Von Willebrand Factor Antigen Levels Research Articles

Patients with aortic stenosis have von Willebrand factor abnormalities and increased proliferation of endothelial colony forming cells

BackgroundPatients with aortic stenosis (AS) can experience bleeding complications including gastrointestinal bleeding from angiodysplastic lesions due to acquired von Willebrand syndrome. Studies have pointed to a role for von Willebrand factor (VWF) in angiogenesis. ObjectiveThe objective of this study was to assess VWF defects in AS patients over time and the impact on angiogenesis using patient‐derived endothelial colony‐forming cells (ECFCs). Patients/MethodsPlasma sample collection and ECFC isolations were performed before valve replacement surgery, 3 to 5 days after, and 6 months after surgery. Plasma VWF antigen, activity, propeptide, collagen binding, multimers, factor VIII coagulant activity, and ADAMTS13 activity (a disintegrin‐like and metalloprotease with thrombospondin type 1 motifs 13) were determined. ECFCs were assessed for VWF and angiopoietin‐2 (Ang‐2) storage and secretion, cell proliferation, and tubule formation in Matrigel. Results and ConclusionsAortic stenosis patients exhibited quantitative and qualitative abnormalities of VWF including significantly increased VWF antigen, activity, and propeptide levels following surgery (P < .01). Increased high molecular weight VWF multimers were observed at all time points and in particular 3 to 5 days after surgery (mean = 14% ± 6%) relative to before (mean = 10% ± 4%), suggesting increased proteolysis by ADAMTS13 pre‐operatively in a shear‐dependent manner. ECFCs from patients with aortic stenosis were more proliferative than controls (P < .05) and had increased retention of Ang‐2 (P < .05) suggesting epigenetic modification of the cells. Overall, there are hemostatic changes in AS patients that are present before valve replacement surgery and these persist long after surgery has occurred. These findings have implications for the current clinical management of AS patients.

Clinical significance of slightly reduced von Willebrand factor activity.

Von Willebrand disease (VWD) is the most common congenital bleeding disorder, with a clinical presentation of mucocutaneous and surgical bleeding varying from mild to severe. It is inherited in an autosomal dominant or autosomal recessive manner. The disease is caused by quantitative or qualitative deficiency of the von Willebrand factor (VWF) and is classified as type 1, 2 (2A, 2B, 2M, 2N), and 3. Although type 1 VWD is the most common form of VWD, the f ormal cutoff for diagnosis remains a subject of debate. In our paper, we present results of studies regarding the clinical and laboratory importance of a new type of bleeding disorder called low VWF. The new guidelines for VWD diagnosis and management suggested that patients with historically type 1 VWD should be divided into 2 subsets: type 1 VWD with a VWF antigen level (VWF:Ag) of less than 30 IU/dl or less than 40 IU/dl, in which about 80% of patients exhibited VWF gene mutations, and low VWF with a VWF:Ag level of 30 to 50 IU/dl or 40 to 50 IU/dl, in which the causative mutation is detected in merely 40% of patients and in most families, inheritance is not dependent on the locus of VWF on chromosome 12. Previously, moderately reduced VWF levels (30-50 IU/dl) were considered a risk factor for bleeding, but not a true bleeding disorder, and this condition was named low VWF. Recently, it was documented in a large group of patients with type 1 VWD and low VWF that bleeding score does not correlate with VWF:Ag and bleeding symptoms in type 1 VWD (<30 IU/dl) and low VWF can change from infrequent and moderate to severe bleeds. Because the plasma concentration of VWF depends on many physiological and pathological factors that may mask the diagnosis of VWD, separation of the group of patients with low VWF (30-50 IU/dl) from those with type 1 VWD may delay or prevent them from receiving appropriate treatment. Diagnosis of VWD in each case, particularly those with a slight decrease in VWF (30-50 IU/dl), should be based primarily on the clinical manifestations and family history of hemorrhagic diathesis.

Elevated Plasma von Willebrand Factor Antigen and Activity Levels Are Associated With the Severity of Coronary Stenosis.

von Willebrand factor (VWF) acts as a bridge between platelets and the subendothelial matrix following vessel damage and plays a vital role in coronary artery disease (CAD). The aim of this study was to investigate the association between VWF and the severity of coronary stenosis quantified by the Gensini score in acute myocardial infarction (AMI), the most dangerous complication of CAD. Plasma VWF antigen (VWF: Ag) and VWF-collagen binding (VWF: CB) in normal controls (n = 123) and in patients with AMI (n = 205) were tested, and then the patients were divided based on Gensini scores. The levels of VWF: Ag and VWF: CB in patients with AMI were significantly higher than those in the control group (P < .001). Plasma levels of VWF: Ag and VWF: CB were positively correlated with both Gensini score and the number of affected vessels. Both VWF: Ag and VWF: CB were independent factors for coronary stenosis, adjusting confounding factors. Thus, the levels of VWF: Ag and VWF: CB were positively correlated with the severity of coronary stenosis. Screening of VWF at time of AMI may have prognostic value in terms of the severity of coronary stenosis.

Expressions of plasma D-dimer, von Willebrand factor antigen and blood coagulation factor VII in lymphoma and their clinical significances

Objective To investigate the clinical value of blood coagulation and fibrinolysis index detection in lymphoma patients. Methods A total of 115 lymphoma patients hospitalized at Zhongshan Hospital of Xiamen University from January 2013 to September 2017 were retrospectively analyzed. According to the diagnostic and therapeutic criteria of lymphoma from World Health Organization (2008), these patients were divided into chemotherapy remission group (76 cases) and chemotherapy non-remission group (39 cases). A total of 138 healthy examination subjects at the same period were selected as the control group. Coagulation factor Ⅶ (FⅦ), D-dimer (D-D), von Willebrand factor antigen (vWF: Ag) and serum lactate dehydrogenase (LDH) levels were measured in all subjects. Kruskal-Wallis test was used to compare the differences of blood coagulation and fibrinolysis indicators in patients with different lymphoma staging and stratified treatment outcomes. Correlation test of D-D and LDH and disease staging was performed by using Spearman correlation analysis. The receiver operating characteristics (ROC) curve was used to analyze the efficacy of D-D in the assisted diagnosis of lymphoma with thrombosis. Results The plasma D-D, vWF: Ag levels and FⅦ activity [the median (interquartile range)] in lymphoma patients were higher than those in healthy controls [1 240 ng/ml (1 610 ng/ml) vs. 250 ng/ml (43 ng/ml), Z = -10.728, P < 0.01; 170 ng/ml (113 ng/ml) vs. 105 ng/ml (28 ng/ml), Z = -6.425, P < 0.01; 120% (26%) vs. 96% (26%), Z = -4.602, P < 0.01]. With the increase of Ann Arbor stage, plasma D-D, vWF: Ag levels and FⅦ activity were also increased gradually (all P < 0.05); plasma D-D, vWF: Ag levels and FⅦ activity in lymphoma with thrombosis group were higher than those in the group without thrombosis (all P < 0.01), D-D and vWF: Ag levels in the chemotherapy remission group were lower than those in the chemotherapy non-remission group (all P < 0.01). Plasma D-D levels were positively correlated with LDH level and Ann Arbor stage (r values were 0.414 and 0.530, respectively, all P < 0.01). When the plasma D-D level was 1 735 ng/ml, the sensitivity of diagnosis of thrombosis in patients with lymphoma was 81.8%, the specificity was 85.7%, the area under the ROC curve was 0.894, and the Youden index was the highest (0.675). Conclusions Clinically, blood coagulation and fibrinolysis in patients with lymphoma can be evaluated by detecting blood coagulation and fibrinolysis indexes such as plasma D-D. Real-time monitoring of plasma D-D level can determine the thrombosis trend of lymphoma patients, and it may play an important role in evaluating the efficacy and prognosis. Key words: Lymphoma; D-dimer; von Willebrand factor antigen; Blood coagulation factor Ⅶ

Histone-induced thrombotic thrombocytopenic purpura in adamts13 -/- zebrafish depends on von Willebrand factor.

Thrombotic thrombocytopenic purpura (TTP) is caused by severe deficiency of ADAMTS13 (A13), a plasma metalloprotease that cleaves endothelium-derived von Willebrand factor (VWF). However, severe A13 deficiency alone is often not sufficient to cause an acute TTP; additional factors may be required to trigger the disease. Using CRISPR/Cas9, we created and characterized several novel zebrafish lines carrying a null mutation in a13−/−, vwf, and both. We further used these zebrafish lines to test the hypothesis that inflammation that results in neutrophil activation and release of histone/DNA complexes may trigger TTP. As shown, a13−/− zebrafish exhibit increased levels of plasma VWF antigen, multimer size, and ability of thrombocytes to adhere to a fibrillar collagen-coated surface under flow. The a13−/−zebrafish also show an increased rate of occlusive thrombus formation in the caudal venules after FeCl3 injury. More interestingly, a13−/− zebrafish exhibit ~30% reduction in the number of total, immature, and mature thrombocytes with increased fragmentation of erythrocytes. Administration of a lysine-rich histone results in more severe and persistent thrombocytopenia and a significantly increased mortality rate in a13−/−zebrafish than in wildtype (wt) ones. However, both spontaneous and histone-induced TTP in a13−/− zebrafish are rescued by the deletion of vwf. These results demonstrate a potentially mechanistic link between inflammation and the onset of TTP in light of severe A13 deficiency; the novel zebrafish models of TTP may help accelerate our understanding of pathogenic mechanisms and the discoveries of novel therapeutics for TTP and perhaps other arterial thrombotic disorders.

Correlations between von Willebrand Factor Antigen Levels and Factor VIII Pharmacokinetics Are Similar across Different FVIII Products in Patients with Severe Hemophilia A

Background: Prophylactic treatment with replacement factor VIII (FVIII) is the standard of care for patients with severe hemophilia A to prevent bleeding and subsequent joint damage and chronic arthropathy. Because of variability in patient characteristics and clinical needs, it is recommended that prophylaxis is tailored to individual patient needs based on their baseline characteristics, bleeding history, and FVIII pharmacokinetic (PK) parameters. Endogenous levels of von Willebrand factor antigen (VWF:Ag) are known to influence the PK of FVIII. This analysis investigates the relationship between FVIII PK parameters and VWF:Ag levels, as well as between VWF:Ag levels and age, in patients with severe hemophilia A treated with various recombinant FVIII products using data obtained from multiple clinical trials. Patients/Methods: This integrated regression analysis is based on data with five FVIII products from six clinical trials. The products were: PEGylated recombinant FVIII BAY 94-9027 (damoctocog alfa pegol, Jivi®); sucrose-formulated recombinant FVIII (rFVIII-FS; Kogenate®-FS); BAY 79-4980 (liposomal version of Kogenate®-FS); unmodified full-length recombinant FVIII BAY 81-8973 (Kovaltry); and recombinant antihemophilic factor plasma/albumin-free method rAHF-PFM (octocog alfa, Advate®). The trials were: NCT01184820, NCT01029340, NCT00623727, NCT01233258, NCT02483208, and NCT01580293. PK parameters were estimated by means of a non-compartmental analysis based on densely sampled single-dose profiles measured by one-stage and chromogenic assays, and following a dose of 25-60 IU/kg. The primary regression analysis was performed using the log-transformed PK parameters (half-life [t1/2], clearance [CL], and dose-normalized area under the curve [AUCnorm]) as dependent variables and the VWF:Ag levels as the independent variable in a mixed model including fixed treatment effects and random patient effects. In addition, the impact of age on VWF:Ag levels was investigated. One-stage assay results are reported. Results based on the chromogenic assay were in line with those reported for the one-stage assay. Results: Data from 104 patients from six studies were used to investigate the relationship of the dose-independent PK-parameter, t1/2 vs VWF:Ag levels, and of VWF:Ag levels vs age. Median age was 31 years (range 12-64 years). For the analyses of the PK parameters AUCnorm and CL, data from 77 patients from five studies were analyzed. Median age was 32 years (range 12-64 years). Some patients participated in multiple studies. The primary regression analyses across all products and studies on the log-transformed PK parameters showed significant associations between PK parameters and VWF:Ag levels. Significant positive correlations vs VWF:Ag levels were observed for t1/2 (n = 155 observations in 104 patients, P &amp;lt; 0.001) and AUCnorm (n = 117 observations in 77 patients, P &amp;lt; 0.001), resulting in a negative correlation with CL (Figure 1). Other factors, such as age and the interaction of treatment with the VWF:Ag-PK effect, were found to be redundant and were not included in the final model. Analysis of VWF:Ag levels vs age using a simple linear regression that did not incorporate the dependencies of multiple observations in patients, revealed a positive correlation between age and VWF:Ag levels (Spearman coefficient , r = 0.290 [P = 0.0029]; Pearson coefficient, r = 0.334 [P = 0.0005]). Comparable results were observed in a mixed model with random patient effects. Conclusions: In this combined analysis across five FVIII products, FVIII PK parameters (t1/2, AUCnorm, and CL) were significantly correlated with endogenous VWF:Ag levels which were also correlated with age. Moreover, while there were differences in the average PK parameters between products (represented by the intercept for treatment), all five FVIII products demonstrated similar VWF-PK relationships. It is well known that a patient's VWF:Ag level influences the PK of rFVIII, but this analysis suggests that the quantitative relationship between FVIII PK and VWF:Ag is similar for modified and unmodified FVIII products. This finding can be used, for example, to support selection of adequate treatment regimens when a patient is switching rFVIII product. Disclosures Maas Enriquez: Bayer: Employment. Delesen:Bayer: Employment. Solms:Bayer: Employment, Equity Ownership. Croteau:Novo Nordisk: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria; Spark Therapeutics: Research Funding; Pfizer: Research Funding; Genentech: Consultancy, Honoraria; Octapharma: Honoraria; CSL Behring: Consultancy, Honoraria; Bayer: Consultancy, Honoraria. Berntorp:Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; CSL Behring: Research Funding; Shire: Consultancy, Honoraria, Research Funding; Sobi/Bioverativ: Research Funding; Octapharma: Consultancy, Honoraria.

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

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 &amp;gt;100, there was a 96.4% NPV for the diagnosis of VWD. For individuals whose initial VWF:Ag and VCWF:RCo were both &amp;gt;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 &amp;gt;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.

The Discordance between Offsite to Onsite Testing for Von Willebrand Disease in Post-Menarchal Females: A Multi-Center Study

The Discordance between Offsite to Onsite Testing for Von Willebrand Disease in Post-Menarchal Females: A Multi-Center Study

P1902Comparison of efficacy of different anticoagulation strategies used during pulmonary vein isolation in left atrial blood samples

Abstract Introduction Ablation of atrial fibrillation is based on the isolation of the pulmonary veins (PVI) from the left atrium. Cerebral thromboembolism is a rare but serious complication of PVI, which highlights the importance of peri-procedural anticoagulation. Aim Studying the coagulation and endothelium activation during PVI in case of using different anticoagulation protocols. Method 52 patients undergoing PVI with cryoballoon technique were involved and were grouped according to three different pre-procedural anticoagulation strategies: in the first group patients received no anticoagulation before the procedure (non anticoag., 24 patients)-, in the second group patients received uninterrupted vitamin K antagonist therapy, and their INR value was in therapeutic range on the day of the procedure (VKA group, 11 patients) and in the third group patients were on uninterrupted dabigatran therapy (dabigatran group, 17 patients). Two blood samples were taken during PVI from the left atrium, the first one before ablation or iv. heparin administration, the second one directly after the last application at the end of the procedure. The samples were used to measure D-dimer, plasmin antiplasmin (PAP) complex, a2 plasmin inhibitor (a2PI), plasminogen, FVIII activity, von Willebrand factor (VWF) antigen levels. Results D-dimer levels increased in all three groups after ablation, but in the samples of patients on dabigatran therapy we detected significantly lower D-dimer levels in the pre- and post-procedural samples compared to patients on other therapeutic strategies (median values before or after PVI: non anticoag.: 0.48 and 1.09 mgFEU/L; VKA: 0.33 and 0.72 mgFEU/L, dabigatran: 0.12 and 0.30 mgFEU/L, p&lt;0.001 non anticoag. vs. dabigatran, p&lt;0.01 VKA vs. dabigatran). PAP complex values were increasing parallel with D-dimer levels and only in the dabigatran group did not increase significantly. VWF antigen and FVIII activity increased significantly in all three groups after ablation, but there was no significant difference between the groups. Conclusions the safest anticoagulation strategy for patients undergoing PVI was uninterrupted dabigatran therapy. The extend of endothelial damage was not affected by anticoagulation.

P13. Abstract Title: Deep Mutational Scan of VWF to Define Mutations that Enhance or Impair Secretion

<h3>Rationale</h3> Von Willebrand Factor (VWF) is a multimeric glycoprotein that recruits platelets to sites of blood vessel injury and serves as a molecular chaperone for coagulation factor VIII (fVIII). Plasma VWF levels are a known risk factor for various thrombotic disorders including VTE, myocardial infarction, and stroke. GWAS studies have identified genetic variants in VWF that contribute to increased and decreased circulating VWF antigen levels. However, it is currently not known why some variants result in high VWF levels while others cause low VWF levels in circulation. We hypothesize that a subset of VWF variants impact its biosynthesis and secretion. <h3>Objective</h3> To identify every possible mutation in VWF that either increase or decrease its biosynthesis and secretion from cells. <h3>Results</h3> We have developed a protein secretion assay in HEK293T-Rex cells based on VWF- eGFP. Mutations in VWF that affect biosynthesis and secretion can be identified based on cell fluorescence intensity using Fluorescence Activated Cell Sorting (FACS). Initial studies have confirmed that cells expressing VWF mutants with known secretion defects have increased intracellular eGFP intensity (eGFP MFI: V1822G=157.72±5.40, C2693Y=184.06±2.10), relative to cells expressing WT VWF (eGFP MFI: WT=69.22±1.87) (p<0.001). This validates that eGFP specific cell retention is a consequence of impaired VWF basal secretion. Mutations of VWF may additionally cause inappropriate VWF secretion in response to physiological stimuli, and exacerbate imbalanced VWF plasma levels under various environmental influences. Cells treated with stimulus show time-dependent stimulation of VWF secretion in cells expressing WT (83.7% MFI reduction, p<0.01) and the C2693Y VWF (46.7% MFI reduction, p<0.01), but minimal response in V1822G VWF expressing cells (8.85% MFI reduction, p<0.05). This suggests that VWF variants may exhibit sensitized/reduced responses to stimulated VWF secretion. <h3>Conclusions</h3> These data provide the proof-of-concept to perform an unbiased deep mutational scan of VWF, to identify every possible mutation that can increase/decrease biosynthesis and secretion. Results of this project will provide a detailed structure/function analysis of VWF in mammalian cells and identify variants in VWF that can impair or enhance its secretion.

Von Willebrand factor antigen levels predict major adverse cardiovascular events in patients with carotid stenosis of the ICARAS study

Background and aimsVon Willebrand factor (VWF) plays an important role in thrombogenesis and mediates platelet adhesion particularly under high shear stress. Such conditions are generally found in stenotic arteries and can eventually cause myocardial infarction or stroke. We aimed to study whether levels of VWF antigen (VWF:Ag) predict future major adverse cardiovascular events (MACE) in patients suffering from carotid artery stenosis. MethodsPatients with atherosclerotic carotid artery disease defined by the presence of nonstenotic plaques or any degree of carotid stenosis were prospectively enrolled. Concentrations of VWF were measured by enzyme immunoassay. ResultsVWF:Ag levels were more stable after 4 freeze-thaw cycles, when compared to VWF activity, and we showed similar concentrations of VWF in citrated plasma and serum (±4%). Levels of VWF:Ag predicted future cardiovascular events in 811 patients with carotid stenosis independent of known cardiovascular risk factors. Patients with VWF:Ag concentrations in the 4th quartile had a 44% event rate after an average 3-year follow up and a hazard ratio of 2.15 (95% confidence interval 1.46–3.16; p < 0.001). ConclusionsHigh concentrations of VWF:Ag predict major cardiovascular events in patients with carotid stenosis, and given their high event rate may be useful for risk stratification of such patients.

Genetic determinants of VWF clearance and FVIII binding modify FVIII pharmacokinetics in pediatric hemophilia A patients

Factor VIII (FVIII) pharmacokinetic (PK) properties show high interpatient variability in hemophilia A patients. Although previous studies have determined that age, body mass index, von Willebrand factor antigen (VWF:Ag) levels, and ABO blood group status can influence FVIII PK, they do not account for all observed variability. In this study, we aim to describe the genetic determinants that modify the FVIII PK profile in a population of 43 pediatric hemophilia A patients. We observed that VWF:Ag and VWF propeptide (VWFpp)/VWF:Ag, but not VWFpp, were associated with FVIII half-life. VWFpp/VWF:Ag negatively correlated with FVIII half-life in patients with non-O blood type, but no correlation was observed for type O patients, suggesting that von Willebrand factor (VWF) half-life, as modified by the ABO blood group, is a strong regulator of FVIII PK. The FVIII-binding activity of VWF positively correlated with FVIII half-life, and the rare or low-frequency nonsynonymous VWF variants p.(Arg826Lys) and p.(Arg852Glu) were identified in patients with reduced VWF:FVIIIB but not VWF:Ag. Common variants at the VWF, CLEC4M, and STAB2 loci, which have been previously associated with plasma levels of VWF and FVIII, were associated with the FVIII PK profile. Together, these studies characterize the mechanistic basis by which VWF clearance and ABO glycosylation modify FVIII PK in a pediatric population. Moreover, this study is the first to identify non-VWF and non-ABO variants that modify FVIII PK in pediatric hemophilia A patients.

Apolipoprotein B100/Low-Density Lipoprotein Regulates Proteolysis and Functions of von Willebrand Factor under Arterial Shear.

Proteolytic cleavage of von Willebrand factor (VWF) by a plasma a disintegrin and metalloproteinase with a thrombospondin type 1 motifs, member 13 (ADAMTS13) is regulated by shear stress and binding of coagulation factor VIII, platelets or platelet glycoprotein 1b, and ristocetin to VWF. Current study aims to identify novel VWF binding partners that may modulate VWF functions under physiological conditions. A deoxyribonucleic acid aptamer-based affinity purification of VWF, followed by tandem mass spectrometry, functional, and binding assays was employed. Apolipoprotein B100/low-density lipoprotein (apoB100/LDL) was identified as a novel VWF-binding partner. Purified apoB100/LDL was able to accelerate the proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. This rate-enhancing activity was dramatically reduced when apoB100/LDL was oxidized. More interestingly, the oxidized apoB100/LDL appeared to compete with native apoB100/LDL for its enhancing activity on VWF proteolysis under shear. As a control, a purified apoA1/high-density lipoprotein (apoA1/HDL) or apoB48 exhibited a minimal or no activity enhancing VWF proteolysis by ADAMTS13 under the same conditions. Both VWF and ADAMTS13 were able to bind native or oxidized apoB100/LDL with high affinities. No binding interaction was detected between VWF (or ADAMTS13) and apoA1/HDL (or apoB48). Moreover, apoB100/LDL but not its oxidized products inhibited the adhesion of platelets to ultra large VWF released from endothelial cells under flow. Finally, significantly reduced ratios of high to low molecular weight of VWF multimers with increased levels of plasma VWF antigen were detected in LDLR-/- mice fed with high cholesterol diet. These results indicate that apoB100/LDL may be a novel physiological regulator for ADAMTS13-VWF functions.

Cryptic non-canonical splice site activation is part of the mechanism that abolishes multimer organization in the c.2269_2270del von Willebrand factor

We report a new pathogenic mechanism in von Willebrand disease involving the use of a non-canonical splicing site. The proband, carrying the homozygous c.2269_2270del mutation previously classified as a type 3 mutation, showed severely reduced plasma and platelet von Willebrand factor antigen levels and functions, and no factor VIII binding capacity. A particular von Willebrand factor multimer pattern emerged in plasma, characterized by the presence of only two oligomers: the dimer and an unusually large band, with no intermediate components. There were von Willebrand factor multimers in platelets, but each band ran more slowly than the normal counterpart. No anti-von Willebrand factor antibodies were detectable. The proband was classified as having severe type 1 von Willebrand disease. Seeking the relationship between phenotype and genotype, we found the c.2269_2270del mutation associated with three different RNA: r.2269_2270del (RNAI), giving a truncated von Willebrand factor protein; r.[2269_2270del;2282_2288del] (RNAII), resulting from activation of a cryptic “AG” splicing site; and r.[2269_2270del;2281_2282insAG] (RNAIII), where the wild-type “AG” acceptor of exon 18 was retained due to the non-canonical 2279-2280 “CG” acceptor splicing site being used. The aberrant RNAII and RNAIII caused the alteration of the furin cleavage and binding sites, respectively, both resulting in a von Willebrand factor protein characterized by the persistence of von Willebrand factor propeptide, as confirmed by western blot analysis of the recombinant mutated von Willebrand factor molecules produced in vitro. Taken together, these findings explain the residual von Willebrand factor synthesis, slower-running multimers, and absent factor VIII binding capacity. The apparently pure gene null mutation c.2269_2270del profoundly alters von Willebrand factor gene splicing, inducing a complex RNA expression pattern.

The role of ADAMTS‐13 and von Willebrand factor in cancer patients: Results from the Vienna Cancer and Thrombosis Study

Unlabelled Box BackgroundCancer‐associated venous thromboembolism (VTE) is an important complication in the course of a malignant disease. Low ADAMTS‐13 (a disintegrin‐like and metalloproteinase with thrombospondin type 1 motif 13) and increased von Willebrand Factor (VWF) levels in cancer patients have been described numerously. ObjectivesInvestigation of the influence of ADAMTS‐13 and VWF on the probability of VTE and survival in malignancy. Patients/MethodsIn the framework of the ongoing prospective Cancer and Thrombosis Study (CATS) ADAMTS‐13 activity and VWF antigen levels were investigated in cancer patients. ResultsIn total, 795 patients with various tumor types (364 female/431 male, median age 62 years) were included; of those, 56 developed VTE and 359 patients died during a median follow‐up time of 730 days. The hazard ratio (HR) of VTE per doubling of VWF level was 1.56 (95% confidence interval [CI] 1.13‐2.16) in multivariable competing risk analysis. ADAMTS‐13 levels showed no correlation with the incidence of VTE in univariate competing risk analysis. The HR of mortality per doubling of VWF level was 1.46 (95% CI 1.28‐1.66) and per SD increment of ADAMTS‐13was 0.90 (95% CI 0.81‐1.00) in multivariable Cox regression analysis. Patients with VWF >75th percentile and concomitant low (<25th percentile) or medium (25‐75th percentile) ADAMTS‐13 values had the highest probability of mortality (HR 4.31 and 4.75, respectively). ConclusionsHigh VWF levels were significantly associated with the risk of developing VTE in cancer patients, whereas ADAMTS‐13 was not. Low ADAMTS‐13 and increased VWF levels were independently associated with worse overall survival.

Therapeutic monoclonal antibody interference in electrophoretic and immunofixation techniques

Consistent with fulminant endothelial cell activation, elevated plasma von Willebrand factor (VWF) antigen levels have been reported in patients with COVID‐19. The multimeric size and function of VWF are normally regulated through A Disintegrin And Metalloprotease with ThrombSpondin Motif type 1 motif, member 13 (ADAMTS‐13)‐‐mediated proteolysis.This study investigated the hypothesis that ADAMTS‐13 regulation of VWF multimer distribution may be impaired in severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) infection contributing to the observed microvascular thrombosis.Patients with COVID‐19 (n = 23) were recruited from the Beaumont Hospital Intensive Care Unit (ICU) in Dublin. Plasma VWF antigen, multimer distribution, ADAMTS‐13 activity, and known inhibitors thereof were assessed.We observed markedly increased VWF collagen‐binding activity in patients with severe COVID‐19 compared to controls (median 509.1 versus 94.3 IU/dl). Conversely, plasma ADAMTS‐13 activity was significantly reduced (median 68.2 IU/dl). In keeping with an increase in VWF:ADAMTS‐13 ratio, abnormalities in VWF multimer distribution were common in patients with COVID‐19, with reductions in high molecular weight VWF multimers. Terminal sialylation regulates VWF susceptibility to proteolysis by ADAMTS‐13 and other proteases. We observed that both N‐ and O‐linked sialylation were altered in severe COVID‐19. Furthermore, plasma levels of the ADAMTS‐13 inhibitors interleukin‐6, thrombospondin‐1, and platelet factor 4 were significantly elevated.These findings support the hypothesis that SARS‐CoV‐2 is associated with profound quantitative and qualitative increases in plasma VWF levels, and a multifactorial down‐regulation in ADAMTS‐13 function. Further studies will be required to determine whether therapeutic interventions to correct ADAMTS‐13‐VWF multimer dysfunction may be useful in COVID‐microvascular thrombosis and angiopathy.

Relationship among HbA1c and Some Markers of Endothelial Damage in Type 2 Diabetes Mellitus

Background: A number of processes regulating the thrombolytic balance are impaired in diabetic patients as a result of dysfunction of endothelial cells leading to a hypercoagulative state. Von Willebrand factor (VWF) is an important marker of endothelial dysfunction. Plasminogen activator inhibitor-1 antigen (PAI-1-Ag), the major physiological inhibitor of tissue plasminogen activator (tPA), is mainly produced by endothelium. The aim of this study is to measure plasma levels of von Willebrand factor, Plasminogen activator inhibitor-1 antigen in type 2 diabetes mellitus patients and to correlate with glycated haemoglobin (HbA1c).&#x0D; Study Design: This prospective cohort study was conducted on 30 diagnosed type 2 DM patients who were about to start treatment.&#x0D; Place and Duration of Study: Medical outpatient (MOP) clinic of Enugu State University of Science and Technology Teaching Hospital (ESUTTH), between January and December 2016.&#x0D; Methodology: We included 30 patients (13 men, 17 women; age range 40-80 years) with type 2 diabetes mellitus. Blood samples were drawn from the patients before they commenced treatment, six months into the treatment and at twelve months of the treatment. Blood samples were also drawn from 25 age matched non diabetic patients. Plasma von Willebrand factor and Plasminogen activator inhibitor-1 antigen levels were determined by Enzyme linked immunosorbent assay. Glycated haemoglobin (HbA1c) and fasting blood sugar (FBS) levels were also evaluated along with them.&#x0D; Results: This study was conducted on 30 type 2 DM patients consisting of 13 males and 17 females. At treatment naïve, mean levels of vWF were significantly increased (45.48 +/- 6.46) in male type 2 Diabetic patients compared to the control (20.45 +/- 0.26). Six months into treatment mean levels of vWF were significantly increased (48.18 +/- 4.99) in female type 2 Diabetic patients compared to the control (37.64 +/- 7.93). The plasma levels of vWF were significantly and positively correlated with HbA1c at six months into treatment in male type 2 DM patients. The plasma levels of vWF were also significantly and positively correlated with PAI-1 at six and twelve months into treatment in both genders.&#x0D; Conclusion: There was strong significant positive correlation between plasma levels of vWF and PAI-1 in type 2 diabetes mellitus patients.

Blood platelet function abnormalities in cirrhotic patients with esophageal varices in relation to the variceal bleeding history*

Objective: The study aimed at assessing the effect of thrombocytopenia and platelet function abnormalities on the occurrence of variceal bleeding in patients with cirrhosis.Methods: The results of impedance aggregometry, von Willebrand factor antigen level and thromboelastometry (TEM) with and without the addition of a platelet inhibitor (FIBTEM®, EXTEM® test, respectively) were compared in two patient groups: Group 1 (n = 32) – patients with moderate or large esophageal or gastric varices, who had never had symptoms of acute gastrointestinal bleeding and Group 2 (n = 26) – patients with history of variceal bleeding.Results: Standard clotting test indicated more hypocoagulable profile in Group 2 compared to Group 1. However, no differences in any TEM component were observed between groups in EXTEM® test. The contribution of platelets to clot strength was significantly higher in Group 2 than in Group 1 [PLT% = 74.2 (67.5–80.4) versus 68.8 (63.7–76.5) %; p = .039]. The aggregation index was also higher in Group 2 compared to Group 1, although not statistically significant [% of healthy = 96.9 (73.2–140.1) versus 67.6 (52.5–118.8) %, p = .195]. No differences in vWF antigen levels were observed between groups.Conclusions: The results of thromboelastometry and aggregometry indicate increased contribution of platelets in clot formation in patients with a history of variceal bleeding compared to cirrhotic patients who never bled. Comparable effectiveness of hemostasis in both groups is most likely associated with the compensatory role of platelets. Increased platelet activity in this group of patients is probably due to a mechanism independent of the von Willebrand factor antigen level.

Elevated Von Willebrand Factor Antigen Levels are an Independent Risk Factor for Venous Thromboembolism: First Report from North India.

Elevated levels of coagulation factor VIII have been associated with increased risk for venous thromboembolism (VTE). The role of Von Willebrand factor (VWF) in VTE is unclear. In this study, we investigated the association of raised levels of VWF and factor VIII in patients with VTE. 100 patients of VTE and 100 age and gender matched controls were tested for levels of VWF antigen, coagulation factor VIII:C, Protein C, Protein S, antithrombin, antiphospholipid antibodies and for the presence of Factor V Leiden mutation. The mean level of VWF was significantly higher amongst patients of VTE (177.3IU/dl) when compared to controls (129.7IU/dl) (P < 0.001). Similarly, the mean level of FVIII:C was significantly higher amongst patients of VTE (199.1IU/dl) when compared to controls (145.2IU/dl) (P < 0.001). On logistic regression analysis, after adjusting for FVIII:C, Protein S, factor V Leiden and trauma, the association of raised VWF with VTE remained significant (P = 0.001). Similarly higher levels of FVIII:C persisted to have an independent association with VTE (P = 0.004). Both, higher levels of VWF and FVIII:C levels, are independently associated with VTE.

Coagulation factor VIII is vital for increasing global coagulation after physical exercise.

In a previous smaller study, we found evidence of a diminished global coagulation capacity after maximal exercise in patients with severe haemophilia A (HA). To validate these results, we repeated the study in a larger cohort. We also examined if the exercise-induced increased levels of von Willebrand factor (VWF) might prolong the effect of factor concentrate administered just before exercise. We studied individual and global coagulation parameters after maximal physical exercise in 10 persons with severe HA and 10 healthy matched control subjects. Blood samples were taken before, 10minutes, 60minutes and 4hours after exercise. Rotational thromboelastometry (ROTEM) and thrombin generation assay-calibrated automated thrombogram (TGA-CAT) showed significantly increased coagulation capacity after maximal exercise in healthy controls but not in patients with severe HA. VWF antigen and activity levels increased significantly in both groups, whereas FVIII:C only showed a significant increase in the control group. No statistically significant differences were seen between FVIII pharmacokinetic results obtained with and without exercise. Our findings do not support the presence of a FVIII-independent mechanism that increases global coagulation, but rather underscores the importance of FVIII in mediating the increased coagulation capacity seen after exercise. Our results could not support the hypothesis that exercise-induced increased levels of VWF for patients with severe HA lead to a prolonged effect of factor concentrate administered just before exercise.