Von Willebrand Factor Ristocetin Cofactor Research Articles

An Automated pre-Dilution Setup for Von Willebrand Factor Activity Assays.

Accurate quantification of von Willebrand factor ristocetin cofactor activity (VWF:RCo) is critical for the diagnosis and classification of von Willebrand disease, the most common hereditary and acquired bleeding disorder in humans. Moreover, it is important to accurately assess the function of von Willebrand factor (VWF) concentrates within the pharmaceutical industry to provide consistent and high-quality biopharmaceuticals. Although the performance of VWF:RCo assay has been improved by using coagulation analyzers, which are specialized devices for blood and blood plasma samples, scientists still report a high degree of intra- and inter-assay variation in clinical laboratories. Moreover, high, manual sample dilutions are required for VWF:RCo determination of VWF concentrates within the pharmaceutical industry, which are a major source for assay imprecision. For the first time, we present a precise and accurate method to determine VWF:RCo, where all critical pipetting and mixing steps are automated. A pre-dilution setup was established on CyBio FeliX (Analytik-Jena) liquid handling system, and an adapted VWF:RCo method on BCS-XP analyzer (Siemens) is used. The automated pre-dilution method was executed on three different, most frequently used coagulation analyzers and compared to manual pre-dilutions performed by an experienced operator. Comparative sample testing revealed a similar assay precision (coefficient of variation = 5.9% automated, 3.1% manual pre-dilution) and no significant differences between the automated approach and manual dilutions of an expert in this method. While no outliers were generated with the automated procedure, the manual pre-dilution resulted in an error rate of 8.3%. Overall, this operator-independent protocol enables standardization and offers an efficient way of fully automating VWF activity assays, while maintaining the precision and accuracy of an expert analyst. Key features • Automated pre-dilution setup for von Willebrand factor concentrates of various natures. • Combination of a liquid handling system (CyBio FeliX) with a coagulation analyzer (BCS-XP). • Simplifies method transfer to other laboratories. • Basic training for CyBio FeliX and BCS-XP is required. Graphical overview VWF:RCo assay principle and measurement setup. Platelets (yellow ellipsoids) with negative surface charge (- - -) are treated with formaldehyde, which partly denatures the cell surface and thus stabilizes platelets for use as assay reagents. Stabilized platelets (dark-yellow-framed yellow ellipsoids) are then brought in contact with ristocetin A (chemical structure shown; black dots), which binds to the platelet surface and facilitates binding of VWF (green circles). The graphs show an example of quantitative determination of platelet agglutination by measurement of light transmission, where increasing amounts of VWF increase light transmission over time. The photo in the left-bottom corner shows the CyBio FeliX setup for VWF sample dilution and the photo in the right-bottom corner displays the BCS-XP system, which is used for VWF:RCo measurements.

Von Willebrand Factor Collagen-Binding Activity and Von Willebrand Factor-Mediated Platelet Adhesion in Patients with Coronary Artery Disease.

In this study, we investigated von Willebrand factor (VWF)-related parameters in 30 patients with stable coronary artery disease (CAD) and 50 patients without CAD. In both groups, the following were measured: the VWF antigen level (VWF:Ag); the VWF ristocetin cofactor activity (VWF:RCo); the VWF collagen-binding activity (VWF:CB); and VWF-mediated platelet adhesion. Platelet adhesion was measured in whole blood at a shear rate of 1300 s-1 using a microfluidic chamber with a collagen-coated surface. VWF:Ag and VWF:RCo were found to be the same in both groups of patients. However, VWF:CB was found to be lower in patients with CAD compared with patients without CAD, with values of 106.7% (82.1; 131.6) and 160.4% (112.5; 218.1), respectively (p < 0.001). The decrease in platelet adhesion after GPIb inhibition was more pronounced in patients with CAD compared with patients of the control group, with recorded values of 76.0% (60.6; 82.1) and 29.3% (0.0; 60.4), respectively (p < 0.001). After adjusting for traditional risk factors, the odds ratio for CAD was found to be 0.98 (95% CI, 0.97-0.99; p = 0.011) per 1% increase in VWF:CB activity, and 1.06 (95% CI, 1.03-1.09; p < 0.001) per 1% decrease in GPIb-mediated platelet adhesion. The findings presented in this paper indicate a possible critical role played by complex VWF-collagen-platelet interactions in the development of CAD.

Hemoglobin concentration and body mass index are determinants of plasma von Willebrand factor and factor VIII levels

BackgroundVon Willebrand Disease (VWD) is the most common inherited bleeding disorder. VWD is characterized by an abnormal quantity or quality of von Willebrand Factor (VWF). Anemia is often found at presentation for a bleeding disorder evaluation due to chronic blood loss. Objectives/HypothesisWe hypothesized that anemia is associated with elevations in both VWF and factor VIII (FVIII) over baseline. We also hypothesized that obesity would be associated with increased levels of VWF. MethodsWe conducted a single-center review of the electronic health record for patients that had proximal von Willebrand profiles and Hb data. ResultsWe identified 4552 unique subjects with VWF studies and a CBC within 24 h. We found that decreasing hemoglobin inversely correlated with VWF antigen, VWF ristocetin cofactor activity, and FVIII activity. We also found that obesity and Black race were independently associated with increased VWF antigen, activity, and FVIII activity. Hb, race, and body mass index (BMI) continued to be determinants of VWF and FVIII levels in multivariable analysis. ConclusionOur study demonstrates that anemia, race, and BMI were found to be associated with elevation of VWF antigen, VWF activity, and FVIII levels. As many individuals with anemia present for evaluation for a bleeding disorder, these variables need to be considered. Key points- Anemia was found to be associated with elevation of VWF antigen, VWF activity and FVIII levels.- Testing von Willebrand factor at times of anemia may mask a diagnosis of von Willebrand Disease.

Evaluation of an automated von Willebrand factor glycoprotein IbM activity assay compared with 3 alternative von Willebrand factor activity assays

BackgroundTo overcome deficiencies of the traditional von Willebrand factor (VWF) ristocetin cofactor activity assay (VWF:RCo), several automated assays for VWF platelet-binding activity have been developed. Information on the performance of these assays and their diagnostic utility remains limited. ObjectivesTo validate the VWF:glycoprotein IbM assay INNOVANCE VWF Ac and compare it with an automated VWF:RCo assay as well as with an automated assay and a manual VWF:Ab assay and to generate reference ranges and analyze reproducibility of the VWF:glycoprotein IbM assay. MethodsClinical sites enrolled healthy subjects and patients representing the intended use population; VWF activity assays were performed, and results were analyzed. The performance of the INNOVANCE VWF Ac assay was also compared between the BCS XP System and the CS-2500 and CS-5100 analyzers. ResultsThe INNOVANCE VWF Ac assay correlated well with the VWF:RCo assay and the automated HemosIL VWF:Ab assay, with Pearson coefficients of >.9 and a predicted bias of ≤5.0 IU/dL at VWF levels of 30 IU/dL and ≤5.8 IU/dL at the levels of 50 IU/dL, but correlation and bias were not as good when compared with the REAADS manual VWF:Ab assay. Reference ranges observed for healthy subjects correlated well with previously published findings. Reproducibility of the INNOVANCE VWF Ac assay on the BCS XP System and the CS analyzers was excellent, as was correlation among devices. ConclusionThe characteristics of the INNOVANCE VWF Ac assay regarding comparability with other VWF activity assays, reference ranges, and precision support the use of this assay for evaluation of patients with concern for von Willebrand disease.

Von Willebrand factor Ristocetin co-factor activity to von Willebrand factor antigen level ratio for diagnosis of acquired von Willebrand syndrome caused by aortic stenosis

BackgroundSevere aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress–dependent cleavage of high molecular weight multimers of von Willebrand factor (VWF). While the current standard diagnostic method is so-called VWF multimer analysis that is western blotting under nonreducing conditions, it remains unclear whether a ratio of VWF Ristocetin co-factor activity (VWF:RCo) to VWF antigen levels (VWF:Ag) of <0.7, which can be measured with an automated coagulation analyzer in clinical laboratories and is used for the diagnosis of hereditary von Willebrand disease. ObjectivesTo evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome. MethodsVWF:RCo and VWF:Ag were evaluated with the VWF large multimer index as a reference, which represents the percentage of a patient’s VWF high molecular weight multimer ratio to that of standard plasma in the VWF multimer analysis. ResultsWe analyzed 382 patients with AS having transaortic valve maximal pressure gradients of >30 mmHg, 27 patients with peripheral artery disease, and 46 control patients free of cardiovascular disease with osteoarthritis, diabetes, and so on. We assumed a large multimer index of <80% as loss of VWF large multimers since 59.0% of patients with severe AS had the indices of <80%, while no control patients or patients with peripheral artery disease, except for 2 patients, exhibited the indices of <80%. The VWF:RCo/VWF:Ag ratios, measured using an automated blood coagulation analyzer, were correlated with the indices (rs = 0.470, P < .001). When the ratio of <0.7 was used as a cut-off point, the sensitivity and specificity to VWF large multimer indices of <80% were 0.437 and 0.826, respectively. ConclusionVWF:RCo/VWF:Ag ratios of <0.7 may indicate loss of VWF large multimers with high specificity, but low sensitivity. VWF:RCo/VWF:Ag ratios in patients with AS having a ratio of <0.7 may be useful for monitoring the loss of VWF large multimers during their clinical courses.

Trend in Von Willebrand Factor Profile in Women With Von Willebrand Disease During Pregnancy and Postpartum

Introduction: Pregnancy is considered a hypercoagulable state, with levels of multiple coagulation factors including Factor VIII (FVIII) and von Willebrand Factor (VWF), progressively increasing throughout, reaching their peak levels during the third trimester. However, in women with von Willebrand disease (VWD), these changes are variable and could be blunted or absent due to the wide heterogeneity of phenotypes and pathophysiological mechanisms linked to this disorder, posing a significant clinical challenge. This study aims to describe the trend in the VWF profile in women with VWD during each trimester of pregnancy and in the postpartum period. Methods: This is a retrospective single-center study that included adult pregnant women diagnosed with VWD, who were followed at Henry Ford Health between 03/2012 and 11/2022. Patients with other bleeding disorders were excluded from the study. Data on baseline characteristics, including age, race, age at childbirth, and parity were collected. Additional data collected included VWF antigen (VWF:Ag) level, FVIII activity level, and VWF ristocetin cofactor (VWF:Rco) activity during the first, second, and third trimesters, as well as in the postpartum period. Results: A total of 33 cases were included in our study. At baseline, the mean VWF:Ag level in our patients was 50.5% (reference range 50%-150%). Compared to baseline, the VWF:Ag level increased by 48.71% in the first trimester, 80.59% in the second trimester, and 168.71% in the third trimester. Postpartum, the VWF:Ag level decreased by 51.29% compared to the third trimester; however, it remained 30.89% higher than baseline levels. While the FVIII level (reference range 50%-150%) was an average of 64.8% at baseline, it increased by 21.91% in the first trimester, 74.23% in the second trimester, and 143.67% in the third trimester, compared to baseline. Postpartum, the FVIII level decreased by 48.45% from the third trimester but remained 25.62% higher than baseline. Additionally, the VWF:Rco level was an average of 44.3% (reference range 51%-215%) at baseline. Compared to baseline, the VWF:Rco level increased by 61.85% in the first trimester, 90.52% in the second trimester, and 142.66% in the third trimester. It decreased by 49.12% postpartum compared to the third trimester but remained 23.48% higher than baseline. Conclusion: In women with VWD, VWF:Ag level, FVIII level, and VWF:Rco levels steadily increase during pregnancy, reaching their maximum levels during the third trimester. All three levels decreased in the postpartum period; however, they remain higher compared to baseline at a median follow up of 105 days after delivery.

Impact of Blood Group O and Von Willebrand Factor on Bleeding Severity in Patients with Von Willebrand Disease and Bleeding Disorder of Unknown Cause

Background: In patients with bleeding, von Willebrand disease (VWD) is diagnosed at von Willebrand factor (VWF) levels of ≤50 IU/dL ( James et al. Blood Adv. 1:280-300. 2021). Nevertheless, the majority of patients with a mild to moderate bleeding disorder (MBD) is diagnosed with bleeding disorder of unknown cause (BDUC), upon exclusion of VWD, platelet function defects (PFD), and coagulation factor deficiencies (CFD). Bleeding severity in patients with VWD does not correlate well with VWF levels in type 1 VWD, suggesting other modifiers of the bleeding tendency, when patients with severe VWD are excluded. Blood group O (BG O) is associated with lower VWF levels and has been found to be overrepresented in VWD. In BDUC patients, BGO is also overrepresented and recognized as an independent risk factor for bleeding severity and recurrent bleeding ( Mehic et al. Blood Adv.20: 5157-5164. 2020; Mehic et al. JTH. 7: 1757-1768. 2023). Aim: This study aimed to comprehensively investigate the clinical phenotype of patients with VWD or BDUC from the Vienna Bleeding Biobank (VIBB) and evaluate the direct effect of ABO BG on bleeding severity beyond its effect on VWF levels. Methods: Patients with VWD or BDUC from the VIBB, a single-center cohort study (EC 603/2009), were categorized by lowest VWF, as measured by VWF:antigen (VWF:Ag) or VWF ristocetin co-factor activity (VWF:RCo), into four groups: &amp;lt;30 IU/dL (very low VWF), 30-50 IU/dL (low VWF), 51-80 IU/dL, and &amp;gt;80 IU/dL. Patients diagnosed with PFD (n=221), CFD (n=28), or other diagnoses (n=4) were excluded. To study the association of BG O with bleeding severity irrespective of VWF levels, we fitted linear regression models adjusting for age, sex, and lowest VWF. We allowed for non-linearity by modelling age and sex with restricted cubic splines. Results: A total of 637 patients, 18 (2.8%) with very low VWF, 55 (8.6%) with low VWF, 237 (37.2%) with VWF levels ranging from 51 to 80 IU/dL and 327 (51.3%) with VWF levels &amp;gt;80 IU/dl, were included in this analysis, as summarized in Table 1. BG O was more prevalent in patients with very low VWF levels (61%), low VWF levels (71%), and VWF levels 51-80 IU/dL (66%) than in patients with VWF levels &amp;gt;80 IU/dL (30%). Patients with low VWF were youngest, followed by very low VWF and those with VWF 51-80 IU/dL. Age was highest in the patient group with VWF levels &amp;gt;80 IU/dL at clinical presentation and might influence VWF levels and bleeding scores. With regard to bleeding severity, patients with very low VWF levels had highest median bleeding scores or number of bleeding manifestations, while the bleeding severity was similar amongst the other three categories (Table 1). VWF levels did not correlate with bleeding severity scores (Vicenza score: Spearman rho=-0.03, p=0.53; ISTH BAT: rho=-0.02, p=0.75; number of bleeding manifestations: rho=-0.02, p=0.66). In linear regression, holding age, sex, and lowest VWF levels constant, predicted mean bleeding scores and number of bleeding manifestations were higher with BG O (Vicenza score mean difference [MD], 0.64; 95% confidence interval [95%CI], 0.12 to 1.25; p=0.015; ISTH BAT MD 0.52; 95%CI, -0.25 to 1.28; p=0.18; number of bleeding manifestations MD, 0.24; 95%CI, -0.06 to 0.54; p=0.12) (Figure 1). Conclusion: Our findings demonstrate an overlap between VWD and BDUC with a similar clinical phenotype and bleeding phenotype, but increased severity in patients with VWF levels &amp;lt;30 IU/dL. Patients with BG O had increased bleeding scores compared to those with BG non-O. BG O appears to be an independent factor in modifying bleeding severity in both patients with VWD or BDUC across all VWF ranges.

Recombinant von Willebrand factor and tranexamic acid for heavy menstrual bleeding in patients with mild and moderate von Willebrand disease in the USA (VWDMin): a phase 3, open-label, randomised, crossover trial

Heavy menstrual bleeding occurs in 80% of women with von Willebrand disease and is associated with iron deficiency and poor response to current therapies. International guidelines indicate low certainty regarding effectiveness of hormonal therapy and tranexamic acid. Although von Willebrand factor (VWF) concentrate is approved for bleeds, no prospective trials guide its use in heavy menstrual bleeding. We aimed to compare recombinant VWF with tranexamic acid for reducing heavy menstrual bleeding in patients with von Willebrand disease. VWDMin, a phase 3, open-label, randomised crossover trial, was done in 13 haemophilia treatment centres in the USA. Female patients aged 13-45 years with mild or moderate von Willebrand disease, defined as VWF ristocetin cofactor less than 0·50 IU/mL, and heavy menstrual bleeding, defined as a pictorial blood assessment chart (PBAC) score more than 100 in one of the past two cycles were eligible for enrolment. Participants were randomly assigned (1:1) to two consecutive cycles each of intravenous recombinant VWF, 40 IU/kg over 5-10 min on day 1, and oral tranexamic acid 1300 mg three times daily on days 1-5, the order determined by randomisation. The primary outcome was a 40-point reduction in PBAC score by day 5 after two cycles of treatment. Efficacy and safety were analysed in all patients with any post-baseline PBAC scores. The trial was stopped early due to slow recruitment on Feb 15, 2022, by a data safety monitoring board request, and was registered at ClinicalTrials.gov, NCT02606045. Between Feb 12, 2019, and Nov 16, 2021, 39 patients were enrolled, 36 of whom completed the trial (17 received recombinant VWF then tranexamic acid and 19 received tranexamic acid then recombinant VWF). At the time of this unplanned interim analysis (data cutoff Jan 27, 2022), median follow-up was 23·97 weeks (IQR 21·81-28·14). The primary endpoint was not met, neither treatment corrected PBAC score to the normal range. Median PBAC score was significantly lower after two cycles with tranexamic acid than with recombinant VWF (146 [95% CI 117-199] vs 213 [152-298]; adjusted mean treatment difference 46 [95% CI 2-90]; p=0·039). There were no serious adverse events or treatment-related deaths and no grade 3-4 adverse events. The most common grade 1-2 adverse events were mucosal bleeding (four [6%] patients during tranexamic acid treatment vs zero during recombinant VWF treatment) and other bleeding (four [6%] vs two [3%]). These interim data suggest that recombinant VWF is not superior to tranexamic acid in reducing heavy menstrual bleeding in patients with mild or moderate von Willebrand disease. These findings support discussion of treatment options for heavy menstrual bleeding with patients based on their preferences and lived experience. National Heart Lung Blood Institute (National Institutes of Health).

Effects of Hypobaric Hypoxia on Coagulation in Healthy Subjects Exposed to 3,500 m Altitude.

Kammerer, Tobias, Anna Walzl, Thomas Müller, Philipp Groene, Giulia Roveri, Rachel Turner, Johanna Roche, Hannes Gatterer, Christoph Siebenmann, and Simon T. Schäfer. Effects of hypobaric hypoxia on coagulation in healthy subjects exposed to 3,500 m altitude. High Alt Med Biol. 24:94-103, 2023. Background: Hypoxia is discussed as a trigger for prothrombotic changes both in intensive care and high altitude medicine. This research study aimed to evaluate the effect of isolated hypobaric hypoxia (HH) on coagulation in females in a highly standardized setting. Methods: Twelve healthy female subjects were studied under HH (equivalent to 3,500 m) and normoxia (NX) during two 4-day sojourns, in a strictly controlled crossover design. Nutrition, fluid intake, hormonal status (i.e., menstrual cycle variation), and physical stress were standardized. Functional coagulation and blood lysis were measured by viscoelastometry and compared between HH and NX. In addition, plasma-based coagulation tests (PBCTs), namely prothrombin time, activated partial thromboplastin time, fibrinogen, factor VIII coagulation activity (FVIII:C), von Willebrand factor antigen (vWF:Ag), and von Willebrand factor ristocetin cofactor activity (vWF:RCo) were measured. Results: Neither for Viscoelastic Haemostatic Assays nor for PBCTs significant changes were found for HH compared with NX (all p > 0.05). Specifically, the lysis ability, as well as clotting time, clot formation, clot amplitude, and maximum clot firmness unchanged were similar between HH and NX. This also applied to all other variables. Conclusion: We demonstrate that moderate HH per se has no influence on blood coagulation in healthy females.

Constructing and evaluating the effect of micron-scale structures on von Willebrand factor damage

The incidence of clinical complication gastrointestinal bleeding has been proved as consequence of von Willebrand factor (VWF) damage after mechanical circulatory support in clinic. Many studies have been conducted to evaluate VWF damage, of which the most studied influencing factors are mechanical factors such as shear stress. However, in addition to mechanical factors, VWF damage may also be affected by interface factors. To address this issue, a roller pump circulation platform was established to investigate the effect of material surface micron-scale structures distribution on VWF damage in flow state. A composite micro-structure combining microngrating and micronpost was designed and constructed on the surface of Si wafer by lithography and reactive ion etching, and detailed characterization of material surfaces was also performed. Then the changes of VWF antigen, VWF ristocetin cofactor activity, and the degradation of high molecular weight VWF on these surfaces were investigated and compared. The results showed that, with the encryption of surface micro-structures arrangement, the material surface tends to be more hydrophobic, which is beneficial to reduce VWF damage. Therefore, in the design of material surface inside the mechanical circulatory support devices, it can be considered to add some surface micro-structures with a certain distribution density to change the hydrophilicity and hydrophobicity, so as to minimize the VWF damage. These results can provide important references for the evaluation of VWF damage caused by interface factors, and aid in designing material surface inside the mechanical circulatory support devices.

Impact of ABO Blood Group on Thromboembolic and Bleeding Complications in Patients with Left Ventricular Assist Devices.

The ABO blood group system is linked to hemostasis via its relationship with von Willebrand factor (VWF) and factor VIII (FVIII). In the current study, we investigated the association of the ABO system with clinical outcomes as well as VWF and platelet function in patients with left ventricular assist devices (LVADs). Bleeding and thromboembolic complications were assessed in 111 patients during 1 year after LVAD implantation. In 67 LVAD patients, VWF antigen, VWF activity, VWF ristocetin cofactor, VWF collagen-binding, and FVIII activity were assessed. Platelet surface P-selectin and activated glycoprotein IIb/IIIa were determined by flow cytometry, and soluble P-selectin was measured with an enzyme-linked immunoassay. Platelet aggregation was assessed by light transmission and impedance aggregometry. Thirty-six patients (32.4%) experienced a bleeding and 22 patients (19.8%) a thromboembolic event. In univariate analyses, patients with blood group O had numerically more bleeding complications and less thromboembolic events as compared to patients with blood group non-O (both p ≥ 0.05). After multivariable adjustment, blood group O was significantly associated with a higher risk of bleeding (hazard ratio 2.42 [95% confidence interval 1.03-5.70], p = 0.044) but not linked to thromboembolic complications. Patients with blood group O had significantly lower levels of VWF and FVIII (all p < 0.05), whereas P-selectin expression in response to thrombin-receptor activating peptide and soluble P-selectin were higher as compared to patients with blood group non-O (both p < 0.05). LVAD patients with blood group O are at an increased bleeding risk, potentially due to lower VWF and FVIII levels.

Polyphosphate Activates von Willebrand Factor Interaction with Glycoprotein Ib in the Absence of Factor VIII In Vitro.

Polyphosphate (polyP), a phosphate polymer released by activated platelets, may modulate various stages of hemostasis by binding to blood proteins. In this context, we previously reported that polyP binds to the von Willebrand factor (VWF). One of the most significant functions of VWF is to bind to and protect the blood circulating Factor VIII (FVIII). Therefore, here, we study the role of polyP in the VWF-FVIII complex in vitro and suggest its biological significance. Surface plasmon resonance and electrophoretic mobility assays indicated that polyP binds dynamically to VWF only in the absence of FVIII. Using the VWF Ristocetin Cofactor assay, the most accepted method for studying VWF in platelet adhesion, we found that polyP activates this role of VWF only at low levels of FVIII, such as in plasmas with chemically depleted FVIII and plasmas from severe hemophilia A patients. Moreover, we demonstrated that FVIII competes with polyP in the activation of VWF. Finally, polyP also increases the binding of VWF to platelets in samples from patients with type 2 and type 3 von Willebrand disease. We propose that polyP may be used in designing new therapies to activate VWF when FVIII cannot be used.

Type 2M VWD Laboratory Defects More Consistent Than Bleeding Symptoms across Family Members

Background Von Willebrand disease (VWD) is an inherited bleeding disorder characterized by a defect or decrease of von Willebrand factor (VWF). Type 2 VWD results from qualitative defects in VWF. Type 2M VWD is defined by defective platelet binding to GPIb and/or collagen. While the inheritance of type 2M VWD is autosomal dominant, the extent of symptoms across family members has not been well characterized. Methods Subjects included index cases with type 2M VWD and all enrolled family members from a large US study of VWD (Zimmerman Program). Index cases were enrolled due to a prior diagnosis of VWD. All available family members were enrolled if willing. VWF testing performed included VWF antigen (VWF:Ag), VWF platelet binding by either VWF ristocetin cofactor activity (VWF:RCo) or VWF:GPIbM, collagen binding with types I, III or IV collagen (VWF:CB1, VWF:CB3, VWF:CB4) and VWF multimer distribution. Bleeding scores were calculated using the ISTH bleeding assessment tool (BAT). DNA sequencing was performed for the full length VWF gene. Statistics were calculated using GraphPad Prism with a p value of <0.05 considered significant. Family members were defined as affected if they had VWF activity levels consistent with type 2M VWD and presence of genetic variant if identified. Results 24 index cases, 51 affected family members and 40 unaffected family members were available for analysis. The mean VWF:RCo/VWF:Ag ratio for index cases was 0.5 and the mean VWF:CB/VWF:Ag ratio was 1.05. The VWF:RCo/VWF:Ag ratio was similar in affected family members (0.44, p=NS) but higher as expected in unaffected members (0.89, p<0.001 for index case vs. unaffected, p<0.001 for affected vs. unaffected). In contrast, there was no difference in VWF:CB/VWF:Ag ratios between index cases and affected family members (0.94, p=NS). Data for VWF:GPIbM, collagens I and IV were available only on index cases, and showed low VWF:GPIbM/VWF:Ag (mean 0.79), higher VWF:CBI/VWF:Ag (mean 1.31) and lower VWF:CBIV/VWF:Ag (mean 0.85). There was a negative correlation between VWF:GPIbM and VWF:CBIV, with subjects having either low VWF:GPIbM and normal VWF:CBIV or low VWF:CBIV and normal VWF:GPIbM. Similar results were seen for VWF:CB1 although only 1 subject was diagnosed with type 2M VWD and low VWF:CB1. Median bleeding scores were highest in index cases (7.5), intermediate in affected family members (3) and normal in unaffected family members (1). There was a significant difference between index cases and affected family members (<0.015), and between index cases and unaffected family members (p<0.001). One subject had a genetic variant in the VWF A3 domain, one in the VWF D3 domain and the remainder of index cases had a defect in the VWF A1 domain. Conclusions These findings suggest that decreases in platelet and collagen binding activity increase bleeding risk in families with type 2M VWD. Index cases had higher bleeding scores as compared to affected family members, suggesting that diagnosis of family members may require more attention. Precise understanding of these defects may assist in better diagnosis and treatment of von Willebrand disease.

Extended coagulation profile of children with Long Covid: a prospective study

Emerging data suggests that endotheliopathy changes can be associated with post covid condition (PCC) in adults. Research on the matter in children is lacking. We analyzed an extended coagulation profile including biomarkers of endothelial damage in children with PCC and compared it with a control group of children that fully recovered post- SARS-CoV-2 infection. A case-control study enrolling children below 18 years of age with previous microbiologically confirmed SARS-CoV-2 infection in a pediatric post-covid unit in Italy ≥ 8 weeks after the initial infection. Samples were taken at 8 and 12 weeks after the SARS-CoV-2 diagnosis and analyzed for coagulation profiling (fibrinogen, prothrombin time, international normalized ratio, activated partial thromboplastin time, d-dimers, factor VIII coagulant activity, plasma von Willebrand factor (VWF) antigen and VWF ristocetin cofactor (RC)). We compared coagulation profiles in samples from children identified with PCC (at least one, or three or more symptoms, which could not be explained by an alternative diagnosis, at the 8- and 12-week follow-up assessment using the pediatric Long Covid International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) survey. Seventy-five children were enrolled, 49.3% were females, the median age was 10.2 (IQR 4.9) years. Forty-six (61%) of the children had at least one persisting symptom at the eight weeks post-onset, (PCC8); 39/75 (52%) had persistent symptoms for more than 12 weeks (PCC12) and 15/75(32%) had at least three persisting symptoms (PCC ≥ 3) at 12 weeks. Children with PCC presented more frequently with abnormal D-Dimer levels above the reference range compared to children that had fully recovered at the 8–12 weeks (39.1% vs. 17.2%, p = 0.04), and 12 week follow up or more (41% vs. 17.2%, p = 0.05), and in children with three or more symptoms at 12 weeks follow up compared to those that had recovered (64.3% vs. 22.2%, p = 0.002). For the other coagulation profiles, there were abnormal values detected for VWF, FVIII, RC and Fibrinogen but no significant differences between children with PCC compared to controls. Although the majority of children in our cohort showed coagulation profile within or close to normal ranges, we found that a higher proportion of children with PCC, and specifically those with a more severe spectrum characterized with three or more persisting symptoms, had abnormal D-dimer levels compared to other children that fully recovered from an acute SARS-CoV-2 infection.

Compound heterozygosity for novel von Willebrand factor genetic variants associated with von Willebrand disease in two Chinese patients.

Von Willebrand factor (VWF) encodes a secreted glycoprotein involved in primary hemostasis. Genetic mutations in this gene leading to either quantitation or qualitative defects of VWF, result in von Willebrand disease (VWD), an inherited bleeding disorder. In this study, two families with VWD were recruited and submitted to a series of clinical and genetic examinations. prothrombin time, activated partial thromboplastin time, thrombin time, factor VIII coagulant activity (FVIII:C), VWF antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo) tests were measured in peripheral blood. F8, F9, and VWF genes were sequenced using next-generation sequencing, and Sanger sequencing was used as a validation method. Both families had a child suffered spontaneous bleeding. Patient 1 showed normal VWF:Ag, severely decreased FVIII:C and VWF:RCo. Patient 2 showed severely decreased FVIII:C, VWF:Ag, and VWF:RCo. Compound heterozygous mutations of VWF gene were identified in both patients. Patient 1 had a novel deletion variant c.1910_1932del (p.Gly637AlafsTer5) and a missense variant c.605G>A (p.Arg202Gln). Patient 2 had a novel missense variant c.4817T>A (p.Met1606Lys) and a novel missense variant c.5983C>T (p.Pro1995Ser). We described clinical and molecular features of VWD caused by compound heterozygous mutations in two Chinese patients. Our results expand the variation spectrum of the VWF gene and deepen the understanding of the relationship between the genotype and clinical characteristics of VWD.

Ristocetin dependent cofactor activity in von Willebrand disease diagnosis: Limitations of relying on a single measure

BackgroundVon Willebrand disease (VWD) is a common inherited bleeding disorder, however the diagnosis can be complicated by a subjective bleeding history and issues with some current von Willebrand factor (VWF) laboratory assays. ObjectivesIn the Zimmerman Program, we sought to determine how often a type 1 diagnosis was based on a single low VWF ristocetin cofactor (VWF:RCo) level resulting from the common genetic variant p.D1472H or an isolated assay issue, if that low value was corroborated by the VWF glycoprotein‐IbM (VWF:GPIbM) assay, and if retesting confirmed original levels. MethodsNew patients being evaluated for bleeding were consented. Analysis included VWF sequencing, bleeding scores, and comparisons of local VWF antigen (VWF:Ag) and VWF:RCo to central VWF:Ag and VWF:GPIbM. ResultsA total of 18% of VWD subjects had a low local VWF:RCo, but normal VWF:Ag and normal central testing including VWF:GPIbM. Seventy percent of the low VWF:RCo cohort had no pathogenic VWF variants; however, 33% carried p.D1472H. Low VWF:RCo subjects with follow‐up local testing within 2 years showed those with p.D1472H continued to have low VWF:RCo and VWF:RCo/VWF:Ag ratio with normal VWF:GPIbM. Subjects without p.D1472H had an increase mean VWF:RCo, resulting in 59% with normal levels on repeat testing. ConclusionsThe diagnosis of VWD based on a single low VWF:RCo but normal VWF:Ag, was often attributed to p.D1472H or variability in VWF:RCo that was eliminated with VWF:GPIbM. Our study suggests that using VWF:RCo alone for diagnostic purposes may be insufficient while repeat VWF:RCo or VWF:GPIbM testing can be valuable in establishing a VWD diagnosis.

Von Willebrand factor activity levels are influenced by driver mutation status in polycythemia vera and essential thrombocythemia patients with well-controlled platelet counts.

von Willebrand factor ristocetin cofactor (vWF activity) and platelet count (PLT) are negatively correlated in patients with polycythemia vera (PV) and essential thrombocythemia (ET). However, vWF activity does not always normalize upon controlling PLT in those patients. To address this issue, we investigated the correlation between vWF activity and PLT in PV and ET patients. The negative correlation between vWF activity and PLT was stronger in calreticulin mutation-positive (CALR+) ET than in Janus kinase 2 mutation-positive (JAK2+) PV or ET groups. When PLT were maintained at a certain level (<600 × 109 /L), low vWF activity (<50%) was more frequently observed in JAK2+ PV patients than in JAK2+ ET (p=.013) or CALR+ ET (p=.013) groups, and in PV and ET patients with ≥50% JAK2+ allele burden than in those with allele burden <50% (p=.015). High vWF activity (>150%) was more frequent in the JAK2+ ET group than in the CALR+ ET group (p=.005), and often associated with vasomotor symptoms (p=.002). This study suggests that some patients with JAK2+ PV or ET have vWF activity outside the standard range even with well-controlled PLT, and that the measurement of vWF activity is useful for assessing the risk of thrombosis and hemorrhage.

ADAMTS13 levels in a plasma-derived FVIII concentrate: A potential therapeutic option for patients with congenital thrombotic thrombocytopenic purpura

IntroductionTreatment of congenital thrombotic thrombocytopenic purpura (cTTP), a disease characterized by the congenital deficiency of ADAMTS13, remains a challenge as there are no specific treatments available yet, other than therapy based on the use of fresh frozen plasma (FFP). Since cTTP is caused by low levels of ADAMTS13 protein, commercially available coagulation factor concentrates have been considered as potential ADAMTS13 source in place of FFP. The study aimed to validate the therapeutic potential of a plasma-derived factor VIII (FVIII) product as a source of ADAMTS13. MethodsThe quantitation of ADAMTS13 activity levels in eight lots of a plasma-derived FVIII product, (Koāte®) was carried out with three different methodologies: a Fluorescence Resonance Energy Transfer (FRET) assay, a chemiluminescence assay, and a chromogenic ELISA. ADAMTS13 protein antigen levels were measured by the FRET technique as well. In addition, von Willebrand factor (VWF) activity (VWF ristocetin cofactor, VWF:RCo, and VWF collagen binding, VWF:CB) and antigen (VWF:Ag) were measured using chemiluminescence assays. Qualification protocols were applied to the methods used. ResultsThe results showed high levels of ADAMTS13 in all eight Koāte® lots analyzed, with antigen and activity levels respectively of 10.72 IU/ml ± 3.94 and 5.62 IU/ml ± 1.39. Despite the significant content of ADAMTS13, VWF integrity seems not to be affected (0.81 ± 0.15 VWF:RCo/VWF:Ag and 0.75 ± 0.15 VWF:CB/VWF:Ag ratios). ConclusionsThese findings suggest that Koāte® could be a potential candidate for the treatment of cTTP, warranting evaluation in a clinical trial.

The VWF binding aptamer rondoraptivon pegol increases platelet counts and VWF/FVIII in type 2B von Willebrand disease

AbstractType 2B von Willebrand disease (VWD) is characterized by an increased binding affinity of von Willebrand factor (VWF) to platelet glycoprotein Ib. This can lead to clearance of high-molecular-weight (HMW) multimers and thrombocytopenia with a resulting moderate-severe bleeding phenotype. Rondoraptivon pegol (BT200) is a pegylated aptamer binding to the A1 domain of VWF with a novel mechanism of action: it enhances VWF/factor VIII (FVIII) levels by decreasing their clearance. To study the potential benefit of rondoraptivon pegol in patients with type 2B VWD, we conducted a prospective phase 2 trial. Patients with type 2B VWD received 3 mg rondoraptivon pegol subcutaneously on study days 1, 4, and 7, followed by 6 to 9 mg every week until day 28. Five patients (male:female ratio = 3:2) were included. Rondoraptivon pegol rapidly tripled platelet counts from a median of 60 to 179 × 10E9/L (P < .001). Circulating VWF antigen increased from a median of 64% to 143%, which doubled FVIII activity levels from 67% to 134%. In all thrombocytopenic patients, plasma levels of VWF:GPIbM normalized, VWF ristocetin cofactor and VWF collagen-binding activity increased, and HMW multimers appeared. These pronounced improvements reversed during washout of the drug, thus demonstrating causality. The A1 domain binding aptamer directly corrects the underlying defect of type 2B VWD, thus providing a novel potential option for prophylaxis and treatment of patients with this VWD type. These data provide the basis for a phase 2b/3 trial in such patients. This trial was registered at www.clinicaltrials.gov as #NCT04677803.

Platelet Dysfunction in Blood Donors Detected by Platelet Function Analyzer PFA-100™.

BackgroundPlatelet transfusions may be indicated to prevent and treat bleeding in patients with quantitative or qualitative platelet defects. Millions of platelet components are transfused worldwide. It is well known that platelet dysfunction predicts blood loss after surgery. Hence, the quality of the platelet donation and the resulting platelet concentrate are critical for the transfusion. The aim of this study is to assess platelet function in well-qualified blood donors.MethodologyBlood samples from 275 blood donors were collected in 0.129 M (3.8%) sodium citrate tubes prior to routine blood donation. Platelet function was assessed by measuring the closure time (CT) on the platelet function analyzer (PFA-100™; Siemens Health Diagnostics, Marburg, Germany) using collagen/epinephrine (CEPI) and collagen/ADP (CDP) cartridges.ResultsUsing the PFA-100™, 20.4% of donors had an abnormal platelet function test, of whom 9.4% had prolonged CT with two cartridges, 7% had only prolonged CEPI CTs consistent with aspirin-like defect, and 4% had prolonged CADP CTs only. We found no closure (>300 seconds) in 6.54% of donors, including 1.45% with the CEPI cartridge, 2.9% with the CADP cartridge, and 2.18% with CEPI and CADP cartridges. Level of von Willebrand factor ristocetin cofactor (vWF: RCo) activity was 112% (56-168%). Of the factors examined (age, sex, cigarette smoking, blood donation type, ABO, and Rhesus blood group), only blood group O was significantly linked with impaired platelet function test in qualified blood donors (p = 0.023; odds ratio = 1.981; 95% confidence interval (1.091-3.595)).ConclusionsSome qualified blood donors present abnormal platelet function results. More research is required to provide greater insight into the impact of platelet dysfunction in blood donors on the clinical efficacy of their platelet components. This study has confirmed that the influence of ABO blood group on the CT PFA-100™is not wholly dependent on vWF.