Type 2A Von Willebrand Disease Research Articles

Characterization and Quantification of Von Willebrand Factor Proteoforms in Von Willebrand Disease Patients and Healthy Controls: Insights from the Win-Study

Background: Von Willebrand disease (VWD) is a commonly inherited bleeding disorder which is characterized by either low (type 1) or absent (type 3) levels of Von Willebrand factor (VWF) or by the occurrence of dysfunctional VWF proteoforms (type 2) in plasma. The complexity of diagnosing and managing VWD arises from limited understanding of the diverse VWF proteoforms present in both healthy individuals as well as in VWD patients. Objective: The aim of this study was to investigate the plasma proteome and to characterize VWF proteoforms in Von Willebrand disease (VWD) patients, that were enrolled in the Willebrand in the Netherlands (WiN) 1 study, compared with healthy controls. Method: We selected type 1, type 2A, type 2M, type 2N and type 3 patients from the WiN cohort for which extensive laboratory- and genotype data were available. Plasma from 64 VWD patients and ten healthy controls was analyzed using a combination of discovery- and targeted mass spectrometry-based approaches. Results: Both discovery-based as well as targeted quantitative proteomics analysis revealed significant variation in VWF levels across the selected VWD patient group, which was in line with reported clinical laboratory data. Elevated VWF-propeptide/VWF ratios were observed in plasma samples from a range of VWD patients, with notable increases observed in VWD type 2A, B, and M, indicating accelerated clearance of these VWF proteoforms. Discovery-based proteomics revealed a strong VWF-F8 relationship in all VWD subtypes and healthy controls, except for type 2N VWD. We were able to identify peptides corresponding with common polymorphisms ( e.g. p.Thr798Ala, p.Gln852Arg, p.Thr1381Ala), for which the observed peptide distribution was in line with the minor allele frequency reported by the Broad institute. In addition, we identified peptides corresponding with 16 pathogenic VWF variants (e.g. p.Arg1306Trp, p. Phe1293Leu, p.Cys1190Arg, p.Arg1374His or p.Tyr1584Cys) of which p.Cys1190Arg wild-type and variant peptide aligned with the zygosity at DNA level (Figure X). Based on spike-in with stable isotope-labeled peptides, the variant-to-wildtype ratios varied among VWD patients with distinct pathogenic variants, ranging from 0.5-2.5 in type 2M (p.Phe1293Leu) and type 1 patients (p.Tyr1584Cys), <1 in type 2A patients (p.Arg1374His), to >8 in type 2B patients (p.Arg1306Trp), suggesting distinct stoichiometry of VWF variants among these patients (Figure 2). Conclusion: Our study highlights the feasibility of VWF proteoform identification and quantification from plasma and suggest a potential role in unravelling their contribution to the biological variations and clinical diversity observed in VWD. 1 De Wee et al., Determinants of bleeding phenotype in adult patients with moderate or severe von Willebrand disease. Thromb Haemost. 2012 Oct;108(4):683-92.

A Feasibility Study Evaluating Von Willebrand Factor in Patients with Gastrointestinal Angiodysplasia

Background: Abnormalities of von Willebrand Factor (VWF), such as those found in von Willebrand Disease (VWD) are associated with the development of abnormal blood vessels known as angiodysplasia. Angiodysplasia occurs primarily in the gastrointestinal (GI) tract, particularly the small bowel. Among patients with VWD, angiodysplasia is significantly overrepresented as a cause of GI bleeding and occurs in all VWD subtypes although it is reported more frequently in those with loss of the high molecular weight multimer of VWF. Recent studies have demonstrated more than a third of patients with VWD and GI bleeding have angiodysplasia when systematically assessed with Video Capsule Endoscopy (VCE) to examine the small bowel. Given the relationship between angiodysplasia and VWD our objective was to assess the feasibility of evaluating patients with GI angiodysplasia for abnormalities of VWF. Methods: We undertook a prospective single center pilot study to assess patients over the age of 18 with GI angiodysplasia for underlying abnormalities of VWF. Patients were identified based on angiodysplasia being present on a VCE study. Patients who consented to enrollment completed a self-administered bleeding assessment tool (BAT) as well as baseline demographic data. VWF testing included VWF antigen (VWF:Ag), VWF functional assay (VWF:GPIbM), Factor VIII coagulant activity (FVIII:C), and VWF pro-peptide (VWFpp) levels. Results: After reviewing 190 consecutive VCE studies performed at our institution between April 2021 and April 2023, we identified 50 patients who had capsule endoscopies demonstrating angiodysplasia of the GI tract. Thirteen of these patients consented to enrollment in the study. The bleeding histories of included patients are detailed in Table 1 along with their baseline clinical, laboratory, and demographic data. Two patients endorsed lifelong bleeding symptoms and three reported a significant family history of bleeding - all in a female relative. ISTH-BAT scores ranged from 0 to 18 with a median score of 2. Since our population is older than other studies that report on bleeding scores, we considered an abnormal bleeding score to be >6 as recent evidence indicates normal BAT scores increase over time. Five patients (38.6%) in our study had an ISTH-BAT abnormal bleeding score. VWF levels were assessed (Table 1) and one patient aged 88 years old with a significant family and lifelong history of bleeding received a new diagnosis of Type 2A VWD. Severe iron deficiency anemia requiring ongoing IV iron was diagnosed in two patients. Conclusion: In our feasibility study, we demonstrated a feasible protocol for recruitment of patients with GI angiodysplasia for clinical and laboratory testing of VWF. A significant proportion of our patients (38.6%) had abnormal bleeding scores and one patient had an underlying Type 2A VWD diagnosis.

Genetic determinants of enhanced von Willebrand factor clearance from plasma

BackgroundEnhanced von Willebrand factor (VWF) clearance from plasma is associated with von Willebrand disease (VWD). However, the genetic background of this disease mechanism is not well defined. ObjectiveTo determine VWF variants that are associated with reduced VWF survival. MethodsTwo hundred fifty-four patients with VWD (type 1 = 50 and type 2 = 204) were investigated, and the results were compared with 120 healthy controls. The patients were comprehensively characterized for phenotypic and genetic features. The ratio of VWF propeptide (VWFpp)/VWF antigen (VWFpp ratio) was used to establish in each patient the VWF clearance state. ResultsOut of 92 variants associated with type 1 (7 were novel) and type 2 VWD, 19 had a VWFpp ratio ranging from 1.7 to 2.2, 24 had a VWFpp ratio between 2.3 and 2.9, and 24 variants had a ratio of ≥3. The VWFpp median ratio in healthy controls was 0.98 (0.55-1.6) so that a cut-off value of >1.6 was considered an indicator of accelerated VWF clearance from plasma. An enhanced VWF clearance was observed in 34% of type 1 cases, 100% of type 1 Vicenza cases, 81% of 2A cases, 77% of 2B cases, 88% of 2M cases, and 36% of 2N cases. ConclusionsAn accelerated VWF clearance was found in most patients with type 2A, 2B, and 2M VWD, with a lower proportion of type 1 and 2N. Sixty-seven different variants alone or in combination with other variants were associated with an increased VWFpp ratio. The variants with the highest VWFpp ratio were mostly located in the D3-A1 VWF domains.

Differences and similarities in endothelial and angiogenic profiles of preeclampsia and COVID-19 in pregnancy

BackgroundCOVID-19 presents a spectrum of signs and symptoms in pregnant women that might resemble preeclampsia. Differentiation between severe COVID-19 and preeclampsia is difficult in some cases.ObjectiveTo study biomarkers of endothelial damage, coagulation, innate immune response, and angiogenesis in preeclampsia and COVID-19 in pregnancy in addition to in vitro alterations in endothelial cells exposed to sera from pregnant women with preeclampsia and COVID-19.Study DesignPlasma and sera samples were obtained from pregnant women with COVID-19 infection classified into mild (n=10) or severe (n=9) and from women with normotensive pregnancies as controls (n=10) and patients with preeclampsia (n=13). A panel of plasmatic biomarkers was assessed, including vascular cell adhesion molecule-1, soluble tumor necrosis factor-receptor I, heparan sulfate, von Willebrand factor antigen (activity and multimeric pattern), α2-antiplasmin, C5b9, neutrophil extracellular traps, placental growth factor, soluble fms-like tyrosine kinase-1, and angiopoietin 2. In addition, microvascular endothelial cells were exposed to patients’ sera, and changes in the cell expression of intercellular adhesion molecule 1 on cell membranes and von Willebrand factor release to the extracellular matrix were evaluated through immunofluorescence. Changes in inflammation cell signaling pathways were also assessed by of p38 mitogen-activated protein kinase phosphorylation. Statistical analysis included univariate and multivariate methods.ResultsBiomarker profiles of patients with mild COVID-19 were similar to those of controls. Both preeclampsia and severe COVID-19 showed significant alterations in most circulating biomarkers with distinctive profiles. Whereas severe COVID-19 exhibited higher concentrations of vascular cell adhesion molecule-1, soluble tumor necrosis factor-α receptor I, heparan sulfate, von Willebrand factor antigen, and neutrophil extracellular traps, with a significant reduction of placental growth factor compared with controls, preeclampsia presented a marked increase in vascular cell adhesion molecule-1 and soluble tumor necrosis factor-α receptor I (significantly increased compared with controls and patients with severe COVID-19), with a striking reduction in von Willebrand factor antigen, von Willebrand factor activity, and α2-antiplasmin. As expected, reduced placental growth factor, increased soluble fms-like tyrosine kinase-1 and angiopoietin 2, and a very high soluble fms-like tyrosine kinase-1 to placental growth factor ratio were also observed in preeclampsia. In addition, a significant increase in C5b9 and neutrophil extracellular traps was also detected in preeclampsia compared with controls. Principal component analysis demonstrated a clear separation between patients with preeclampsia and the other groups (first and second components explained 42.2% and 13.5% of the variance), mainly differentiated by variables related to von Willebrand factor, soluble tumor necrosis factor-receptor I, heparan sulfate, and soluble fms-like tyrosine kinase-1. Von Willebrand factor multimeric analysis revealed the absence of von Willebrand factor high-molecular-weight multimers in preeclampsia (similar profile to von Willebrand disease type 2A), whereas in healthy pregnancies and COVID-19 patients, von Willebrand factor multimeric pattern was normal.Sera from both preeclampsia and severe COVID-19 patients induced an overexpression of intercellular adhesion molecule 1 and von Willebrand factor in endothelial cells in culture compared with controls. However, the effect of preeclampsia was less pronounced than the that of severe COVID-19. Immunoblots of lysates from endothelial cells exposed to mild and severe COVID-19 and preeclampsia sera showed an increase in p38 mitogen-activated protein kinase phosphorylation. Patients with severe COVID-19 and preeclampsia were statistically different from controls, suggesting that both severe COVID-19 and preeclampsia sera can activate inflammatory signaling pathways.ConclusionAlthough similar in in vitro endothelial dysfunction, preeclampsia and severe COVID-19 exhibit distinctive profiles of circulating biomarkers related to endothelial damage, coagulopathy, and angiogenic imbalance that could aid in the differential diagnosis of these entities.

Does Age Affect Desmopressin Responsiveness in Patients with Von Willebrand Disease?

Background: Patients with von Willebrand Disease (VWD) typically undergo a desmopressin acetate (DDAVP) responsiveness challenge at diagnosis to assess whether their coagulation deficits are sufficiently reversed following DDAVP administration. If responsive, patients can be treated with DDAVP in future when at increased risk of bleeding. Current practice assumes that DDAVP responsiveness remains constant over the adult lifetime, and therefore many adult patients are not re-tested. In the normal population, VWF plasma levels (VWF:Ag),VWF ristocetin activity (VWF:RCO) and coagulation factor VIII (FVIII:C) increase with age. This relationship has also been observed in patients with type 1 VWD. We hypothesized that DDAVP responsiveness also increased with age, and set out to investigate this relationship in patients with type 1 VWD.Methods: We conducted a retrospective chart review of 106 patients enrolled at our center since 1990 who met diagnostic criteria for VWD and had undergone at least 1 DDAVP challenge. We recorded baseline, 1 hour, 2 hour and 4 hour post-DDAVP VWF related parameters for every patient when available. Baseline values were taken prior to DDAVP challenge, but may not reflect nadir levels required to diagnose VWD. We excluded patients on medications known to impact VWF and FVIII levels, and patients with types 2B, 2M and 3 VWD due to contraindication or lack of benefit with DDAVP. We stratified analyses by gender to control for the impact of estrogen on VWF and FVIII:C levels. Our primary outcome was DDAVP responsiveness in type 1 VWD, measured as changes in VWF:Ag, VWF:RCo, and FVIII:C levels one hour after DDAVP. As a secondary outcome, we determined these changes in type 2A VWD patients. Analyses were conducted using descriptive statistics, and Pearson and Spearman correlations were used to evaluate for the presence of linear and monotonic relationships, respectively.Results: We had 90 patients (66 female) at our center with type 1 and 16 with type 2A (10 female) VWD who had undergone at least 1 DDAVP challenge. Blood type was available for 66 patients with type 1 VWD; 47 were type O (33 female) and 19 were non-type O (18 female). Mean age at DDAVP challenge for women with type 1 VWD was 23.2 years (SD 15.6) and 27.4 years (SD 19.8) for men. Mean DDAVP dose was approximately 0.3 mcg/kg, with no dose cap. Mean baseline VWF:RCo for type 1 VWD women was 0.39 IU/ml (SD 0.16) and 0.25 IU/ml (SD 0.14) for men, with concordant VWF:Ag mean levels. Mean baseline FVIII:C was 0.74 IU/ml (SD 0.33) and 0.49 IU/ml (SD 0.32) for the women and men, respectively. Blood type was available for 11 patients with type 2A VWD; 6 were type O (5 female) and 5 were non-type O (4 female). In the type 2A group, mean age at DDAVP challenge was 23.7 years (SD 19.0) in women and 50.7 years (SD 18.9) in men. Mean baseline VWF related parameters in type 2A VWD patients were, for women and men, respectively: VWF:Ag 0.48 IU/ml (SD 0.33), VWF:RCo 0.20 IU/ml (SD 0.09), FVIII:C 0.56 IU/ml (SD 0.14) and VWF:Ag 0.50 IU/ml (SD 0.30), VWF:RCo 0.18 IU/ml (SD 0.09), FVIII:C 0.57 IU/ml (SD 0.33).We excluded eleven type 1 VWD patients and one type 2A VWD patient due to medication use. The correlation between age and VWF-related responsiveness to DDAVP at 1 hour in women with type 1 VWD did not reveal a meaningful linear (VWF:Ag, Pearson -0.08, p = 0.62; FVIII:C Pearson 0.04, p=0.77) or monotonic (VWF:Ag, Spearman -0.03, p = 0.86; FVIII:C Spearman 0.12, p=0.40) relationship. However, when we explored the relationship graphically there was an indication of an age-related effect in women of child-bearing years for VWF:Ag, VWF:RCo and FVIII:C (see Figure 1). This finding was not observed in men with type 1 VWD, or in type 2A patients. Regression models including non-linear age terms may assist in explaining this trend.Conclusion: In women with type 1 VWD, we found that responsiveness to DDAVP appears to be amplified around child-bearing years. This trend is reminiscent of the well-established relationship between serum estrogen levels and coaguability. To our knowledge, we have shown for the first time that post-pubertal women with type 1 VWD may have an enhanced ability to respond dynamically to DDAVP but only around this time period. We will confirm our findings with advanced statistical modeling. Future studies should assess whether these age-related changes of coagulation studies translate into meaningful lifecycle-related changes in the bleeding phenotype of type 1 VWD. [Display omitted] DisclosuresJames:Bayer: Research Funding; Shire: Research Funding; CSL Behring: Research Funding. Sholzberg:Shire: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; NovoNordisk: Honoraria; Octapharma: Honoraria, Research Funding.

Type 2A and 2M von Willebrand Disease: Differences in Phenotypic Parameters According to the Affected Domain by Disease-Causing Variants and Assessment of Pathophysiological Mechanisms.

Type 2A and 2M von Willebrand disease (VWD) broadly show similar phenotypic parameters, but involve different pathophysiological mechanisms. This report presents the clinical and laboratory profiles of type 2A and type 2M patients genotypically diagnosed at one large center. Higher bleeding score values and a higher incidence of major bleeding episodes were observed in type 2A compared with type 2M, potentially reflective of the absence of large and intermediate von Willebrand factor (VWF) multimers in 2A. In type 2A, most of disease-causing variants (DCVs) appeared to be responsible for increased VWF clearance and DCV clustered in the VWF-A1 domain resulted in more severe clinical profiles. In type 2M, DCV in the VWF-A1 domain showed different laboratory patterns, related to either reduced synthesis or shortened VWF survival, and DCV in the VWF-A2 domain showed patterns related mainly to shortened survival. VWF-type 1 collagen binding/Ag (C1B/Ag) showed different patterns according to DCV location: in type 2A VWD, C1B/Ag was much lower when DCVs were located in the VWF-A2 domain. In type 2M with DCV in the VWF-A1domain, C1B/Ag was normal, but with DCV in the VWF-A2 domain, C1B/Ag was low. The higher frequency of major bleeding in VWD 2M patients with DCV in the VWF-A2 domain than that with DCV in the VWF-A1 domain could be a summative effect of abnormal C1B/Ag, on top of the reduced VWF-GPIb binding. In silico modeling suggests that DCV impairing the VWF-A2 domain somehow modulates collagen binding to the VWF-A3 domain. Concomitant normal FVIII:C/Ag and VWFpp/Ag, mainly in type 2M VWD, suggest that other nonidentified pathophysiological mechanisms, neither related to synthesis/retention nor survival of VWF, would be responsible for the presenting phenotype.

Emicizumab improves thrombus formation of type 2A von willebrand disease under high shear condition.

Type 2A von Willebrand disease (VWD) is common in type-2 group caused by qualitative deficiency of von Willebrand factor (VWF). Emicizumab is a bispecific antibody that mimics activated factor VIII (FVIIIa) cofactor function, and emicizumab prophylaxis substantially reduces bleeding in patients with haemophilia A. It is unknown whether emicizumab affects thrombus formation in type 2A VWD characterized by not only low FVIII levels but also the impaired platelet adhesion and aggregation. To examine the coagulant potential of emicizumab in type 2A VWD. Perfusion chamber experiments combined with immunostaining were performed using whole blood from 5 patients with type 2A VWD under high shear condition (2500s-1 ). The addition of FVIII to type 2A VWD whole blood did not augment thrombus formation, whilst supplementation with VWF or FVIII/VWF enhanced. FVIII appeared to contribute to thrombus height rather than surface coverage. The addition of emicizumab enhanced thrombus formation in type 2A VWD compared with FVIII, but this potency was less than the presence of VWF. The effect on thrombus formation mediated by emicizumab appeared to be more rapid than that by FVIII for non-requirement of activation step of FVIII, whilst that by FVIII showed more impact on thrombus formation at the late phase. Emicizumab-induced enhancing effects of thrombus formation, independent on VWF, may be useful as an alternative therapy for type 2A VWD patients. These results supported a critical role for the FVIII-VWF complex facilitating thrombus formation under high shear.

Correcting dominant‐negative von Willebrand disease

Correcting dominant‐negative von Willebrand disease

In vivo modulation of a dominant‐negative variant in mouse models of von Willebrand disease type 2A

Unlabelled Box AbstractBackgroundTreatment options for patients suffering from von Willebrand disease (VWD) are limited. Von Willebrand factor (VWF) is a polymeric protein that undergoes regulated dimerization and subsequent multimerization during its biosynthesis. Numerous heterozygous variants within the VWF gene display a dominant‐negative effect and result in severe VWD. Previous studies have suggested that preventing the assembly of wild‐type and mutant heteropolymers using siRNAs may have beneficial effects on VWF phenotypes in vitro. ObjectivesTo study heterozygous dominant‐negative variants in vivo, we developed a mouse model of VWD‐type 2A and tested two independent strategies to modulate its detrimental effect. MethodsThe p.P1127_C1948delinsR deletion/variant, causing defective VWF multimerization, was expressed in mice as a model of VWD‐type 2A variant. Two corrective strategies were applied. For the first time in a mouse model of VWD, we applied siRNAs selectively inhibiting translation of the mutant transcripts and we combined the VWD‐type 2A deletion with the Cys to Arg substitution at position 2773, which is known to prevent dimerization. ResultsThe RNA silencing approach induced a modest but consistent improvement of the VWF multimer profile. However, due to incomplete efficiency, the dominant‐negative effect of the original variant could not be completely prevented. In contrast, the DNA approach resulted in increased antigen levels and restoration of a normal multimer profile. ConclusionsOur data showed that preventing the detrimental impact of dominant‐negative VWF variants by independent molecular mechanisms has beneficial consequences in vivo, in mouse models of dominant VWD.

Shear stress induced activation of von Willebrand factor may predispose to gastrointestinal bleeding in syndrome of Heyde

Abstract Introduction Von Willebrand factor (vWF) changes conformation from globular to fibrillar in the range of shear rates above 5000 s-1. High shear rates, observed in severe aortic stenosis, create conditions for activation of vWF, which opens up access for platelets and coagulation factors to the previously hidden domains of the molecule. At the same time, vWF undergoes increased degradation by metalloproteinase ADAMTS13. Proteolytic cleavage of vWF leads to deficiency of hemostatically active high molecular weight multimers (HMWM) of vWF, while its mass concentration remains unaltered. This results in the development of acquired von Willebrand disease type 2A (vWD) and concomitant gastrointestinal bleeding. The combination of acquired vWD 2A and gastrointestinal bleeding, developed due to severe aortic stenosis, is called Heyde's syndrome. The correlation of shear stress activation of vWF and gastrointestinal bleeding in patients with Heyde's syndrome remains poorly studied. The aim of the study was to measure vWF-mediated platelet adhesion to fibrinogen-coated surfaces under shear rates higher than 5000 s-1 in whole blood samples of healthy volunteers and patients with Heyde's syndrome. Methods A microfluidic system simulating blood flow in vessels was used to assess platelet adhesion. Platelet adhesion was measured by an increase in the intensity of laser radiation scattered from a fibrinogen-coated surface during a 15 minutes circulation of whole blood samples through a flow chamber under shear rates higher than 5000 s-1. Platelets in whole blood samples were activated with 5 μM ADP prior to measurement. The study included 5 patients with Heyde's syndrome 55–80 years old. The control group included 6 healthy volunteers 25–55 years old. vWF-mediated platelet adhesion was detected by blocking platelet-vWF binding with anti-GPIb monoclonal antibody (mAb). Fibrinogen-mediated platelet adhesion was detected by blocking platelet GPIIb/IIIa receptors with mAb. Result The inhibition of GPIb vWF-receptor reduced platelet adhesion by 7.6±3.5% (p<0.05) in patients with Heyde's syndrome, and by 16.5±3.3% (p<0.05) in healthy volunteers. The inhibition of GPIIb/IIIa fibrinogen receptor reduced platelet adhesion by 96±7% (p<0.05) in patients with Heyde's syndrome, and by 80.2±6.6% (p<0.05) in healthy volunteers. Conclusion Significantly reduced contribution of vWF to platelet adhesion under shear rates higher than 5000 s-1 may indicate a decrease in hemostatically active HMWM of vWF. Shear stress activation of vWF in the range of high shear rates and its subsequent inactivation by ADAMTS13 may lead to functional vWF deficiency and the development of gastrointestinal bleeding in Heyde's syndrome. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): The Russian Science Foundation

Acquired von Willebrand Syndrome Associated with Cardiovascular Diseases.

The blood glycoprotein von Willebrand factor (VWF) plays an important role in hemostasis and thrombosis. VWF is produced and secreted as large multimers by endothelial cells and megakaryocytes. It is then cleaved in a sheer-stress dependent manner by a specific protease, ADAMTS13, into multimers consisting of 2–80 subunits. Among VWF multimers, high molecular weight (HMW) multimers play important roles in platelet aggregation. Therefore, their loss induces a hemostatic disorder known as von Willebrand disease (VWD) type 2A. Various cardiovascular diseases, such as aortic stenosis, hypertrophic obstructive cardiomyopathy (HOCM), and several congenital structural diseases, as well as mechanical circulatory support systems, generate excessive high shear stress in the bloodstream. These cause excessive cleavage of VWF multimers resulting in a loss of HMW multimers, known as acquired von Willebrand syndrome (AVWS), a hemostatic disorder similar to VWD type 2A. Bleeding often occurs in the gastrointestinal tract since a fragile angiodysplasia develops associated with these diseases. Radical treatment for AVWS is to remove the pathological high shear causing AVWS.

Differences in von Willebrand factor function in type 2A von Willebrand disease and left ventricular assist device‐induced acquired von Willebrand syndrome

BackgroundPatients with von Willebrand disease (VWD) type 2A or acquired von Willebrand syndrome (aVWS) as a consequence of implantation of left ventricular assist devices (LVAD) are both characterized by a loss of von Willebrand factor (VWF) function. Loss of VWF function is however more severe in VWD type 2A than in LVAD patients. ObjectivesTo compare VWF function in patients with VWD type 2A and LVAD‐induced aVWS to highlight the differences in VWF activity and to stress the importance of VWF multimer analysis for correct diagnosis of aVWS in LVAD patients. Patients/MethodsPlasma samples from nine VWD type 2A, nine LVAD patients, and 20 healthy donors (HD) were analyzed for VWF function (VWF:CB/VWF:Ag and VWF:RCo/VWF:Ag) and loss of high molecular weight (HMW) VWF multimers. ResultsA severely impaired VWF function was indeed confirmed in all VWD 2A patients. HMW VWF multimers were severely reduced compared to HD (0% [0, 12.29] vs 34.19% [31.68, 38.88] for HD, P<0.001) and this loss was reflected by VWF:CB/VWF:Ag and VWF:RCo/VWF:Ag ratios <0.7. In contrast, VWF function was less affected in LVAD patients. Although HMW VWF multimers were reduced in all patients (20.31% [15.84, 21.71], vs 34.19% [31.68, 38.88] for HD, P<0.001), six out of nine LVAD patients had normal VWF:CB/VWF:Ag or VWF:RCo/VWF:Ag ratios (>0.7). ConclusionsVWF:CB/VWF:Ag or VWF:RCo/VWF:Ag analysis allows detection of impaired VWF function in VWD type 2A but not always in LVAD‐induced aVWS patients. In contrast, VWF multimeric analysis allows detection of the loss of HMW VWF multimers in both groups of patients. Hence, performing VWF multimer analysis is crucial to detect aVWS in LVAD patients.

Correction of a dominant‐negative von Willebrand factor multimerization defect by small interfering RNA‐mediated allele‐specific inhibition of mutant von Willebrand factor

Background Treatment of the bleeding disorder von Willebrand disease (VWD) focuses on increasing von Willebrand factor (VWF) levels by administration of desmopressin or VWF-containing concentrates. Both therapies leave the production of mutant VWF unhindered, which may have additional consequences, such as thrombocytopenia in patients with VWD type 2B, competition between mutant and normal VWF for platelet receptors, and the potential development of intestinal angiodysplasia. Most cases of VWD are caused by dominant-negative mutations in VWF, and we hypothesize that diminishing expression of mutant VWF positively affects VWD phenotypes. Objectives To investigate allele-specific inhibition of VWF by applying small interfering RNAs (siRNAs) targeting common single-nucleotide polymorphisms (SNPs) in VWF. This approach allows allele-specific knockdown irrespective of the mutations causing VWD. Methods Four SNPs with a high predicted heterozygosity within VWF were selected, and siRNAs were designed against both alleles of the four SNPs. siRNA efficiency, allele specificity and siRNA-mediated phenotypic improvements were determined in VWF-expressing HEK293 cells. Results Twelve siRNAs were able to efficiently inhibit single VWF alleles in HEK293 cells that stably produce VWF. Transient cotransfections of these siRNAs with two VWF alleles resulted in a clear preference for the targeted allele over the untargeted allele for 11 siRNAs. We also demonstrated siRNA-mediated phenotypic improvement of the VWF multimerization pattern of the VWD type 2A mutation VWF p.Cys2773Ser. Conclusions Allele-specific siRNAs are able to distinguish VWF alleles on the basis of one nucleotide variation, and are able to improve a severe multimerization defect caused by VWF p.Cys2773Ser. This holds promise for the therapeutic application of allele-specific siRNAs in dominant-negative VWD.

Characterization of Von Willebrand Factor Multimer Structure in Patients With Severe Aortic Stenosis.

Acquired von Willebrand syndrome (AVWS) associated with severe aortic stenosis (AS) has been frequently subclassified into a subtype 2A based on the deficiency of high-molecular-weight (HMW) multimers as it is seen in inherited von Willebrand disease (VWD) type 2A. However, the multimeric phenotype of VWD type 2A does not only include an HMW deficiency but also a decrease in intermediate-molecular-weight (IMW) multimers and an abnormal inner triplet band pattern. These additional characteristics have not been evaluated in AVWS associated with severe AS. Therefore, we recruited N = 31 consecutive patients with severe AS and performed a high-resolution Western blot with densitometrical band quantification to characterize the von Willebrand factor (VWF) multimeric structure and reevaluate the AVWS subtype classification. Study patients showed an isolated HMW VWF multimer deficiency without additional abnormalities of the IMW portions and the inner triplet structure in 65%. In conclusion, the multimeric pattern of AVWS associated with severe AS does neither resemble that seen in AVWS type 2A nor that seen in inherited VWD type 2A. Therefore, a subclassification into a type 2A should not be used.

Life-threatening subdural hematoma after aortic valve replacement in a patient with Heyde syndrome: a case report

BackgroundHeyde syndrome is known as a triad of calcific aortic stenosis, anemia due to gastrointestinal bleeding from angiodysplasia, and acquired type 2A von Willebrand disease. This acquired hemorrhagic disorder is characterized by the loss of the large von Willebrand factor multimers due to the shear stress across the diseased aortic valve. The most frequently observed type of bleeding in these patients is mucosal or skin bleeding, such as epistaxis, followed by gastrointestinal bleeding. On the other hand, intracranial hemorrhage complicating Heyde syndrome is extremely rare.Case presentationA 77-year-old woman presented to our hospital with severe aortic stenosis and severe anemia due to gastrointestinal bleeding and was diagnosed with Heyde syndrome. Although aortic valve replacement was performed without recurrent gastrointestinal bleeding, postoperative life-threatening acute subdural hematoma occurred with a marked midline shift. Despite prompt surgical evacuation of the hematoma, she did not recover consciousness and she died 1 month after the operation.ConclusionsPostoperative subdural hematoma is rare, but it should be kept in mind as a devastating hemorrhagic complication, especially in patients with Heyde syndrome.

P3277Distinct differences in laboratory findings of patients with von Willebrand disease type 2A versus patients with LVAD-induced acquired von Willebrand syndrome

P3277Distinct differences in laboratory findings of patients with von Willebrand disease type 2A versus patients with LVAD-induced acquired von Willebrand syndrome

Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism

The large multimeric glycoprotein von Willebrand Factor (VWF) plays a pivotal adhesive role during primary hemostasis. VWF is cleaved by the protease ADAMTS13 as a down-regulatory mechanism to prevent excessive VWF-mediated platelet aggregation. For each VWF monomer, the ADAMTS13 cleavage site is located deeply buried inside the VWF A2 domain. External forces in vivo or denaturants in vitro trigger the unfolding of this domain, thereby leaving the cleavage site solvent-exposed and ready for cleavage. Mutations in the VWF A2 domain, facilitating the cleavage process, cause a distinct form of von Willebrand disease (VWD), VWD type 2A. In particular, the VWD type 2A Gly1629Glu mutation drastically accelerates the proteolytic cleavage activity, even in the absence of forces or denaturants. However, the effect of this mutation has not yet been quantified, in terms of kinetics or thermodynamics, nor has the underlying molecular mechanism been revealed. In this study, we addressed these questions by using fluorescence correlation spectroscopy, molecular dynamics simulations, and free energy calculations. The measured enzyme kinetics revealed a 20-fold increase in the cleavage rate for the Gly1629Glu mutant compared with the wild-type VWF. Cleavage was found cooperative with a cooperativity coefficient n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites of the VWF multimer at the same time. According to our simulations and free energy calculations, the Gly1629Glu mutation causes structural perturbation in the A2 domain and thereby destabilizes the domain by ∼10 kJ/mol, promoting its unfolding. Taken together, the enhanced proteolytic activity of Gly1629Glu can be readily explained by an increased availability of the ADAMTS13 cleavage site through A2-domain-fold thermodynamic destabilization. Our study puts forward the Gly1629Glu mutant as a very efficient enzyme substrate for ADAMTS13 activity assays.

De novo mutation and somatic mosaicism of gene mutation in type 2A, 2B and 2M VWD

BackgroundVon Willebrand disease (VWD) is not uncommon in Taiwan. In type 2 or type 3 VWD hemorrhagic symptoms are severer and laboratory data relatively more distinctive. De novo mutation and somatic mosaicism of type 2 VWD gene were rarely reported. Therefore clinical, laboratory and genetic studies of only type 2A, 2B and 2M VWD will be presented and issues of de novo mutation and somatic mosaicism will be explored.MethodsFifty-four patients belonging to 23 unrelated families from all around the country in whom type 2 VWD exclusive of type 2N has been diagnosed not only by clinical and routine laboratory studies but also by genetic confirmation during 1990–2015 were investigated. A novel technique named amplification refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR) was used to confirm the presence of somatic mosaicism. Informed consent was obtained for study.Results De novo mutation was identified in 4 families among 15 families (26.7 %) in whom family members including parents were available for examination. All their parents were free from bleeding symptoms and had no similar mutation as their respective affected daughter.An interesting example of somatic mosaicism of VWF gene mutation was found in a large family with type 2A VWD. The father carrying a mutated VWF gene, p.Arg1597Trp, transmitted this mutation to his 3 daughters, 1 son, 3 granddaughters and 2 grandsons. However, the father had normal laboratory findings and experienced no abnormal bleeding, while his offspring who inherited the mutation showed abnormal laboratory findings compatible with type 2A VWD and had history of abnormal bleedings. ARMS-qPCR revealed that the father had only 25.5 % mutant in his blood cells and 31.1 % mutant in his oral mucosal cells, while all his offspring had about 49 % mutant in their blood cells.Conclusion De novo mutation of type 2 VWD gene was identified in 4 out of 15 families (26.7 %) examined. Since only one child was affected in each family, germline mosaicism was not likely. A somatic mosaicism of type 2A VWD gene was documented in a big family by a newly in-house developed technique ARMS-qPCR.

Usefulness of the Total Thrombus-Formation Analysis System (T-TAS) in the diagnosis and characterization of von Willebrand disease.

The heterogeneity of von Willebrand disease (VWD) makes its diagnosis a difficult task. We report here on the usefulness of a microchip-based flow-chamber system, the total thrombus-formation analysis system (T-TAS), in the identification and characterization of VWD. Thirty VWD patients and 20 healthy subjects were studied with the T-TAS platelet (PL) and atherome (AR) microchips developed for the in vitro assessment of platelet thrombus formation and fibrin-rich platelet thrombus formation respectively. Samples from severe type 1 VWD, characterized by von Willebrand factor (VWF) levels below 10 U dL-1 , failed to occlude either the PL or the AR chip capillaries, while the occlusion times were normal in patients with mild type 1 VWD (VWF above 25 U dL-1 ). PL and/or AR chip occlusion occurred, but took longer than normal, for samples from type Vicenza and type 1 VWD patients, whose VWF levels ranged between 10 and 25 U dL-1 . No PL or AR chip capillary occlusion was seen for samples from patients with type 2A or 2B VWD featuring the absence of large VWF multimers, whereas no abnormalities emerged for type 2B patients with normal multimer patterns. The T-TAS appears to be sensitive mainly to plasma VWF concentration and the presence of large multimers. Failure of the PL and AR chips to become occluded points to a lack of large VWF multimers, or type 1 VWD with VWF levels below 10 U dL-1 . Although the T-TAS does not assure a precise VWD diagnosis, it does point us in the right direction, and thus seems a useful global preliminary test.

Type 2M and Type 2A von Willebrand Disease: Similar but Different.

Analogous to the differentiation between hemophilia A and B, respectively, reflecting deficiency in factor VIII (FVIII) and FIX, and increasing being recognized as reflecting clinically different disorders, types 2A and 2M von Willebrand disease (VWD) can also be shown to express both similarities and differences in their prevalence, genetic defects, laboratory test results, clinical features, and treatment responses. In this narrative review, we explore these two "subtypes" of type 2 VWD, identifying parallels and dissimilarities in various aspects of their presentation to clinicians and to scientists/laboratories. This differential will become increasingly important as we strive to provide personalized approaches to future management of patients with VWD, particularly in the emerging landscape of recombinant von Willebrand factor.