Von Willebrand Factor Activity Levels Research Articles

The severe von Willebrand disease variant p.M771V leads to impaired anterograde trafficking of von Willebrand factor in patient-derived and base-edited endothelial colony-forming cells

Backgroundvon Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects in von Willebrand factor (VWF). The p.M771V VWF variant leads to a severe bleeding phenotype in homozygous patients. However, the exact molecular mechanism remains unclear, which prevents personalized treatment of those VWD patients. ObjectivesThis study aimed to characterize the underlying molecular mechanisms of the p.M771V variant in multiple representative ex vivo cell models. MethodsEndothelial colony-forming cells (ECFCs) were isolated from venous blood of VWD patients from the Willebrand in the Netherlands cohort carrying homozygous and heterozygous p.M771V VWF variants. The p.M771V variant was also introduced in cord blood-derived ECFCs (CB-ECFCs) through adenine base editing and was overexpressed in HEK293 cells. Biosynthesis, storage, and secretion of VWF was studied using biochemical methods and confocal microscopy. ResultsTwo unrelated homozygous p.M771V patients presented with very low VWF activity and antigen levels in plasma. Patient ECFCs showed impaired uncleaved VWF processing into mature VWF, with secreted VWF being severely reduced when compared to ECFCs of healthy donors. Multimer analysis of p.M771V ECFCs showed a deficiency of high molecular weight VWF multimers. Immunofluorescent staining revealed VWF retention in the endoplasmic reticulum; this was confirmed in various populations of base-edited CB-ECFCs harboring the p.M771V variant. ConclusionThe severe endothelial phenotype observed in patient-derived p.M771V ECFCs, HEK293 cells, and an original base-edited CB-ECFC modeling system show that endoplasmic reticulum retention of VWF and failure to undergo subsequent proteolytic processing underpins the severe bleeding phenotype of patients with homozygous variants at M771.

Misleading antigenic von Willebrand factor levels in acquired von Willebrand syndrome secondary to monoclonal gammopathy of undetermined significance.

In the diagnosis and treatment of acquired von Willebrand syndrome (AVWS), von Willebrand factor (VWF) antigen levels (VWF:Ag) are helpful for quantifying blood VWF-protein levels. Most clinical laboratories measure VWF:Ag by latex immunoassay (LIA), but underlying diseases of AVWS may influence LIA results. A 60year-old AVWS patient with immunoglobulin G (IgG) kappa-type monoclonal gammopathy of undetermined significance (MGUS) showed reduced VWF activity but normal levels of VWF:Ag. His VWF multimers were broadly decreased, which represented a large discrepancy with VWF:Ag. To investigate the mechanism of this discrepancy, we measured the patient's plasma VWF:Ag by in-house enzyme-linked immunosorbent assay (ELISA) and LIA. We also purified the IgG fraction from the patient's serum and measured VWF:Ag in VWF-deficient plasma supplemented with this fraction. VWF:Ag measured by in-house ELISA (VWF:AgELISA) was much lower than that measured by LIA (VWF:AgLIA), which indicated reduced VWF-protein volume in blood. Indeed, VWF:Ag was detected by LIA in VWF-deficient plasma spiked with a patient-derived IgG fraction. These results suggest that LIA detected a non-specific immunoreaction and overestimated the patient's VWF:AgLIA. Clinicians should be aware that underlying diseases of AVWS could influence the LIA system, and interpret VWF:Ag cautiously.

Pregnancy loss in individuals with von Willebrand disease and unspecified mucocutaneous bleeding disorders: a multicenter cohort study

BackgroundWhile bleeding around pregnancy is well described in von Willebrand disease (VWD), the risk of pregnancy loss is less certain. ObjectivesWe aimed to describe the frequency of pregnancy loss in females with VWD compared with those with a similar mucocutaneous bleeding phenotype and no VWD or compared with nonbleeding disorder controls. MethodsFemale patients were consecutively approached in 8 specialty bleeding disorder clinics between 2014 and 2023. The VWD group was defined as having von Willebrand factor (VWF) antigen and VWF activity levels, each <0.50 IU/mL on ≥2 occasions, and a condensed MCMDM-1 score of ≥4. The non-VWD mucocutaneous bleeding disorder group had VWF levels ≥ 0.50 IU/mL on ≥2 occasions and an MCMDM-1 score ≥ 4. A nonbleeding disorder control group was recruited in pregnancy from a low-risk maternity clinic. ResultsThere were 150 females in the VWD group, 145 in the non-VWD mucocutaneous bleeding disorder group, and 137 in the control group. There was a similar frequency of individuals with ≥1 loss in the VWD group (45.3%, 68/150), the non-VWD group (56.6%; 82/145; −11.2%; 97.5% CI, −24.2%, 1.8%), and the nonbleeding disorder control group (37.2%; 51/137; 8.1%; 97.5% CI, −4.9%, 21.1%). Using a logistic regression, the odds ratio of pregnancy losses in the VWD group vs the non-VWD group was 0.94 (95% CI 0.65, 1.36). All groups experienced more recurrent losses compared with the literature. ConclusionThere was no statistically significant difference in risk of pregnancy loss between females with VWD, females with a similar mucocutaneous bleeding phenotype, and nonbleeding disorder controls.

Evaluating the Role of Silymarin in Coordinating the Relationship between Inflammation and Thrombosis by Affecting Endothelial Cells

Background: A widespread crosstalk between inflammation and coagulation has been shown in numerous studies. This suggests that coagulation can trigger an inflammatory response which ultimately leads to coagulation activation. Previous research has shown that polyphenols can affect blood pressure and endothelial dysfunction, resulting in reduced risk of cardiovascular diseases. This study aimed to investigate whether Silymarin, a flavonolignans, could play a role in the interaction between inflammation and coagulation by influencing endothelial cells. Materials and Methods: In this experimental study, human umbilical vein endothelial cells (HUVECs) were seeded with and without various concentrations of silymarin. In vivo, treatment with silymarin was also carried out. Coagulative and fibrinolytic factors, including Von Willebrand factor (VWF) and Factor VIII (FVIII), tissue plasminogen activator-1 (TPA-1), and inflammatory factors, including interleukin 8 (IL-8) and tumor necrosis factor-alpha (TNF-α), were evaluated by flow cytometry, Real-Time Polymerase Chain Reaction (qPCR), Enzyme-Linked Immunosorbent Assay (ELISA) and Immunocytochemistry (ICC). Results: Silymarin increased the gene expression, release, and storage of VWF while diminishing the gene expression, release, and storage of TPA-1 (P ˂ 0.05). The activity of FVIII was dramatically increased, and IL-8 and TNF-α levels were augmented. The in vivo study also indicated an elevated plasma level of VWF and IL-8 by silymarin administration. Conclusion: The results showed that, although silymarin reduces inflammatory factors, it can affect coagulation factors by increasing the levels of VWF and FVIII activity and inhibiting TPA-1 production, thereby making thrombosis probable. Consequently, it is advisable to prescribe this medication with caution for individuals who are susceptible to thrombotic events.

Early Online.

Long-haul flights have been associated with a two- to four-fold increased risk of aviation-related thrombosis (ART). Several studies have investigated the extent to which hypoxic hypobaric exposure, dehydration and prolonged immobilisation during air travel induce changes in haemostasis. To investigate the role of high altitude as a risk factor for ART. Healthy volunteers aged ≥18 years (N=40), without risk factors for venous thromboembolism, were exposed to an exacerbated altitude of 18 000 feet (5 486 m) for 1 hour. During the flight, the oxygen (O2) levels of the participants, who received supplemental O2, were measured by pulse oximetry and maintained at >92%. Venous blood and urine samples were collected prior to departure and immediately after flying in an unpressurised twin-engine airplane. D-dimer levels, thromboelastography (TEG) parameters, von Willebrand factor (VWF) activity and urine osmolality were measured. The participants were 19 men and 21 women, with a mean (standard deviation) age of 46 (14) years. A significant difference in D-dimer levels, VWF activity, urine osmolality and TEG parameters (reaction (R) time, kinetic (K) time and maximum amplitude (MA)) before and after the 1-hour flight was observed (p<0.001). Urine osmolality correlated positively with VWF activity levels (r=0.469; p<0.002). Air travel at high altitude induced a hypercoagulable state in healthy volunteers. Future research should focus on whether thromboprophylaxis can significantly obviate the activation of coagulation in response to high altitude.

Efficacy of a 1:1 ratio VWF/FVIII concentrate in patients with von Willebrand disease.

Patients with von Willebrand disease (VWD) require administration of von Willebrand factor (VWF) concentrates peri-operatively. Concerns about FVIII accumulation after repetitive injections of a 1:1 ratio VWF/FVIII clotting factor concentrate (CFC) led this study to explore the recovery and FVIII accumulation over time. This monocentre study examined patients with VWD receiving perioperative 1:1 ratio CFC infusions. CFC dosing was based on body weight and endogenous VWF/FVIII activity. FVIII and VWF activity was monitored at T0 (baseline), T1 (15 min postinfusion), and trough levels at T2-T6 (24-120h). We included 125 patients, undergoing 125 procedures (63 major surgeries, 62 minor), with a median of two CFC infusions (IQR 1-3). With a mean administered dose of 35.7IU/kg CFC, recovery rates of FVIII and VWF were 2.6IU/dL per IU/kg and 2.4IU/dL per IU/kg, respectively. Mean FVIII levels at T0 were 62 (SD 51.9), T1: 164 (SD 80.4), T2: 155 (SD 62.8), T3: 162 (SD 59.8), T4: 124 (SD 78.4), and T5: 120 (SD 65.3) IU/dL. Mean VWF activity levels at T0 were 29 (SD 25.0), T1: 133 (SD 43.7), T2: 92 (SD 37.2), and T3: 86 (SD 37.5) IU/dL. Subgroup analysis in 47 patients with more than three infusions, showed no accumulation of mean FVIII levels. This perioperative study demonstrated excellent FVIII and VWF recovery of a 1:1 ratio VWF product in patients with VWD. Stable FVIII and VWF activity levels were observed after repeated infusions, without accumulation. Most major surgeries required only three CFC infusions.

A comparative study of anti–ADAMTS-13 antibody dynamics in immune-mediated thrombotic thrombocytopenic purpura

BackgroundThrombotic thrombocytopenic purpura, particularly its immune-mediated variant (iTTP), necessitates accurate diagnostic approaches for effective management. ObjectivesTo compare a chemiluminescence immunoassay (CLIA) and an enzyme-linked immunosorbent assay (ELISA) for testing ADAMTS-13 activity and detecting anti–ADAMTS-13 autoantibodies (AAbs) in patients with iTTP. MethodsThis study involved 31 paired samples from 12 iTTP patients. ADAMTS-13 activity was measured using the HemosIL AcuStar (Instrumentation Laboratory, CLIA) and Technozym (Technoclone) activity assay (ELISA). The presence of AAbs was assessed using Technozym ADAMTS-13-INH assay (ELISA) and HemosIL AcuStar activity (CLIA) within a Bethesda assay following mixing with normal pool plasma. von Willebrand factor (VWF) multimers were analyzed using the HYDRASYS-2 SCAN system and the HYDRAGEL 5- or 11-VW Multimer kits (Sebia). VWF activity levels were measured with the HemosIL AcuStar VWF:GPIbR on the ACL AcuStar Analyzer (IL). ResultsFor ADAMTS-13 activity, a strong linear relationship and no bias between CLIA and ELISA were confirmed (slope = 1.01 [0.91, 1.11], intercept = 0.00 [−0.47, 0]). However, significant discrepancies were found in AAb detection during remission phases with ADAMTS-13 activity between 10% and 50%, with CLIA and ELISA showing significant divergence (P < .001, Cohen’s g = 0.34). Consistently, VWF multimers and activity levels exhibited significantly different values between remission samples with ADAMTS-13 activity below 50% and above 50%. In longitudinal analysis of patients with multiple iTTP relapses, positivity to CLIA appears to precede ELISA in predicting exacerbations. ConclusionWhile CLIA and ELISA might be interchangeable for assessing ADAMTS-13 activity, they are not equivalent for detecting AAbs, particularly in patients in clinical remission with ADAMTS-13 activity between 10% and 50%.

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.

Nanobody activator improves sensitivity of the von Willebrand factor activity assay to multimer size

BackgroundThe activity of von Willebrand factor (VWF) in facilitating platelet adhesion and aggregation correlates with its multimer size. Traditional ristocetin-dependent functional assays lack sensitivity to multimer sizes. Recently, nanobodies targeting the autoinhibitory module and activating VWF were identified. ObjectivesTo develop an assay that can differentiate the platelet-binding activity of VWF multimers. MethodsA novel enzyme-linked immunosorbent assay (nanobody-triggered glycoprotein Ib binding assay [VWF:GPIbNab]) utilizing a VWF-activating nanobody was developed. Recombinant VWF, plasma-derived VWF (pdVWF), and selected gel-filtrated fractions of pdVWF were evaluated for VWF antigen and activity levels. A linear regression model was developed to estimate the specific activity of VWF multimers. ResultsOf the 3 activating nanobodies tested, 6C11 with the lowest activation effect exhibited the highest sensitivity for high-molecular-weight multimers (HMWMs) of VWF. VWF:GPIbNab utilizing 6C11 (VWF:GPIbNab6C11) produced significantly higher activity/antigen ratios for recombinant VWF (>2.0) and HMWM-enriched pdVWF fractions (>2.0) than for pdVWF (∼1.0) or fractions enriched with shorter multimers (<1.0). The differences were much larger than those produced by VWF:GPIbNab utilizing other nanobodies, VWF:GPIbM, VWF:GPIbR, or VWF:CB assays. Linear regression analysis of 5 pdVWF fractions of various multimer sizes produced an estimated specific activity of 2.7 for HMWMs. The analysis attributed >90% of the VWF activity measured by VWF:GPIbNab6C11 to that of HMWMs, which is significantly higher than all other activity assays tested. ConclusionThe VWF:GPIbNab6C11 assay exhibits higher sensitivity to HMWMs than ristocetin-based and collagen-binding assays. Future studies examining the application of this assay in clinical settings and any associated therapeutic benefit of doing so are warranted.

Monitoring of Hemostatic Status and Treatment Response in Von Willebrand Disease Using a Microchip Flow Chamber Assay

Introduction: Von Willebrand factor (VWF) carries out its hemostatic roles by interacting with both platelets and FVIII, and its appropriate active multimeric conformation is strongly affected by blood flow shear. Our aim was to assess the hemostatic status and the response to treatments in VW disease (VWD) using a flow chamber-based thrombus formation analysis system (T-TAS ®, Zacros) that allows evaluation of both primary and secondary hemostasis in a scenario closer to in vivo conditions. Methods: Blood samples from patients diagnosed with VWD type 1 (n=3), type 2A (n=2), type 2B (n=1) and type 3 (n=8, two of them with inhibitory antibodies) were collected before and after in vivo administration of the prescribed treatment with desmopressin (DDAVP ®) or plasma-derived (pd)VWF/FVIII concentrates (Table-1). The effect of ex vivo administration of 100, 150 and 300 IU/dL of pdVWF/FVIII (Fanhdi ®, Grifols) was also evaluated. Thrombus formation analysis was performed according to the manufacturer's standardized protocols: Blood samples collected in tubes containing BAPA (inhibitor of FXa and thrombin) were loaded onto type-1 collagen-coated platelet (PL)-chips to assess platelet-dependent thrombus formation. Citrated blood samples were recalcified in presence of CTI and loaded onto collagen/tissue factor-coated atherome (AR)-chips to assess thrombus formation mediated by both platelet and coagulation activation. An optimized flow rate is automatically adjusted for each chip. Area under the flow-pressure curve (AUC) values were collected. Platelet count, fibrinogen levels, prothrombin time (PT), activated partial thromboplastin time (aPTT), aPTT ratio, antigenic VWF (VWF:Ag), VWF activity by recombinant GPIb binding assay (VWF:GPIbR) and FVIII activity (FVIII:C) were also analyzed. Results: All VWD patient samples tested, including type-1 patients, showed a deficient platelet function (PL_AUC) and a reduced coagulation-dependent thrombus formation (AR_AUC) prior to treatment (Table-1). VWD type-1 responded to desmopressin with a high increase of FVIII activity (FVIII:C &amp;gt;200%) and normalization of VWF activity (VWF:GPIbR &amp;gt;100%) and AUC levels. Treatment with pdVWF/FVIII increased VWF:GPIbR levels (&amp;gt;60% after treatment) in all cases, but failed to completely normalize the AUC values, which showed a slight increase (Table-1). A high correlation (r&amp;gt;0.8) was observed between plasma levels of VWF or FVIII activity and AUC values obtained both before and after treatment Ex vivo administration of pdVWF/FVIIIa resulted in a concentration-dependent increased of PL_ and AR_AUC, with values within the normal range at the highest dose tested (300 IU/dL, equivalent to 150 IU/kg) in all cases, except for VWD type-3 samples with inhibitor and for VWD type-2B sample with PL-chip assay (Figure-1). Conclusions: Monitoring treatment efficacy in VWD using a flow chamber-based thrombus formation analysis system could be a reliable and standardized method to assess the response to desmopressin and pdVWF/FVIII concentrates in patients with VWD, allowing an optimal personalized therapeutic dosing and a better prevention of bleeding episodes in these patients. Funding: Grifols; Zacros; Fundación Rodríguez Pascual; Instituto de Salud Carlos III (ISCIII) (PI22/01461), co-funded by the European Union.

Clinical and Laboratory Presentation and Underlying Mechanism in Patients with Low VWF.

Low von Willebrand factor (VWF) refers to subjects with plasma levels of 30 to 50 IU/dL. The mechanism of low VWF is poorly understood. We chose to determine the clinical presentation, laboratory phenotype, and underlying mechanisms of low VWF. We included 250 patients characterized with low VWF. The International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT) was used to assess clinical symptoms. To determine the underlying mechanisms of low VWF, we used as markers the VWF propeptide (VWFpp) assay and FVIII:C/VWF:Ag ratio for VWF synthesis and the VWFpp/VWF:Ag ratio for VWF clearance. Results were compared with those of 120 healthy controls. Cases with abnormal screening tests were further evaluated for coagulation factor levels and platelet disorders. The median age of the cohort was 35 years (range 3-85), 21% were children (n = 53), 34% were adult males (n = 85), and 45% (n = 112) were adult females. According to the ISTH-BAT, abnormal bleeding was found in 35% of children, 47% of males, and 49% of females. No association was found between VWF activity levels and ISTH-BAT. Patients showed an overall decreased VWF synthesis/secretion and an enhanced VWF clearance was identified in 33% of them. In 89 patients (36%), there were other hemostasis-related defects, but there was no difference in the ISTH-BAT between the two groups. Our findings indicate that reduced VWF synthesis/secretion and enhanced VWF clearance are major mechanisms of low VWF levels. Patients with low VWF have significant bleeding manifestations. While other hemostasis defects occurred together with low VWF, this combination did not exacerbate clinical symptoms.

Effect of Factor VIII and Von Willebrand Factor Activities on Number of Bleeding Events in Previously Treated Patients with Von Willebrand Disease on Regular Prophylaxis with a Plasma-Derived Von Willebrand Factor/Factor VIII Concentrate

Background: von Willebrand disease (VWD) is characterized by a deficiency or reduction of activity in von Willebrand factor (VWF). As VWF has a role in stabilizing coagulation factor VIII (FVIII), VWD patients often suffer from secondary FVIII deficiency. Current guidelines recommend that VWD patients with a history of severe and frequent bleeds should use long-term prophylaxis with factor replacement therapy. The WIL-31 study demonstrated the efficacy of prophylaxis with a plasma-derived VWF/factor VIII concentrate containing VWF and FVIII in a 1:1 activity ratio (pdVWF/FVIII; wilate ®) in adults and children with VWD. The primary endpoint was met, showing a decrease of 84% in the mean total annualized bleeding rate (ABR). As elevated levels of FVIII are associated with an increased risk for thrombosis, it is important to determine whether repeated dosing with pdVWF/FVIII leads to accumulation of FVIII over time. Aims: To investigate the activity levels of VWF and FVIII in patients with VWD receiving regular prophylaxis with pdVWF/FVIII. Methods: WIL-31 (NCT04052698) was a prospective, non-controlled, international, multicenter phase 3 trial that enrolled male/female patients, aged ≥6 years old with VWD type 1 (VWF:RCo &amp;lt;30 IU/dL), type 2 (except 2N) or type 3. Prior to entering the WIL-31 study, all patients had received on-demand treatment with a pdVWF/FVIII concentrate during a 6-month, prospective, observational, run-in study (WIL-29). Patients in WIL-31 received regular pdVWF/FVIII prophylaxis 2-3 times per week at a dose of 20-40 IU/kg for 12 months. VWF activity (VWF:RCo) and FVIII activity (measured by the chromogenic [Chr] and one-stage [OS] assays) were performed at baseline and throughout the study. Full pharmacokinetic (PK) analyses were performed in all patients aged 6-16 years, a single dose of 60 ± 10 IU/kg pdVWF/FVIII was administered. Samples were collected at up to 1 hour prior to injection and then at multiple time points post-injection (1-72 hours). Safety, including the occurrence of thrombotic events, was monitored throughout the study. Results: The overall study population included 33 patients, with a median age of 18 years consisting of 9 (27.3%) patients aged 6-11 years, 6 (18.2%) patients aged 12-16 years, and 18 (54.5%) patients aged ≥17 years. Six (18.2%) patients had severe type 1 VWD, 5 (15.2%) patients had type 2, and 22 (66.7%) patients had type 3. Baseline FVIII and VWF activities in WIL-31 were similar regardless of age group, and as expected were lower in patients with type 3 VWD compared with severe type 1 and type 2 (Figure 1). There was a negative correlation of baseline FVIII and VWF activity with the number of spontaneous bleeding episodes (BEs) during prophylaxis. Patients who had no spontaneous BEs during prophylaxis had higher baseline FVIII and VWF levels compared with those who had spontaneous BEs. Mean peak and trough FVIII and VWF activity levels remained consistent during the study and there was no accumulation of either factor over the 12-month prophylaxis period (Figure 2). Full PK assessments were performed according to protocol in 13 patients aged 6-16 years. The mean ± standard deviation in vivo half-life was 16.1±6.7, 16.2±7.7, and 9.8±7.7 hours, for FVIII Chr, FVIII OS, and VWF:RCo, respectively. No thrombotic events were observed. Conclusion: No accumulation of FVIII or VWF in the plasma was reported in 33 patients who received regular prophylaxis with pdVWF/FVIII for 12 months, regardless of VWD type and age.

Abacavir use is associated with increased prothrombin conversion.

There is ongoing debate as to whether abacavir (ABC) increases the risk for cardiovascular disease(CVD) in people living with HIV (PLHIV) and the mechanisms underlying this possible association. We recently showed that the use of an ABC-containing regimen was independently associated with increased thrombin generation (TG). In the present study, we aim to explore these findings further, by studying the mechanistical processes that underly the global thrombin generation test via thrombin dynamics analysis. Thrombin dynamics analysis can pinpoint the cause of increased thrombin generation associated with ABC-use either to the procoagulant prothrombin conversion pathway or the anticoagulant thrombin inactivation pathway. In this cross-sectional study, 208 virally suppressed PLHIV were included, of whom 94 were on a ABC-containing regimen, 92 on a tenofovir disoproxil fumarate (TDF)-containing regimen, and the remainder on other regimens. We used Calibrated Automated Thrombinography to measure thrombin generation and perform thrombin dynamics analysis. The total amount of prothrombin conversion, as well as the maximum rate of prothrombin conversion were significantly increased in PLHIV on an ABC containing regimen compared to other treatment regimens. The levels of pro- and anticoagulant factors were comparable, indicating that the ABC-induced changes affect the kinetics of prothrombin conversion rather than procoagulant factor levels. Moreover, Von Willebrand Factor (VWF), active VWF and VWF pro-peptide levels were significantly higher in PLHIV than controls without HIV. However, they did not differ between ABC and non-ABC treated participants.

Von Willebrand factor: from figurant to main character in the scene of inflammation

von Willebrand factor: from figurant to main character in the scene of inflammation

Analysis of thrombogenicity under flow reveals new insights into the prothrombotic state of patients with post-COVID syndrome

BackgroundPost-COVID syndrome (PCS) affects millions of people worldwide, causing a multitude of symptoms and impairing quality of life months or even years after acute COVID-19. A prothrombotic state has been suggested; however, underlying mechanisms remain to be elucidated. ObjectivesTo investigate thrombogenicity in PCS using a microfluidic assay, linking microthrombi, thrombin generation, and the von Willebrand factor (VWF):a Disintegrin and Metalloproteinase with a Thrombospondin Type 1 motif, member 13 (ADAMTS13) axis. MethodsCitrated blood was perfused through microfluidic channels coated with collagen or an antibody against the VWF A3 domain, and thrombogenicity was monitored in real time. Thrombin generation assays were performed and α(2)-antiplasmin, VWF, and ADAMTS13 activity levels were also measured. ResultsWe investigated thrombogenicity in a cohort of 21 patients with PCS with a median time following symptoms onset of 23 months using a dynamic microfluidic assay. Our data show a significant increase in platelet binding on both collagen and anti-VWF A3 in patients with PCS compared with that in controls, which positively correlated with VWF antigen (Ag) levels, the VWF(Ag):ADAMTS13 ratio (on anti-VWF A3), and inversely correlated with ADAMTS13 activity (on collagen). Thrombi forming on collagen presented different geometries in patients with PCS vs controls, with significantly increased thrombi area mainly attributable to thrombi length in the patient group. Thrombi length positively correlated with VWF(Ag):ADAMTS13 ratio and thrombin generation assay results, which were increased in 55.5% of patients. α(2)-Antiplasmin levels were normal in 89.5% of patients. ConclusionTogether, these data present a dynamic assay to investigate the prothrombotic state in PCS, which may help unravel the mechanisms involved and/or establish new therapeutic strategies for this condition.

Repeated Testing Unfolds the Challenge of Diagnosing Von Willebrand Disease: Longitudinal Data from the Vienna Bleeding Biobank

Background: Diagnosis of von Willebrand disease (VWD) in patients with a mild to moderate bleeding tendency (mild bleeding disorders, MBD) bears special challenges, as von Willebrand factor (VWF) levels are influenced by several factors, such as age, acute phase reaction and blood group, and might be unstable. Unstable VWF levels might lead to under- or over-diagnosis of VWD in MBD patients, and repeated measurements to identify VWD patients might be required. Aims: We aimed to identify clinical and epidemiological factors associated with unstable VWF levels, leading to change of the diagnosis based on the recommended diagnostic cut-off levels. Furthermore, we wanted to investigate cut-off of baseline VWF levels with a low probability to decrease below the diagnostic cut-off and, thus, not require subsequent VWF measurements. Methods: All patients enrolled into the Vienna bleeding biobank (VIBB, EC no. 603/2009) before October 2021 with at least two separate assessments of VWF levels, in particular VWF antigen (VWF:Ag) and activity (VWF:GPIbM and/or ristocetin-cofactor activity [VWF:RCo]), were included in this analysis. Detailed inclusion and exclusion criteria of the VIBB, a single-center cohort study on patients with MBD, have been published previously (Gebhart et al. Haemophilia.2018). For this analysis, pathological VWF levels were defined as any measurement of VWF:Ag, VWF:GPIbM or VWF:RCo ≤50 IU/dL, according to current guidelines (James et al. Blood Adv. 2021). We defined 3 subgroups of patients according to lowest VWF (VWF:Ag or VWF:GPIbM or VWF:RCo) at baseline and at each measurement: patients with steadily pathological measurements ≤50 IU/dL ("steady pathological"), patients with repeatedly normal measurements >50 IU/dL ("steady normal") and patients crossing the 50 IU/dL threshold between study visits ("changing"). Measurements were excluded from the analysis if an evident VWF influence such as pregnancy, infection, surgery or acute phase reaction was identified. Results: In total, 281 of 691 patients (40.7 %) from the VIBB study who had records of at least two VWF measurements of VWF antigen and activity levels were included in this analysis. Patients had a median number of 2 VWF measurements (Q1-Q3, 2-2) over a median follow-up time of 781 days (Q1-Q3, 105-1679) for a total of 660 measurements. 212 patients had 2 measurement of VWF antigen and activity, followed by 3 (n=47), 4 (n=11), 5 (n=5) and 6 (n=1) measurements. Of all analyzed patients, 217 patients (77.2 %) had VWF levels consistently above the cut-off of 50 IU/dL ( "steady normal"), 28 patients (10.0%) had repeatedly values ≤50 IU/dL ("steady pathological"), while 36 patients (12.8%) had changing results with values both below and above the cut-off of 50 IU/dL ( "changing"; Figure 1). The clinical and laboratory characteristics of each subgroup are summarized in Table 1. Patients in the "steady pathological” and "changing” groups compared to the "steady normal” group were younger and more commonly had blood group O. The values of VWF activity (VWF:RCo or VWF:GPIbM) at inclusion appeared to be more unstable than the VWF:Ag values (Figure 1). Seventeen (47.2%) patients in the changing group had pathological VWF activity values but VWF:Ag values above the cut-off of 50 IU/dL at study inclusion. VWF antigen and activity values at baseline within the changing group ranged from 36 IU/dL to 101 IU/dL and 28 IU/dL to 86 IU/dL, respectively. In a logistic regression model, modeling changing status as a binary outcome and lowest VWF at baseline using linear splines with a knot set at 50 IU/dL, the probability of changing was 37.2% (95%CI, 25.7-50.3), 18.6% (95%CI, 12.2-27.4) and 1.1% (95%CI, 0.2-4.8) at 40, 60 and 80 IU/dL, respectively (Figure 2). The added value of the clinical predictors age, sex, the Vicenza score and blood type O beyond lowest VWF at baseline, however, was only weak (χ2 5.0, p = 0.290). Conclusion: Based on our data, the reproducibility of VWF measurements is poor in patients with MBD. Re-evaluation of suspected patients for VWD who do not clearly fulfill the diagnostic criteria should be considered, especially if VWF levels are close to the cut-off level, as targeted treatment before surgical interventions might be warranted. Further studies are needed to better understand the reproducibility of VWF measurements and improve the diagnosis of VWD in patients with MBD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Von Willebrand Disease-Specific Aspects in Women.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder, which results from a deficiency or dysfunction of von Willebrand factor (VWF). The major symptoms of patients affected by VWD include mucocutaneous and gastrointestinal bleeding, easy bruising, and prolonged provoked bleeding due to injury or surgery. Although women and men are equally likely to be affected by VWD, women continue to be disproportionately affected by the bleeding challenges. Women with VWD suffer from sex-specific symptoms, such as menorrhagia, and are at higher risk of reproductive problems and recurrent miscarriage. Furthermore, pregnant women with VWD are more likely at higher risk of suffering from primary and secondary peripartal hemorrhage and anemia and the need for transfusions. Despite being affected by gynecologic and obstetrical bleeding, women face multiple barriers in obtaining an accurate diagnosis. This constitutes a problem that needs to be addressed, and early appropriate medical care should be ensured. There are several effective treatment options for women with VWD that can significantly improve their quality of life, including desmopressin, VWF concentrates, hormonal therapy, and antifibrinolytic therapy. During pregnancy, the monitoring of VWF activity levels is essential. The peripartal management depends on the type of VWD and on the measured levels of VWF levels and activity prior to delivery.

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.

Population-wide persistent hemostatic changes after vaccination with ChAdOx1-S.

Various vaccines were developed to reduce the spread of the Severe Acute Respiratory Syndrome Cov-2 (SARS-CoV-2) virus. Quickly after the start of vaccination, reports emerged that anti-SARS-CoV-2 vaccines, including ChAdOx1-S, could be associated with an increased risk of thrombosis. We investigated the hemostatic changes after ChAdOx1-S vaccination in 631 health care workers. Blood samples were collected 32 days on average after the second ChAdOx1-S vaccination, to evaluate hemostatic markers such as D-dimer, fibrinogen, α2-macroglobulin, FVIII and thrombin generation. Endothelial function was assessed by measuring Von Willebrand Factor (VWF) and active VWF. IL-6 and IL-10 were measured to study the activation of the immune system. Additionally, SARS-CoV-2 anti-nucleoside and anti-spike protein antibody titers were determined. Prothrombin and fibrinogen levels were significantly reduced after vaccination (−7.5% and −16.9%, p < 0.0001). Significantly more vaccinated subjects were outside the normal range compared to controls for prothrombin (42.1% vs. 26.4%, p = 0.026) and antithrombin (23.9% vs. 3.6%, p = 0.0010). Thrombin generation indicated a more procoagulant profile, characterized by a significantly shortened lag time (−11.3%, p < 0.0001) and time-to-peak (−13.0% and p < 0.0001) and an increased peak height (32.6%, p = 0.0015) in vaccinated subjects compared to unvaccinated controls. Increased VWF (+39.5%, p < 0.0001) and active VWF levels (+24.1 %, p < 0.0001) pointed toward endothelial activation, and IL-10 levels were significantly increased (9.29 pg/mL vs. 2.43 pg/mL, p = 0.032). The persistent increase of IL-10 indicates that the immune system remains active after ChAdOx1-S vaccination. This could trigger a pathophysiological mechanism causing an increased thrombin generation profile and vascular endothelial activation, which could subsequently result in and increased risk of thrombotic events.