Von Willebrand Factor Expression in Endothelium in a Co-Culturing System of Acute Lymphoblastic Leukemia

Abstract Background: Acute lymphoblastic leukemia (ALL) is the most common cause of cancer and death from cancer among children, with an incidence of 1 in 1,750 children. A previous Dutch cohort study highlighted that 7.6% of children with ALL also developed venous thromboembolism (VTE; n=55/778). VTE increases the risk of morbidity and mortality in cancer patients. However, the mechanisms of hemostatic abnormality in ALL is not well understood. Von Willebrand factor (vWF) is involved in thrombosis and is a well-established marker for endothelial dysfunction. High vWF levels and qualitative abnormality of the protein have been documented in patients with ALL and may contribute to the prothrombotic state observed in these patients. Objective: To determine whether co-culturing cancerous ALL cells with healthy human umbilical vein endothelial cells (HUVECs) may increase vWF secretion from endothelial cells. Methods: HUVECs were obtained from Lonza Bioscience Solutions (Basel, Switzerland) and leukemic B-lymphoblasts (ALL cells) derived from bone marrow were obtained from ATCC (Manassas, Virginia, USA). Passages 3 to 5 were used in this study and cell lines were grown as per manufacturer's instruction. A confluent monolayer of HUVECs was established before the co-culturing experiments. Firstly, ALL cells and HUVECs were grown together (co-cultured) at a 1:1 ratio. Then, the conditioned media and cell lysates were harvested at three different time points and the vWF levels were quantitated by a vWF-ELISA. To further evaluate the effect of leukemic cells on the endothelium, ALL cells were also co-cultured at a 10:1 ratio to endothelial cells. All experiments were conducted in biological triplicates. Results: Cells were monitored throughout the study period and no morphological changes to the endothelial cells were observed. An accumulation of vWF was recorded over time in both the conditioned media and cell lysates. The differences in vWF levels between the co-culture group and HUVEC-only group at three time points (24, 48 and 72 hours) were not statistically significant. In addition, 10-fold differences in ratio between leukemic and endothelial cells did not have a meaningful effect on vWF levels. Discussions: In an in vitro co-culture system, ALL cells do not appear to induce an increase vWF secretion in HUVECs. Contrary to prior evidence, this study suggests that increased vWF secretion from endothelial cells may not be a contributor to the prothrombotic state observed in ALL. The circulating lymphoblasts may have a more pronounced effect on other endothelial layers where vWFs are highly expressed such as the pulmonary endothelium. On the other hand, peripheral blood pediatric leukemic lymphoblasts have been demonstrated to produce hemostatic factors, which may contribute the hemostatic imbalance in the progression of disease. In addition, the pathogenesis of thrombosis in ALL is likely to be therapeutically related since thrombosis rarely occurs at presentation. Alternative mechanisms, such as ADAMST13 and hemostatic factors' level and function or the use of chemotherapeutic agents, ought to be explored. Figure 1 Figure 1. Disclosures Matino: Bayer: Membership on an entity's Board of Directors or advisory committees, Other: research grants and personal fees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: research grants and personal fees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Other: research grants and personal fees; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: research grants and personal fees; Spark: Other: research grants; Octopharma: Membership on an entity's Board of Directors or advisory committees, Other: research grants and personal fees; Sobi: Membership on an entity's Board of Directors or advisory committees, Other: personal fees.

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HSCT-Associated Hepatic VOD Is Initiated With Preceding Appearance Of Unusually Large Von Willebrand Factor Multimers In Patient Plasmas

Blood ◽

10.1182/blood.v122.21.3625.3625 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3625-3625

Author(s):

Masaki Hayakawa ◽

Masanori Matsumoto ◽

Yumi Yoshii ◽

Hideo Yagi ◽

Hiroshi Kimura ◽

...

Keyword(s):

Body Weight ◽

Board Of Directors ◽

Plasma Levels ◽

Combination Regimen ◽

Adamts13 Activity ◽

Advisory Committees ◽

Von Willebrand ◽

Willebrand Factor ◽

Frozen Plasma ◽

Platelet Transfusions

Abstract Backgrounds and Aims Hepatic veno-occlusive disease (VOD) associated with hematopoietic stem cell transplantation (HSCT) is characterized by a clinical triad of jaundice (total bilirubin, >2 mg/mL), hepatomegaly with right upper quadrant pain, and ascites and/or unexplained body weight gain (>5% of baseline) within 30 days after operation. Although the pathogenesis of hepatic VOD has not been fully elucidated, the common pathological features are thrombi formed in hepatic central vein. We previously reported that a significant decrease of plasma ADAMTS13 activity was noted in patients undergoing HSCT, who subsequently developed VOD (Park et al, BMT 2002). Then, we showed that plasma antigen levels of von Willebrand factor (VWF) has been kept higher in HSCT-patients with VOD than in those without, and in fact prophylactic infusions of fresh frozen plasma (FFP) with a dose of 10 ml/kg body weight 3 times per week were effective to reduce the frequency of VOD occurrence in high risk patients (Matsumoto et al, BMT 2007). However, more recent studies by ours indicate that FFP infusion alone is not enough to totally eliminate the occurrence (unpublished). Recently, it has been shown that the treatment with recombinant soluble thrombomodulin (rTM) is sometimes highly efficient to reverse VOD progression. But its pharmacokinetics and regimen for the treatment has not been established. As a first step to elucidate a possible combination regimen of FFP and rTM to VOD patients, here we have analyzed the transitional changes of unusually large VWF multimers (UL-VWFMs). The UL-VWFMs are released from damaged endothelial cells and induce platelet hyperagglutination under high shear stress generated in microvasculatures, and are often observed in patient plasmas undergoing HSCT. Patients and Methods During 2011-2012, 45 patients were received allogenic HSCT in the second internal medicine department of our university hospital. None of these patients ,however, were received planned prophylactic FFP infusions, and as a result six patients undergoing allogenic cord blood transplantation (CBT) developed VOD. Clinical features of these 6 patients are shown in Table 1. Under approval of Ethics Committee of Nara Medical University, we consecutively collected patient's citrated plasmas (ca 2.5 ml) and stored at -80°C until use. Using these deep-frozen plasma samples, we here extensively analyzed plasma levels of ADAMTS13 activity (ADAMTS13:AC), VWF antigen (VWF:Ag), and VWF collagen binding activity (VWF: CBA), together with VWF multimer analysis. More importantly, we also measured the corrected platelet count increment (CCI) to evaluate the efficiency of Platelet trsnsfusions. Then, we comprehensively evaluated these data with routine clinical and laboratory findings. Results and Discussion 1) Plasma levels of ADAMTS13:AC were moderately but consistently decreased during two months after HSCT, whereas those of VWF:Ag were kept high, usually more than 200% and often 500% of the normal. 2) The UL-VWFMs appeared soon after HSCT, and continued at least until absolute neutrophil count (ANC) increased to >500 (usually 20-30 days after HSCT). 3) Until platelet engraftment (usually 40-60 days after HSCT), platelet transfusions (every 2-3 days interval) are usually performed to prevent the bleeding complications. During that period, the CCI values were consistently low, but those values were significantly increased during the administration of rTM. 4) Excess platelet transfusions before platelet engraftment induced the consumption of larger VWFMs in patient's plasmas, and often almost simultaneously hepatic VOD developed. Thus, platelet transfusions during the appearance of UL-VWFMs in patient's plasmas may induce platelet clumping in microvasculatures, and lead to the development of thrombotic complications including hepatic VOD. 5) The measurement of plasma levels of VWF:CB activity appeared to well predict the presence of UL-VWFMs. A representative case is shown in Figure. Thus, a combination regimen of FFP and rTM might be advisable when the patients show the early clinical signs of hepatic VOD and the laboratory data such as VWF:CBA suggest the presence of UL-VWFMs in patient's plasmas. Disclosures: Matsumoto: Alexion Pharma: Membership on an entity’s Board of Directors or advisory committees. Fujimura:Alexion Pharma: Membership on an entity’s Board of Directors or advisory committees; Baxter International Inc: Membership on an entity’s Board of Directors or advisory committees.

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Prolonged Circulation of Ultra-Large Von Willebrand Factor and a Reduction in ADAMTS13 Activity Promotes Microvascular Disease Following Traumatic Injury

Blood ◽

10.1182/blood-2019-124531 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 444-444 ◽

Cited By ~ 1

Author(s):

William E Plautz ◽

Mitchell Dyer ◽

Margaret V. Ragni ◽

Shannon Haldeman ◽

Wyeth E Alexander ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Traumatic Injury ◽

Microvascular Disease ◽

Severe Trauma ◽

Trauma Patients ◽

Adamts13 Activity ◽

Advisory Committees ◽

Von Willebrand ◽

Willebrand Factor

Introduction: Increases in plasma von Willebrand Factor (vWF) levels, accompanied by decreases in its respective metalloprotease, ADAMTS13, have been demonstrated in diseases of microvascular injury. We hypothesized that following severe trauma, a burst of ultra-large vWF (UL-vWF) is released into the bloodstream by damaged endothelium, resulting in increased thrombogenicity due to circulating vWF multimers. We further hypothesized that traumatic injury would lead to a deficit of ADAMTS13, promoting the accumulation of UL-vWF forms and, ultimately, the increased risk of microvascular disease, such as acute kidney injury (AKI). Methods: A cohort of 37 severely injured trauma patients was analyzed for antigen levels of plasma vWF and ADAMTS13 at 0- and 24-hours after admission. Circulating vWF multimeric composition from both time points was determined by vertical agarose gel electrophoresis. Multivariate analyses were performed with data abstracted from the electronic medical records to identify further dependences. A similar analysis was also performed on plasma from a cohort of 8 patients with trauma induced AKI at 0-, 24-, and 72-hours after admission; these patients were well matched against trauma patients without AKI. Finally, we utilized a murine model of polytrauma and hemorrhage, in conjunction with qRT-PCR of ADAMTS13 in total liver RNA, to specifically address how the expression of ADAMTS13 is altered by the systemic effects of traumatic injury. Results: Circulating vWF levels were increased in severe trauma patients when compared to healthy controls at presentation (189% (110-263) vs. 95% (74-120)) and persisted through 24-hours (213% (146-257) vs. 132% (57-160)). Ultra-large vWF forms were elevated at both 0- and 24-hours when compared to pooled normal plasma ((10.0% (8.9-14.3) and 11.3% (9.1-21.2), respectively, vs 0.6%). The largest vWF forms within trauma patient plasma circulated at 33±4 dimers vs 18±1 dimer in length within pooled normal plasma. Severe trauma patient ADAMTS13 activity was decreased at 0-hours (66% (47-86) vs. 100% (98-100)) and at 24-hours (72.5% (56-87.3) vs 103% (103-103)) when compared to healthy patients. Furthermore, the proportion of circulating low molecular weight multimeric (LMWM) vWF to total circulating vWF forms was directly dependent upon ADAMTS13 activity at 24-hours (Decreased ADAMTS13 Activity: 20.4% (15.0-22.7) LMWM vWF vs Normal Activity: 25.8% (22.7-35.2) LMWM vWF). Strikingly, ADAMTS13 activity independently predicted the development of coagulopathy, correlating with presentation INR (ρ =-0.63), activated clotting time of thromboelastography (TEG) (ρ=-0.36), and TEG maximum amplitude (ρ=0.36). ADAMTS13 activity also closely correlated with injury severity (ISS) (ρ=-0.34) and blood product transfusion (ρ =-.45). The cohort of 8 trauma patients who went on to develop AKI showed a 1.54-fold (1.02-2.05) increase in plasma vWF antigen levels between 0 and 72 hours, while those who did not develop AKI showed no change in vWF levels over this time period. Furthermore, those who developed AKI demonstrated a smaller proportion of LMWM vWF in plasma than those who did not (25.4% (23.4-28.0) vs 31.2% (27.2-35.6)), suggesting the increased thrombogenicity of their circulating vWF forms. Finally, qRT-PCR of total liver RNA in 6 mice demonstrated a 2-fold decrease in ADAMTS13 RNA expression levels between the times immediately before and 24-hours after trauma. Altogether, these data indicate that both circulating ADAMTS13 and its production are deficient in the days following severe injury. Conclusions: Severe traumatic injury alters the circulating composition of ADAMTS13 and its target, vWF, shifting their equilibrium to one that promotes thrombosis. Not only is the concentration of circulating ADAMTS13 decreased following traumatic injury, but hepatic expression of the enzyme is lacking as well. In the immediate moments following injury, these mechanisms contribute to life-saving hemostasis; however, as these changes extend into the following days, the early hemostatic benefit quickly shifts to burden that may exacerbate microvascular disease. Disclosures Ragni: Bioverativ/Sanofi: Consultancy, Research Funding; Sangamo: Research Funding; Shire/Takeda: Consultancy, Other: Study drug; Alnylam/Sanofi: Consultancy, Research Funding; Bayer: Consultancy; Spark Therapeutics: Consultancy, Research Funding; ICER: Consultancy; OPKO: Research Funding; Biomarin: Consultancy, Research Funding. Rollins-Raval:Bayer, Inc: Membership on an entity's Board of Directors or advisory committees. Raval:Sanofi: Membership on an entity's Board of Directors or advisory committees; Bayer, Inc: Research Funding. Neal:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

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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 ◽

10.1182/blood-2018-99-114408 ◽

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.

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

Blood ◽

10.1182/blood-2020-140435 ◽

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.

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

Blood ◽

10.1182/blood-2019-127258 ◽

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.

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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 ◽

10.1182/blood-2019-131727 ◽

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.

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Antiphospholipid Antibodies Increase Von Willebrand Factor-Platelet String Formation From Human Endothelial Cells Under Physiologic Flow Conditions

Blood ◽

10.1182/blood.v122.21.2305.2305 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2305-2305

Author(s):

Christopher J. Ng ◽

Keith R. McCrae ◽

Junmei Chen ◽

Michael Wang ◽

Marilyn J. Manco-Johnson ◽

...

Keyword(s):

Endothelial Cells ◽

Board Of Directors ◽

Antiphospholipid Antibodies ◽

Research Funding ◽

Umbilical Vein ◽

Shear Stresses ◽

Advisory Committees ◽

String Formation ◽

Umbilical Vein Endothelial Cells ◽

Von Willebrand

Abstract Background The antiphospholipid syndrome (APS) is characterized by a predisposition to arterial and venous thromboembolism. The underlying mechanism of thrombosis in APS is uncertain, but is thought to involve a multifactorial activation of various components of the blood and vasculature. The role of von Willebrand factor (VWF), a plasma protein expressed by endothelial cells in response to endothelial cell damage or activation, is not clearly defined in APS. Previous studies have demonstrated that antiphospholipid antibodies increase the release of soluble VWF from human umbilical vein endothelial cells. However, these experiments were performed in static conditions and not in a flow based model. Recent investigations of expressed ultra large VWF (ULVWF) under flow have led to a new model of VWF-platelet strings that remain anchored to endothelial cells and provide a nidus for the formation of thrombi. Consistent with this, increases in VWF and variations in ADAMTS13, the enzyme that modulates ULVWF, have been associated with venous and arterial thrombosis. Objectives We hypothesized that antiphospholipid antibodies would induce VWF-platelet strings on endothelial cells and that this mechanism contributes to APS-associated thrombosis. Methods Human umbilical vein endothelial cells were seeded in flow chambers prepared with a collagen substrate. All experiments were performed with cells of passage 5 or under. Confluent cells were incubated in serum-free medium with APS-derived IgG or anti-β2-GPI antibodies vs. control IgG. After incubation, the slides were perfused with lyophilized platelets and washed with Tyrode’s buffer prior to image acquisition. Shear stresses were varied from 2-10 dyn/cm2 to assess for shear stress dependent variations in string formation. Pre-determined sites of imaging were set via a standardized method with relief contrast brightfield microscopy. Between 50-100 images were collected per condition. A VWF-platelet string-unit was defined as a 25 µm length of an individual VWF-platelet string. Images were analyzed for the number of VWF-platelet string units per image; quantification of string units was performed blinded to sample vs. control IgG treatment status. Data are shown as means +/- SEM, and significance was determined as p<0.05 by paired t-test. Results Endothelial cells treated with human antiphospholipid antibodies demonstrated increased VWF-platelet strings at two different shear stresses, one mimicking venous flow and one mimicking arterial flow. At 2 dyn/cm2, cells incubated for 1 hour with 100 µM of APS patient–derived IgG demonstrated 23.04 +/- 2.139 (SEM) VWF-platelet string units/image vs. 17.64 +/- 1.203 in cells treated with 100 µM control human IgG (p=0.0247). At 10 dyn/cm2, cells incubated for 6 hours with 600 nM of human anti-β2-GPI antibodies demonstrated 1.848 +/- 0.2866 (SEM) VWF-platelet string units/image vs 1.048 +/- 0.2350 in cells treated with 600 nM control human IgG (p=0.0395). Conclusions Antiphospholipid antibodies, in aggregate (IgG from an APS patient) or specific (anti-β2-GPI), induce the formation of VWF-platelet strings from human endothelial cells. This finding suggests the potential role of VWF-platelet strings in thrombosis associated with APS. The clinical predisposition to arterial and venous thrombi in APS and the observation of increased VWF-platelet strings at arterial and venous shear stresses suggest that modulation of VWF-platelet strings may provide a future therapeutic target. Disclosures: Manco-Johnson: Bayer HealthCare: Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Baxter BioScience: Membership on an entity’s Board of Directors or advisory committees; Biogen Idec: Membership on an entity’s Board of Directors or advisory committees; Novo Nordisk: Membership on an entity’s Board of Directors or advisory committees; Eisai: Research Funding. Di Paola:Pfizer: DSMB, DSMB Other; CSL Behring: Consultancy.

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Von Willebrand Factor to Prevent Postpartum Hemorrhage

Blood ◽

10.1182/blood.v128.22.1400.1400 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1400-1400 ◽

Cited By ~ 1

Author(s):

Nicoletta Machin ◽

Margaret V. Ragni ◽

Andra H. James ◽

Craig D. Seaman ◽

Lynn M. Malec ◽

...

Keyword(s):

Blood Loss ◽

Board Of Directors ◽

Research Funding ◽

Structured Interviews ◽

First Line ◽

Vascular Medicine ◽

Advisory Committees ◽

Thrombosis Risk ◽

Von Willebrand ◽

Willebrand Factor

Abstract Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is characterized by deficient and/or defective von Willebrand factor (VWF) which results in spontaneous and traumatic mucosal bleeding. In women with VWD, pregnancy is associated with excess blood loss and poor quality of life. Recently, a prospective cohort study by James et al Haemophilia 2015, determined that even when treatment is given, women with VWD had lower VWF levels, greater blood loss at delivery, and lower hematocrit than controls without VWD. The reason for this finding remains unknown. Current VWF dosing is weight-based, but does not account for the ~1.4-1.5-fold increase in blood volume during pregnancy. To address this, we conducted a feasibility study for a prospective, randomized phase III trial comparing weight-based, 50 IU/kg, with volume-based, 80 IU/kg, VWF to prevent postpartum hemorrhage (PREVENT PPH Trial). Methods: To establish trial feasibility: 1) we compared pre-pregnancy and 8th-month VWF levels in women with VWD with and without PPH (≥500 cc blood loss in the 1st 24 hours) following vaginal delivery; 2) we evaluated VWF dosing in women with PPH in the James study; 3) we assessed thrombosis risk and VWF concentrate dose by literature review; 4) we surveyed current practice regarding VWF concentrate use at delivery by U.S. hemophilia treatment center (HTC) MDs; and 5) we conducted structured interviews of MDs and VWD patients to determine trial acceptability. Analysis was by Student's t-test for continuous data, and Fisher's exact test for discrete data. Results:In the Retrospective VWD-PPH Study of 16 women with VWD (14 type 1; 2 type 2) undergoing vaginal delivery, PPH was associated with higher pre-delivery weight, pre: 88.1 vs. 67.6 kg; 8thmonth: 99.9 vs. 75.0 kg; and delivery: 104.0 vs. 78.6 kg, all p<0.005; and a family bleeding history, 75.0% vs. 12.5%, p=0.041. Women with PPH had lower pre-pregnancy VWF:RCo, 0.34 IU/ml vs. 0.48 IU/ml, non-significant, p=0.067; similar 8th month VWF:RCo, 1.31 IU/ml vs. 1.45 IU/ml, p=0.484, and were more likely to be treated with VWF, 75.0% vs. 37.5%, p=0.315. Pre-pregnancy bleeding score (BS) correlated directly with EBL (blood loss) at delivery, r=0.663, p=0.005. VWF Dosing Data from James co-authors indicated a mean VWF dose of 46 IU/kg (median 50 IU/kg), range 28-83 IU/kg, at delivery, followed by 3.8 days (median 3 days), range 2-19 days, postpartum treatment. The Literature Review identified 13 publications between 1992-2015 reporting pharmacokinetic, safety, and/or efficacy studies in a total of 570 patients (none pregnant) at VWF doses 10-222 IU/kg. Thrombosis rate was low, 0.4%, including 2 of 353 (0.6%) receiving plasma-derived (pd) VWF and none of 95 (0%) receiving recombinant (r) VWF: these two patients included one with injection-site phlebitis and one with a remote postoperative VTE, each receiving VWF≤100 IU/kg. An HTC Survey distributed to 70 MDs, 19 (27.1%) of whom responded, 16/18 (88.9%) reported VWF was first-line therapy at delivery, mean dose 50 IU/kg; DDAVP was the most common second-line therapy in 7/17 (41.2%), and anti-fibrinolytic therapy was third-line, 8/10 (80.0%). All 19 indicated interest and willingness to participate in a trial comparing weight-based, 50 IU/kg, vs. 1.6-fold higher volume-based, 80 IU/kg, VWF at delivery. In Structured Interviews of 18 MDs and 18 VWD patients, the trial was acceptable to MDs if staff costs are covered, if the drug is safe re thrombosis, if there are sufficient patients, and if there is obstetrician collaboration to facilitate in-hospital dosing and blood draws. The trial was also acceptable to VWD patients if the drug is safe for mother and child, if childcare and travel costs are covered, and if visits are minimized in the postpartum period. Conclusions: The findings of this feasibility study indicate that pre-pregnancy VWF:RCo may be a better predictor of PPH than 8thmonth VWF:RCo. High pre-delivery weight which is associated with high blood volume may increase PPH, possibly through dilution of VWF levels. A VWF dose of ~50 IU/kg, which is the first line of therapy at delivery and the current standard of practice, may not prevent PPH. Thrombosis risk with VWF in published studies is low even at doses >80 IU/kg. Drug safety, local obstetric collaboration, and coverage of staff, travel, and childcare costs are critical issues to address in trial design. Disclosures Ragni: SPARK: Research Funding; Genentech: Research Funding; Tacere Benitec: Consultancy; Vascular Medicine Institute: Research Funding; OPKO: Research Funding; Novo Nordisk: Research Funding; Alnylam Pharmaceuticals: Consultancy, Research Funding; Baxalta: Research Funding; Biogen: Consultancy, Research Funding; Biomarin: Consultancy; Shire: Consultancy; CSL Behring: Research Funding. James:Intramural University of Ghana Research Fund: Research Funding; Vanderbilt University Medical Center Gift Funds: Research Funding. Malec:Biogen: Consultancy; Vascular Medicine Institute: Research Funding; Biogen: Research Funding; Baxalta: Research Funding. Kessler:Octapharma: Consultancy, Research Funding; Novo Nordisk: Consultancy, Research Funding; Grifols: Consultancy; Genentech: Consultancy, Research Funding; Biogen: Consultancy; Bayer: Consultancy, Research Funding; Pfizer: Consultancy; Baxalta: Consultancy, Research Funding; LFB: Other: Member of DSMB. Kouides:CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxalta: Consultancy. Neff:Shire: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: DSMB Chair for research study; ABIM: Other: Hematology Exam committee; CSL Behring: Membership on an entity's Board of Directors or advisory committees; HEMA Biologics: Membership on an entity's Board of Directors or advisory committees. Philipp:Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: Data Safety Monitoring Board. Brooks:Gilead Sciences: Research Funding.

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Platelet Activation In Sitosterolemia Mice

Blood ◽

10.1182/blood.v116.21.2022.2022 ◽

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.

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Pharmacokinetics of a Recombinant Von Willebrand Factor in Patients with Severe Von Willebrand Disease

Blood ◽

10.1182/blood.v126.23.2293.2293 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2293-2293

Author(s):

Margaret V Ragni ◽

Giancarlo Castaman ◽

Joan Cox Gill ◽

Peter Kouides ◽

Miranda Chapman ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Phase 1 ◽

Pharmacokinetic Profile ◽

Phase 3 ◽

Advisory Committees ◽

Fviii Activity ◽

Von Willebrand ◽

Dose Levels ◽

Willebrand Factor

Abstract Introduction Recombinant von Willebrand factor (rVWF, vonicog alfa, BAX 111), which is manufactured using a plasma-free method (Turecek et al Semin Thromb Hemost 2010), may provide an important alternative replacement therapy for VWD subjects. In contrast to plasma-derived (pd) VWF concentrates, BAX 111 contains a high ratio of high molecular weight (HMW) and ultralarge VWF multimers (ULM), which improve platelet and collagen binding and enhance FVIII stabilization (Kragh et al Thromb Res 2014), and may therefore be associated with better hemostatic outcomes and simplify bleeding management in VWF patients for a variety of clinical conditions. As BAX 111 is a pure VWF product, it is also not restricted to co-administration with coagulation factor VIII (FVIII). The pharmacokinetic profile of BAX 111 has been evaluated in two prospective trials (phase 1 and phase 3) in adults with severe VWD. Methods The pharmacokinetic profile of BAX 111 (with vs. without rFVIII; and BAX 111:rFVIII compared with a pdVWF:pdFVIII) in subjects with VWD, evaluated in a non-bleeding state, was determined in two clinical trials by assessment of ristocetin co-factor activity (VWF:RCo), VWF antigen (VWF: Ag); collagen-binding activity (VWF:CB), FVIII activity, and VWF multimer activity. Lower doses were examined in the first-in-human phase 1 dose-finding study, and the expected therapeutic doses (as recommended for licensed pdVWF-containing concentrates) of 50 IU VWF:RCo/kg and 80 IU VWF:RCo/kg (for major bleeding episodes) were selected for further evaluation in phase 3. PK assessments were also repeated after 6 months with the 80 IU VWF:RCo/kg dose. Results The PK profile of BAX 111 has been evaluated in a total of 56 unique subjects with severe VWD. A rapid increase in VWF:RCo was observed after a single infusion of BAX 111. There was no apparent dose-dependency at a wide range of dose levels (20 IU, 50 IU and 80 IU VWF:RCo/kg) and BAX 111 PK was not affected by administration together with rFVIII in the crossover study with the 50 IU VWF:Co/kg dose (Figure 1). A tendency toward a longer mean terminal half-life (T1/2) of VWF:RCo for BAX 111:rFVIII as compared with a pdVWF:pdFVIII was observed in the phase 1 study (23.6 h for 20 IU BAX 111:rFVIII, 19.3h for 50 IU BAX 111:rFVIII) and the mean T1/2 at 50 IU VWF:RCo/kg in the phase 3 study (19.6 h for BAX 111:rFVIII and 21.9 h for BAX 111) is also considerably longer than that of pdVWF products (Batlle et al Blood Coagul Fibrinolysis 2009). PK parameters were similar for the first (19.1 h) and second (21.2 h) assessment in the repeated PK study at 80 IU VWF:RCo/kg. VWF:CB parameters were comparable to VWF:RCo across dose levels in the phase 1 study (15.7 h for 20 IU BAX 111:rFVIII, 24.4h for 50 IU BAX 111:rFVIII) and phase 3 study (19.3h for BAX 111:rFVIII and 18.3h for BAX 111 at 50 IU; 18.8h for 80 IU in the first assessment and 20.9h 80 IU in the second assessment). ULMs were detected after 15 minutes post-infusion of BAX 111 (earliest time point measured), followed by a substantial decline due to rapid degradation by endogenous ADAMTS13 between 12 and 24 hours. A substantial increase in FVIII activity was observed after infusion with BAX 111 and BAX 111:rFVIII, followed by a secondary rise in FVIII activity after 12 hours due to the stabilization of endogenous FVIII, which was more pronounced for BAX 111:rFVIII than for pdVWF:pdFVIII (geometric mean ratio for AUC0-inf = 1.36 [90% Clopper-Pearson confidence interval: 0.93; 1.99]), and similar for BAX 111 alone as compared with BAX 111:rFVIII, confirming the ability of BAX 111 to stabilize endogenous FVIII:C. Conclusion BAX 111 has an enhanced PK profile compared to plasma-derived concentrates and there is no apparent dose dependency over a wide dose range (20 IU, 50 IU, 80 IU VWF:RCo/kg). The ULM present in BAX 111 may be more effective in stabilizing endogenous FVIII, and therefore BAX 111 may have the potential for less-frequent administration compared with plasma-derived VWF concentrates, and for FVIII-independent dosing after the initial infusion or in non-emergency bleeds. Disclosures Ragni: Biogen: Research Funding; Biomarin: Research Funding; Genentech Roche: Research Funding; Vascular Medicine Institute: Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; SPARK: Research Funding; Alnylam: Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Research Funding; Tacere Benitec: Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Dimension: Research Funding; CSL Behring: Research Funding; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Castaman:Bayer, Baxalta, CSL Behring, Kedrion, Novo Nordisk, and Pfizer: Membership on an entity's Board of Directors or advisory committees. Gill:Bayer: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees. Kouides:CSL Behring: Membership on an entity's Board of Directors or advisory committees. Chapman:Baxalta: Employment. Sytkowski:Baxalta: Employment. Obermann-Slupetzky:Baxalta: Employment. Presch:Baxalta: Employment. Fritsch:Baxalta: Employment. Ewenstein:Baxalta: Employment.

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