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

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 161-161
Author(s):  
Tom E. Howard ◽  
Bernadette W. Luu ◽  
Marco Hofmann ◽  
Marcio A. Almeida ◽  
Satish Kumar ◽  
...  
Keyword(s):  
Factor Viii ◽  
Board Of Directors ◽  
Von Willebrand Factor ◽  
Human Leukocyte ◽  
Class Ii ◽  
Hla Class Ii ◽  
Proteolytic Processing ◽  
Advisory Committees ◽  
Von Willebrand ◽  
Willebrand Factor

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<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<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<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.

scholarly journals Treatment of Haemophilia a (HA) Patients with Inhibitors: Immune Tolerance Induction with a Single Factor VIII/Von Willebrand Factor Concentrate in an Observational Immune Tolerance Induction Study (ObsITI)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2482-2482
Author(s):  
C. Escuriola Ettingshausen ◽  
Erik Berntorp ◽  
Yesim Dargaud ◽  
Zeynep Gutowski ◽  
Claude Negrier ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Von Willebrand Factor ◽  
Immune Tolerance ◽  
Research Funding ◽  
Tolerance Induction ◽  
Advisory Committees ◽  
Immune Tolerance Induction ◽  
Inhibitor Titre ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Introduction and objectives: Development of neutralising inhibitors against factor VIII (FVIII) is one of the most serious and costly complications in the treatment of HA. An ongoing international, open-label, uncontrolled, multicentre observational study, ObsITI (ClinicalTrials.gov. NCT 02207894) started in 2005 to assess immune tolerance induction (ITI), the standard of care in patients with inhibitors. The study evaluates patient- and therapy-related variables on ITI course, outcome and morbidity in HA patients with inhibitors. ObsITI satellite studies additionally look at other factors related to tolerisation. Methods and Materials: As of February 2018, 193 patients from 20 countries undergoing ITI have been recruited in ObsITI. 152 patients completed the study and 41 are ongoing. A subgroup of more than 80 prospective patients were treated exclusively during the complete ITI course with a single plasma-derived (pd) FVIII concentrate that contains von Willebrand factor (VWF) in a VWF/FVIII ratio of 0.4 (Octapharma AG). According to the recommended Bonn protocol, low responders at ITI start received 50-100 IU FVIII kg-1 daily, or every other day; high responders received 100 IU FVIII kg-1 every 12 hours. Results: In this ongoing study, the majority of patients treated with the pdFVIII/VWF product achieved a negative inhibitor titre. ITI outcome was significantly correlated with the bleeding rate during ITI, the peak titre during ITI, the inhibitor titre at start of ITI >10 BU, and the number of poor prognosis factors. Conclusion: Treatment with this particular pdFVIII/VWF concentrate, mainly according to the Bonn protocol, resulted in a high ITI success rate in HA patients with inhibitors and corroborates previously published success rates (77.1% complete/partial success in 48 inhibitor patients undergoing ITI with the same product). Disclosures Escuriola Ettingshausen: SOBI: Honoraria, Research Funding; Shire: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Novo Nordisk: Honoraria; Roche: Honoraria; Grifols: Honoraria; Pfizer: Honoraria; LFB: Honoraria. Berntorp:Octapharma: Consultancy; CSL Behring: Consultancy; Shire: Consultancy, Other: honoraria for lecturing . Negrier:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Octapharma: Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Alnylam: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; LFB: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sobi/Bioverativ: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta/Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Research Funding. Pavlova:Novo Nordisk: Honoraria; Octapharma: Honoraria. Oldenburg:Chugai: Honoraria, Membership on an entity's Board of Directors or advisory committees; Grifols: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Biogen Idec: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biotest: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals Traumatic Hyphema in a Type 1 Von Willebrand Patient: Clinical Case and Management

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-30
Author(s):  
Jason Lewis ◽  
Jatinder Dhami ◽  
Michael Langue ◽  
Gayle Smink
Keyword(s):  
Intraocular Pressure ◽  
Board Of Directors ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Advisory Committees ◽  
Increased Risk ◽  
Elevated Intraocular Pressure ◽  
Von Willebrand ◽  
Willebrand Factor

Von Willebrand disease (vWD) is the most common inherited congenital bleeding disorder. Type 1 is a deficiency, but not absence, of von Willebrand factor (vWF) and is the most common type. Bleeding episodes in patients with type 1 vWD vary and are related to the relative amount of von Willebrand factor produced. We report a case of a 15-year-old male with mild von Willebrand disease type 1 (vWF: 35%) who presented with a right eye grade lll hyphema and elevated intraocular pressure (IOP) following a traumatic injury. Despite medical management with Diamox, ophthalmic steroids, vWF replacement with Humate-P, the patient had a repeat bleeding event and worsening of his elevated intraocular pressure. Due to concern for corneal blood staining, the patient required surgical intervention with an anterior chamber washout. Von Willebrand factor replacement was given on presentation and prior to the procedure with goal von Willebrand factor (vWF) activity and Factor Vlll (FVlll) levels of 80-100 IU/dL. Factor replacement for a traumatic hyphema is necessary to reduce bleeding, but could lead to increased risk of thrombosis formation and increase risk of corneal staining. Balance between bleeding and thrombosis can lead to treatment challenges in patients with bleeding disorders including vWD. Disclosures Smink: Forma Therapeutics:Membership on an entity's Board of Directors or advisory committees;Highmark Insurance:Membership on an entity's Board of Directors or advisory committees.

scholarly journals Obtaining a Von Willebrand Evaluation at Time of Acute Heavy Menstrual Bleeding Presentation Leads to Overestimation of Von Willebrand Levels

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 627-627
Author(s):  
Megan C. Brown ◽  
Michael H. White ◽  
Robert F. Sidonio
Keyword(s):  
Factor Viii ◽  
Board Of Directors ◽  
Research Funding ◽  
Heavy Menstrual Bleeding ◽  
Menstrual Bleeding ◽  
Quality Improvement Initiative ◽  
Advisory Committees ◽  
Von Willebrand ◽  
Willebrand Factor

Background: Acute heavy menstrual bleeding (HMB) is common for adolescent females, with about a quarter of menstruating females seeking care for HMB over a 3-year time period (O'Brien et al, Blood 2018). Inherited bleeding disorders are common in this adolescent population, identified in 24.6% referred for hematologic evaluation (Zia et al, Blood 2016). The timing and contents of the hemostatic workup for acute HMB in adolescents is extrapolated from adults, although the causes of acute HMB varies significantly between adult women and adolescents. A consensus statement by the American College of Obstetrics and Gynecology recommends obtaining a variety of hemostatic tests including CBC, von Willebrand studies, factor VIII, prothrombin time, partial thromboplastin time, fibrinogen, and thyroid stimulating hormone at the time of presentation (Committee Opinion 557, ACOG 2011). Factor VIII and Von Willebrand studies are known to be increased in the setting of physiologic stress and supplemental estrogen use, questioning their diagnostic accuracy in the setting of acute bleeding. Repeat testing is often required for diagnosis of von Willebrand disease A von Willebrand factor antigen (VWF:Ag) or von Willebrand factor ristocetin cofactor level over 100IU/dL has been shown to have a negative predictive value (NPV) of 95%.(Doshi et al, ASH 2018). Methods: As part of a quality improvement initiative to improve the evaluation and management of adolescents with HMB at Children's Healthcare of Atlanta (CHOA), we instituted an acute HMB protocol for emergency department (ED) and inpatient use. This protocol was implemented at all CHOA emergency departments in metropolitan Atlanta. Subjects were included if they presented with acute HMB as determined by an adapted Philip Menorrhagia Screening Tool. Subjects with a previously diagnosed bleeding disorder, ITP, active rheumatologic disease, cancer, or anticoagulant use were excluded. Descriptive statistics were used to summarize demographics and clinical characteristics. Patients with a positive Philip screen underwent a uniform bleeding inventory and a standardized set of laboratory tests based on the adult consensus statement. Inpatient and outpatient treatments were standardized by hemoglobin level and symptomology. Follow up with hematology and gynecology was encouraged for all. Data was extracted using various heavy menstrual bleeding ICD-10 codes from January 1, 2017 to December 31, 2018. Individuals with von Willebrand studies at baseline and follow up were identified. T-tests and Wilcoxon rank sum tests were utilized to compare VWF:Ag, VWF:RCo and Factor VIII as baseline and follow up. Results: Over a 2-year period, 232 adolescent girls were seen in CHOA EDs for acute HMB with 88 (37.9%) requiring admission and 6 (2.6%) requiring intensive care. The population was primarily African American (63%) with a median age at presentation of 14.8 years (IQR 13.1-16.7). The majority of adolescents had the core hemostatic labs drawn (55.6%) as described per protocol. Thirty-six individuals had baseline and follow up VWD studies. Those with repeat VWD studies were younger (median 13.2 years vs 15.0 years), more commonly white (44.4% vs 21.2%), were more likely to have been admitted (83.3% vs 29.6%) and more likely to have had a hematology follow up appointment (63.4% vs 7.8%). Mean and median VWF:Ag, VWF:RCo and Factor VIII were significantly higher at presentation with HMB than at follow up. Of those with a baseline VWF:Ag and/or VWF:RCo >100, there was a 96.4% NPV for the diagnosis of VWD. For individuals whose initial VWF:Ag and VCWF:RCo were both >100, there was 100% NPV. Conclusions: Among the adolescents cared for at our institution with acute HMB who had confirmatory VWD testing, initial VWF:Ag and VWF:RCo >100 ruled out VWD based on repeat testing. However, poor adherence with hematology or gynecology follow-up may give false reassurance against a diagnosis of VWD. Further improvements of our quality improvement initiative will include a limited hemostatic workup at presentation with a focus on improved adherence to follow up and subsequent hemostatic evaluation. Disclosures White: National Hemophilia Foundation: Other: Shire Clinical Fellowship Program. Sidonio:Kedrion: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Grifols: Membership on an entity's Board of Directors or advisory committees, Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees; Uniqure: Membership on an entity's Board of Directors or advisory committees.

N-Linked Glycans on Therapeutic Factor VIII (FVIII) Proteins Attenuate Immunogenicity Potential: Evidence from Independent HLA-Class-II/FVIII (HLAcII/FVIII) Peptidomes

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 29-30
Author(s):  
Vincent P. Diego ◽  
Bernadette W. Luu ◽  
Hari Movva ◽  
Marco Hofmann ◽  
Marcio A. Almeida ◽  
...  
Keyword(s):  
Protective Effect ◽  
Factor Viii ◽  
Board Of Directors ◽  
Hla Class Ii ◽  
Proteolytic Processing ◽  
Statistical Analyses ◽  
Advisory Committees ◽  
Asparagine Residue ◽  
Peptidomic Profiling ◽  
Combined Data

The role played by N-linked glycans (NLGs) in the immunogenicity of therapeutic Factor VIII (tFVIII) proteins is poorly understood. Our study addresses this question using peptidomic profiling data on HLA-class-II (HLAcII)-presented peptides from dendritic cell (DC)-protein processing and presentation assays (PPPAs) performed across three independent experiments reported in the literature (Sorvillo et al. (2016), Peyron et al. (2018), and Diego et al. (2020)). Assuming that the number of peptides presented on HLAcII molecules is directly proportional to immunogenicity potential (IP), we asked if the NLGs on tFVIII proteins provide some degree of protection from proteolytic processing within DCs for the amino acid (AA) residues in their immediate vicinity. If NLGs are protective, then we expect an attenuated IP, which would reflect in lower counts of associated AAs. We examined the effects of NLGs both pointwise (i.e., at the single glycated asparagine residue) and at the glycosylation umbrella (GUMB), a term we defined as being −5 to +5 AAs from the glycated asparagine residue of all NLGs. Our first step in addressing these effects was to construct 2×2 contingency tables of the number of AA residues in the HLAcII bound and unbound fractions, and the number of AA residues falling either within the set of NLGs or within the set of GUMBs. We statistically evaluated our question by using Fisher's Exact tests of the hypothesis of no association, and calculated the odds ratio (OR) and its 95% confidence interval (CI). Results from the pointwise tests of the effect of NLGs reported in Figure 1 suggest that overall NLGs exert a protective effect in that non-glycated AAs were at significantly greater risk of being in the fraction of peptides bound to and presented by HLAcII molecules. The results from these statistical analyses-listed as OR (95% CI LB, 95% CI UB)-were: 7.0 (2.6, 21.9) for Diego et al.; 5.6 (0.9, 230.6) for Sorvillo et al. with 25 nM tFVIII; 6.5 (1.0, 269.0) for Sorvillo et al. with 50 nM tFVIII; 3.7 (1.1, 19.8) for Peyron et al.; and 4.4 (2.3, 9.3) for all data combined. The data from Sorvillo et al. (2016; for their experiments using 50 nM tFVIII but not for their experiments using 25 nM tFVIII), Peyron et al. (2018), and Diego et al. (2020), as well as the combined data, all revealed significantly greater risk of non-glycated AAs being in the HLAcII bound/presented fraction relative to glycated AAs. While the results from Sorvillo et al. with 25 nM tFVIII was not statistically significant, this was most likely due to the effects on power of its "small sample size" as the data was trending. The results for the GUMB-level tests reported in Figure 2 are even more robust with the data suggesting a protective effect of the GUMBs in that the AAs not located in their immediate vicinity (as defined above) were at significantly greater risk of being in the bound fraction of peptides presented on HLAcII molecules in all experiments, as well as in the combined data. The results from these statistical analyses-listed as OR (95% CI LB, 95% CI UB)-were: 6.0 (4.5, 8.2) for Diego et al.; 19.9 (6.7, 97.6) for Sorvillo et al. with 25 nM tFVIII; 7.3 (3.9, 15.6) for Sorvillo et al. with 50 nM tFVIII; 2.5 (1.8, 3.6) for Peyron et al.; and 3.8 (3.2, 4.6) for all data combined. In conclusion, these results-from the tFVIII proteins tested using peptidomic profiling of HLAcII-presented peptides from DC-PPPAs performed across three independent experiments-demonstrate NLGs provided significant protection from either proteolytic processing in DCs or peptide binding to HLAcII molecules, or both, and thus lower IP. Our results support the conclusion that NLGs play a significant role in the immunogenicity of tFVIII proteins. Disclosures Luu: Haplogenics Corporation: Current Employment. Hofmann:CSL Behring: Current Employment. Dinh:Haplogenics Corporation: Current Employment. Powell:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees. Mead:CSL Behring: Current Employment. Escobar:Takeda: 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; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: 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; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maraskovsky:CSL Behring: Current Employment. Howard:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees.

Platelet Activation In Sitosterolemia Mice

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2022-2022
Author(s):  
Taisuke Kanaji ◽  
Sachiko Kanaji ◽  
Shailendra B. Patel ◽  
Peter J. Newman
Keyword(s):  
Board Of Directors ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Plant Sterols ◽  
Integrin Subunit ◽  
Plasma Samples ◽  
Advisory Committees ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Deficient Mice

Abstract Abstract 2022 Introduction: Sitosterolemia is a rare, autosomal recessive disorder characterized by the accumulation of plant sterols in blood and tissues, and is caused by mutations in one of the adenosine triphosphate-binding cassette (ABC) transporter ABCG5 or ABCG8 genes. Patients with mutations in either of these sterol transport proteins, which normally form a heterodimer sterol egress channel, frequently develop tendon and cutaneous xanthomas and, most importantly, are at risk of developing premature coronary atherosclerosis. Other clinical manifestations include hematological abnormalities such as hemolytic anemia, macrothrombocytopenia, and loss of ristocetin-induced platelet agglutination – a measure of the ability of platelet glycoprotein (GP) Ib to function as a adhesion receptor for von Willebrand factor (VWF). Mice genetically deficient in ABCG5 or ABCG8 fully recapitulate the macrothrombocytopenia and loss of platelet function seen in human sitosterolemia, a condition that can be corrected by treatment with the sterol-absorption inhibitor, ezetimibe. Because the mechanism by which accumulated plant sterols affects platelet size, production, and function is incompletely understood, we further analyzed these traits in Abcg5- and Abcg8-deficient mice in an animal model of sitosterolemia. Methods: Blood was collected every 1–2 weeks and CBC monitored in Abcg5- and Abcg8-deficient mice that had been fed either a high or low plant sterol diet. Expression of platelet receptors was analyzed by both flow cytometry and Western blotting. GPIbα null mice were used as controls since they have similarly enlarged platelets. Platelet microparticles were analyzed by labeling with a mAb specific for GPIIb and Annexin V. Plasma samples were analyzed for von Willebrand factor (VWF) antigen and multimer patterns. Results: Following onset of a high sterol diet, the platelet count decreased over a two week period to less than 30% of that of normal controls in both Abcg5-/- and Abcg8-/- mice. At the same time, the mean platelet volume gradually increased over a 4 week period from a normal value of approximately 7 fl to approximately 10 fl. In addition, an increased number of platelet-derived microparticles were detected in Abcg5-/- and Abcg8-/- mice kept on high sterol diet over a 9 week period, suggesting that low-grade platelet activation was occurring in these mice. Unexpectedly, the surface expression of the GPIIb integrin subunit was decreased in both Abcg5-/- and Abcg8-/- mouse platelets compared to that of similarly large platelets derived from GPIbα-null (Bernard-Soulier) platelets. Intracellular staining revealed that the GPIIb in Abcg8-/- platelets was being internalized and retained within the cell. Both surface-bound and total platelet fibrinogen was increased in Abcg5-/- and Abcg8-/- mice fed a high sterol diet. Finally, plasma VWF antigen levels decreased by approximately 50% within two weeks following switching Abcg5-/- mice from a low-fat to a high-fat diet. Multimer analysis showed loss of high molecular weight multimers in some of the plasma samples. Conclusions: This study demonstrates a heightened state of platelet activation in a murine model of sitosterolemia, leading to VWF and fibrinogen binding, with concomitant internalization of GPIIb. This might explain, at least in part, the cause of macrothorombocytopenia and absence of ristocetin-induced platelet aggregation in sitosterolemia patients. Platelet activation may also lead to calpain activation, resulting filamin A and talin degradation and microparticle production. Future studies aims at identifying the mechanism by which plant sterols or their metabolites elicit these effects on platelets may provide important clues to the ability of dietary lipids to affect platelet activation and the development of atherosclerosis. Disclosures: Newman: New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Children's Hospital of Boston: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Novel Insights into the Clinical Phenotype and Pathophysiology Underlying Low VWF Levels: The Low Von Willebrand Factor in Ireland Cohort (LoVIC) Study

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 873-873
Author(s):  
Michelle Lavin ◽  
Sonia Aguila ◽  
Sonja Schneppenheim ◽  
Niall Dalton ◽  
Jamie M O'Sullivan ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Von Willebrand Factor ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Assessment Tools ◽  
Bleeding Tendency ◽  
Advisory Committees ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Cohort studies have identified VWF gene mutations in ~65% of patients with type 1 Von Willebrand Disease (VWD). However, in patients with mild reductions in Von Willebrand Factor (VWF) levels, VWF mutations are less common and linkage studies suggest that the reduced plasma VWF levels are often independent of the VWFgene. Important clinical questions therefore remain unanswered regarding diagnosis and management of patients with Low VWF (levels 30-50 IU/dL). Evidence-based diagnostic criteria are needed for establishing the diagnosis of Low VWF, and the relationship between Low VWF levels and bleeding phenotype needs to be defined. Furthermore, the molecular mechanisms responsible for the reduced plasma VWF levels remain unclear. To address these questions, we established the Low VWF Ireland Cohort (LoVIC) study. In contrast to previous Type 1 VWD studies, this prospective longitudinal cohort study recruited only patients with Low VWF levels, (defined by bleeding history and lowest VWF levels 30-50 IU/dL on two occasions, three months apart) and recruited 140 adult Irish patients. Although Bleeding Assessment Tools (BATs) have been used in type 1 VWD, their utility has not been studied in patients with Low VWF. As determined using either the ISTH BAT or the Condensed MCMDM-1 VWD score, we observed significant bleeding histories in the majority of LoVIC patients. For example, among female patients (n=112), 77% had positive ISTH BAT scores (≥6) and 72% had positive MCMDM-1 scores (≥4). Importantly, bleeding tendency did not correlate with plasma VWF levels within the 30-50 IU/dL range. To further investigate this bleeding phenotype, hemostatic studies (including platelet aggregation and coagulation factor assays) were performed. Additional mild coagulation defects were identified in only 10 subjects. Furthermore, abnormal multimer patterns were identified in only 3 patients. To investigate the pathophysiology underlying Low VWF levels, plasma FVIII:C and VWF propeptide (VWF:pp) levels were defined. Interestingly, plasma FVIII:C/VWF:Ag ratios were significantly increased in LoVIC patients compared to normal controls (mean 1.3 versus 1.07; p<0.001). In contrast, increased plasma VWF:pp/VWF:Ag ratios > 3 were observed in only 6% of the total cohort. Taken together, these data demonstrate that the reduced plasma VWF levels in patients with Low VWF are predominantly attributable to decreased VWF synthesis and/or secretion rather than enhanced VWF clearance. To determine whether quantitative and/or qualitative abnormalities in platelet (plt)-VWF may influence bleeding phenotype in patients with Low VWF, we studied plt-VWF:Ag and VWF:CB in 50 consecutive LoVIC patients. In keeping with the hypothesis that reduced VWF synthesis plays a key role in the pathogenesis underlying Low VWF levels, plt-VWF:Ag and plt-VWF:CB levels were both significantly reduced in LoVIC patients compared to controls (mean plt-VWF:Ag 0.16 versus 0.21 IU/109/L, p<0.05; mean plt-VWF:CB 0.18 versus 0.34 IU/109/L, p<0.001). To further study the molecular mechanisms underlying Low VWF levels, plasma VWF:Ag and VWF:RCo responses following desmopressin (DDAVP) administration were examined. In 88% of patients, the post-DDAVP peak levels exceeded 100 IU/dL. Importantly, the response to DDAVP was also sustained with both VWF:Ag and VWF:RCo above 100 IU/dL after 4 hours in 72% subjects. These DDAVP responses demonstrate that Weibel Palade body stores of VWF are maintained in patients with Low VWF, and that the regulated pathway of VWF secretion is intact. Furthermore, the sustained plasma VWF response observed following DDAVP supports the hypothesis that enhanced VWF clearance does not play a major role in the pathogenesis of Low VWF levels. In conclusion, we demonstrate that Low VWF levels are associated with a significant bleeding phenotype. In addition, we further show that in the majority of patients with Low VWF levels, the bleeding phenotype cannot be explained by the presence of concomitant bleeding disorders. Finally, our novel data demonstrate that Low VWF levels are due in large part to reductions in VWF synthesis and/or constitutive secretion. Although enhanced VWF clearance may contribute to the pathophysiology in a minority of individuals with Low VWF, the absolute reduction in VWF plasma half-life is usually mild and not sufficient to significantly impact upon the duration of DDAVP-induced VWF response. Disclosures Lavin: Baxalta: Consultancy, Research Funding. O'Sullivan:Pzifer: Research Funding. O'Connell:Baxalta: Consultancy, Research Funding. James:Octapharma: Research Funding; Bayer: Research Funding; CSL Behring: Research Funding; Biogen: Consultancy; Basalt: Consultancy. Di Paola:CSL BEhring: Consultancy; Biogen: Consultancy. O'Donnell:Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Leo Pharma: Speakers Bureau; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
Author(s):  
Suzuki Suzuki ◽  
Morio Arai ◽  
Kagehiro Amano ◽  
Kazuhiko Kagawa ◽  
Katsuyuki Fukutake
Keyword(s):  
Complex Formation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Domain Specificity ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

Molecular Biology of Factor VIII/von Willebrand Factor

1978 ◽  
Vol 40 (02) ◽  
pp. 245-251 ◽  
Author(s):  
D Meyer ◽  
P A Mc Kee ◽  
L W Hoyer ◽  
T S Zimmerman ◽  
H R Gralnick
Keyword(s):  
Molecular Biology ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Von Willebrand ◽  
Willebrand Factor
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