Von Willebrand Factor Propeptide Research Articles

Von Willebrand factor antigen, von Willebrand factor propeptide and ADAMTS13 activity in TIA or ischaemic stroke patients changing antiplatelet therapy

Data are limited on the impact of commencing antiplatelet therapy on von Willebrand Factor Antigen (VWF:Ag) or von Willebrand Factor propeptide (VWFpp) levels and ADAMTS13 activity, and their relationship with platelet reactivity following TIA/ischaemic stroke.In this pilot, observational study, VWF:Ag and VWFpp levels and ADAMTS13 activity were quantified in 48 patients ≤4 weeks of TIA/ischaemic stroke (baseline), and 14 days (14d) and 90 days (90d) after commencing aspirin, clopidogrel or aspirin+dipyridamole. Platelet reactivity was assessed at moderately-high shear stress (PFA-100® Collagen-Epinephrine / Collagen-ADP / INNOVANCE PFA P2Y assays), and low shear stress (VerifyNow® Aspirin / P2Y12, and Multiplate® Aspirin / ADP assays).VWF:Ag levels decreased and VWFpp/VWF:Ag ratio increased between baseline and 14d and 90d in the overall population (P ≤ 0.03). In the clopidogrel subgroup, VWF:Ag levels decreased and VWFpp/VWF:Ag ratio increased between baseline and 14d and 90d (P ≤ 0.01), with an increase in ADAMTS13 activity between baseline vs. 90d (P ≤ 0.03). In the aspirin+dipyridamole subgroup, there was an inverse relationship between VWF:Ag and VWFpp levels with both PFA-100 C-ADP and INNOVANCE PFA P2Y closure times (CTs) at baseline (P ≤ 0.02), with PFA-100 C-ADP, INNOVANCE PFA P2Y and C-EPI CTs at 14d (P ≤ 0.05), and between VWF:Ag levels and PFA-100 INNOVANCE PFA P2Y CTs at 90d (P = 0.03). There was a positive relationship between ADAMTS13 activity and PFA-100 C-ADP CTs at baseline (R2 = 0.254; P = 0.04).Commencing/altering antiplatelet therapy, mainly attributed to commencing clopidogrel in this study, was associated with decreasing endothelial activation following TIA/ischaemic stroke. These data enhance our understanding of the impact of VWF:Ag and VWFpp especially on ex-vivo platelet reactivity status at high shear stress after TIA/ischaemic stroke.

Current Diagnosis of von Willebrand Disease in Italy: 3 Years Following the Release of the International Guidelines.

The American Society of Hematology-International Society on Thrombosis and Haemostasis-National Hemophilia Foundation-World Federation of Hemophilia 2021 International Guidelines (IGL) on von Willebrand disease (VWD) have pointed out many challenges, mainly in the diagnostic approach of VWD patients. To determine the impact of these IGL on the current clinical and laboratory diagnosis of Italian VWD patients, we have recently conducted a survey among 43 centers affiliated with the Italian Association of Hemophilia Centers (AICE). Directors and colleagues responsible for the management of VWD patients were invited to report in a detailed questionnaire how IGL recommendations about the assessment of the specific activities of von Willebrand Factor (VWF) could be applied at their local sites. Results from such a survey showed that bleeding assessment tools, VWF antigen, and factor VIII procoagulant are currently in use in all centers. The automated assays for platelet-dependent VWF activity with or without ristocetin described in IGL have been used since 2021 in 37/43 (86%) centers. Among other laboratory tests, VWF collagen binding, ristocetin-induced platelet agglutination, multimeric analysis, VWF propeptide, VWF:FVIII binding assay were available in 49, 63, 26, 7, and 28% of AICE, respectively. Analyses of VWF gene defects are available only at 3/43 (7%) centers. Desmopressin (DDAVP) infusion trials at diagnosis, with measurements of VWF activities at 1 and 4 hours post-DDAVP, is currently performed at 38/43 (88%) centers. Based on this information, a simplified clinical diagnosis using a few automated tests before and after DDAVP has been proposed. Such a diagnostic approach will be validated prospectively in a large cohort of Italian VWD patients.

A noncanonical splicing variant c.875-5 T > G in von Willebrand factor causes in-frame exon skipping and type 2A von Willebrand disease

IntroductionA novel variant involving noncanonical splicing acceptor site (c.875-5 T > G) in propeptide coding region of von Willebrand factor (VWF) was identified in a patient with type 2A von Willebrand disease (VWD), who co-inherited with a null variant (p.Tyr271*) and presented characteristic discrepancy of plasma level of VWF antigen and activity, and a selective reduction of both intermediate-molecular-weight (IMWMs) and high-molecular-weight VWF multimers (HMWMs). Materials and methodsVWF mRNA transcripts obtained from peripheral leukocytes and platelets of the patients were investigated to analyze the consequence of c.875-5 T > G on splicing. The impact of the variant on expression and multimer assembly was further analyzed by in vitro expression studies in AtT-20 cells. The intracellular processing of VWF mutant and the Weibel-Palade bodies (WPBs) formation was evaluated by immunofluorescence staining and electron microscopy. ResultsThe mRNA transcript analysis revealed that c.875-5 T > G variant led to exon 8 skipping and an in-frame deletion of 41 amino acids in the D1 domain of VWF (p.Ser292_Glu333delinsLys), yielding a truncated propeptide. Consistent with the patient's laboratory manifestations, the AtT-20 cells transfected with mutant secreted less VWF, with the VWF antigen level in conditioned medium 47 % of wild-type. A slight retention in the endoplasmic reticulum was observed for the mutant. Almost complete loss of IMWMs and HMWMs in the medium and impaired WPBs formation in the cell, indicating truncated VWF propeptide lost its chaperon-like function for VWF multimerization and tubular storage. ConclusionsThe VWF splicing site variant (c.875-5 T > G) causes propeptide truncation, severely compromising VWF multimer assembly and tubular storage.

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.

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>A (p.R854Q), c.2572 T>A (p.C858S), and c.2635 G>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.

Clinical and Laboratory Presentation and Underlying Mechanism in Patients with Low VWF.

Low von Willebrand factor (VWF) refers to subjects with plasma levels of 30 to 50 IU/dL. The mechanism of low VWF is poorly understood. We chose to determine the clinical presentation, laboratory phenotype, and underlying mechanisms of low VWF. We included 250 patients characterized with low VWF. The International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT) was used to assess clinical symptoms. To determine the underlying mechanisms of low VWF, we used as markers the VWF propeptide (VWFpp) assay and FVIII:C/VWF:Ag ratio for VWF synthesis and the VWFpp/VWF:Ag ratio for VWF clearance. Results were compared with those of 120 healthy controls. Cases with abnormal screening tests were further evaluated for coagulation factor levels and platelet disorders. The median age of the cohort was 35 years (range 3-85), 21% were children (n = 53), 34% were adult males (n = 85), and 45% (n = 112) were adult females. According to the ISTH-BAT, abnormal bleeding was found in 35% of children, 47% of males, and 49% of females. No association was found between VWF activity levels and ISTH-BAT. Patients showed an overall decreased VWF synthesis/secretion and an enhanced VWF clearance was identified in 33% of them. In 89 patients (36%), there were other hemostasis-related defects, but there was no difference in the ISTH-BAT between the two groups. Our findings indicate that reduced VWF synthesis/secretion and enhanced VWF clearance are major mechanisms of low VWF levels. Patients with low VWF have significant bleeding manifestations. While other hemostasis defects occurred together with low VWF, this combination did not exacerbate clinical symptoms.

VWF–ADAMTS13 axis dysfunction in children with sickle cell disease treated with hydroxycarbamide vs blood transfusion

AbstractPrevious studies have reported elevated von Willebrand factor (VWF) levels in patients with sickle cell disease (SCD) and demonstrated a key role for the VWF-ADAMTS13 axis in the pathobiology of SCD vaso-occlusion. Although blood transfusion is the gold standard for stroke prevention in SCD, the biological mechanisms underpinning its improved efficacy compared with hydroxycarbamide are not fully understood. We hypothesized that the improved efficacy of blood transfusion might relate to differences in VWF–ADAMTS13 axis dysfunction. In total, 180 children with a confirmed diagnosis of SCD (hemoglobin SS) on hydroxycarbamide (n = 96) or blood transfusion (n = 84) were included. Despite disease-modifying treatment, plasma VWF and VWF propeptide were elevated in a significant proportion of children with SCD (33% and 47%, respectively). Crucially, all VWF parameters were significantly higher in the hydroxycarbamide compared with the blood transfusion cohort (P < .05). Additionally, increased levels of other Weibel-Palade body–stored proteins, including factor VIII (FVIII), angiopoietin-2, and osteoprotegerin were observed, indicated ongoing endothelial cell activation. Children treated with hydroxycarbamide also had higher FVIII activity and enhanced thrombin generation compared with those in the blood transfusion cohort (P < .001). Finally, hemolysis markers strongly correlated with VWF levels (P < .001) and were significantly reduced in the blood transfusion cohort (P < .001). Cumulatively, to our knowledge, our findings demonstrate for the first time that despite treatment, ongoing dysfunction of the VWF–ADAMTS13 axis is present in a significant subgroup of pediatric patients with SCD, especially those treated with hydroxycarbamide.

OC 13.1 Scavenger Receptor Stabilin-2 is a Major Regulator of VWF Propeptide Clearance

OC 13.1 Scavenger Receptor Stabilin-2 is a Major Regulator of VWF Propeptide Clearance

Dynamic and functional linkage between von Willebrand factor and ADAMTS-13 with aging: an Atherosclerosis Risk in Community study

Backgroundvon Willebrand factor (VWF) is a multimeric glycoprotein critically involved in hemostasis, thrombosis, and inflammation. VWF function is regulated by its antigen levels, multimeric structures, and the state of enzymatic cleavage. Population studies in the past have focused almost exclusively on VWF antigen levels in cross-sectional study designs. ObjectiveTo identify subjects in the Atherosclerosis Risk in Community study who had persistently low and high VWF antigen over 10 years and to quantify longitudinal changes in the biological activities and cleavage of VWF in these subjects. MethodsWe measured VWF antigen, propeptide, adhesive activities, and cleavage by ADAMTS-13 quantified using a mass spectrometry method that detected the cleaved VWF peptide EQAPNLVY, as well as coagulation factor VIII activity. ResultsWe determined the mean subject-specific increase in VWF to be 22.0 International Units (IU)/dL over 10 years, with 95% between -0.3 and 59.7 IU/dL. This aging-related increase was also detected in VWF propeptide levels, ristocetin cofactor activity, and VWF binding to collagen. We identified 4.1% and 25.0% of subjects as having persistently low (<50 IU/dL) and high (>200 IU/dL) VWF antigen, respectively. Subjects with persistently low VWF had enhanced ristocetin cofactor activity, whereas those with persistently high VWF had elevated levels of ADAMTS-13, resulting in a comparable rate of VWF cleavage between the 2 groups. ConclusionsThese results provide new information about the effects of aging on VWF antigens and adhesive activity and identify a functional coordination between VWF and the rate of its cleavage by ADAMTS-13.

Preparation and Application of Monoclonal Antibody Against Human von Willebrand Factor Propeptide

To develop monoclonal antibodies that can specifically recognize human von Willebrand factor (VWF) propeptide (VWFpp) in plasma, and establish a rapid and reliable method for the detection of VWFpp antigen in plasma by using the double-antibody sandwich ELISA with the obtained anti-VWFpp monoclonal antibody. The recombinant human VWFpp (D1 and D2 regions) protein expressed in eukaryotic cells was used as immunogen to immunize BALB/c mice with routine method, so as to obtain clones of fusion cells. After screening and identification, hybridoma cell lines secreting monoclonal antibodies against VWFpp were selected, and then double-antibody sandwich ELISA assay was used to construct VWFpp antigen detection kit for the determination of VWFpp in human plasma. The levels of VWFpp antigen in plasma of 12 leukemia patients who underwent bone marrow transplantation were dynamically detected. Two hybridoma cell lines that can be subcultured continuously and secrete monoclonal antibodies against VWFpp were obtained and named SZ175 and SZ176 respectively. Identified by ELISA and Western blot, the antibodies could both specifically recognize VWFpp but couldn't recognize mature VWF (without propeptide). Based on the principle of double-antibody sandwich ELISA, monoclonal antibodies SZ175 and SZ176 were successfully made into a kit for detecting VWFpp antigen. The plasma VWFpp levels of leukemia patients before and after bone marrow transplantation were dynamically detected. The results showed that the plasma VWFpp levels of the patients after transplantation were significantly higher than those before transplantation. Two monoclonal antibodies against VWFpp were successfully prepared, and a double-antibody sandwich ELISA detection kit for VWFpp antigen was constructed, which provides a powerful tool for further study on the biological function of VWFpp, the clinical diagnosis and classification of von Willebrand disease (VWD), and the prognostic monitoring of endothelial injury-related diseases.

Impact of transcatheter aortic valve implantation on circulating von Willebrand factor in patients with severe aortic stenosis.

Severe aortic stenosis (AS) can lead to degradation of high molecular weight (HMW) von Willebrand factor (VWF) which can result in haemostatic abnormalities. While studies have explored changes in VWF profiles before and after surgical aortic valve replacement (SAVR), the longer-term changes in VWF profiles pre- and post-transcatheter aortic valve implantation (TAVI) are less understood. Our primary objective was to identify differences in VWF multimer profiles and VWF function pre-TAVI and 1-month post-TAVI. Our secondary objective was to correlate VWF markers with measures of AS severity. Adult patients with severe AS referred for TAVI at our institution were prospectively enrolled in this cohort study. Blood samples were collected for plasma analysis at three time points for all patients: 1day pre-TAVI, 3days post-TAVI, and 1-month post-TAVI. VWF antigen, activity, propeptide, collagen binding, multimers, and factor VIII coagulant activity were determined at each time point. Correlations between VWF parameters and severity of AS were assessed. Twenty participants (15 males, five females) with severe AS were recruited for the study. There was a significant increase in HMW VWF between pre-procedure and 1-month post-TAVI (p<.05). There was a transient increase in VWF antigen levels and activity at 3-days post TAVI that decreased to pre-TAVI levels at 1-month. There were no statistically significant correlations between VWF markers and AS severity. This is the first study to elucidate longer-term (>1week) improvements in HMW VWF after a TAVI procedure in severe AS patients.

Abacavir use is associated with increased prothrombin conversion.

There is ongoing debate as to whether abacavir (ABC) increases the risk for cardiovascular disease(CVD) in people living with HIV (PLHIV) and the mechanisms underlying this possible association. We recently showed that the use of an ABC-containing regimen was independently associated with increased thrombin generation (TG). In the present study, we aim to explore these findings further, by studying the mechanistical processes that underly the global thrombin generation test via thrombin dynamics analysis. Thrombin dynamics analysis can pinpoint the cause of increased thrombin generation associated with ABC-use either to the procoagulant prothrombin conversion pathway or the anticoagulant thrombin inactivation pathway. In this cross-sectional study, 208 virally suppressed PLHIV were included, of whom 94 were on a ABC-containing regimen, 92 on a tenofovir disoproxil fumarate (TDF)-containing regimen, and the remainder on other regimens. We used Calibrated Automated Thrombinography to measure thrombin generation and perform thrombin dynamics analysis. The total amount of prothrombin conversion, as well as the maximum rate of prothrombin conversion were significantly increased in PLHIV on an ABC containing regimen compared to other treatment regimens. The levels of pro- and anticoagulant factors were comparable, indicating that the ABC-induced changes affect the kinetics of prothrombin conversion rather than procoagulant factor levels. Moreover, Von Willebrand Factor (VWF), active VWF and VWF pro-peptide levels were significantly higher in PLHIV than controls without HIV. However, they did not differ between ABC and non-ABC treated participants.

Quantitative super-resolution imaging of platelet degranulation reveals differential release of von Willebrand factor and von Willebrand factor propeptide from alpha-granules

BackgroundVon Willebrand factor (VWF) and VWF propeptide (VWFpp) are stored in eccentric nanodomains within platelet alpha-granules. VWF and VWFpp can undergo differential secretion following Weibel-Palade body exocytosis in endothelial cells; however, it is unclear if the same process occurs during platelet alpha-granule exocytosis. Using a high-throughput 3-dimensional super-resolution imaging workflow for quantification of individual platelet alpha-granule cargo, we studied alpha-granule cargo release in response to different physiological stimuli. ObjectivesTo investigate how VWF and VWFpp are released from alpha-granules in response to physiological stimuli. MethodsPlatelets were activated with protease-activated receptor 1 (PAR-1) activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, secreted protein acidic and cysteine rich (SPARC), and fibrinogen were imaged using 3-dimensional structured illumination microscopy, followed by semiautomated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content. ResultsVWFpp overlapped with VWF in eccentric alpha-granule subdomains in resting platelets and showed a higher degree of overlap with VWF than SPARC or fibrinogen. Activation of PAR-1 (0.6-20 μM PAR-1 ap) or glycoprotein VI (GPVI) (0.25-1 μg/mL CRP-XL) signaling pathways caused a dose-dependent increase in alpha-granule exocytosis. More than 80% of alpha-granules remained positive for VWF, even at the highest agonist concentrations. In contrast, the residual fraction of alpha-granules containing VWFpp decreased in a dose-dependent manner to 23%, whereas SPARC and fibrinogen were detected in 60% to 70% of alpha-granules when stimulated with 20 μM PAR-1 ap. Similar results were obtained using CRP-XL. Using an extracellular anti-VWF nanobody, we identified VWF in postexocytotic alpha-granules. ConclusionWe provide evidence for differential secretion of VWF and VWFpp from individual alpha-granules.

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.

Heterogeneity in the half-life of factor VIII concentrate in patients with hemophilia A is due to variability in the clearance of endogenous von Willebrand factor

BackgroundPrevious studies have reported marked interindividual variation in factor VIII (FVIII) clearance in patients with hemophilia (PWH) and proposed a number of factors that influence this heterogeneity. ObjectivesTo investigate the importance of the clearance rates of endogenous von Willebrand factor (VWF) compared with those of other FVIII half-life modifiers in adult PWH. MethodsThe half-life of recombinant FVIII was determined in a cohort of 61 adult PWH. A range of reported modifiers of FVIII clearance was assessed (including plasma VWF:antigen and VWF propeptide levels; VWF-FVIII binding capacity; ABO blood group; and nonneutralizing anti-FVIII antibodies). The FVIII-binding region of the VWF gene was sequenced. Finally, the effects of variation in FVIII half-life on clinical phenotype were investigated. ResultsWe demonstrated that heterogeneity in the clearance of endogenous plasma VWF is a key determinant of variable FVIII half-life in PWH. Both ABO blood group and age significantly impact FVIII clearance. The effect of ABO blood group on FVIII half-life in PWH is modulated entirely through its effect on the clearance rates of endogenous VWF. In contrast, the age-related effect on FVIII clearance is, at least in part, VWF independent. In contrast to previous studies, no major effects of variation in VWF-FVIII binding affinity on FVIII clearance were observed. Although high-titer immunoglobulin G antibodies (≥1:80) were observed in 26% of PWH, these did not impact FVIII half-life. Importantly, the annual FVIII usage (IU/kg/y) was significantly (p = .0035) increased in patients with an FVIII half-life of <12 hours. ConclusionOur data demonstrate that heterogeneity in the half-life of FVIII concentrates in patients with hemophilia A is primarily attributable to variability in the clearance of endogenous VWF.

The Experimental Gene Therapy on Type 2A/Iic VWD Mouse Model with AAV Vector Delivering Only VWF Propeptide Expression cassette4

Type 2A/IIC von Willebrand Disease (VWD) is caused by mutations in the propeptide of von Willebrand Factor (VWF), and characterized by the impaired multimer assembly. The structural analysis revealed that VWF propeptide (D1D2 domain) serves as a pH-sensing template for VWF multimerization and tubular storage, which implies that D1D2 expression "in trans" has the potential to restore the multimerization defect of type 2A/IIC mutatns. The gene therapy of VWD is challenging due to the huge size of VWF gene, which cannot be packed to the virus vectors, particularly recombinant adeno-associated virus (rAAV). In the pilot studies, we had co-transfected AtT20 cell with plasmid encoding full length VWF, containing the Y87S mutation on propeptide region and causing type 2A VWD phenotype, and plasmid expressing only wild type propeptide of VWF. The co-transfection rescued the multimer formation in a concentration dependent manner (Figure 1A )and corrected the functional defects caused by the Y87S mutation (Figure 1B). Inspired by the results of in vitro studies, we thus have designed a novel gene therapy strategy for type 2A VWD utilizing the machinery of VWF multimer assembling. Instead of trying to squeeze the VWF gene into the AAV vector, we had prepared the AAV vector containing the expression cassette of propeptide region directed by endothelial cell specific promoter FLT-1. The mouse model representing natural occurring VWF Y87S mutation identified in type 2A VWD patient was established by criper9 gene editing technique. The AAV vectors was administered via tail vein injection at a dose of 5Ⅹ1011 genome copies to 6 weeks old male mice to deliver the propeptide of VWF gene and the plasma of the treated mice was collected retroorbital pretreatment and at 2- ,4- and 6-weeks post vector injection. The antigen level of VWF was determined by ELISA and the function activity of VWF was measured by collagen binding assay. The status of VWF multimerization was examined by agarose gel electrophoresis. Although the Y87S mutation led to significant reduce of high molecule weight multimer in mice, unlike observed in mutation harboring patient, the mice still had medium and low molecular weight multimer, maintaining ~30% of activity. The expression of propeptide by AAV delivered vector improved the VWF activity significantly to ~70% of wild type control (Figure 1C), and restored the formation of high molecular weight multimer (Figure 1D). The experimental gene therapy conducted on VWD mouse model showed that it would be efficient to express the propeptide alone to correct the type 2A/IIC VWD defect and the preliminary results provide a novel strategy to overcome technique limits on gene therapy of VWD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Blood Transfusion Ameliorates Free Haem-Induced Loss of Thrombomodulin and Endothelial Cell Activation in Paediatric Sickle Cell Anaemia

Background Sickle cell anaemia (SCA) is responsible for major paediatric morbidity including risk of stroke in up to 10% of children. Mainstays of therapy include hydroxyurea (HU) and chronic blood transfusion (BT), with BT considered gold standard for stroke prevention. However, the biological mechanisms through which BT is significantly superior to HU in reducing stroke risk have not been clearly defined. SCA complications are due in part to free haem-mediated damage, leading to endothelial cell (EC) activation, tissue factor (TF) expression and release of von Willebrand Factor (VWF), which binds platelets and sickle RBCs. Recently loss of EC Thrombomodulin (TM) has been shown to promote EC activation and VWF release and soluble TM (sTM) plays a role in disseminated intravascular coagulation and COVID-19. Hypothesis We hypothesize free haem alters EC TM expression, promoting EC activation and VWF release. Intravascular hemolysis and free haem may be reduced in children on chronic BT, maintaining EC quiescence and enhancing protection against stroke compared with HU. Methods Following ethical approval and informed consent 180 SCA children (median age 12 years, 49% male) were recruited. Steady-state plasma samples were collected prior to BT (n=84) or at outpatient clinic for HU (n=96). Paired crisis samples were obtained in a subset of HU (n=30). EC activation markers and Weibel Palade Body (WPB) proteins were evaluated by ELISA, VWF multimers were assessed by 1.8% agarose gels and ADAMTS13 activity assessed by FRET. Free haem levels were quantified and thrombin generation (TG) assays performed. Factor VIII:C was measured in the National Coagulation Centre at St James's Hospital. Human umbilical vein endothelial cells (HUVEC) were exposed to cell-free haem (Hemin), hydroxyurea, cytokines (IL-1β and TNFα) or patient plasma; VWF was measured in the supernatant and HUVEC TM expression assessed by flow cytometry. Results VWF:Ag and VWF propeptide (VWFpp) levels were significantly increased in HU compared to BT children, suggesting acute EC activation (median plasma VWF:Ag levels 1.29 v 1.03 IU/ml; p=0.0005, median VWFpp levels 1.63 v 1.17 IU/ml p=0.006). Furthermore, plasma levels of other WPB stored proteins were also significantly elevated in the HU cohort, including Angiopoietin-2 (Ang-2) (4214 v 2337 pg/ml, p<0.0001 and Osteoprotegerin (OPG) (1825 v 1507 pg/ml p=0.006). Abnormalities of high-, intermediate- and low molecular weight VWF multimers were noted in HU but not BT. While putative inhibitors of ADAMTS13 were increased in HU, including Platelet Factor 4 and Interleukin-6, ADAMTS13 activity did not differ between groups. FVIII:C levels were significantly elevated in 65% of the total cohort and were higher in HU than BT (1.62 IU/ml v 1.30 IU/ml p<0.0001). Free haem levels were increased in HU versus BT (12.5 v 9.5μM p=0.0003), as well as levels of LDH and reticulocyte %, pointing to increased intravascular hemolysis. EC markers and FVIII:C correlated strongly with hemolytic indices. In addition to increased EC activation, increased endogenous thrombin potential (ETP), peak thrombin and velocity index were observed in HU versus BT, pointing to enhanced TG, possibly driven by elevated circulating TF. TG parameters correlated with plasma FVIII:C levels. Exposure of HUVEC to free haem in vitro resulted in a significant decrease in surface TM expression and increased VWF secretion (p<0.05). Conversely exposure to hydroxyurea or cytokines did not alter TM expression. Exposure of HUVEC to plasma from crisis but not paired steady-state plasma induced EC activation and VWF release compared with control plasma. Importantly, free haem levels ≥20 μM were observed in 25% HU children in steady state and 40% in crisis, compared with 0% of BT. Furthermore, significantly elevated sTM levels in SCA children with a history of cerebrovascular disease compared to those without (5.7 v 4.6 ng/ml p=0.02), suggesting that sTM may predict end-organ dysfunction. Conclusions We have shown in a large cohort of SCA children that EC activation, TG and intravascular haemolysis are increased in HU-treated children compared with BT. Free haem induces loss of EC TM and VWF release in vitro, leading to EC activation. Elevated sTM levels were a biomarker of end-organ damage. These data suggest that reduction of free haem in children undergoing regular BT is a key mechanism through which EC quiescence is maintained in paediatric SCA.

Von Willebrand factor and cancer: Another piece of the puzzle

Von Willebrand factor and cancer: Another piece of the puzzle

Von Willebrand factor propeptide variants lead to impaired storage and ER retention in patient‐derived endothelial colony‐forming cells

Backgroundvon Willebrand factor (VWF) is synthesized by vascular endothelial cells and megakaryocytes. The VWF propeptide is critical for multimerization and acts as an intra‐molecular chaperone for mature VWF in sorting to its storage organelles, Weibel‐Palade bodies (WPBs). In the Canadian Type 3 VWD study, almost half of the identified variants were in the VWF propeptide and these were associated with an increased bleeding phenotype. ObjectiveTo investigate VWF propeptide variants that cause quantitative von Willebrand disease (VWD) by utilizing patient‐derived endothelial colony‐forming cells (ECFCs). Patients/MethodsEndothelial colony‐forming cells were isolated from five Type 3 VWD patients from four families with the following variants: (1) homozygous p.Asp75_Gly178del (deletion of exons 4 and 5 deletion; Ex4‐5del), (2) homozygous p.Cys633Arg, (3) homozygous p.Arg273Trp, and (4) p.Pro293Glnfs*164 and p.Gln419* inherited in the compound heterozygous state. Additionally, ECFCs were isolated from six family members (two Type 1 VWD, four unaffected). ResultsEndothelial colony‐forming cells from the Type 3 patient with the compound heterozygous genotype exhibited a true null VWF cellular phenotype, with negligible VWF detected. In contrast, the other three propeptide variants presented a similar expression pattern in homozygous ECFCs where VWF was synthesized but not packaged in WPBs, and variant VWF had an increased association with the endoplasmic reticulum (ER) marker, protein disulfide‐isomerase (PDI), indicating an ER‐retention phenotype. The biosynthetic phenotype was similar but to a lesser degree in heterozygous ECFCs expressing the non‐null variants. ConclusionThis study further elucidates the importance of the VWF propeptide in the VWD phenotype using patient‐derived cells.

Enhanced VWF clearance in low VWF pathogenesis: limitations of the VWFpp/VWF:Ag ratio and clinical significance

AbstractIncreased von Willebrand factor (VWF) clearance plays a key role in the pathogenesis of type 1 and type 2 von Willebrand disease (VWD). However, the pathological mechanisms involved in patients with mild to moderate reductions in plasma VWF:Ag (range, 30-50 IU/dL; low VWF) remain poorly understood. In this study, we investigated the hypothesis that enhanced VWF clearance may contribute to the pathobiology of low VWF. Patients with low VWF were recruited to the LoVIC study after ethics approval and receipt of informed consent. Desmopressin was administered IV in 75 patients, and blood samples were drawn at baseline and at the 1-hour and 4-hour time points. As defined by recent ASH/ISTH/NHF/WFH guidelines, 20% of our low-VWF cohort demonstrated significantly enhanced VWF clearance. Importantly, from a clinical perspective, this enhanced VWF clearance was seen after desmopressin infusion, but did not affect the steady-state VWF propeptide (VWFpp)-to-VWF antigen (VWF:Ag) ratio (VWFpp/VWF:Ag) in most cases. The discrepancy between the VWFpp/VWF:Ag ratio and desmopressin fall-off rates in patients with mild quantitative VWD may have reflected alteration in VWFpp clearance kinetics. Finally, bleeding scores were significantly lower in patients with low VWF with enhanced VWF clearance, compared with those in whom reduced VWF biosynthesis represented the principle pathogenic mechanism. This trial was registered at http://www.clinicaltrials.gov as #NCT03167320.