Defects Of Von Willebrand Factor Research Articles

Outcomes Following Total Hip Arthroplasty in Patients Who Have Von Willebrand Disease Depend on Postoperative Anticoagulation

BackgroundVon Willebrand disease (VWD) is the most common congenital bleeding disorder. This autosomal dominant condition arises from quantitative or qualitative defects of Von Willebrand factor. To our knowledge, this study leveraged a national database to characterize the largest VWD cohort of total hip arthroplasty (THA) patients to date, assessing 90-day postoperative adverse events and 5-year revision-free survival. MethodsAdult patients who underwent primary THA for osteoarthritis were identified from January 2010 to October 2021 in a nationwide database. Patients who had and did not have VWD were matched (4:1) on age, sex, and Elixhauser Comorbidity Index and compared with multivariable logistic regression. Patients were then categorized based upon venous thromboembolism (VTE) chemoprophylaxis prescription patterns to compare bleeding and thrombotic adverse events. ResultsOf 544,851 THA patients, VWD was identified in 309 patients (0.06%). The matched cohorts contained 1,221 patients who did not have VWD and 306 patients who have VWD. On multivariable analysis, VWD patients had increased odds of 90-day VTE (odds ratio [OR] = 1.86) and hematoma (OR = 3.40) (P < .05 for all). No difference in 5-year revision-free survival was found. The VWD patients receiving aspirin or no prescriptions had greater odds of VTE (OR = 2.39, P = .048). Those on other chemoprophylaxis agents had greater odds of hematoma (OR = 4.84, P = .006). ConclusionsPatients with VWD undergoing THA had increased odds of 90-day VTE if using aspirin or no prescriptions, or hematoma if using other chemoprophylaxis. There is a delicate balancing act of clotting versus bleeding that must be considered in managing such patients, but it was reassuring that no difference in overall 5-year revision-free survival was found.

Von Willebrand disease: A guide for the internist.

von Willebrand disease (VWD), the most common inherited bleeding disorder, results when patients either do not make enough von Willebrand factor (VWF) or make defective VWF. The pathophysiology of this disorder is complex but needs to be understood to interpret the diagnostic tests. Most patients have mild to moderate symptoms and can be adequately counseled and managed by a general internist, but some need to consult a hematologist. We review the pathophysiology of VWD, its subtypes, common presentations of each subtype, diagnostic testing, and management of mild as well as severe clinical manifestations of VWD.

Functional Assessment of VWD Type 2N Variants Using Mammalian Cell Displayed Surface-Tethered Von Willebrand Factor

Background von Willebrand factor (VWF) is a large plasma protein critical in hemostasis and thrombosis. Defects in VWF cause the inherited autosomal bleeding disorder von Willebrand disease (VWD). One critical function of VWF is to bind circulating coagulation factor VIII (FVIII) and protect it from rapid clearance. Type 2N VWD is a recessive form of VWD characterized by low FVIII levels due to impairment in VWF-FVIII binding. Type 2N VWD clinically is similar to mild-to-moderate hemophilia A, an X-linked bleeding disorder caused by deficiency in FVIII. It is critical to distinguish type 2N VWD from hemophilia A because the treatments are different. VWF genotyping can inform accurate diagnoses, treatment, and testing in relatives. With the rapid expansion of DNA sequencing, a growing number of DNA variants are being identified in the VWF gene. However, the interpretation of significance of these variants is often challenged by insufficient evidence to inform confident classification regarding pathogenicity. In vitro functional assays provide an avenue to develop the evidence needed to accurately assign functional significance to DNA variants. We adapted multiplexed surface tethering of extracellular proteins (MultiSTEP), our mammalian cell surface display system for secreted proteins, to display VWF and test the functional impact of VWF variants on VWF-FVIII binding. Methods We constructed a VWF cDNA of common VWF coding sequences by mutating rare variants to the more common nucleotides. The regions encoding the VWF propeptide were deleted (∆Pro-VWF). ∆Pro-VWF was cloned into our MultiSTEP cassette that provides a C-terminal flexible linker, Strep II tag, and CD28 transmembrane domain. Three VWF missense variants implicated in type 2N VWD (Table 1) that had varying levels of evidence to support classification of pathogenicity were chosen for study - VWF c.2561 G&amp;gt;A (p.R854Q), c.2572 T&amp;gt;A (p.C858S), and c.2635 G&amp;gt;A (p.D879N). Variants were subcloned into our ∆Pro-VWF cassette and then recombined into the genomically integrated MultiSTEP landing pad in HEK293 Freestyle cells. Following induction of expression, VWF-displaying cells were incubated with recombinant FVIII. VWF expression was measured using a polyclonal anti-VWF antibody, and VWF-FVIII binding was detected with two different monoclonal antibodies to FVIII. Cells were incubated with fluorescently-conjugated secondary antibodies and cell surface VWF and FVIII abundance determined by flow cytometry. Results We show that ∆Pro-VWF robustly displays in our MultiSTEP system and binds FVIII. All three VWF missense variants express VWF at levels similar to ∆Pro-VWF. Relative to ∆Pro-VWF all three variants show marked impairment in VWF-FVIII binding (Figure 1), providing strong in vitro evidence for pathogenicity in type 2N VWD useful in variant reinterpretation (bold column in Table 1). Conclusions We have developed an in vitro mammalian cell surface display system for the interrogation of VWF variants and adapted it to study VWF-FVIII binding. The Pathogenic variant p.R854Q shows decreased VWF-FVIII binding in this assay, as expected. The Likely Pathogenic variant p.D879N and the Variant of Uncertain Significance p.C858S both exhibit a profound loss of VWF-FVIII binding in this assay. Our results provide evidence for reclassification of p.C858S to Likely Pathogenic for type 2N VWD. We expect our system can be used at scale to similarly interrogate the functional significance of a multiplexed library of VWF variants and improve variant interpretation in VWD.

The Impacts of Genetic Polymorphisms on the Von Willebrand Factor Level in Type 1 Von Willebrand Disease

Background: von Willebrand disease (VWD) is the most common inherited bleeding disease, which results from deficiency or defect of von Willebrand factor (VWF). In type 1 VWD, there are only 53-81% patient whose mutations of VWF gene can be identified. Some studies reported that variants outside the VWF gene may also contribute to the VWF level. There are 7 genetic variants of single nucleotide polymorphism ( SNP ) reported to be associated with the variation of VWF levels, including CLEC4M, STXBP5, SCARA5, ABO, VWF, STAB2 and UFM1. The aims of this study were to explore the frequency of SNPs of the 7 genes among type 1 VWD patients and the effects of the 7 genetic variants on the VWF levels in type 1 VWD patients. Patients and methods: There were 62 patients ( the ratio of M/F: 1 ) with type 1 VWD enrolled in this study, including 35 (56.5%) patients with found mutations of VWF gene. Each patient's bleeding symptoms were assessed by the MCMDM-1 VWD bleeding score. The results of VWF levels: VWF:Ag and VWF activity ( VWF:ACL or VWF:RCo ) and FVIII: C at the VWD diagnosis were retrospectively collected, as shown in Table 1. Each enrolled patient's genomic DNA was extracted and the SNPs of the 7 gene were targeted by PCR amplification followed by direct sequencing. Results: There was high correlations between VWF:Ag and VWF activity ( r = 0.73, p &amp;lt; 0.0001 ) and between VWF:Ag and FVIII:C ( r = 0.60, p &amp;lt; 0.0001 ), respectively. The patients without variant ( A/A) of UFM1 has significantly lower median VWF:Ag of 33% than 44.7% of patients with heterozygous variant ( A/C ) type ( p = 0.033 ). Patients without variant (A/A ) of STXBP gene had both significantly lower median value of VWF:Ag ( 31.9% vs 46.1%, p = 0.017 ) and VWF activity (26.55% vs 30.4%, p = 0.0467 ) than those patients with heterozygous variant ( A/G ). Four alleles substitution of SNP of UFM1( C&amp;gt;A ) and STXBP genes ( G&amp;gt;A ) variant has remarkably lowest median VWF antigen level of 27.4%, which was significantly lower than 43.05% of the three alleles substitution ( p = 0.0014 ) and the 48.6% of the two alleles substitution ( p = 0.0279 ), as shown in Figure 1. Conclusions: Our study demonstrates that both C to A variant of UFM1 gene and G to A variant of STXBP5 gene were associated with lower VWF: Ag level than those without variant, respectively. The combination of four allelic variant of UFM1 and STXBP5 genes had the most reducing effect on VWF: Ag level.

Laboratory Testing for von Willebrand Factor: Factor VIII Binding for the Diagnosis or Exclusion of Type 2N von Willebrand Disease: An Update.

von Willebrand factor (VWF) is a large adhesive plasma protein that expresses several functional activities. One of these activities is to bind coagulation factor VIII (FVIII) and to protect it from degradation. Deficiency of, and/or defects in, VWF can give rise to a bleeding disorder called von Willebrand disease (VWD). The defect in VWF that affects its ability to bind to and protect FVIII is captured within type 2N VWD. In these patients, FVIII is produced normally; however, plasma FVIII quickly degrades as it is not bound to andprotected by VWF. These patients phenotypically resemble those with hemophilia A, where instead, FVIII is produced in lower amount. Both hemophilia A and 2N VWD patients therefore present with reduced levels of plasma FVIII relative to VWF level. However, therapy differs, since patients with hemophilia A are given FVIII replacement products, or FVIII mimicking products; instead, patients with 2N VWD require VWF replacement therapy, since FVIII replacement will only be effective for a short term, given this replacement product will quickly degrade in the absence of functional VWF. Thus, 2N VWD needs to be differentiated from hemophilia A. This can be achieved by genetic testing or by use of a VWF:FVIII binding assay. The current chapter provides a protocol for the performance of a commercial VWF:FVIII binding assay.

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.

Quality of Life in Patients with Clinically Severe Von Willebrand Disease: Interim Analysis of Athn 9: A Natural History Study for People with Severe Von Willebrand Disease (VWD)

Background: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder, which results from either a quantitative (Type 1 or 3) or qualitative (Type 2) defect in von Willebrand Factor (VWF). Prior studies, largely focused on individuals with mild VWD and bleeding symptoms, have shown significant reductions in quality of life in patients with Von Willebrand disease. Because of the rarity less is known about individuals with severe forms of VWD or those with a severe bleeding phenotype. Aim: To report quality of life measures in participants with clinically severe VWD in the United States (US). Study Design and Methods: ATHN 9 Severe VWD Natural History Study is sponsored by the American Thrombosis and Hemostasis Network (ATHN) and is being conducted at 25 ATHN-affiliated sites across the US. ATHN 9 is an observational study focused on persons with clinically severe VWD treated with VWF factor on demand or for prophylaxis. Participants are identified by site investigators with a projected goal to enroll 130 individuals. Patients with severe VWD defined as type 3 VWD, or a VWF:RCo, VWF:GPIbM or VWF:Ag ≤ 30%, or persons with "clinically severe VWD" defined by VWF:RCo, VWF:GPIbM or VWF:Ag ≤ 40% and requiring recurrent use of factor concentrates, and prior enrollment in the ATHNdataset national surveillance data collection project are included. Participants with platelet-type or acquired VWD are excluded. Quality of life was assessed using PROMIS measures (PROMIS 25, PROMIS 25 Proxy, PROMIS 29) and the VWIQ at enrollment, annually, and at study exit. PROMIS-29 assesses seven different quality of life domains (physical function, anxiety, depression, fatigue, sleep disturbance, ability to participate in social roles, and pain interference) and has been validated in adult populations. PROMIS measures in the general US population have a mean of 50 and standard deviation of 10. PROMIS-25 is a pediatric questionnaire assessing the same domains (except for sleep disturbance) and an accompanying proxy form for caregivers of participants <8 years of age. Baseline enrollment results are reported here. Results: Enrollment (baseline) PROMIS-29 forms were completed by 43 participants. At baseline, mean physical function was lower than the general population at 45.7 (p=0.005), and fatigue and pain interference were greater than the general population at 53.2 and 53 respectively (p=0.036 and 0.049 respectively). Individuals on continuous VWF factor prophylaxis (n=18) had significantly decreased physical function (p<0.01), and decreased ability to participate in social roles (p<0.01) than individuals on episodic treatment (n=19). Enrollment PROMIS-25 forms were completed for 26 participants, while PROMIS-25 proxy forms were completed for 19 participants. Mean enrollment scores for PROMIS-25 depression symptoms was lower than the general population at 44.4 (p=0.0004) and peer relationships were better than the general population at 54 (p=0.042). Mean enrollment scores for PROMIS-25 Proxy were lower than the general population for depression, fatigue, and pain interference at 41, 42.9, and 45.4 respectively (p<0.0001, p=0.002, and p=0.045 respectively). In contrast to the adult participants, no statistically significant differences were seen between pediatric participants on VWF factor prophylaxis versus episodic treatment. Discussion: Pediatric participants with clinically severe VWD reported less depression and better peer relationships than the general population, and parents of pediatric participants with VWD reported less depression, fatigue, and pain interference in their children compared to the general population. Patient-reported outcomes in adult participants were less encouraging with worse physical function, and more fatigue and pain interference compared to the general population. Adult participants on VWF factor prophylaxis had decreased physical function, and decreased ability to participate in social roles compared to adults treated episodically, perhaps secondary to disease severity and accumulation of bleed-related morbidity. No statistically significant differences were seen in quality of life outcomes between pediatric patients on VWF factor prophylaxis and those on episodic treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Compound heterozygosity for novel von Willebrand factor genetic variants associated with von Willebrand disease in two Chinese patients.

Von Willebrand factor (VWF) encodes a secreted glycoprotein involved in primary hemostasis. Genetic mutations in this gene leading to either quantitation or qualitative defects of VWF, result in von Willebrand disease (VWD), an inherited bleeding disorder. In this study, two families with VWD were recruited and submitted to a series of clinical and genetic examinations. prothrombin time, activated partial thromboplastin time, thrombin time, factor VIII coagulant activity (FVIII:C), VWF antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo) tests were measured in peripheral blood. F8, F9, and VWF genes were sequenced using next-generation sequencing, and Sanger sequencing was used as a validation method. Both families had a child suffered spontaneous bleeding. Patient 1 showed normal VWF:Ag, severely decreased FVIII:C and VWF:RCo. Patient 2 showed severely decreased FVIII:C, VWF:Ag, and VWF:RCo. Compound heterozygous mutations of VWF gene were identified in both patients. Patient 1 had a novel deletion variant c.1910_1932del (p.Gly637AlafsTer5) and a missense variant c.605G>A (p.Arg202Gln). Patient 2 had a novel missense variant c.4817T>A (p.Met1606Lys) and a novel missense variant c.5983C>T (p.Pro1995Ser). We described clinical and molecular features of VWD caused by compound heterozygous mutations in two Chinese patients. Our results expand the variation spectrum of the VWF gene and deepen the understanding of the relationship between the genotype and clinical characteristics of VWD.

Analysis of von Willebrand Disease in the "Heart of Europe".

Background von Willebrand disease (VWD) is a genetic bleeding disorder caused by defects of von Willebrand factor (VWF), quantitative (type 1 and 3) or qualitative (type 2). The laboratory phenotyping is heterogenic making diagnosis difficult. Objectives Complete laboratory analysis of VWD as an expansion of the previously reported cross-sectional family-based VWD study in the Czech Republic (BRNO-VWD) and Slovakia (BRA-VWD) under the name “Heart of Europe,” in order to improve the understanding of laboratory phenotype/genotype correlation. Patients and Methods In total, 227 suspected VWD patients were identified from historical records. Complete laboratory analysis was established using all available assays, including VWF multimers and genetic analysis. Results A total of 191 patients (from 119 families) were confirmed as having VWD. The majority was characterized as a type 1 VWD, followed by type 2. Multimeric patterns concordant with laboratory phenotypes were found in approximately 83% of all cases. A phenotype/genotype correlation was present in 84% (77% type 1, 99% type 2, and 61% type 3) of all patients. Another 45 candidate mutations (23 novel variations), not found in the initial study, could be identified (missense 75% and truncating 24%). An exon 1–3 gene deletion was identified in 14 patients where no mutation was found by direct DNA sequencing, increasing the linkage up to 92%, overall. Conclusion This study provides a cross-sectional overview of the VWD population in a part of Central Europe. It is an addition to the previously published BRNO-VWD study, and provides important data to the International Society of Thrombosis and Haemostasis/European Association for Haemophilia and Allied Disorders VWD mutation database with identification of novel causal mutations.

Evaluation of different diagnostic tools for detection of acquired von Willebrand syndrome in patients with polycythemia vera or essential thrombocythemia

IntroductionPatients with myeloproliferative neoplasms (MPNs) are at increased risk of both thromboembolic and hemorrhagic complications. Among the risk factors for bleeding is the development of an acquired qualitative von Willebrand factor defect with loss of larger VWF plasma multimers, resulting in acquired von Willebrand syndrome (aVWS). The diagnosis of aVWS is challenging, because no single automated test is sufficient to prove or exclude aVWS. We aimed to compare different diagnostic tools used for the detection of MPN-associated aVWS in daily practice. MethodsPatients with polycythemia vera (PV) or essential thrombocythemia (ET), who had been routinely assessed for quantitative and qualitative abnormalities of plasma VWF, were retrospectively studied. ResultsSixty-four patients (37 with PV and 27 with ET) were analyzed. Using multimer analysis of plasma VWF, aVWS with a loss of VWF high-molecular-weight multimers was detected in 51.4 % and 55.6 % of PV and ET patients, respectively. A VWF:GPIbM/Ag threshold of <0.8 was defined as useful screening tool for aVWS, with acceptable sensitivity (88.2 %) and specificity (76.7 %) and good agreement with VWF multimer analysis (κ = 0.653). VWF:GPIbM/Ag showed better performance than VWF:CB/Ag. Regarding hematological parameters, there was no difference in hematocrit between patients with and without aVWS. However, leukocyte and platelet counts were significantly increased in the aVWS cohort. ConclusionFor the first time, we identified a VWF:GPIbM/Ag ratio of <0.8 as simple screening tool for aVWS in patients with PV or ET. This observation, however, should be confirmed in a larger patient cohort.

Von Willebrand Disease-Associated Angiodysplasia: Presentation of a Paediatric Case

Von Willebrand disease (VWD) is a bleeding disorder, resulting from a quantitative or qualitative defect in von Willebrand factor (VWF). A regulatory role for VWF in angiogenesis was postulated upon the clinical observation that qualitative or quantitative VWF defects are associated with the frequent occurrence of neoangiogenesis, particularly in the gastrointestinal (GI) tract. Vascular malformations of the GI tract are a cause of digestive bleeding in the form of either acute or chronic haemorrhage and represent a heterogeneous group of lesions, including angiodysplasias and telangiectasias. The management of these patients is challenging due to recurrent and severe episodes of GI bleeding. The mainstay of treatment of angiodysplasia is replacement therapy when this abnormality causes GI bleeding in patients with congenital VWD. When bleeding episodes recur frequently, regular prophylaxis should be implemented, leading to an acceptable degree of prevention of bleeding. The authors had a difficult experience in a 14-year-old adolescent with Type 3 VWD, who had presented with extremely serious recurrent bleeding secondary to duodenal angiodyplasia.

Evaluating Performance of Contemporary and Historical von Willebrand Factor (VWF) Assays in the Laboratory Identification of von Willebrand Disease (VWD): The Australasian Experience.

von Willebrand disease (VWD) is a common bleeding disorder that arises from deficiency and/or defects of von Willebrand factor (VWF). Appropriate diagnosis of VWD, including differential identification of qualitative (types 2A, 2B, 2M, 2N VWD) versus quantitative (types 1 and 3 VWD) defects remains problematic but has important management implications, given differential therapy. Complete assessment for VWD in a patient with a bleeding history requires comprehensive test panels, including VWF activity and antigen. We describe the Australasian experience, using data from the Royal College of Pathologists of Australasia (RCPA) Quality Assurance Program (QAP) related to VWF testing in their VWD test module. The RCPAQAP has been providing samples for VWF testing since 1998, representing 25 years of proficiency testing related to VWD diagnosis. A total of 109 samples have been dispatched to participants over these years, with current assessment involving dispatches of two samples (=4 samples) per year. Samples have represented all types of VWD, as well as normal or other samples, including acquired von Willebrand syndrome and plasma VWF concentrates as used in VWD therapy. Different VWF assays and activity/antigen ratios show different utility in VWD and type identification. In the past 9 years of data capture, a total of 166 errors were identified from a total of 1,839 interpretations, representing a base error rate of 9.0%. Identification errors were highest for type 2 VWD samples (15.3%), intermediate for type 1 VWD samples (7.5%), and lowest for normal samples (2.4%). Errors can be linked to assay limitations, including assay variability and low-level VWF detection limits, as well as laboratory issues (including test result misinterpretation, which accounts for approximately 40% of all errors for type 2 VWD). For test-associated errors, VWF:RCo and VWF:GPIbM were associated with the highest variability and error rate, which was up to 10x higher than that using VWF:CB. As a test group, chemiluminescence-based procedures were associated with lowest inter-laboratory variability, best low-level VWF detection (down to <1 U/dL), and least errors overall. These findings inform on reasons behind high rates of errors associated with VWD diagnosis, with some assays and methodologies performing substantially better than others.

Defective VWF secretion due to expression of MYH9-RD E1841K mutant in endothelial cells disrupts hemostasis

AbstractMutations in MYH9, the gene encoding the heavy chain of nonmuscle myosin IIa (NMII-A), cause MYH9-related disease (MYH9-RD), which is an autosomal-dominant thrombocytopenia with bleeding tendency. Previously, we showed that NMII-A in endothelial cells (ECs) is critical for hemostasis via regulating von Willebrand factor (VWF) release from Weibel-Palade bodies (WPBs). The aim of this study was to determine the role of the expression of MYH9 mutants in ECs in the pathogenesis of the MYH9-RD bleeding symptom. First, we expressed the 5 most common NMII-A mutants in ECs and found that E1841K mutant-expressing ECs secreted less VWF than the controls in response to a cyclic adenosine monophosphate (cAMP) signaling agonist. Then, we generated 2 knockin mouse lines, 1 with Myh9 E1841K in ECs and the other in megakaryocytes. Endothelium-specific E1841K mice exhibited impaired cAMP-induced VWF release and a prolonged bleeding time with normal platelets, whereas megakaryocyte-specific E1841K mice exhibited macrothrombocytopenia and a prolonged bleeding time with normal VWF release. Finally, we presented mechanistic findings that E1841K mutation not only interferes with S1943 phosphorylation and impairs the peripheral distribution of Rab27a-positive WPBs in Ecs under quiescent condition but also interferes with S1916 phosphorylation by disrupting the interaction with zyxin and CKIIα and reduces actin framework formation around WPBs and subsequent VWF secretion under the stimulation by a cAMP agonist. Altogether, our results suggest that impaired cAMP-induced endothelial VWF secretion by E1841K mutant expression may contribute to the MYH9-RD bleeding phenotype.

The Lesson Learned from the New c.2547-1G > T Mutation Combined with p.R854Q: When a Type 2N Mutation Reveals a Quantitative von Willebrand Factor Defect.

Type 2N is a rare von Willebrand disease (VWD) variant involving an impairment in the factor VIII (FVIII) carrier function of von Willebrand factor (VWF). It has a phenotype that mimics hemophilia A, and FVIII binding to VWF (VWF:FVIIIB) is tested to differentiate between the two disorders. Type 2N VWF defects may also be associated with quantitative VWF mutations (type 2N/type 1), further complicating the identification of cases. We report on a new quantitative VWF mutation (c.2547-1G > T) revealed by a p.R854Q type 2N mutation acting as homozygous despite being carried as a heterozygous defect. The proband had near-normal VWF levels (initially ruling out a defective VWF synthesis) and slightly reduced FVIII levels, while a VWF:FVIIIB test showed significantly reduced binding. Routine tests on type 2N homozygotes or heterozygotes combined with quantitative VWF defects in our cohort showed reduced FVIII levels in both groups, but it was only in the former that the FVIII/VWF antigen (VWF:Ag) ratio was always significantly reduced. The two tests are therefore not enough to identify all forms of type 2N VWD. While relatives of type 2N homozygotes usually have normal FVIII levels and FVIII/VWF:Ag ratios, relatives of type 2N/type 1 may have high FVIII/VWF:Ag ratios, but their VWF:FVIIIB and/or VWF:FVIIIB/VWF:Ag ratios are always low. Measuring FVIII and VWF levels may therefore suggest type 2N VWD in patients carrying type 2N mutations alone, but not in type 2N combined with quantitative VWF defects. The VWF:FVIIIB test should consequently be included when exploring VWF function, whatever VWD patient's phenotype.

Phenotypic and Genotypic Signatures of VWF Exon 18 in Eastern Saudi Patients Previously Diagnosed with Type 1 von Willebrand Disease.

Introductionvon Willebrand disease (VWD) is the most prevalent bleeding disease, which is associated with either low levels of von Willebrand factor (VWF) or abnormality in its structure. Three types of the disease have been described; type 1 (VWD1) and 3 (VWD3) are caused by deficiency of VWF and type 2 (VWD2) is caused by production of defective VWF. The aim of the current study was to characterize gene variants of VWF gene; exon 18 in particular, in a cohort of Saudi families as well as healthy control subjects.MethodsA total of 19 families comprising 60 subjects of type 1 VWD were enrolled in the study. Participants were divided into 22 index cases, 21 affected family members and 17 unaffected family members ranging in age from 6 to 70 years. Blood samples were collected from all participants to measure activated partial thromboplastin time test (APTT), von Willebrand antigen level (VWF:Ag), Factor VIII activity (FVIII:C) and ristocetin cofactor activity (VWF:RCo), platelet count, determining the ABO blood group and for genetic analysis by Sanger sequencing.ResultsThe results indicated that VWD1 patients have lower levels of VWF and factor VIII than the non-affected family members and the control subjects. In addition, five gene variants were reported in VWF exon 18; of these, c.2365A>G and c.2385T>C were more common in the control group and might be protective from VWD.DiscussionIn conclusion, VWF levels are influenced by blood group, and there was no association between variants in exon 18 of VWF gene reported in all groups and the disease status; however, blood group analysis and genome-wide genotyping could help to highlight high-risk groups and improve clinical management of VWD.

Towards novel treatment options in von Willebrand disease.

Deficiency or dysfunction of von Willebrand factor (VWF) is associated with a bleeding disorder known as von Willebrand disease (VWD). The clinical manifestations of VWD are heterogeneous, and are in part dictated by the structural or functional defects of VWF. The tools to control bleeding in VWD are dominated by VWF concentrates, desmopressin and antifibrinolytic therapy. In view of these treatments being considered as effective, it is surprising that quality-of-life studies consistently demonstrate a significant mental and physical burden in VWD patients, particularly in women. Apparently, the current weaponry to support the management of VWD is insufficient to fully address the needs of the patients. It is important therefore to continue to search for innovative treatment options which could better serve the VWD patients. In this short review, two of such options are discussed in more detail: emicizumab to correct for the deficiency of factor VIII (FVIII), and the pegylated aptamer BT200 to increase endogenous levels of the VWF/FVIII complex.

Is pharmacokinetic-guided dosing of desmopressin and von Willebrand factor-containing concentrates in individuals with von Willebrand disease or low von Willebrand factor reliable and feasible? A protocol for a multicentre, non-randomised, open label cohort trial, the OPTI-CLOT: to WiN study

IntroductionVon Willebrand disease (VWD) is a bleeding disorder, caused by a deficiency or defect of von Willebrand factor (VWF). In case of medical procedures or bleeding, patients are treated with...

Primary hemostatic disorders drive early and late major bleedings of patients with atrial fibrillation after transcatheter aortic valve replacement

Patients with atrial fibrillation (AF) are likely to have multiple co-morbidities which lead to a poor prognosis including bleeding events. Closure time adenosine diphosphate (CT-ADP) is a primary hemostasis point-of-care test used as a surrogate marker of high molecular weight multimers defect of Von Willebrand factor. Our prior studies suggest that prolongation of CT-ADP (> 180 sec) after TAVR is a determinant of major/life-threatening bleeding complications (MLBCs). To evaluate the impact of post-procedural CT-ADP > 180 sec on bleeding events in TAVR patients with AF. We included 878 patients from our prospective TAVR registry. Bleeding complications were assessed according to the VARC-2 criteria. Major adverse cardiac and cerebrovascular events (MACCE) was defined as a composite of all-cause death, myocardial infarction, stroke, and heart failure hospitalization after TAVR. Primary endpoint was 1-year MLBCs and the second endpoint was 1-year MACCE. Patients with AF had a higher incidence of all-cause mortality (15% vs. 8%, P = 0.002), MACCE (29% vs. 20%, P = 0.002), and MLBCs (20% vs. 12%, P = 0.001) within 1-year compared to non-AF patients. When the cohort was split into 4 subgroups according to AF and CT-ADP > 180 sec, patients with AF and CT-ADP > 180 sec had the highest risk of MLBCs (log-rank test; P < 0.001) ( Fig. 1 ). Multivariate Cox regression analysis confirmed that the patients with AF and CT-ADP > 180 sec had 4.6-fold higher risk of MLBCs within 1 year compared to non-AF patients with CT-ADP ≤ 180 sec (hazard ratio: 4.60; 95% confidence interval: 2.18-9.68; P < 0.001) ( Fig. 1 ). Among TAVR patients, AF with post-procedural CT-ADP > 180 sec was identified as a strong predictor of MLBCs at 1-year follow-up. Our study suggest that persistent primary haemostasis disorders contribute to a higher risk of bleeding events particularly in AF patients and may be considered for a tailored and risk-adjusted antithrombotic therapy after TAVR.

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.

Bleeding in Patients with Clinically Severe Von Willebrand Disease: Interim Analysis of Athn 9: A Natural History Study for People with Severe Von Willebrand Disease (VWD)

Background:Von Willebrand Disease (VWD) is the most common inherited bleeding disorder, occurring in ~0.1% of the population. VWD results from either a quantitative (Type 1 or 3) or qualitative (Type 2) defect in von Willebrand Factor (VWF), a multimeric plasma protein involved in platelet adhesion and aggregation at the vascular injury site. Accurate diagnosis of VWD is complex due to pre-analytical variables, a wide coefficient of variation in testing, and incomplete penetrance. Though individuals with mild VWD and bleeding symptoms are common, less is known regarding persons with VWD and a clinically severe bleeding phenotype.Aims:To characterize the bleeding phenotype and safety of treatment regimens in participants with clinically severe VWD in the United States (US).Study Design and Methods:ATHN 9 is sponsored by the American Thrombosis and Hemostasis Network (ATHN) and is being conducted at 25 ATHN-affiliated sites across the US. Participants are identified by the site investigators with a projected goal to enroll 130 individuals. Patients with severe VWD defined as type 3 VWD, or a VWF:RCo, VWF:GPIbM or VWF:Ag ≤ 30%, or persons with “clinically severe VWD” defined by VWF:RCo, VWF:GPIbM or VWF:Ag ≤ 40% and requiring recurrent use of factor concentrates and prior enrollment in the ATHNdataset national surveillance data collection project are included. Participants with platelet-type or acquired VWD are excluded. Laboratory assessments including a standardized diagnostic battery, VWF genetic analysis (Next-gen sequencing), and inhibitor testing, performed by a central laboratory. Bleeding tendency is assessed by International Society for Thrombosis and Haemostasis (ISTH) Bleeding Assessment Tool (BAT) (abnormal BS is ≥4 in adult males, ≥6 in adult females and ≥3 in children) and the Pictorial Bleeding Assessment Chart (PBAC; heavy menstrual blood loss ≥100) if applicable.Results:Initial data on 81 participants was analyzed. Given that the enrollment occurred during the pandemic, the completion of data varied as follows: Baseline Demographics Forms 81 completed, Baseline History Forms 73 completed, and VWD Diagnostic Testing Results Forms 38 completed. Most were adult (53%), female (58%), Caucasian (82%) and non-Hispanic (82%). Approximately half (38/81, 47%) have undergone central lab diagnostic testing (Table 1) while the remaining had previous diagnostic studies. About half of the patients had Type 1, a quarter have Type 3, and the remaining had Type 2 VWD or unknown. The majority of patients (91%) had VWF:GPIbM activity <30IU/dL and (61%) had a VWF:Ag <30IU/dL. One participant had an inhibitor (1/22, 4%) to VWF. Of the participants with a baseline history form submission (73 in total), more than half (42/73, 57%) had undergone surgery, the most common being nasal cautery (11/42, 26.2%) and dental extraction (11/42, 26.2%); complicated by bleeding most commonly during or following dental extraction (2/11, 18.2%) and endoscopy/colonoscopy (2/4, 50%). Few participants (6/73, 8%) reported the presence of a target joint at enrollment, ankle being most common. The bleeding phenotype was variable with a mean ISTH BAT score of 10 (range 0-39) with the first bleeding event commonly occurring prior to age 10 years (51%), with 20% occurring prior to age 12 months (20%). The PBAC was performed on 6 of the 47 female participants in reference to their last period and was abnormal(mean 247; range 0-754). The majority (70 participants, 96%) utilized factor concentrates for prophylaxis or on-demand treatment; 27 participants (39%) were on continuous prophylaxis, while 8 (11%) were on event-based while less (10%) were on heavy menstrual bleeding (HMB) prophylaxis, and the remainder (40%) received episodic treatment. Participants used plasma derived VWF concentrate most commonly (64.3%) with the remainder using recombinant VWF or DDAVP/antifibrinolytics.Discussion: Initial evaluation of 81 participants with clinically severe VWD were diagnostically determined to be type 1 VWD with a majority having a bleeding phenotype (mean (ISTH BAT 10) and HMB (mean PBAC 247). In contrast to patients with mild disease where antifibrinolytics and desmopressin are frequently used, factor replacement was the most common treatment modality. Future analysis will focus on response to factor replacement therapy, genotype-phenotype correlation and quality of life. DisclosuresWeyand: Novo Nordisk: Research Funding; Genentech: Consultancy; Sanofi: Consultancy, Research Funding; Takeda: Consultancy. Friedman: Siemens: Consultancy; Bayer: Consultancy; Alexion: Speakers Bureau; Genentech: Consultancy; Instrumentation Laboratories: Consultancy; Sanofi: Consultancy. Haley: American Thrombosis and Hemostasis Network: Research Funding. He: ATHN: Ended employment in the past 24 months. Roberts: Takeda; Speakers Bureau: Novo Nordisk, Octapharma, Sanofi, Takeda.: Research Funding; Genentech, Novo Nordisk, Octapharma, Pfizer, Sanofi, Takeda, uniQure: Consultancy. Wynn: Sanofi: Research Funding; Takeda: Research Funding; Genentech: Research Funding. Sidonio: Sanofi, Takeda, Octapharma, Bayer, Biomain, Grifols, Kedrion, Genentech. Catalyst, Guardian Therapeutics, Novo Nordisk, Hema Biologics, Uniqure.: Consultancy, Honoraria. OffLabel Disclosure:Vonvendi (recombinant VWF) does not have a product indication for prophylaxis