Absence Of Von Willebrand Factor Research Articles

Unfolded von Willebrand factor binds protein S and reduces anticoagulant activity

AbstractThe critical plasma anticoagulant protein S (PS) circulates in 2 functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP; anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we used biochemical approaches and human patient plasma samples to identify an interaction between PS and von Willebrand factor (VWF), which causes free PS deficiency and reduced PS anticoagulant activity. We first identified a shear-dependent interaction between PS and VWF by mass spectrometry. Consistently, PS and VWF could be crosslinked together in plasma, and plasma PS and VWF comigrated in gel electrophoresis. The PS/VWF interaction was blocked by and tissue factor pathway inhibitor but not activated protein C, suggesting an interaction with the sex hormone binding globulin region of PS. Microfluidic systems demonstrated that PS stably binds VWF because VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation–based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in patients with COVID-19 correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data indicate that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. Because many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.

Prevalence and characterization of anti-VWF antibodies in a population of patients with type 3 VWD

Abstractvon Willebrand disease (VWD) is an inherited bleeding disorder caused by quantitative or qualitative defects in the von Willebrand factor (VWF) protein. Type 3 VWD has a severe bleeding phenotype caused by the absence of VWF, in which treatment usually involves replacement therapy with VWF-containing products. The immune system can react to the VWF product and form anti-VWF antibodies to neutralize or clear the VWF, which can compromise efficacy of treatment or lead to anaphylaxis. Current diagnostic testing is limited to the detection of anti-VWF antibodies that neutralize VWF binding to platelets by using a ristocetin cofactor assay. We set out to develop assays to identify both neutralizing and nonneutralizing antibodies to screen, quantify, and characterize anti-VWF antibodies in samples from the Zimmerman Program, a large multicenter study of patients with VWD. We detected anti-VWF immunoglobulin G (IgG) or IgM antibodies in 18% of 49 unrelated individuals with type 3 VWD. The antibodies ranged in concentration and consisted of 33% nonneutralizing and 67% neutralizing to factor VIII, collagen III, platelet glycoprotein Ib alpha (GPIbα), and/or collagen IV binding. Of the positive type 3 VWD samples, 8 of 9 were IgG, which were further subclassified into mostly IgG1 and IgG4 antibodies. Through a series of testing methods, we identified VWF-specific antibodies in 9 unrelated individuals with type 3 VWD with varying demographics, bleeding phenotypes, and genetic variants. This anti-VWF antibody testing strategy provides a useful tool to assess risk and better navigate treatment options for patients with type 3 VWD.

Novel GPIb-independent platelet aggregation induced by botrocetin: implications for diagnosis and antithrombotic therapy

BackgroundSnake venom botrocetin facilitates von Willebrand factor (VWF) binding to platelet GPIbα and has been widely used for the diagnosis of von Willebrand disease and GPIb-related disorders. Botrocetin is also commonly employed for the development/characterization of antithrombotics targeting the GPIb-VWF axis. ObjectivesTo explore the alternative receptor(s)/mechanisms that participate in botrocetin-induced platelet aggregation. MethodsThe effects of botrocetin on platelet aggregation were examined using platelets from wild-type, VWF- and fibrinogen-deficient, GPIbα-deficient, IL4Rα/GPIbα-transgenic and αIIbβ3-deficient mice and Bernard–Soulier syndrome and healthy human samples. Platelet-fibrinogen and platelet-VWF interaction were measured using flow cytometry. GPIbα-VWF binding was evaluated utilizing enzyme-linked immunosorbent assay. Botrocetin-αIIbβ3 and botrocetin-GPIbα interactions were measured using enzyme-linked immunosorbent assay and fluorescence anisotropy assays. Heparinized whole blood from healthy donors was examined for thrombus formation and growth in a perfusion chamber. ResultsBotrocetin could induce aggregation of platelets from a Bernard–Soulier syndrome patient and GPIbα-deficient mice as well as platelets lacking the N-terminal extracellular domain of GPIbα. Botrocetin could interact with αIIbβ3 and facilitated αIIbβ3-VWF interaction independent of GPIb. Botrocetin competitively bound to the ligand-binding domain of activated rather than resting αIIbβ3. Although botrocetin-induced platelet aggregation requires VWF, strikingly, in the absence of VWF, botrocetin blocked fibrinogen and other ligand binding to αIIbβ3 and inhibited platelet aggregation and thrombus formation. Consistently, recombinant botrocetin defective in VWF binding inhibited αIIbβ3- and GPIb-mediated platelet aggregation, spreading, and thrombus formation. ConclusionOur study provides insights into avoiding the misdiagnosis of GPIb-related disorders and developing botrocetin mutants as potential new antithrombotics that may simultaneously target both αIIbβ3 and GPIbα.

Development of a rapid and fully automated factor VIII inhibitor assay, insensitive to emicizumab, and a lowest level of quantification of 0.2 BU/mL

BackgroundFactor (F)VIII inhibitors are measured using labor- and resource-expensive Nijmegen or Bethesda assays, which lack sensitivity for low-titer inhibitors and show high variations in quality surveys, mainly because of manual assay procedures. ObjectivesThe goal of this study was the development of a fast and fully automated FVIII inhibitor assay by using recombinant (r)FVIII as substrate and dedicated equipment for execution of the test. MethodsA new rapid, fully automated, FVIII inhibitor assay is presented, the core of which is use of full-length recombinant FVIII (rFVIII; Kovaltry, Bayer) as inhibitor substrate instead of plasma FVIII, resulting in rapid binding of inhibitors to rFVIII due to absence of von Willebrand factor. Dramatic shortening of incubation time facilitated full automation on an analyzer capable of 3 subsequent sample dilution steps and 3 reagent additions. Equal volume mixtures of sample and rFVIII (1.0 U/mL) were incubated for 10 minutes at 37 °C, whereafter remaining FVIII activity was analyzed with a kinetic chromogenic assay, allowing inhibitor activity calculation without preceding FVIII activity calibration, using a Ceveron s100 analyzer (Technoclone). ResultsMean titer in 60 nonhemophiliacs was 0.0 BU/mL (SD, 0.1), yielding a limit of blank of 0.1 BU/mL and lower limit of quantification of 0.2 BU/mL. Analyses were performed with the new method and a Nijmegen assay in 28 inhibitor-positive clinical samples, 14 containing emicizumab and 14 without. Correlation coefficient in emicizumab-free type I inhibitor samples was 1.0. Emicizumab dependency of the method was excluded in spiking experiments with inhibitor-positive samples. Reproducibility was tested by analyzing 7 samples in 3 laboratories for 5 days, twice daily; coefficients of variation of all samples were <15%. ConclusionWe present development data of a sensitive and specific rapid, automated FVIII inhibitor assay generating results within 20 minutes that is less resource-intensive than standard assays with potential to improve assay variability.

Cryoprecipitate-Poor Plasma Instead of Fresh Frozen Plasma As Replacement Therapy in Thrombotic Thrombocytopenic Purpura: A Systematic Review and Meta-Analysis

Background: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening condition characterized by the presence of small-vessel thrombi enriched with platelets, leading to thrombocytopenia, thrombotic microangiopathy (TMA), and on occasion, resulting in end-organ damage. The standard therapeutic plasma exchange (TPE) approach, initiated after suspicion of TTP, is done with fresh frozen plasma (FFP). Yet, the use of cryoprecipitate-poor plasma (CPP) has been suggested as alternative in recent decades given its absence of von Willebrand factor, which would benefit the pathophysiology of TTP. However, its efficacy over FFP remains unclear. Therefore, we aimed to conduct a meta-analysis to compare the safety and efficacy of CPP versus FFP in patients with TTP. Methods: We systematically searched PubMed, Cochrane Central and Embase for clinical studies published up to June 2023, comparing CPP to FFP as replacement fluids in TPP or TMA patients. Outcomes assessed were overall mortality, relapse rate, response to treatment and number of TPE sessions. We pooled outcomes as odds ratios (OR) and mean differences (MD) with 95% confidence intervals (CI). We also performed a subgroup analysis with randomized controlled trials (RCTs). Statistical analysis was performed with Review Manager version 5.4.1. Heterogeneity was assessed with I 2 statistics. The protocol was registered in PROSPERO (ID: CRD42023440665). Results: A total of eight articles met inclusion criteria, of which three were RCTs. The combined dataset involved 290 patients, of which 144 (49.7%) were in CPP group and 146 (50.3%) in the FFP group. CPP significantly reduced overall mortality compared to FFP (OR 0.27; 95% CI 0.13-0.59; p=0.001; I 2=0%; Figure 1A). However, our subgroup analysis of RCTs showed no significant difference in overall mortality between groups (OR 0.49; 95% CI 0.13-1.84; p=0.29; I 2=0%; Figure 1B). Additionally, there were no significant differences between groups regarding relapses (OR 0.81; 95% CI 0.37-1.77; p=0.60; I 2=0%; Figure 1C), response to treatment (OR 2.20; 95% CI 0.69-7.07; p=0.18; I 2=44%; Figure 1D) or number of TPE sessions (MD 1.63; 95% CI -2.83 - 6.08; p=0.47; I 2=55%; Figure 1E). Conclusion: Our meta-analysis shows that the safety and efficacy of CPP in TTP patients is at least similar to FFP in reducing mortality, relapse rate, and improving response to treatment. The reduced mortality with CPP found in the whole pool of studies, along with the trend towards reduced mortality found in the RCT-only analysis indicates the potential benefit of CPP over FFP. However, larger population-based randomized trials should contribute to narrow confidence intervals and decrease bias of observational studies, providing clearer conclusions for treatment efficacy in the future.

Novel gene mutation causing type 3 Von Willebrand disease: a case report and review of literature

Von Willebrand disease (VWD) Type 3 is an uncommon bleeding disorder, resulting from the near absence of Von Willebrand factor (VWF) and extremely low factor-VIII levels. It is a close differential diagnosis of hemophilia. A wide heterogeneity of VWD mutations are reported in the literature. We report a 16-year-old girl with hemarthrosis, finally diagnosed with Type 3 VWD. Clinical exome sequencing confirmed the diagnosis, revealing a homozygous mutation c.4387G&gt;T (p. Glu1463Ter) in exon 28 of the VWF gene, a unique mutation not yet reported in the literature.

Von Willebrand factor and cancer: Another piece of the puzzle

Von Willebrand factor and cancer: Another piece of the puzzle

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

BackgroundType 3 von Willebrand Disease (VWD) is a rare and severe form of VWD characterized by the absence of von Willebrand factor (VWF). ObjectivesAs part of the Zimmerman Program, we sought to explore the molecular pathogenesis, correlate bleeding phenotype and severity, and determine the inheritance pattern found in type 3 VWD families. Patients/Methods62 index cases with a pre‐existing diagnosis of type 3 VWD were analyzed. Central testing included FVIII, VWF:Ag, VWF:RCo, and VWFpp. Bleeding symptoms were quantified using the ISTH bleeding score. Genetic analysis included VWF sequencing, comparative genomic hybridization and predictive computational programs. Results75% of subjects (46) had central testing confirming type 3, while 25% were re‐classified as type 1‐Severe or type 1C. Candidate VWF variants were found in all subjects with 93% of expected alleles identified. The majority were null alleles including frameshift, nonsense, splice site, and large deletions, while 13% were missense variants. Additional studies on 119 family members, including 69 obligate carriers, revealed a wide range of heterogeneity in VWF levels and bleeding scores, even amongst those with the same variant. Co‐dominant inheritance was present in 51% of families and recessive in 21%, however 28% were ambiguous. ConclusionThis report represents a large cohort of VWD families in the U.S. with extensive phenotypic and genotypic data. While co‐dominant inheritance was seen in approximately 50% of families, this study highlights the complexity of VWF genetics due to the heterogeneity found in both VWF levels and bleeding tendencies amongst families with type 3 VWD.

Von Willebrand factor propeptide and pathophysiological mechanisms in European and Iranian patients with type 3 von Willebrand disease enrolled in the 3WINTERS‐IPS study

BackgroundType 3 von Willebrand disease (VWD) is a severe bleeding disorder caused by the virtually complete absence of von Willebrand factor (VWF). Pathophysiological mechanisms of VWD like defective synthesis, secretion, and clearance of VWF have previously been evaluated using ratios of VWF propeptide (VWFpp) over VWF antigen (VWF:Ag) and factor (F)VIII coagulant activity (FVIII:C) over VWF:Ag. ObjectiveTo investigate whether the VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios may also be applied to understand the pathophysiological mechanism underlying type 3 VWD and whether VWFpp is associated with bleeding severity. MethodsEuropean and Iranian type 3 patients were enrolled in the 3WINTERS‐IPS study. Plasma samples and buffy coats were collected and a bleeding assessment tool was administered at enrolment. VWF:Ag, VWFpp, FVIII:C, and genetic analyses were performed centrally, to confirm patients’ diagnoses. VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios were compared among different variant classes using the Mann‐Whitney test. Median differences with 95% confidence intervals (CI) were estimated using the Hodges‐Lehmann method. VWFpp association with bleeding symptoms was assessed using Spearman’s rank correlation. ResultsHomozygosity/compound heterozygosity for missense variants showed higher VWFpp level and VWFpp/VWF:Ag ratio than homozygosity/compound heterozygosity for null variants ([VWFpp median difference, 1.4 IU/dl; 95% CI, 0.2–2.7; P = .016]; [VWFpp/VWF:Ag median difference, 1.4; 95% CI, 0–4.2; P = .054]). FVIII:C/VWF:Ag ratio was similarly increased in both. VWFpp level did not correlate with the bleeding symptoms (r = .024; P = .778). ConclusionsAn increased VWFpp/VWF:Ag ratio is indicative of missense variants, whereas FVIII:C/VWF:Ag ratio does not discriminate missense from null alleles. The VWFpp level was not associated with the severity of bleeding phenotype.

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.

Snake Venom Botrocetin Induces GPIb-Independent Platelet Aggregation

AbstractIntroduction：Snake venom-derived botrocetin facilitates von Willebrand factor (VWF) binding to GPIbα, and has been used clinically for the detection of von Willebrand disease (VWD) and GPIb-related disorders. Botrocetin has also been widely used experimentally for the development and characterization of potential antithrombotic drugs targeting the GPIb-VWF axis. Although compelling evidence suggests GPIb is responsible for botrocetin-induced VWF binding and platelet aggregation, some reports suggest that botrocetin could induce platelet aggregation in some Bernard-Soulier syndrome (BSS) patients who lack a functional GPIb complex. However, the alternative mechanism for botrocetin-induced BSS platelet aggregation and the receptor(s) mediating this action are unclear.Methods: Botrocetin was purified from the lyophilized venom of Bothrops jararaca using ion-exchange column chromatography. Light transmission aggregometry assay was performed using platelet-rich plasma (PRP) from human, wild type (WT) mice, GPIbα-deficient mice, αIIbβ3-deficient mice and VWF-deficient mice, or CHO cells stably transfected with αIIbβ3 integrin. O-sialoglycoprotein endopeptidase (OSGE) was used to cleave the N-terminal extracellular domain of GPIbα. The binding of botrocetin, VWF and fibrinogen to platelets from WT or the gene-deficient mice were measured by flow cytometry. Antibodies against GPIbα (SZ2, NIT A) and integrin αIIbβ3 (abciximab, JON/A, M1, PSI E1) were used to investigate the binding site of botrocetin. Perfusion chamber assay was used to measure thrombus formation under different shear stresses.Results: We discovered that botrocetin induced aggregation of human platelets lacking the N-terminal extracellular domain of GPIbα and platelets from GPIbα-deficient mice in the presence of VWF. This VWF-dependent, GPIbα-independent platelet aggregation induced by botrocetin was inhibited by αIIbβ3 antagonists. Botrocetin also induced aggregation of CHO cells stably transfected with αIIbβ3 in VWF-dependent manner. Further experiments with gel-filtered platelets showed that botrocetin competitively bound to the ligand-binding area exposed on αIIbβ3 and blocked fibrinogen and other ligands from binding to the active state of αIIbβ3 in the absence of VWF. Botrocetin inhibited platelet aggregation and thrombus formation in VWF-deficient mice.Conclusion: Integrin αIIbβ3 is the alternative receptor that mediates VWF-dependent, GPIb-independent platelet aggregation induced by botrocetin. However, via targeting αIIbβ3, botrocetin itself inhibits platelet aggregation in the absence of VWF. These results demonstrate versatility in the mechanism of botrocetin, which may provide snakes containing this toxin the adaptability necessary to aggregate platelets/thrombocytes of different prey or predators. Our data reveals a previously unknown role of botrocetin in the integrin-VWF interaction and also provides insight into developing new antithrombotic drugs that target the active conformation of integrin αIIbβ3. The target switching of botrocetin between GPIb-VWF and αIIbβ3-VWF may explain the possible misdiagnosis of the GPIb-related congenital disorders evaluated by botrocetin. DisclosuresNo relevant conflicts of interest to declare.

Genotype-Phenotype Relationship and the Role of Alloantibodies in Type 3 VWD in the Zimmerman Program

Type 3 von Willebrand Disease (VWD) is the rarest and most severe form of VWD and is characterized by the complete absence of von Willebrand factor (VWF). Inheritance is autosomal recessive although heterozygous carriers may be affected with type 1 VWD. As part of the Zimmerman Program for the Molecular and Clinical Biology of VWD (Zimmerman Program) we sought to explore the molecular pathogenesis, correlate bleeding phenotype and severity, and determine the presence and impact of alloantibodies in type 3 VWD.57 index cases (IC) with a pre-existing diagnosis of type 3 VWD by their local treatment center were enrolled into the Zimmerman Program. Clinical VWF testing for phenotypic analysis was done in a central laboratory at BloodCenter of Wisconsin and included FVIII, VWF:Ag, VWF:RCo, VWFpp, VWF:CB and multimer analysis. Full-length exonic VWF sequencing was performed, including intron/exon boundaries. If two sequence variants (SV) were not identified, cases were further investigated by array comparative genomic hybridization (aCGH) to identify any large deletions or duplications in the VWF gene. Bleeding symptoms were quantified using the ISTH bleeding score (ISTH BS). The presence of VWF alloantibodies was determined in type 3 index cases and affected family members (AFM) using an ELISA based assay that detected IgM and IgG antibodies.Of the 57 IC with type 3 VWD enrolled, 42 (74%) had central laboratory testing confirming type 3 (absence of VWF), 7 subjects (12%) had detectable levels and were re-classified as type 1-severe, and 8 subjects (14%) had a significantly increased VWFpp/VWF:Ag and were re-classified as type 1C. Sequencing results were available on 40 of the confirmed 42 type 3 IC. SV were identified in 37 of these subjects (93%): 31 subjects had 2 SVs, 6 subjects had only 1, and 3 had none; 68 of the 80 SVs expected were identified (85%). Of the SVs identified, 37% were frameshift, 29% nonsense, 15% missense, 9% splice site, 7% large deletions detected only by aCGH, and 3% intronic. The large deletions included heterozygous deletions of exon 1-3, exon 4-5, exon 18 and exon 35-38 in combination with another SV. One case only had 2 different deletions (exon 1-3 and exon 4-5) on separate alleles.An equal number (21) of females and males comprised the type 3 cohort with a mean age at enrollment of 24 (range 0-61). Median bleeding score in this group was 15, with no difference between males and females (15). Adults (n=25) had a median ISTH BS of 18 that was significantly increased (p<0.0005) compared to a score of 11 in pediatric subjects (<18 years old, n=17). No difference in ISTH BS was observed according to number of SV or between those with or without null alleles. The highest median bleeding subscores reported were hemarthrosis (3), followed by nose bleeds (2), bruising (2), and oral cavity bleeding (2) and bleeding from minor wounds (1).51 type 3 subjects, including 42 IC and 9 AFM, were tested for alloantibodies to VWF. 16 subjects showed positivity on two separate assays to either IgM or IgG using a 3 SD cutoff above the mean of normal pooled plasma: 4 were markedly positive and 12 were slightly positive. Of the 4 markedly positive subjects, 1 subject had IgM antibodies and 3 had IgG. 3 of the subjects with alloantibodies had no SVs (acquired VWD ruled out), while 1 subject had 2 nonsense variants. ISTH BS was not different between the 16 subjects with an alloantibody (16.5) compared to those without (16). Longitudinal studies will determine the correlation between treatment with VWF concentrate and development of alloantibodies in type 3 VWD.In this type 3 VWD cohort, we have found that some type 1-severe and type 1C patients have been misdiagnosed as type 3. We have also observed that not all type 3 subjects have 2 SV and in fact, 15% of type 3 single alleles did not have an identifiable SV. Null alleles including nonsense, frameshift and large deletions account for 73% of the variant alleles in this cohort. Bleeding symptoms are severe in type 3 and are predominantly due to hemarthrosis, nose bleeds, bruising, oral cavity bleeding and minor wound bleeding. While alloantibodies have been previously associated mainly with homozygous large deletions, our data suggest that type 3 VWD, regardless of the genetic cause, carries a risk of alloantibody formation. DisclosuresFlood:CSL Behring: Consultancy; Shire: Consultancy. Gill:Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Involvement of platelet-derived VWF in metastatic growth of melanoma in the brain.

BackgroundThe prognosis of patients with brain metastases (BM) is poor despite advances in our understanding of the underlying pathophysiology. The high incidence of thrombotic complications defines tumor progression and the high mortality rate. We, therefore, postulated that von Willebrand factor (VWF) promotes BM via its ability to induce platelet aggregation and thrombosis.MethodsWe measured the abundance of VWF in the blood and intravascular platelet aggregates of patients with BM, and determined the specific contribution of endothelial and platelet-derived VWF using in vitro models and microfluidics. The relevance for the brain metastatic cascade in vivo was demonstrated in ret transgenic mice, which spontaneously develop BM, and by the intracardiac injection of melanoma cells.ResultsHigher levels of plasma VWF in patients with BM were associated with enhanced intraluminal VWF fiber formation and platelet aggregation in the metastatic tissue and peritumoral regions. Platelet activation triggered the formation of VWF multimers, promoting platelet aggregation and activation, in turn enhancing tumor invasiveness. The absence of VWF in platelets, or the blocking of platelet activation, abolished platelet aggregation, and reduced tumor cell transmigration. Anticoagulation and platelet inhibition consistently reduced the number of BM in preclinical animal models.ConclusionsOur data indicate that platelet-derived VWF is involved in cerebral clot formation and in metastatic growth of melanoma in the brain. Targeting platelet activation with low-molecular-weight heparins represents a promising therapeutic approach to prevent melanoma BM.

Von Willebrand factor increases in experimental cerebral malaria but is not essential for late‐stage pathogenesis in mice

BackgroundCerebral malaria (CM) is the most severe complication of malaria. Endothelial activation, cytokine release, and vascular obstruction are essential hallmarks of CM. Clinical studies have suggested a link between von Willebrand factor (VWF) and malaria pathology. ObjectivesTo investigate the contribution of VWF in the pathogenesis of experimental cerebral malaria (ECM). MethodsBoth Vwf+/+ and Vwf−/− mice were infected with Plasmodium berghei ANKA (PbANKA) to induce ECM. Alterations of plasma VWF and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), platelet count, neurological features, and accumulation of platelets and leukocytes in the brain were examined following infection. ResultsPlasma VWF levels significantly increased upon PbANKA infection in Vwf+/+ animals. While ADAMTS13 activity was not affected, high molecular weight VWF multimers disappeared at the end‐stage ECM, possibly due to an ongoing hypercoagulability. Although the number of reticulocytes, a preferential target for the parasites, was increased in Vwf−/− mice compared to Vwf+/+ mice early after infection, parasitemia levels did not markedly differ between the two groups. Interestingly, Vwf−/− mice manifested overall clinical ECM features similar to those observed in Vwf+/+ animals. At day 8.5 post‐infection, however, clinical ECM features in Vwf−/− mice were slightly more beneficial than in Vwf+/+ animals. Despite these minor differences, overall survival was not different between Vwf−/− and Vwf+/+ mice. Similarly, PbANKA‐induced thrombocytopenia, leukocyte, and platelet accumulations in the brain were not altered by the absence of VWF. ConclusionsOur study suggests that increased VWF concentration is a hallmark of ECM. However, VWF does not have a major influence in modulating late‐stage ECM pathogenesis.

Discovery of Type 3 von Willebrand Disease in a Cohort of Patients with Suspected Hemophilia A in Côte d'Ivoire.

BackgroundType 3 von Willebrand disease (VWD) is the most severe form of VWD, characterized by a near-total absence of von Willebrand factor (vWF), leading to a massive deficiency in plasmatic factor VIII (FVIII). VWD may be confused with hemophilia A, sometimes leading to misdiagnosis. The purpose of this work was to finalize the biological diagnosis of patients with FVIII activity deficiency in Abidjan in order to guide the best type of management.MethodsWe conducted a cross-sectional descriptive study from June 2018 to April 2019. Forty-nine patients, all of whom had lower FVIII levels or had been referred for a bleeding disorder, were monitored in the clinical hematology service. The pro-coagulant activity of coagulation factors was performed in Abidjan. The assays for von Willebrand antigen and activity were performed at Nîmes University Hospital in France.ResultsThe mean age of patients was 13.8 years (1 – 65) and 86% were Ivorian. FVIII deficiency was discovered during a biological checkup, circumcision or post-traumatic bleeding, in 33%, 31% and 29% respectively. The FVIII deficiency of patients was classified as severe (89.8%), moderate (8.2%) and mild (2%). Only one patient had a quantitative deficiency of von Willebrand factor (vWF: Ag <3%) with undetectable von Willebrand factor activity (vWF: Ac) and an FVIII level <1%.ConclusionsNot all of the congenital deficiency of FVIII are represented by hemophilia A. It was crucial to assess the Willebrand factor of these patients followed in Côte d’Ivoire for whom hemophilia A had been suspected.

The Dendritic Cell HLA-Class-II/Therapeutic Factor VIII (FVIII) Peptidome Is Influenced in Unanticipated Ways By the B-Domain of FVIII and the FVIII Chaperon Protein, Von Willebrand Factor: The Outrigger and Glycosylation-Umbrella (GUMB) Hypotheses

Background: Patients with the X-linked bleeding disorder hemophilia A have impaired blood clotting due to deficient or absent factor VIII (FVIII) coagulant activity. Bleeding can be managed by infusions of any of several plasma-derived (pd) or recombinant (r) therapeutic FVIII proteins (tFVIIIs). The efficacy of tFVIIIs can be eliminated, however, if neutralizing anti-tFVIII-antibodies called "inhibitors" develop. Since the development of inhibitors is T-helper-cell dependent, human leukocyte antigen (HLA)-class-II (HLAcII) molecules comprise an important early determinant. Accumulating evidence suggests that the presence of the FVIII chaperon protein, von Willebrand factor (VWF), in either pdFVIII or rFVIII concentrates, decreases the immunogenicity of these tFVIIIs by reducing their uptake by antigen presenting cells, especially dendritic cells (DCs). Objectives: Use a native (i.e., non-engineered) full-length (FL) tFVIII without ((−)) or with ((+)) pdVWF in DC-protein processing and presentation assays (PPPAs) followed by mass-spectrometric and peptide-proteomic analyses to identify and quantify the DP-, DQ-, and DR-bound/tFVIII-derived-peptides in individual HLAcII repertoires. Compare the number of peptides in the subset from any of the five globular domains (A1, A2, A3, C1 and C2) or three acidic-residue-rich connecting segments (a1, a2 and a3), which we collectively refer to as the non-B-domain (NBD) portion of a tFVIII, with the number of peptides from its non-globular "outrigger like" B-domain (BD) that contains ~80% of the N-linked glycans of a FL-FVIII molecule despite containing only 908 amino acid residues, i.e. slightly less than 40% of its 2,332 total residues. Methods: DC-PPPAs were performed using monocyte-derived (Mo)DCs obtained from 12 healthy blood donors. The tFVIII tested was a FL-rFVIII (Advate®) used (−) or (+) pdVWF (i.e., FL-rFVIII − pdVWF and FL-rFVIII + pdVWF) and the resulting data consists of counts of tFVIII-derived peptides presented on and extracted from HLAcII molecules. Difference of proportion tests were used to compare the effect of pdVWF as well as the NBD- and BD-regions of the FL tFVIII on the peptide counts. Results: FL-rFVIII − pdVWF yielded significantly more peptides (p&amp;lt;0.05) than FL-rFVIII + pdVWF from the NBD portions but not the BD. Interestingly, for the FL-rFVIII − pdVWF preparation, the NBD portions yielded significantly more peptides (p&amp;lt;0.05) than the BD, but this pattern was reversed for the FL-rFVIII + pdVWF preparation in that the NBD portions yielded significantly less peptides (p&amp;lt;0.05) than the BD. Conclusions: The Outrigger Hypothesis posits that in the presence of VWF the heavily glycosylated BD acts as an "outrigger" and renders this portion of a FL tFVIII relatively more likely to be internalized, proteolytically processed and HLAcII-presented. However, in the absence of VWF, the N-linked glycans individually act to protect the amide bonds in the underlying peptide bond backbone of a tFVIII from proteolytic processing, as posited by the GUMB Hypothesis. Our results support both hypotheses as important determinants in the pathogenesis of inhibitor development. Disclosures Howard: Haplogenics Corporation: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Luu:Haplogenics Corporation: Employment. Hofmann:CSL Behring: Employment. Dinh:Haplogenics Corporation: Employment. Mead:CSL Behring: Employment. Powell:Haplogenics: Membership on an entity's Board of Directors or advisory committees. Escobar:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees. Eugene:CSL Behring: Employment.

Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells

BackgroundSynthesis of the hemostatic protein von Willebrand factor (VWF) drives formation of endothelial storage organelles called Weibel‐Palade bodies (WPBs). In the absence of VWF, angiogenic and inflammatory mediators that are costored in WPBs are subject to alternative trafficking routes. In patients with von Willebrand disease (VWD), partial or complete absence of VWF/WPBs may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF using VWD patient–derived blood outgrowth endothelial cells (BOECs) have reported conflicting results due to the intrinsic heterogeneity of patient‐derived BOECs. ObjectiveTo generate a VWF‐deficient endothelial cell model using clustered regularly interspaced short palindromic repeats (CRISPR) genome engineering of blood outgrowth endothelial cells. MethodsWe used CRISPR/CRISPR‐associated protein 9 editing in single‐donor cord blood–derived BOECs (cbBOECs) to generate clonal VWF−/− cbBOECs. Clones were selected using high‐throughput screening, VWF mutations were validated by sequencing, and cells were phenotypically characterized. ResultsTwo VWF−/− BOEC clones were obtained and were entirely devoid of WPBs, while their overall cell morphology was unaltered. Several WPB proteins, including CD63, syntaxin‐3 and the cargo proteins angiopoietin (Ang)‐2, interleukin (IL)‐6, and IL‐8 showed alternative trafficking and secretion in the absence of VWF. Interestingly, Ang‐2 was relocated to the cell periphery and colocalized with Tie‐2. ConclusionsCRISPR editing of VWF provides a robust method to create VWF‐ deficient BOECs that can be directly compared to their wild‐type counterparts. Results obtained with our model system confirmed alternative trafficking of several WPB proteins in the absence of VWF and support the theory that increased Ang‐2/Tie‐2 interaction contributes to angiogenic abnormalities in VWD patients.

Characterization of the mutation spectrum in a Pakistani cohort of type 3 von Willebrand disease.

Type 3 von Willebrand disease (VWD), a severe autosomal recessive hereditary bleeding disorder, is described by the virtual absence of von Willebrand factor (VWF). In consanguineous populations, for example Pakistan, the disease is reported with a higher incidence rate than the worldwide prevalence. This study aims to characterize molecular pathology and clinical profile of type 3 VWD cohort of Pakistani origin. In total, 48 patients were enrolled in the current study. Initially, the index patients (IPs) were evaluated by a standardized questionnaire for recording bleeding manifestations and by performing conventional coagulation tests. The diagnosis of VWD type 3 was confirmed by VWF antigens less than 5IU/dL. Direct sequencing of VWF gene (VWF) was carried out to identify causative gene variations. We evaluated the potential consequence of novel splice site and missense variations by predictive computational programs and in silico structural analysis. VWF mutations were detected in 46 out of 48 IPs (95.8%), predominantly as homozygous variants. In total, twenty-nine different gene defects were characterized in this cohort from which 10 (34.5%) are novel. The majority of the mutations were null alleles (66%; including gene conversions, nonsense, splice site variations, small deletions and insertions), and 34% of them were missense substitutions. Herein, we reported for the first time, the pattern of gene defects in Pakistani type 3 VWD cohort. We identified a wide heterogeneous mutation spectrum along with variability in the type of bleeding episodes.

The endothelial lectin clearance receptor CLEC4M binds and internalizes factor VIII in a VWF‐dependent and independent manner

Essentials CLEC4M is an endocytic receptor for factor FVIII. CLEC4M interacts with FVIII in a VWF-dependent and independent manner. CLEC4M binds to mannose-containing glycans on FVIII. CLEC4M internalization of FVIII involves clathrin coated pits. SUMMARY: Background von Willebrand factor (VWF) and factor VIII (FVIII) circulate in the plasma as a non-covalent complex, and the majority of FVIII is likely to be cleared by VWF-dependent pathways. Clearance of VWF-free FVIII is rapid and underlies the pathological basis of some quantitative FVIII deficiencies. The receptor pathways that regulate the clearance of VWF-bound and VWF-free FVIII are incompletely uncharacterized. The human liver-expressed endothelial lectin CLEC4M has been previously characterized as a clearance receptor for VWF, although its influence on FVIII is unknown. Objective The interaction between FVIII and CLEC4M was characterized in the presence or absence of VWF. Methods FVIII interactions with CLEC4M were evaluated by invitro cell-based and solid phase binding assays. Interactions between FVIII and CLEC4M or liver sinusoidal endothelial cells were evaluated invivo by immunohistochemistry. Results CLEC4M-expressing HEK 293 cells bound and internalized recombinant and plasma-derived FVIII through VWF-dependent and independent mechanisms. CLEC4M binding to recombinant FVIII was dependent on mannose-exposed N-linked glycans. CLEC4M mediated FVIII internalization via a clathrin-coated pit-dependent mechanism, resulting in transport of FVIII from early and late endosomes for catabolism by lysosomes. In vivo hepatic expression of CLEC4M after hydrodynamic liver transfer was associated with a decrease in plasma levels of endogenous murine FVIII:C in normal mice, whereas infused recombinant human FVIII was associated with sinusoidal endothelial cells in the presence or absence of VWF. Conclusions These findings suggest that CLEC4M is a novel clearance receptor that interacts with mannose-exposed glycans on FVIII in the presence or absence of VWF.

Molecular Aggregation of Marketed Recombinant FVIII Products: Biochemical Evidence and Functional Effects.

Background Recombinant (rec-) coagulation factor VIII concentrates available for hemophilia A (HA) treatment differ in cell line production and structure, which could affect their pharmacodynamics and immunogenicity. Clinical trials showed that previously untreated patients with severe HA present higher rates of inhibitor development if treated with rec-FVIII products and that differences do exist as to inhibitor's formation among different rec-FVIII products. This finding could arise from several causes, such as absence of von Willebrand factor, different glycosylation profiles, or processes of molecular aggregation of the recombinant FVIII molecules. Objectives/Methods In this study, using size exclusion high-performance liquid chromatography (SE-HPLC), dynamic light scattering (DLS) spectroscopy, and functional biochemical assays, we investigated the purity grade, FX activating ability, and aggregation status of three recombinant marketed products (Advate [Baxalta], Refacto AF [Pfizer], and Kogenate [Bayer]). Results The overall analysis of the results obtained with SE-HPLC and DLS spectroscopy showed that the three recombinant FVIII concentrates contain low but significant amounts of molecular aggregates. This phenomenon was less evident for the Advate product. Molecular aggregation negatively affects the in vitro pharmacodynamics of the concentrates with higher aggregates' content. Conclusions This study shows that the three pharmaceutical formulations of recombinant FVIII contain variable amounts of molecular aggregates after their reconstitution at therapeutic concentrations. This phenomenon negatively affects the in vitro potency of the products with higher aggregates' content and might be invoked as a contributing cause of their increased risk to induce the formation of FVIII inhibitors.