High Molecular Weight Multimers Research Articles

Investigation of von Willebrand factor multimer abnormalities before and after aortic valve replacement using the Hydragel-5 assay

BackgroundSevere aortic stenosis (sAS) is associated with acquired von Willebrand syndrome (AVWS) by loss of high-molecular-weight multimers (HMWM) of von Willebrand factor (VWF), potentially resulting in perioperative bleeding. Analysis of VWF multimers remains challenging. Recently, the new, rapid Hydragel 5 assay has been developed, using electrophoretic protein separation for dividing VWF-multimers into low (LMWM), intermediate (IMWM), and HMWM, the hemostatically active part of VWF. Here, we evaluated its impact on predicting blood loss in presence of AVWS after surgical aortic valve replacement (SAVR). MethodsWe prospectively examined 52 patients (age: 68 ± 7 years; 54 % male) admitted to SAVR. They were divided in two groups (A: normal VWF, n = 28; B: abnormal VWF, n = 24, defined as VWF-activity/antigen (VWF:Ac/Ag)-ratio < 0.7 and/or HMWM loss). Blood samples and echocardiographic data were collected before, seven days and three months after SAVR. Blood loss and transfusions were recorded. ResultsBaseline characteristics and clinical data were similar in both groups. HMWM loss was present in 38.5 % of all patients. HMWM, the VWF:Ac/Ag- and HMWM/(IMWM+LMWM)-ratios were significantly decreased preoperatively in group B but normalized after SAVR. Bleeding, re-thoracotomy and transfusion rates were comparable. HMWM loss was inversely correlated with the peak aortic gradient (Pmax) and positively with the aortic valve area (AVA), while HMWM/(IMWM+LMWM)-ratio negatively correlated with the mean aortic gradient (Pmean). ConclusionHMWM and HMWM/(IMWM+LMWM)-ratio inversely correlate with severity of AS and normalize after SAVR. The Hydragel-5 assay's might be valuable for routine diagnostics to assess bleeding risk and postoperative normalization of AS and VWF abnormalities in SAVR patients.

In vitro study on device-induced damage to blood cellular components and degradation of von Willebrand factor in a CentriMag pump-assisted circulation.

High mechanical shear stress (HMSS) generated by blood pumps during mechanical circulatory support induces blood damage (or function alteration) not only of blood cell components but also of plasma proteins. In the present study, fresh, healthy human blood was used to prime a blood circuit assisted by a CentriMag centrifugal pump at a flow rate of 4.5 L/min under three pump pressure heads (75, 150, and 350 mm Hg) for 4 h. Blood samples were collected for analyses of plasma-free hemoglobin (PFH), von Willebrand factor (VWF) degradation and platelet glycoprotein (GP) IIb/IIIa receptor shedding. The extent of all investigated aspects of blood damage increased with increasing cross-pump pressure and duration. Loss of high-molecular-weight multimers (HMWM)-VWF in Loop 2 and Loop 3 significantly increased after 2 h. PFH, loss of HMWM-VWF, and platelet GPIIb/IIIa receptor shedding showed a good linear correlation with mean shear stress corresponding to the three pump pressure heads. HMSS could damage red blood cells, cause pathological VWF degradation, and induce platelet activation and platelet receptor shedding. Different blood components can be damaged to different degrees by HMSS; VWF and VWF-enhanced platelet activation may be more susceptible to HMSS.

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.

A career in solving clinical-pathological conundrums: Heyde syndrome, anti-platelet factor 4 disorders, and microvascular limb ischemic necrosis.

Hematology is a clinical specialty with strong roots in the laboratory; accordingly, the lab can help solve perplexing clinical problems. This review highlights clinical-pathological conundrums addressed during my 35-year hematology career at McMaster University. Heyde syndrome is the association between aortic stenosis and bleeding gastrointestinal (GI) angiodysplasia where the bleeding is usually cured by aortic valve replacement; the chance reading of a neonatal study showing reversible deficiency of high-molecular-weight (HMW) multimers of von Willebrand factor (vWF) following surgical correction of congenital heart disease provided the key insight that a subtle deficiency of HMW multimers of vWF explains Heyde syndrome. The unusual immunobiology of heparin-induced thrombocytopenia (HIT)-a highly prothrombotic, antibody-mediated, anti-platelet factor 4 (PF4) disorder featuring rapid appearance and then disappearance (seroreversion) of the pathological heparin-dependent platelet-activating antibodies-permitted identification of key clinical features that informed development of a scoring system (4Ts) to aid in HIT diagnosis. Atypical clinical presentations of HIT prompted identification of heparin-independent anti-PF4 antibodies, now recognized as the explanation for vaccine-induced immune thrombotic thrombocytopenia (VITT), as well as VITT-like disorders triggered by adenovirus infection. Another unusual feature of HIT is its strong association with limb ischemia, including limb necrosis secondary to deep-vein/microvascular thrombosis (venous limb gangrene). The remarkable observation that supratherapeutic warfarin anticoagulation predisposes to HIT- and cancer-associated venous limb gangrene provided insight into disturbed procoagulant/anticoagulant balance; these concepts are relevant to microvascular thrombosis in critical illness (symmetrical peripheral gangrene), including a pathophysiological role for proximate "shock liver" (impaired hepatic synthesis of natural anticoagulants).

Multi-Method Investigation of Blood Damage Induced By Blood Pumps in Different Clinical Support Modes.

To investigate the effects of blood pumps operated in different modes on nonphysiologic flow patterns, cell and protein function, and the risk of bleeding, thrombosis, and hemolysis, an extracorporeal blood pump (CentriMag) was operated in three clinical modalities including heart failure (HF), venous-venous (V-V) extracorporeal membrane oxygenation (ECMO), and venous-arterial (V-A) ECMO. Computational fluid dynamics (CFD) methods and coupled hemolysis models as well as recently developed bleeding and thrombosis models associated with changes in platelet and von Willebrand factor (vWF) function were used to predict hydraulic performance and hemocompatibility. The V-A ECMO mode had the highest flow losses and shear stress levels, the V-V ECMO mode was intermediate, and the HF mode was the lowest. Different nonphysiologic flow patterns altered cell/protein morphology and function. The V-A ECMO mode resulted in the highest levels of platelet activation, receptor shedding, vWF unfolding, and high molecular weight multimers vWF (HMWM-vWF) degradation, leading to the lowest platelet adhesion and the highest vWF binding capacity, intermediate in the V-V ECMO mode, and opposite in the HF mode. The V-A ECMO mode resulted in the highest risk of bleeding, thrombosis, and hemolysis, with the V-V ECMO mode intermediate and the HF mode lowest. These findings are supported by published experimental or clinical statistics. Further studies found that secondary blood flow passages resulted in the highest risk of blood damage. Nonphysiologic blood flow patterns were strongly associated with cell and protein function changing, blood damage, and complications.

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.

A Feasibility Study Evaluating Von Willebrand Factor in Patients with Gastrointestinal Angiodysplasia

Background: Abnormalities of von Willebrand Factor (VWF), such as those found in von Willebrand Disease (VWD) are associated with the development of abnormal blood vessels known as angiodysplasia. Angiodysplasia occurs primarily in the gastrointestinal (GI) tract, particularly the small bowel. Among patients with VWD, angiodysplasia is significantly overrepresented as a cause of GI bleeding and occurs in all VWD subtypes although it is reported more frequently in those with loss of the high molecular weight multimer of VWF. Recent studies have demonstrated more than a third of patients with VWD and GI bleeding have angiodysplasia when systematically assessed with Video Capsule Endoscopy (VCE) to examine the small bowel. Given the relationship between angiodysplasia and VWD our objective was to assess the feasibility of evaluating patients with GI angiodysplasia for abnormalities of VWF. Methods: We undertook a prospective single center pilot study to assess patients over the age of 18 with GI angiodysplasia for underlying abnormalities of VWF. Patients were identified based on angiodysplasia being present on a VCE study. Patients who consented to enrollment completed a self-administered bleeding assessment tool (BAT) as well as baseline demographic data. VWF testing included VWF antigen (VWF:Ag), VWF functional assay (VWF:GPIbM), Factor VIII coagulant activity (FVIII:C), and VWF pro-peptide (VWFpp) levels. Results: After reviewing 190 consecutive VCE studies performed at our institution between April 2021 and April 2023, we identified 50 patients who had capsule endoscopies demonstrating angiodysplasia of the GI tract. Thirteen of these patients consented to enrollment in the study. The bleeding histories of included patients are detailed in Table 1 along with their baseline clinical, laboratory, and demographic data. Two patients endorsed lifelong bleeding symptoms and three reported a significant family history of bleeding - all in a female relative. ISTH-BAT scores ranged from 0 to 18 with a median score of 2. Since our population is older than other studies that report on bleeding scores, we considered an abnormal bleeding score to be &amp;gt;6 as recent evidence indicates normal BAT scores increase over time. Five patients (38.6%) in our study had an ISTH-BAT abnormal bleeding score. VWF levels were assessed (Table 1) and one patient aged 88 years old with a significant family and lifelong history of bleeding received a new diagnosis of Type 2A VWD. Severe iron deficiency anemia requiring ongoing IV iron was diagnosed in two patients. Conclusion: In our feasibility study, we demonstrated a feasible protocol for recruitment of patients with GI angiodysplasia for clinical and laboratory testing of VWF. A significant proportion of our patients (38.6%) had abnormal bleeding scores and one patient had an underlying Type 2A VWD diagnosis.

Influence of Aortic Valve Stenosis and Wall Shear Stress on Platelets Function.

Aortic valve stenosis (AS) is a common heart valve disease in the elderly population, and its pathogenesis remains an interesting area of research. The degeneration of the aortic valve leaflets gradually progresses to valve sclerosis. The advanced phase is marked by the presence of extracellular fibrosis and calcification. Turbulent, accelerated blood flow generated by the stenotic valve causes excessive damage to the aortic wall. Elevated shear stress due to AS leads to the degradation of high-molecular weight multimers of von Willebrand factor, which may involve bleeding in the mucosal tissues. Conversely, elevated shear stress has been associated with the release of thrombin and the activation of platelets, even in individuals with acquired von Willebrand syndrome. Moreover, turbulent blood flow in the aorta may activate the endothelium and promote platelet adhesion and activation on the aortic valve surface. Platelets release a wide range of mediators, including lysophosphatidic acid, which have pro-osteogenic effects in AS. All of these interactions result in blood coagulation, fibrinolysis, and the hemostatic process. This review summarizes the current knowledge on high shear stress-induced hemostatic disorders, the influence of AS on platelets and antiplatelet therapy.

Development and validation of a mathematical model for evaluating shear-induced damage of von Willebrand factor

PurposeTo develop a mathematical model for predicting shear-induced von Willebrand factor (vWF) function modification which can be used to guide ventricular assist devices (VADs) design, and evaluate the damage of high molecular weight multimers (HMWM)-vWF in VAD patients for reducing clinical complications. MethodsMathematical models were constructed based on three morphological variations (globular vWF, unfolded vWF and degraded vWF) of vWF under shear stress conditions, in which parameters were obtained from previous studies or fitted by experimental data. Different clinical support modes (pediatric vs. adult mode), different VAD operating states (pulsation vs. constant mode) and different clinical VADs (HeartMate II, HeartWare and CentriMag) were utilized to analyze shear-induced damage of HMWM-vWF based on our vWF model. The accuracy and feasibility of the models were evaluated using various experimental and clinical cases, and the biomechanical mechanisms of HMWM-vWF degradation induced by VADs were further explained. ResultsThe mathematical model developed in this study predicted VAD-induced HMWM-vWF degradation with high accuracy (correlation with experimental data r2 > 0.99). The numerical results showed that VAD in the pediatric mode resulted in more HMWM-vWF degradation per unit time and per unit flow rate than in the adult mode. However, the total degradation of HMWM-vWF is less in the pediatric mode than in the adult mode because the pediatric mode has fewer times of blood circulation than the adult mode in the same amount of time. The ratio of HMWM-vWF degradation was lower in the pulsation mode than in the constant mode. This is due to the increased flushing of VADs in the pulsation mode, which avoids prolonged stagnation of blood in high shear regions. This study also found that the design feature, rotor size and volume of the VADs, and the superimposed regions of high shear stress and long residence time inside VADs affect the degradation of HMWM-vWF. The axial flow VADs (HeartMate II) showed higher degradation of HMWM-vWF compared to centrifugal VADs (HeartWare and CentriMag). Compared to fully magnetically suspended VADs (CentriMag), hydrodynamic suspended VADs (HeartWare) produced extremely high degradation of HWMW-vWF in its narrow hydrodynamic clearance. Finally, the study used a mathematical model of HMWM-vWF degradation to interpret the clinical statistics from a biomechanical perspective and found that minimizing the rotating speed of VADs within reasonable limits helps to reduce HWMW-vWF degradation. All predicted conclusions are supported by the experimental and clinical data. ConclusionThis study provides a validated mathematical model to assess the shear-induced degradation of HMWM-vWF, which can help to evaluate the damage of HMWM-vWF in patients implanted with VADs for reducing clinical complications, and to guide the optimization of VADs for improving hemocompatibility.

Acquired von Willebrand disease in children undergoing extracorporeal membrane oxygenation: a prospective observational study

BackgroundExtracorporeal membrane oxygenation (ECMO) provides cardiopulmonary support for children with severe cardiac and/or pulmonary failure. The incidence of bleeding complications during ECMO support is high. Acquired von Willebrand disease (AVWD) might contribute to the development of bleeding complications. ObjectiveTo study the incidence and longitudinal profile of AVWD during the first 14 days of ECMO support in children and to investigate the association between AVWD and bleeding complications. MethodsThis prospective observational study included pediatric patients (0-17 years) receiving ECMO. Blood was sampled prior to and after ECMO start, daily and 12 to 24 hours after stopping ECMO. von Willebrand factor (VWF) parameters and multimer patterns were determined. Clinical data were collected for each patient. AVWD was defined as loss of high-molecular weight multimers (ie, decreased compared with baseline) or a VWF:collagen binding/VWF: antigen (Ag) ratio or VWF:activity/VWF:Ag ratio below 0.7. ResultsAll of 50 (100%) patients developed AVWD during ECMO. The VWF:collagen binding /VWF:Ag ratio, VWF:activity/VWF:Ag ratio, and high-molecular weight multimers decreased during the initial days and recovered to baseline level within 24 hours after stopping ECMO. The incidence and longitudinal profile of AVWD were similar in patients with and without major bleeding complications. ConclusionChildren receiving ECMO support commonly develop AVWD. AVWD develops rapidly after ECMO initiation and recovers quickly after ECMO cessation. Importantly, AVWD appears to be independent of major bleeding.

P107: In Vitro, Benchtop Testing Indicates Improved Hemocompatibility with a New Magnetically Levitated VAD

Purpose: Ventricular assist devices (VADs) improve heart failure outcomes, yet the challenge of reducing bleeding, stroke, and infection persists. Biomarkers related to these adverse events were evaluated with a next-generation, totally magnetically suspended (TMS) VAD in comparison to other blood pumps. Method: Bovine whole blood or human plasma was circulated through benchtop flow loops with an Abbott CentriMag (CM) or HeartMate II (HMII), or a CH Biomedical USA BrioVAD (N = 5 tests). Flows were maintained at 4.5 L/min against an afterload of 75 mmHg. Samples were collected hourly for 6 hours. Plasma free hemoglobin was measured by spectroscopy. Normalized index of hemolysis (NIH) was calculated as described in ASTM F1841-19. VWF function (VWF:CB/Ag) was quantified using VWF:CB and VWF:Ag assay kits; VWF multimer distribution was determined using agarose-gel electrophoresis and in-gel immunofluorescent staining. Statistical significance was determined using an ANOVA (NIH) or regression analysis (VWF:CB/Ag) followed by a post-hoc Tukey test for significance between specific devices. Results: The CM and BrioVAD exhibited significantly lower hemolysis than the HMII (NIH: BrioVAD = 0.006 g/100L, CM = 0.002 g/100L, HMII = 0.029 g/100L; p < 0.01). The BrioVAD preserved VWF function after 6 hours compared to both HMII and CM (Fig. 1; VWF:CB/Ag, 6 h: BrioVAD = 94% retained, CM = 75%, HMII = 74%; p < 0.01). Similarly, the BrioVAD preserved a greater percentage of high molecular weight multimers (HMWM) than HMII and CM (BrioVAD = 96%, CM = 84%, HMII = 76%) with a higher ratio of HMWM to low molecular weight multimers (LMWM) (HMWM/LMWM: BrioVAD = 1.69, CM = 1.22, HMII = 1.09). Conclusion: Both the BrioVAD and CM achieved lower hemolysis than the HMII. However, the BrioVAD alone preserved VWF function and HMWM better than the CM and HMII. The inference that magnetically levitated centrifugal pumps are more hemocompatible than axial flow pumps with mechanical bearings is supported. The observation of improved In Vitro hemocompatibility with a newer magnetically levitated pump is encouraging and warrants further study.Figure 1. VWF function measured over the course of 6 hours.

P14: Degradation of High Molecular Weight Multimers of von Willebrand Factor under Whole Blood Conditions in High Turbulent Flow: Implications for Continuous Flow Left Ventricular Assist Devices

Background: High turbulent flow often found in continuous flow left ventricular assist devices (cf-LVADs) is believed to cause acquired von Willebrand Syndrome (AvWS). Our group recently published a study on the kinetic and dynamic effects of various flows on the loss of high molecular weight multimers (HMWMs) of von Willebrand factor (vWF). In the study, we applied high shear to citrated human platelet-poor plasma (PPP) and characterized the flow by Reynolds number (Re). The present study aims to extend upon our previous findings by investigating the degradation of HMWM under whole blood conditions at higher flow conditions to be characterized by energy dissipation rate and clinically relevant exposure times previously employed. Methods: Diluted ovine blood with a hematocrit of 25%, dynamic viscosity of 3.0 cP and density of 1030 kg/m3 was flowed through a custom high shear rotary device. The device is designed to provide a fully controlled exposure time, texp and flow regime. The flow rate was set at 1.75 ml/sec, 0.76 ml/sec, or 0.38 ml/sec, resulting in an exposure time of 22 ms, 50 ms, or 100 ms, respectively. The dynamic flow condition was controlled by the speed of the rotor, ω, and characterized by the energy dissipation rate per unit mass, ε. The ω of 10k, 12k, 15k, 18k, 20k, and 22k RPM were applied, and the corresponding ε were estimated by running a series of large eddy simulations (LES). After each run, a 0.5 ml sample solution was collected, frozen in dry ice immediately, and shipped to the Blood Center of Wisconsin for multimer analysis. The amount of multimer degradation at each testing condition was assessed with respect to the mean of the initial concentration of HMWM of vWF in the controls. Results: The results show that the loss of HMWM at all given exposure times increases in a logarithmic manner within the extended flow conditions (Fig. 1A). The loss of HMWM versus energy dissipation rate at given texp is shown in Fig. 1B. It is noted that as the flow becomes more chaotic, the loss of HMWM appears to be primarily dominated by the energy dissipation rate while the exposure time to chaotic flow becomes less significant. It is not clear if the less significant effect of kinetics at relatively high flow conditions (>18k RPM) is because there are no more HMWM left to be cleaved by ADAMTS13, an enzyme that cleaves vWF. The results are consistent with our previous observations using human PPP, in that both flow conditions and exposure time play crucial roles in the destruction of HMWM. Conclusion: This study demonstrated the relative roles of flow and exposure time using whole blood to characterize the loss of HMWM in terms of energy dissipation rate and exposure time. The findings have the potential to assist in the design and optimization of blood pumps, ultimately leading to improved clinical outcomes for patients with AvWS.

PEDS9: Bleeding in Pediatric Patients on Extracorporeal Membrane Oxygenation-Use of Procoagulant Factors

Background: Bleeding complications remain common in extracorporeal membrane oxygenation (ECMO) and can be associated with adverse outcomes. Effect of excessive shear stress on vasculature may increase bleeding risk associated with preferential loss of high molecular weight von Willebrand factor (high molecular weight (vWF) multimers; acquired vW syndrome, AVWS) and may also be associated with platelet dysfunction. Some pediatric patients may have a pre-existing increased risk of shear-related abnormalities which may further increase bleeding risk on ECMO. We aimed to review bleeding events requiring intervention in a pediatric ECMO cohort. Methods: Retrospective review of pediatric ECMO patients who underwent treatment of bleeding beyond simple transfusion in conjunction with ECMO anticoagulation consultation (EAC) between June 2020 and December 2022 was conducted. Results: Fifty patients required ECMO for 53 runs (52 VA, 2 VV, 1 VAV). Median weight was 4.3 kg (2.4-131 kg). Mean duration of ECMO was 11 days (2-97). 60% (30/50) of patients had congenital heart disease (CHD), and within these 30 patients, 27% of them had bleeding requiring procedural intervention. Ten patients with bleeding were screened for AVWS guided by EAC with 100% meeting criteria for AVWS. Platelet transfusions were administered as first line bleeding therapy with thrombocytopenia or platelet dysfunction and plasma or prothrombin complex concentrate (PCCC) in volume-sensitive patients for coagulopathy. With ongoing bleeding despite transfusion, antihemophilic factor/vWF complex (Humate P) was administered to 4 patients guided by EAC (figure 1). Of these 4 patients, 75% had CHD and 100% had valve abnormalities with 50% having new mechanical valves placed within 1 month prior to ECMO. Initial Humate P dose was given on mean day 14, with mean number of 8 doses. No circuit or mechanical valve thrombosis occurred. Platelet counts and function improved with a decrease in platelet transfusions and clinical bleeding in all patients following Humate P. Conclusion: Bleeding remains a common complication but is often multifactorial and may be difficult to determine specific etiologies. AVWS and thrombocytopenia commonly occur during pediatric ECMO. However, not all patients have clinical bleeding, such that additional risk factors may contribute to increased bleeding risk in these patients. Given impact of recurrent transfusions due to bleeding on morbidity and mortality, as well as cost consideration and potential thrombosis risk of vWF or PCCC supplementation, development of an expert-guided (EAC) algorithm for screening and treatment criteria for such agents appears to be safe, cost-effective and may be associated with decreased bleeding and transfusions. Further research into etiologies and consequences of ECMO-induced bleeding as well as treatment is needed.Figure 1. Algorithm for Assessment of Persistent Bleeding and Consideration of Humate P Administration (pilot).

A comparative study in patients with type 2 von Willebrand disease using 4 different platelet-dependent von Willebrand factor assays

BackgroundSeveral assays are now available to evaluate platelet-dependent von Willebrand factor (VWF) activity. ObjectiveTo report the results obtained using 4 different assays in patients with von Willebrand disease (VWD) carrying variants mainly in the A1 domain, which is critical for VWF binding to glycoprotein Ib (GPIb) and ristocetin. MethodsWe evaluated 4 different assays, 2 gain-of-function mutant GPIb binding (VWF:GPIbM) and 2 ristocetin cofactor (VWF:RCo) assays, in 76 patients with type 2 VWD. Patients and healthy controls were tested using VWF:GPIbM enzyme-linked immunosorbent assay (ELISA), VWF:GPIbM automated, VWF:RCo aggregometric, and VWF:RCo automated assays. ResultsThere was a good correlation (Pearson’s r>0.82) and agreement (Bland-Altman plots assessment) between the 4 assays, although several outliers existed among the type 2B without high-molecular-weight multimers (HMWM). The VWF activity/VWF:antigen ratios, calculated for each assay, were used to establish the percentage of a correct diagnosis of type 2 (ratio<0.60) in these patients: VWF:RCo aggregometric, 2A(100%), 2M(78%), 2M/2A(100%), 2B(68%); VWF:RCo automated, 2A(88%), 2M(89%), 2M/2A(100%), 2B(63%); VWF:GPIbM ELISA, 2A(96%), 2M(67%), 2M/2A(67%), 2B(0%); VWF:GPIbM automated, 2A(73%), 2M(44%), 2M/2A(75%), 2B(84%). In type 2B patients with HMWM, all assays gave a ratio ≥0.60. ConclusionThe VWF:GPIbM-automated assay is the most effective to diagnose as type 2 the 2B variants, whereas the VWF:RCo assays are the most effective in detecting 2M and 2M/2A variants. The VWF:GPIbM ELISA greatly overestimates the activity of the type 2B patients lacking HMWM. In this study, the use of a VWF activity/VWF:antigen ratio cut-off of 0.70 halved the number of misdiagnosed patients.

A nanobody against the VWF A3 domain detects ADAMTS13-induced proteolysis in congenital and acquired VWD

Abstractvon Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A–group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high–molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.

Von Willebrand factor in diagnostics and treatment of cardiovascular disease: Recent advances and prospects.

Von Willebrand factor (VWF) is a large multimeric glycoprotein involved in hemostasis. It is essential for platelet adhesion to the subendothelium of the damaged endothelial layer at high shear rates. Such shear rates occur in small-diameter arteries, especially at stenotic sites. Moreover, VWF carries coagulation factor VIII and protects it from proteolysis in the bloodstream. Deficiency or dysfunction of VWF predisposes to bleeding. In contrast, an increase in the concentration of high molecular weight multimers (HMWM) of VWF is closely associated with arterial thrombotic events. Severe aortic stenosis (AS) or hypertrophic obstructive cardiomyopathy (HOCM) can deplete HMWM of VWF and lead to cryptogenic, gastrointestinal, subcutaneous, and mucosal bleeding. Considering that VWF facilitates primary hemostasis and a local inflammatory response at high shear rates, its dysfunction may contribute to the development of coronary artery disease (CAD) and its complications. However, current diagnostic methods do not allow for an in-depth analysis of this contribution. The development of novel diagnostic techniques, primarily microfluidic, is underway. Such methods can provide physiologically relevant assessments of VWF function at high shear rates; however, they have not been introduced into clinical practice. The development and use of agents targeting VWF interaction with the vessel wall and/or platelets may be reasonable in prevention of CAD and its complications, given the prominent role of VWF in arterial thrombosis.

Kinetic and Dynamic Effects on Degradation of von Willebrand Factor.

The loss of high molecular weight multimers (HMWM) of von Willebrand factor (vWF) in aortic stenosis (AS) and continuous-flow left ventricular assist devices (cf-LVADs) is believed to be associated with high turbulent blood shear. The objective of this study is to understand the degradation mechanism of HMWM in terms of exposure time (kinetic) and flow regime (dynamics) within clinically relevant pathophysiologic conditions. A custom high-shear rotary device capable of creating fully controlled exposure times and flows was used. The system was set so that human platelet-poor plasma flowed through at 1.75 ml/sec, 0.76 ml/sec, or 0.38 ml/sec resulting in the exposure time ( texp ) of 22, 50, or 100 ms, respectively. The flow was characterized by the Reynolds number (Re). The device was run under laminar (Re = 1,500), transitional (Re = 3,000; Re = 3,500), and turbulent (Re = 4,500) conditions at a given texp followed by multimer analysis. No degradation was observed at laminar flow at all given texp . Degradation of HMWM at a given texp increases with the Re. Re ( p < 0.0001) and texp ( p = 0.0034) are significant factors in the degradation of HMWM. Interaction between Re and texp , however, is not always significant ( p = 0.73).

The Experimental Gene Therapy on Type 2A/Iic VWD Mouse Model with AAV Vector Delivering Only VWF Propeptide Expression cassette4

Type 2A/IIC von Willebrand Disease (VWD) is caused by mutations in the propeptide of von Willebrand Factor (VWF), and characterized by the impaired multimer assembly. The structural analysis revealed that VWF propeptide (D1D2 domain) serves as a pH-sensing template for VWF multimerization and tubular storage, which implies that D1D2 expression "in trans" has the potential to restore the multimerization defect of type 2A/IIC mutatns. The gene therapy of VWD is challenging due to the huge size of VWF gene, which cannot be packed to the virus vectors, particularly recombinant adeno-associated virus (rAAV). In the pilot studies, we had co-transfected AtT20 cell with plasmid encoding full length VWF, containing the Y87S mutation on propeptide region and causing type 2A VWD phenotype, and plasmid expressing only wild type propeptide of VWF. The co-transfection rescued the multimer formation in a concentration dependent manner (Figure 1A )and corrected the functional defects caused by the Y87S mutation (Figure 1B). Inspired by the results of in vitro studies, we thus have designed a novel gene therapy strategy for type 2A VWD utilizing the machinery of VWF multimer assembling. Instead of trying to squeeze the VWF gene into the AAV vector, we had prepared the AAV vector containing the expression cassette of propeptide region directed by endothelial cell specific promoter FLT-1. The mouse model representing natural occurring VWF Y87S mutation identified in type 2A VWD patient was established by criper9 gene editing technique. The AAV vectors was administered via tail vein injection at a dose of 5Ⅹ1011 genome copies to 6 weeks old male mice to deliver the propeptide of VWF gene and the plasma of the treated mice was collected retroorbital pretreatment and at 2- ,4- and 6-weeks post vector injection. The antigen level of VWF was determined by ELISA and the function activity of VWF was measured by collagen binding assay. The status of VWF multimerization was examined by agarose gel electrophoresis. Although the Y87S mutation led to significant reduce of high molecule weight multimer in mice, unlike observed in mutation harboring patient, the mice still had medium and low molecular weight multimer, maintaining ~30% of activity. The expression of propeptide by AAV delivered vector improved the VWF activity significantly to ~70% of wild type control (Figure 1C), and restored the formation of high molecular weight multimer (Figure 1D). The experimental gene therapy conducted on VWD mouse model showed that it would be efficient to express the propeptide alone to correct the type 2A/IIC VWD defect and the preliminary results provide a novel strategy to overcome technique limits on gene therapy of VWD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Acquired von Willebrand syndrome (AVWS) type 2, characterized by decreased high molecular weight multimers, is common in children with severe pulmonary hypertension (PH).

Emerging evidence suggests that increased degradation of von Willebrand factor and decrease in high molecular weight multimers occurs in patients with pulmonary hypertension (PH). However, the link between acquired von Willebrand Syndrome (AVWS) type 2 and PH remains poorly understood. We retrospectively evaluated the charts of 20 children with PH who underwent bilateral lung transplantation (LuTx) between 2013 and 2022. Von Willebrand variables were determined in 14 of these patients; 11 patients had complete diagnostics including multimer analysis. We confirmed AVWS in 82% of the children studied (9 of 11 patients by multimer analysis). The two remaining patients had suspected AVWS type 2 because of a VWF:Ac/VWF:Ag ratio of <0.7. Platelet dysfunction or suspicion of VWD type 1 were found in two separate patients. All but one of the 14 children with severe PH had a coagulation disorder. Most patients (9 proven, 2 suspected) had AVWS type 2. Notably, 3 of 5 patients (60%) with normal VWF:Ac/VWF:Ag ratio >0.7 had abnormal VWF multimers, indicating AVWS type 2. Hemostatic complications were observed in 4 of 12 (33%) patients with VWS and 3 of 6 (50%) patients without diagnostics and therapy. For children with moderate to severe PH, we recommend systematic analysis of von Willebrand variables, including multimer analysis, PFA-100 and platelet function testing. Awareness of the diagnosis "AVWS" and adequate therapy may help to prevent these patients from bleeding complications in case of surgical interventions or trauma.

Resistance to unfolding by acidic pH and resistance to lysosomal degradation explains disease-association of HLA-B27 subtypes

Several hypotheses have been proposed to explain the high rate of disease association of HLA-B27 with ankylosing spondylitis (AS), including formation of disulfide-bonded dimers and misfolding of the heavy chain (HC), involving formation of high molecular weight (HMW) multimers. Recently, we have shown that the HMW entities of non-disease associated (non-DA) subtypes cause activation of endosomal-lysosomal pathways, while disease-associated (DA) subtypes of HLA-B27 cause activation of autophagy and unfolded protein response (UPR) pathways. In this paper, we seek an explanation for the failure of these pathways to degrade the HMW entities of DA subtypes of HLA-B27, using a combination of in vitro assays, using extracellular domains of heavy chains (EDHC), as well as in vivo assays, using stable transfectants of the full lengths of heavy chains (FLHC) of DA and non-DA subtypes. Our data shows that both DA and non-DA subtypes form HMW entities. However, non-DA HMW entities display far greater levels of degradation than DA HMW species. Non-DA EDHC display greater loss of structure at lysosomal pH in vitro. This was confirmed by experiments showing that (i) DA FLHCs co-localize with LAMP1, and (ii) induction of autophagy by rapamycin causes significant decrease in levels of non-DA HMW entities, but not that of DA HMW entities. These results point towards lack of facile lysosomal clearance of FLHCs of DA subtypes, suggesting that disease association of HLA-B27 subtypes is correlated with higher persistence of HMW entities in the low pH of lysosomes, with higher potential to trigger immune response.