In contrast, there was no difference in VWF:CB/VWF:Ag ratios between index cases and affected family members (0.94, p=NS). Data for VWF:GPIbM, collagens I and IV were available only on index cases, and showed low VWF:GPIbM/VWF:Ag (mean 0.79), higher VWF:CBI/VWF:Ag (mean 1.31) and lower VWF:CBIV/VWF:Ag (mean 0.85). There was a negative correlation between VWF:GPIbM and VWF:CBIV, with subjects having either low VWF:GPIbM and normal VWF:CBIV or low VWF:CBIV and normal VWF:GPIbM. Similar results were seen for VWF:CB1 although only 1 subject was diagnosed with type 2M VWD and low VWF:CB1. Median bleeding scores were highest in index cases (7.5), intermediate in affected family members (3) and normal in unaffected family members (1). There was a significant difference between index cases and affected family members (<0.015), and between index cases and unaffected family members (p<0.001). One subject had a genetic variant in the VWF A3 domain, one in the VWF D3 domain and the remainder of index cases had a defect in the VWF A1 domain. Conclusions These findings suggest that decreases in platelet and collagen binding activity increase bleeding risk in families with type 2M VWD. Index cases had higher bleeding scores as compared to affected family members, suggesting that diagnosis of family members may require more attention. Precise understanding of these defects may assist in better diagnosis and treatment of von Willebrand disease.

The VWF binding aptamer rondoraptivon pegol increases platelet counts and VWF/FVIII in type 2B von Willebrand disease

AbstractType 2B von Willebrand disease (VWD) is characterized by an increased binding affinity of von Willebrand factor (VWF) to platelet glycoprotein Ib. This can lead to clearance of high-molecular-weight (HMW) multimers and thrombocytopenia with a resulting moderate-severe bleeding phenotype. Rondoraptivon pegol (BT200) is a pegylated aptamer binding to the A1 domain of VWF with a novel mechanism of action: it enhances VWF/factor VIII (FVIII) levels by decreasing their clearance. To study the potential benefit of rondoraptivon pegol in patients with type 2B VWD, we conducted a prospective phase 2 trial. Patients with type 2B VWD received 3 mg rondoraptivon pegol subcutaneously on study days 1, 4, and 7, followed by 6 to 9 mg every week until day 28. Five patients (male:female ratio = 3:2) were included. Rondoraptivon pegol rapidly tripled platelet counts from a median of 60 to 179 × 10E9/L (P < .001). Circulating VWF antigen increased from a median of 64% to 143%, which doubled FVIII activity levels from 67% to 134%. In all thrombocytopenic patients, plasma levels of VWF:GPIbM normalized, VWF ristocetin cofactor and VWF collagen-binding activity increased, and HMW multimers appeared. These pronounced improvements reversed during washout of the drug, thus demonstrating causality. The A1 domain binding aptamer directly corrects the underlying defect of type 2B VWD, thus providing a novel potential option for prophylaxis and treatment of patients with this VWD type. These data provide the basis for a phase 2b/3 trial in such patients. This trial was registered at www.clinicaltrials.gov as #NCT04677803.

Investigation into the effect of the aspect ratio of cylindrical surface microstructure on von Willebrand factor damage.

Hemorrhagic episodes in patients carrying mechanical circulatory support represent a severe clinical complication. These bleeding episodes may originate from a reduced functionality of von Willebrand factor (VWF), a multimer protein pertinent to form a hemostatic plug. The reduced functionality is due to increased loss of high molecular weight von Willebrand factor multimers (HMWM-VWF), a phenomenon that is facilitated by device-induced increases in shear stress to which VWF is exposed. However, in addition to the mechanics factors, VWF damage may also be affected by interface factors, including the properties of bulk material and the surface characteristics. In this study, the effect of cylindrical surface microstructure topography on VWF damage was investigated. In the 1 to 9 range, the high aspect ratio surface features were constructed on the polycarbonate (PC) films. The topographic surfaces were fabricated by 3D printing casting on a template. A roller pump circulation platform was built to conduct in vitro experiments. VWF antigen (VWF-Ag) and VWF ristocetin cofactor activity (VWF-Rico) on these topographic surfaces were quantified by enzyme-linked immunosorbent assay (ELISA), the loss of HMWM-VWF was quantified by immunoblotting. The lower loss of HMWM-VWF was observed on surfaces with high aspect ratio compared to the pristine PC templates and surfaces with low aspect ratio, while VWF-Ag was nearly unchanged. The topographical parameters found to significantly reduce the loss of HMWM-VWF were high aspect ratio structures of more than 5. The results signify that topographical manipulation of surfaces is a feasible approach for reducing the loss of HMWM-VWF.

Impact of obesity on factor VIII and von Willebrand factor levels in patients with Type 1 von Willebrand disease and low von Willebrand factor: An analysis of the ATHNdataset.

Obesity is associated with endothelial dysfunction, haemostatic and fibrinolytic disturbances, however the impact of obesity on von Willebrand factor (VWF) is unclear. The aim of this study was to test our hypothesis that the prevalence of obesity is higher among participants with low VWF (LVWF) compared to type 1 von Willebrand disease (T1VWD). A retrospective review of the ATHNdataset as of March 2018 was performed. Participants were categorized as T1VWD if their VWF ristocetin cofactor activity was 30IU/dL and LVWF if the values were 30-50IU/dL, and by the NIH definitions for body mass index (BMI) for adult participants (≥18 years of age) or BMI z-score for paediatric participants (<18 years). The prevalence of obesity was not significantly different between adults with T1VWD (n=186) and LVWF (n=362) (32%vs 36%; p=.345). The mean factor VIII (FVIII) increased with increasing BMIs in both groups. In the paediatric cohort (T1VWD, n=583; LVWF, n=1702), there was no difference in the prevalence of obesity, but BMI was positively correlated with mean FVIII (p<.001). Children<10 years were 27.6% more likely to be diagnosed with T1VWD compared to>10 years. Among participants in the ATHNdataset, the prevalence of obesity was similar among those with LVWF and T1VWD. However, higher BMI levels were associated with elevated FVIII. Further research is needed to evaluate the impact of obesity on bleeding phenotype and treatment practices.

IX international curse of continuing formation in haemophilia and other congenital coagulopathies. The role of the Laboratory in coagulation disorders. Diagnosis of von Willebrand disease.

Von Willebrand disease (VWD) is the most frequent inherited bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (VWF). This protein far from simplicity constitutes a very complex molecular model, remaining unravelled yet many aspects of it, even though the VWF gene (VWF) was cloned already in 1985 and the structure of VWF well defined. VWD diagnosis is difficult to achieve in a significant proportion of patients due to both disease heterogeneity and limitations in existing test processes. The cornerstone of diagnosis relies on interpretation of VWF test results, the presence of clinical manifestations of bleeding, especially mucocutaneous, and (in most cases) a positive family history. However, even with a significant bleeding history, a family history may not be positive due to factors of incomplete penetrance and variable expressivity that affect genetic changes. The laboratory diagnosis of VWD can be difficult, as the disease is heterogeneous and an array of assays is required to describe the phenotype. Basic classification of quantitative (type 1 and 3) and qualitative (type 2 variants) VWD requires determination of VWF antigenic (VWF:Ag) levels and assaying of VWF ristocetin cofactor (VWF:RCo) activity. The latter is required for identifying and subtyping VWD, but the assay is poorly standardized. For that reason, novel VWF activity assays have been developed awaiting more extensive comparison data between different methodologies and requiring validation on larger patient series. The qualitative type 2 VWF deficiency can be further divided into four different subtypes (A, B, M and N) using specific assays that measure other activities or the size distribution of VWF multimers. However, frequently, it may be difficult to correctly classify the VWD phenotype, and genetic analysis is through mutation identification may provide a tool to clarify the disorder.

The Von Willebrand Disease Aging and Bleeding Correlation (VWD ABC) Study

IntroductionIt is well established that von Willebrand factor (VWF) levels increase with age among healthy adults. Recently, there is emerging research demonstrating this may also occur in patients with von Willebrand disease (VWD), particularly type 1 VWD, and may be related to comorbidities. Despite increasing VWF levels, it remains unclear as to whether or not this alters bleeding phenotype. It is also unclear why this occurs most commonly in patients with type 1 VWD, but VWF mutation status may play a role.Older patients commonly undergo invasive procedures, and all VWD patients require periprocedural VWD-specific therapy to ensure appropriate hemostasis. If older type 1 VWD patients have experienced normalization of VWF levels, and no longer have an increased risk of bleeding, VWD-specific therapy may increase thrombosis risk, especially among patients with underlying cardiovascular disease or related risk factors, subject the patient to other adverse reactions such as hyponatremia, and is unnecessarily costly. For these reasons, investigation into the effect of age on VWF levels and bleeding risk in type 1 VWD patients is sorely needed.MethodsThis is an NHLBI-funded K23 multicenter, cross-sectional study to determine the effect of age on VWF levels and bleeding risk in patients with type 1 VWD, and to determine if pathogenic VWF mutations alter this effect.Individuals with a new or historical diagnosis of type 1 VWD (defined as clinical symptoms consistent with VWD and VWF antigen level or ristocetin cofactor activity <0.50 IU/mL) and age 18 or older are enrolled during routine clinic visits at participating Hemophilia Treatment Centers (HTCs). Following enrollment, pertinent medical history is obtained; the condensed MCMDM-1 VWD Bleeding Assessment Tool is administered, with bleeding history based on bleeding symptoms during the past 5 years; and blood samples are collected for the following: VWF antigen (VWF:Ag) level, VWF ristocetin cofactor activity, factor VIII activity, blood type, and VWF gene sequencing.We hypothesize age is associated with increased VWF:Ag levels and lower condensed MCMDM-1 VWD bleeding scores in patients with type 1 VWD, and this association is weaker among those with a pathogenic VWF mutation. In addition, we hypothesize multimorbidity partially explains the association between age and VWF:Ag levels, and VWF:Ag levels partially explain the association between age and condensed MCMDM-1 VWD bleeding scores in patients with type 1 VWD. A sample size of 250 participants provides 90% power to detect an effect size of Beta=±0.032 points per year of age, which is much smaller than the observed effect size, Beta=-0.080, from preliminary data. The primary analyses will be based on multivariable linear regression models with adjustment for blood type O, exogenous estrogen therapy, multimorbidity (defined as 2 or more of the core set of 20 chronic conditions, i.e., cancer, hypertension, stroke, etc., as selected by the United States Department of Health and Human Services), and medications (aspirin, nonsteroidal antiinflammatory drugs, and anticoagulants). In the regression models, two-sided t-tests will be used with an alpha=0.05.ResultsThis multicenter, cross-sectional study consists of seven HTCs: Hemophilia Center of Western Pennsylvania, Children's Hospital of Pennsylvania, Mary M. Gooley Hemophilia Center, Ohio State University, Bleeding & Clotting Disorders Institute, Mayo Clinic, and University of California, San Diego. During the first year of the study, site initiation visits were conducted, regulatory approval obtained, and contracts executed. Delays in these activities occurred in large part due to the COVID-19 pandemic. As the first year of the study concludes, all sites are now active and enrolling participants. Thus far, 43 participants have been enrolled (Table 1). No barriers to enrollment have been encountered and very few patients have declined study participation.DiscussionIn conclusion, this ongoing multicenter, cross-section study seeks to determine the effect age has on VWF levels and bleeding risk in patients with type 1 VWD while exploring the role of VWF mutations and multimorbidity in this process. The results will be used to justify a longitudinal study, which is the ideal approach to research the effects of aging in this population. [Display omitted] DisclosuresSeaman: Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; HEMA Biologics: Consultancy, Honoraria. Ragni: Bioverativ (Sanofi): Membership on an entity's Board of Directors or advisory committees; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Takeda Therapeutics: Membership on an entity's Board of Directors or advisory committees; Alnylam (Sanofi): Membership on an entity's Board of Directors or advisory committees; University of Pittsburgh: Research Funding; BioMarin Pharmaceutical: Membership on an entity's Board of Directors or advisory committees. Kraut: Uniqure: Consultancy. Pruthi: CSL Behring: Honoraria; Bayer Healthcare AG: Honoraria; Instrumentation Laboratory: Honoraria; Genentech: Honoraria; HEMA Biologics: Honoraria; Merck: Honoraria. Raffini: CSL Behring: Consultancy; HEMA Biologics: Consultancy; Genentech: Consultancy; Bayer: Consultancy; XaTek: Consultancy. Roberts: Takeda; Speakers Bureau: Novo Nordisk, Octapharma, Sanofi, Takeda.: Research Funding; Genentech, Novo Nordisk, Octapharma, Pfizer, Sanofi, Takeda, uniQure: Consultancy. von Drygalski: Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; uniQure: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Biomarin: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Research Funding; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hematherix, Inc: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Super FVa.

Contribution of Select ISTH-BAT Subscores to the Total Score in Type 1 VWD

Background:von Willebrand Disease (VWD) is the most common inherited bleeding disorder with partial quantitative deficiency in von Willebrand Factor (VWF); Type 1 comprises 80% of VWD. Clinical manifestations include mucocutaneous bleeding, prolonged bleeding after surgery or injury and excessive menorrhagia in females which have also been reported in normal subjects. A diagnosis of Type 1 VWD is based upon reduced plasma VWF, abnormal mucocutaneous bleeding symptoms and usually a family history of bleeding and VWD. Evaluating abnormal bleeding symptoms involves a detailed bleeding history, which can be quite subjective and time consuming. To address these challenges, the Vicenza-based Bleeding Assessment Tool (BAT) was developed as a validated score and has evolved into the current International Society on Thrombosis and Haemostasis-Bleeding Assessment Tool (ISTH-BAT). A large multi-center NIH supported study (The Zimmerman Program for the Molecular and Clinical Biology of VWD), began in 2005 to correlate clinical laboratory testing, bleeding severity determined by the ISTH-BAT and genetic variability in VWD. In this current study, we investigated which bleeding symptoms were most sensitive in applying the ISTH-BAT to the diagnosis of Type I VWD.Methods:Subjects were enrolled in the Zimmerman Program . Each symptom in the validated ISTH-BAT questionnaire was scored from 0-4 (no symptoms to symptom intervention) and included epistaxis, bruising, oral bleeding, hematuria, gastrointestinal (GI) bleeding, head bleeding, hemarthrosis, hematoma and prolonged bleeding with minor wounds, tooth extraction, surgery, menstruation and child birth. Other scored bleeding symptoms included bleeding at circumcision, excessive umbilical stump bleeding, venipuncture bleeding, vacuum extraction delivery bleeding, ovulatory bleeding and cephalohematoma. The scores of each symptom were analyzed in both healthy controls and subjects diagnosed with Type 1 VWD. Only subjects with VWF antigen (VWF:Ag) and/or VWF ristocetin cofactor activity (VWF:RCo) <30 IU/dL were considered in the comparative analysis. VWF levels were performed by a central laboratory, including VWF:Ag, VWF:RCo, VWF propeptide (VWFpp) and VWF collagen binding with type III collagen (VWF:CB). Statistical analysis included Mann-Whitney test, Chi-square or a Fisher's exact test, Pearson correlations and Spearman correlations. Results were considered significant for a two-sided p-value <0.05. A regression analysis was performed to examine which subscores contributed the most to the total score of the ISTH-BAT.Results:Analysis included 383 subjects (Female=262, Male=121) enrolled in the Zimmerman Program . Of these subjects, 244 were healthy controls (mean age of 38.93 [range 10-73 years of age]) and 139 had Type 1 VWD (mean age of 19.46 [range 1-76 years of age]). Within the Type 1 subjects, there were more females >12 years of age (no gender difference < 12 years of age) and greater number of Caucasians and younger subjects. Type 1 VWD subjects scored significantly higher than the healthy control group in both ISTH-BAT total scores (p<0.0001) as well as all subscores (p<0.001). In comparing bleeding symptoms from healthy controls with Type 1 VWD subjects: epistaxis 4% vs 46%, bruising 16% vs 64%, minor wound bleeding 1% vs 42%, oral bleeding 3% vs 35%, tooth extraction bleeding 3% vs 18%, GI bleeding 3% vs 11%, hematuria 0% vs 11%, surgical bleeding 4% vs 23%, menstrual bleeding 60% vs 85%, bleeding after delivery 2% vs 12%, other bleeding 0% vs 11% (47% were circumcision bleeding), head bleeding 0% vs 4%, hemarthrosis 0% vs 11%, and hematoma 0% vs 7%. Regression analysis revealed that epistaxis, bruising, excessive bleeding with minor wounds, oral bleeding, and menstrual bleeding subscores contributed the most to the ISTH-BAT total score (p<0.016).Conclusion:The ISTH-BAT currently is strongly predictive of bleeding symptoms in the diagnosis of Type 1 VWD. Though all ISTH-BAT subscores correlated with a diagnosis of Type 1 VWD, some subscores contributed more significantly to the total score than others and were more prevalent than in normal subjects. Accordingly, the sensitivity and specificity of ISTH-BAT subscores are critical to the assessment of bleeding in the diagnosis of Type 1 VWD. DisclosuresFlood:Shire: Consultancy; CSL Behring: Consultancy. Gill:Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. James:CSL Behring: Research Funding; Shire: Research Funding; Bayer: Research Funding.

Active von Willebrand Factor in patients with a bleeding diathesis

IntroductionIncreased levels of circulating von Willebrand Factor (VWF) in its active, platelet-binding conformation have been implicated in the pathogenesis of several thrombotic conditions as well as bleeding conditions characterized by severe thrombocytopenia. However, it is unclear whether the proportion of activated VWF in the circulation also plays a role in patients with mild to moderate bleeding without thrombocytopenia. MethodsCitrated plasma samples were collected from 145 patients with a bleeding diathesis with unknown cause. Active VWF levels were measured with an in-house developed ELISA assay. In addition, VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo) and (flow-cytometric) platelet-VWF binding (Plt:VWF) were determined. ResultsActive VWF levels were on average mildly, but not significantly, lowered in patients with a bleeding diathesis compared to the reference interval (especially in individuals with non-O blood groups). Active VWF was not significantly different for subjects with (median 107.4%, IQR 18.3) versus without (median 111.1%, IQR 32.3%) an increased bleeding score, nor between subjects with suspected VWD (median 104%, IQR 20.6%) versus other suspected causes of bleeding diathesis (median 111.7%, IQR 33.3%). ConclusionIn this clinically heterogeneous population of patients with a mild bleeding phenotype, quantification of active VWF levels does not have added diagnostic value to VWF:Ag and VWF activity assays in the diagnosis of unexplained bleeding disorders.

A Novel VWD Variant Identified in a Multi-Generational Canadian Ojibway Kindred with a Type 2B VWD Phenotypic Expression

Introduction: Type 2 VWD is caused by variants in the von Willebrand factor (VWF) gene leading to impaired function. Distinction between subtypes of Type 2 has traditionally relied on a panel of assays including VWF antigen (VWF:Ag), VWF ristocetin-cofactor activity (VWF:RCo), VWF multimers, and Factor VIII (FVIII:C). In type 2A, there is either impaired synthesis or accelerated degradation of the more adhesive VWF high molecular weight multimers (HMWMs). In type 2B, enhanced VWF-platelet interactions result in binding and removal of the HMWMs, impairing platelet adhesion. Type 2B VWD can manifest spontaneous platelet agglutination and thrombocytopenia. The inheritance patterns of Type 2A and 2B VWD are generally described to be autosomal dominant and fully penetrant. We identified a multigenerational Canadian First Nations Ojibway kindred with VWD harboring a missense variant in exon 28 (A&amp;gt;T transversion at nucleotide 4898), leading to a single amino acid substitution (p.Asn1633Ile). Some individuals in the kindred are heterozygous, and some homozygous, for the causative variant. Objectives: The objective of this case series are to describe the genetic basis and pattern of inheritance of this variant of VWD, and to characterize the laboratory hemostasis characteristics in the affected individuals. Methods: A retrospective case review was conducted on all affected individuals in the kindred known or suspected to harbour this variant. There were 20 individuals seen at our centre over 32 years who were included in this study. We collected clinical and laboratory data to characterize the bleeding phenotype. Results: Sixteen of the 20 individuals in the study were known or inferred to be heterozygous, and 4 were homozygous. We were able to examine laboratory hemostasis in ten individuals in the study (Table 1). The platelet count, FVIII:C and VWF:Ag levels were within or slightly above reference range for all individuals. In the heterozygous individuals, VWF:RCo was mildly reduced, VWF multimer testing showed loss of HMWMs, and ristocetin-induced platelet aggregation (RIPA) testing normal aggregation to a higher dose (1.25 mg/ml), a pattern consistent with mild Type 2A VWD (Table 1). In homozygous individuals, VWF:RCo was markedly reduced, and while RIPA was minimal with low-dose ristocetin, aggregation to the higher dose was normal despite the very low ristocetin cofactor activity in the plasma. The PFA-100 (collagen-epinephrine) closure times were markedly prolonged in the homozygous subjects and in one heterozygous subject, exceeding 300 seconds. Discussion/Conclusion: This novel VWF variant confers a laboratory phenotype consistent with type 2A VWD in the heterozygotes. Homozygotes have more severely impaired hemostatic function. The conventional hallmark of type 2B VWD (hyperresponsiveness to low-dose ristocetin) was not seen, but the fact that full aggregation was obtained in homozygotes despite ristocetin cofactor activities of only 5-6% in the plasma suggests some degree of hyperaggregability, and hence a phenotype more in keeping with Type 2B than 2A. In the future, it would be interesting to see how this missense variant affects GP1b binding to VWF, as RIPA and VWF:RCo are not entirely comparable assays. Disclosures No relevant conflicts of interest to declare.

Von Willebrand Factor Concentrate Administration for Acquired Von Willebrand Syndrome- Related Bleeding During Adult Extracorporeal Membrane Oxygenation

To review the use of Von Willebrand Factor (VWF) concentrate for treatment of acquired Von Willebrand syndrome (VWS)-related bleeding in adult extracorporeal membrane oxygenation (ECMO) patients and determine if it was associated with improved VWF laboratory parameters. Retrospective observational cohort study. Tertiary care academic medical center. Adult ECMO patients who received VWF concentrate for treatment of acquired VWS- related bleeding. None, observational study. Ten adult ECMO patients received VWF concentrate for treatment of bleeding with evidence of acquired VWS over a 15-month period. Six patients were on veno-arterial ECMO and 4 were on veno-venous ECMO. The most common site of bleeding was airway or tracheal bleeding. The mean dose of VWF concentrate was 41 IU/kg. Mean VWF antigen was 263 ± 93 IU/dL before treatment and 394 ± 54 after treatment. Mean ristocetin cofactor activity was 127 ± 47 IU/dL before treatment and 240 ± 33 after treatment. The mean VWF ristocetin cofactor activity antigen ratio increased from 0.52 ± 0.14 before treatment to 0.62 ± 0.04 after treatment. Four of 10 patients had complete resolution of their bleeding within 24 hours, and 6 of 10 had complete resolution of their bleeding within 2- to- 4 days. There were 3 patients who had thrombotic events potentially related to VWF concentrate administration. No patient had an arterial thrombosis, stroke, or myocardial infarction. VWF concentrate administration increases VWF function in adult ECMO patients, but also may be associated with increased thrombotic risk. Larger studies are needed to determine VWF concentrate's safety, efficacy, and optimal dosing in adult ECMO patients.

Acquired von Willebrand Syndrome and Platelet Function Defects during Extracorporeal Life Support (Mechanical Circulatory Support).

Patients with ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) suffer from an increased risk for thromboembolic events as well as for hemorrhages. High shear stress in the mechanical device results in acquired von Willebrand syndrome (AVWS), characterized by a loss of high-molecular-weight multimers of von Willebrand factor (VWF) leading to an increased bleeding risk. Onset of AVWS occurs within hours, persists during the whole period of mechanical support, and subsides rapidly after explantation. Patients with the older HeartMate II exhibit more severe AVWS than those with the newer HeartMate III, thanks to lower shear stress in the latter. All ECMO and VAD patients exhibit thrombocytopathia and often thrombocytopenia which further increases the bleeding risk. Etiological models for AVWS are increased cleavage by the metalloproteinase ADAMSTS13, mechanical destruction of VWF, and shear-induced VWF binding to platelets. Platelet secretion defects may be caused by transient platelet activation leading to degranulation. AVWS can be diagnosed by detection of VWF multimers using gel-electrophoresis and functional assays of varying sensitivity (VWF ristocetin cofactor activity, VWF activity, VWF collagen binding). Platelet dysfunction is monitored using light transmission aggregometry and secretion defects are detectable using flow cytometry. Modest use of anticoagulants and a target-controlled therapy based on VWF parameters and other coagulation and platelet parameters are shown to be beneficial in this patient group. Persistent hemorrhages may be controlled with tranexamic acid and platelet concentrates. Prompt weaning from the device, when indicated, is the best therapeutic option to prevent recurrent bleeding.

VWF and FVIII Levels after Desmopressin Are Associated with the Bleeding Phenotype in Type 1 VWD

Introduction The bleeding phenotype of type 1 von Willebrand disease (VWD) patients is very heterogeneous. We hypothesized that part of this heterogeneity is explained by variability in response of von Willebrand factor (VWF) and factor VIII (FVIII) levels to stress during hemostatic challenges. Patients who have a strong increase in VWF and FVIII levels during hemostatic challenges, may have less frequent or less severe bleeding episodes compared to patients who have a smaller increase in VWF and FVIII levels. Therefore, we investigated whether VWF and FVIII levels after desmopressin, which mimic in vivo hemostatic response during hemostatic challenges, are associated with the bleeding score, and therefore may explain part of the heterogeneity in bleeding phenotype of type 1 VWD patients. Methods In this retrospective cohort study, we included type 1 VWD patients of all ages who were diagnosed and treated in the Erasmus University Medical Center. All patients had a hemorrhagic diathesis or family history of VWD and historically lowest VWF antigen (VWF:Ag) and/or VWF Ristocetin Cofactor activity (VWF:RCo) ≤ 0.30 IU/mL with VWF activity/ VWF antigen ratio above 0.60. All patients participated in the Willebrand in the Netherlands (WiN) study, in which a self-administered Tosetto bleeding score was assessed. Results We identified 173 eligible type 1 VWD patients in our treatment center who participated in the WiN study. After exclusion of 50 patients who did not receive a desmopressin test dose, and 1 patient who did not have a bleeding score assessment, we included 122 type 1 VWD patients. Table 1 shows the patient characteristics. Higher FVIII activity during the complete time course (i.e. one, three and 5-6 hours) after desmopressin, and higher VWF and FVIII levels combined at three hours after desmopressin, were associated with a lower bleeding score, respectively β=-0.9 (95% CI: -1.7; -0.1) and β=-1.2 (-1.9; -0.5), both adjusted for age, sex, BMI and comorbidities. FVIII:C at three hours after desmopressin was significantly associated with a lower bleeding score: β=-1.5 (-3.0; -0.1), whereas VWF levels were not associated (Figure 1). Moreover, patients with VWF and FVIII levels in the highest quartile three hours after desmopressin administration had a lower bleeding score compared to other patients (Figure 2). The largest difference was found for FVIII:C: β=-5.1 (-8.4; -1.8), adjusted for age, sex, BMI, comorbidities and baseline FVIII:C. These patients also less often had an abnormal bleeding score: OR=0.1 (0.0-0.5), adjusted for age, sex, BMI, comorbidities and baseline FVIII:C. Lastly, patients with an increased clearance of VWF, defined as VWF propeptide/VWF:Ag ratio &gt;2.2, had lower VWF:Ag (β=-0.3, -0.6; -0.1), VWF:Act (β=-0.5, -0.7; -0.2), VWF:CB (β=-0.5, -1.0; -0.1) and FVIII:C (β=-0.7, -1.0; -0.4) at three hours after desmopressin administration compared to other type 1 VWD patients, all adjusted for age, sex, BMI and comorbidities. An increased clearance of VWF was also associated with a higher bleeding score: β=3.2 (0.2; 6.2), adjusted for age, sex, BMI, comorbidities and baseline VWF and FVIII levels. Discussion In type 1 VWD patients, the desmopressin induced increase of VWF and FVIII levels is associated with a lower bleeding score. Especially higher VWF and FVIII levels at three hours after desmopressin were associated with a milder bleeding phenotype, suggesting that sustained VWF and FVIII levels after a hemostatic challenge may be important to prevent bleeding. This was strengthened by an association between increased clearance of VWF and a higher bleeding score. Furthermore, patients with VWF and FVIII levels in the highest quartile three hours after desmopressin had 5 points lower bleeding score, and patients with FVIII:C in the highest quartile had 10 times less often an abnormal bleeding score. These results indicate that a good hemostatic response may have important and clinically relevant consequences on the bleeding phenotype, and may compensate for the low baseline VWF and FVIII levels in type 1 VWD patients. In conclusion, VWF and FVIII levels after desmopressin administration, which mimic in vivo hemostatic response to hemostatic challenges, are associated with the bleeding phenotype in type 1 VWD patients. This may partly explain the well-known variability in bleeding phenotype of type 1 VWD patients. Disclosures Atiq: Sobi: Other: Travel grant (ISTH 2019); Professor Heimburger Award 2018 (CSL Behring): Research Funding. Schütte:CSL Behring: Other: Travel grant. Boender:Sobi: Employment; Professor Heimburger Award 2016 (CSL Behring): Research Funding. Kruip:Bayer: Research Funding; Pfizer: Research Funding; Daiichy Sankyo: Research Funding; Boehringer Ingelheim: Research Funding. Leebeek:Shire/Takeda: Consultancy; UniQure: Consultancy; Novo Nordisk: Consultancy; Shire/Takeda: Research Funding; Sobi: Other: Travel grant; CSL Behring: Research Funding.

Analytical characterization and reference interval of an enzyme-linked immunosorbent assay for active von Willebrand factor.

BackgroundInteraction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF A1 domain, enabling platelet glycoprotein Ibα binding. Quantification of this ‘‘active” conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction.MethodsWe developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF A1 domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters.ResultsIntra- and inter-assay CVs were between 2.4–7.2% and 4.1–9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103±3%. The assay was linear in the range of 90.1–424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6–154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF:GP1bM binding.ConclusionsWe developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.

Bleeding Symptoms and von Willebrand Factor Levels: 30-Year Experience in a Tertiary Care Center.

Correlations between bleeding symptoms and von Willebrand factor (VWF) levels may help to predict hemorrhagic severity in the Westerners with von Willebrand disease (VWD), but data in Asians are lacking. In this study, Thai patients with VWF levels <50 IU/dL without any secondary causes were enrolled from 1988 to 2018 to determine the relationship between VWF levels and hemorrhagic manifestations. According to the current concept, we reclassified VWD and low VWF by VWF levels ≤30 and 30 to 50 IU/dL, respectively. Type 2 VWD was diagnosed if VWF activity to antigen ratio was ≤0.6. Bleeding severity was determined by the condensed MCMDM-1VWD bleeding score (BS). Among 83 patients, VWF activities showed negative correlations with BS (P = .001), which were higher in type 2 (median: 7, interquartile range [IQR]: 5-11) compared with type 1 VWD (median: 3, IQR: 2-4) and low VWF (median: 4, IQR: 2-8). Bleeding symptoms were indistinguishable between type 1 VWD and low VWF using the 30 IU/dL cutoff point. However, VWF ristocetin cofactor activity or gain-of-function mutant glycoprotein Ib binding activity <36.5 IU/dL and VWF collagen binding activity <34.5 IU/dL could predict increased bleeding risk (BS ≥3) by 92.3% specificity and 70.0% sensitivity (P < .0001).

Elevated Von Willebrand Factor May Abrogate Bleeding Phenotype in a Male with a Non-Null F8 Mutation

Introduction: Although FVIII activity (FVIII:C) correlates with phenotype in most Hemophilia A males, some have bleeding phenotypes milder than predicted by FVIII:C. Analysis of these individuals may provide additional insight into the relationship between F8 genotype and bleeding phenotype, identify additional factors that modulate FVIII, refine the classification of Hemophilia A and improve clinical management.We present a male subject who carries a c.5999G>C mutation in F8. This mutation has been reported in an individual with a severe bleeding phenotype in the EAHAD F8 Variant Database (www.factorviii-db.org). Our subject was serendipitously diagnosed at age 80, and has never had abnormal bleeding despite numerous challenges, including multiple dental extractions, adult circumcision, internal hemorrhoid ligation and cardiac catheterization. In an effort to resolve this genotype-phenotype discrepancy, we performed FVIII and von Willebrand Factor (VWF) assays, quantitated the extent of alternative splicing in F8, and screened for mutations in additional genes known to impact hemostasis. We validated a partial splicing defect that is consistent with mild Hemophilia A, but surprisingly cannot explain the lack of a bleeding phenotype.Methods: Peripheral blood was tested by one-stage FVIII:C and chromogenic FVIII assays, FVIII antigen ELISA, VWF antigen and activity assays, and von Willebrand Disease (VWD) Type 2 Normandy binding assay.To evaluate F8 splice variants, quantitative RT-PCR (qRT-PCR) was performed on RNA isolated from peripheral blood using primers that distinguish FVIII isoforms. Samples were tested in quadruplicate, with sample input normalized relative to GAPDH. Screening for exonic mutations in F8, F5, F2 and VWF genes was undertaken by Next Generation Sequencing (NGS).Results and Conclusions: In contrast to the previously reported severe c.5999G>C patient, this subject has reduced FVIII activity consistent with mild Hemophilia A measured by both FVIII activity assays. FVIII antigen assessment by ELISA gave similar results (0.35 IU/mL).To better understand FVIII:C levels in this individual, we sought to characterize F8 transcript isoforms. The c.5999G>C resides within the splice acceptor site of exon 19 and may result in two FVIII isoforms. One isoform produces a full-length FVIII containing a missense mutation (p.Gly2000Ala). The second isoform skips the 117 bp exon 19, but is predicted to retain an open reading frame; however, its location in the A3 domain likely disrupts secretion or function (Donadon et al., Haematologica 2018). The relative abundance of F8 variants evaluated by qRT-PCR demonstrated that despite a modest overall increase in F8 transcript relative to a normal control (1.6 fold higher expression of F8 at exon 18), the predominant isoform in this subject lacks exon 19, resulting in substantial depletion of the full-length isoform (0.3 relative to a normal control). These data indicate the full-length protein likely retains function, with transcript levels that explain residual FVIII activity.In an effort to explain absent bleeding phenotype, NGS was performed. The c.5999G>C F8 mutation was confirmed, while the thrombophilic Factor V Leiden and Prothrombin G20210A mutations were excluded. Sequencing also excluded mutations implicated in Type 2 Normandy VWD. Additional clinical testing may provide an explanation. VWF was elevated as indicated by VWF antigen and Ristocetin cofactor activity (>400%, >100%, respectively). Furthermore, VWF:FVIII binding activity by ELISA showed a higher than normal binding ratio (>1.42). VWF multimer analysis indicated normal distribution. Therefore, we propose elevated VWF abrogates this subject's bleeding phenotype.Altogether, our study expands our understanding of the functional consequences of this mutation by demonstrating that this mutation can result in FVIII levels predicting mild Hemophilia A. This case also underscores the complexities of predicting phenotype for mutations that result in partial splicing defects. Perhaps more intriguing is the lack of bleeding phenotype in this elderly individual with numerous hemostatic challenges, that might be related to increased VWF. These data support additional studies investigating the role of VWF in ameliorating bleeding risk in Hemophilia A. DisclosuresNo relevant conflicts of interest to declare.