High Molecular Weight Von Willebrand Factor Research Articles

Purified VWF Corrects Dysfunctional Primary Hemostasis in Patients with Extracorporeal Life Support. in Vitro Studies Against Cryoprecipitate

INTRODUCTION. Extracorporeal membrane oxygenation (ECMO) is a mechanical assistance system used to temporarily support patients with life-threatening cardiac and/or respiratory dysfunction when traditional treatment fails. Bleeding is one of the main complications causing significant morbidity and mortality. Development of acquired von Willebrand disease (AVWD) within 1-6 hours after ECMO implantation has been described, with recovery 3-24 hours after device explant. There is no consensus on the most suitable treatment for these patients. OBJECTIVE. To investigate the improvement in primary hemostasis after the in vitro addition of VWF-containing concentrates in samples from patients with ECMO. MATERIAL AND METHODS: During 2023, 7 adults with ECMO (n=2 venous-venous, and n=5 veno-arterial) due to pulmonary or cardiovascular diseases were included in the study. Samples were collected within the first 72 hours after implantation and 5 days after removal (n=16 samples).Hematocrit, platelet counts, and ADAMTS13 activity, by FRETS assay, were determined.Platelet function was evaluated by PFA-200 ( Siemens Healthineers®) with collagen-epinephrine and collagen-ADP cartridges, and platelet aggregation with ristocetin 1 mg/mL ( Helena BioSciences APACT4 ®). VWF function was assessed by antigen (VWF:Ag), activity (VWF:GPIbM), and VIII factor (VIIIF) quantification ( Siemens Atellica COAG360®), collagen (VWF:CB) and VIIIF binding (VWF:VIIIF) to VWF ( Asserachrom ®and Thermo Scientific MultiSkan FC ®) and VWF multimeric analysis (agarose gels and immunoblotting). Total-TAS ®( Zacros) was used to analyze hemostasis before and after in vitro addition of VWF concentrate ( Haemate-P ® ) or cryoprecipitate (Cp) in each sample. The cohort was divided into two groups according to the time when the samples were collected. Only the subjects showing a correction of the occlusion times at Total-TAS® were included in the comparative analysis. RESULTS. Mucocutaneous bleeding and pulmonary embolism were reported in n=3.One patient died before device removal. Within the first 72 hours after implantation PFA-200 was prolonged (>300s), VWF:Ag, VWF:GPIbM, and VWF:CB values increased, and hematocrit, platelet counts, ristocetin aggregation and VWF:VIIIFdecreased, as reported in Table 1.VIIIF was within normal range. Loss of high molecular weight VWF multimers was observed in all samples, and VWF:GPIbM/VWF:Ag ratio was <0.70 in 29% patients. These findings persisted within 5 days after removal, except for PFA-200 and platelet counts that normalized in 29% and 50%, respectively, while VIIIF values increased. In the samples collected after ECMO removal, the addition of Haemate-P® significantly reduced Total-TAS ® occlusion times (573.00±280.91 vs 821.20±586.54 p=0.043, post- vs pre-). Correction of the occlusion time with Cp was not statistically significant. The hemostatic effect of Haemate-P® was superior to Cp (790.80 ± 582.02, p=0.043). No differences were observed between basal values and the addition of neither Haemate-P ® nor Cp in samples collected during ECMO, Figure 1. CONCLUSIONS. Treatment with ECMO alters primary hemostasis in association with AVWD. In our cohort, these effects were seen early after implantation and, at least, up to 5 days after device removal. Most patients requiring ECMO treatment exhibit a complex medical history and are exposed to multiple drugs affecting numerous analytical values, making the interpretation of primary hemostasis complicated. The addition of Haemate-P ® in vitro significantly improved the hemostatic efficiency compared to Cp only when ECMO was discontinued. More studies are needed to evaluate the role of VWF-containing concentrates in the treatment of these patients, being Total-TAS ® a potentially useful mechanism to investigate it. GRANTS: López-Borrasca Scholarship funded by FETH/SETH, with the collaboration of CSL Behring (Spain) and Zacros (Fujimori Kogyo Co, Ltd, Japan), and 2021-SGR-01118, Agencia de Gestió de Ajuts Universitaris i de Recerca (AGAUR), Generalitat de Catalunya.

PB0831 Novel VWF Exon 9-10 Duplication in a Family with Inherited Bleeding, High VWF:Ag and FVIII Levels, and Loss of High Molecular Weight VWF Multimers

PB0831 Novel VWF Exon 9-10 Duplication in a Family with Inherited Bleeding, High VWF:Ag and FVIII Levels, and Loss of High Molecular Weight VWF Multimers

Impact of transcatheter aortic valve implantation on circulating von Willebrand factor in patients with severe aortic stenosis.

Severe aortic stenosis (AS) can lead to degradation of high molecular weight (HMW) von Willebrand factor (VWF) which can result in haemostatic abnormalities. While studies have explored changes in VWF profiles before and after surgical aortic valve replacement (SAVR), the longer-term changes in VWF profiles pre- and post-transcatheter aortic valve implantation (TAVI) are less understood. Our primary objective was to identify differences in VWF multimer profiles and VWF function pre-TAVI and 1-month post-TAVI. Our secondary objective was to correlate VWF markers with measures of AS severity. Adult patients with severe AS referred for TAVI at our institution were prospectively enrolled in this cohort study. Blood samples were collected for plasma analysis at three time points for all patients: 1day pre-TAVI, 3days post-TAVI, and 1-month post-TAVI. VWF antigen, activity, propeptide, collagen binding, multimers, and factor VIII coagulant activity were determined at each time point. Correlations between VWF parameters and severity of AS were assessed. Twenty participants (15 males, five females) with severe AS were recruited for the study. There was a significant increase in HMW VWF between pre-procedure and 1-month post-TAVI (p<.05). There was a transient increase in VWF antigen levels and activity at 3-days post TAVI that decreased to pre-TAVI levels at 1-month. There were no statistically significant correlations between VWF markers and AS severity. This is the first study to elucidate longer-term (>1week) improvements in HMW VWF after a TAVI procedure in severe AS patients.

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.

Immune and Hereditary Thrombotic Thrombocytopenic Purpura: Can ADAMTS13 Deficiency Alone Explain the Different Clinical Phenotypes?

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy caused by a hereditary or immune-mediated deficiency of the enzyme ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). TTPs are caused by the following pathophysiological mechanisms: (1) the presence of inhibitory autoantibodies against ADAMTS13; and (2) hereditary mutations of the ADAMTS13 gene, which is present on chromosome 9. In both syndromes, TTP results from a severe deficiency of ADAMTS13, which is responsible for the impaired proteolytic processing of high-molecular-weight von Willebrand factor (HMW-VWF) multimers, which avidly interact with platelets and subendothelial collagen and promote tissue and multiorgan ischemia. Although the acute presentation of the occurring symptoms in acquired and hereditary TTPs is similar (microangiopathic hemolytic anemia, thrombocytopenia, and variable ischemic end-organ injury), their intensity, incidence, and precipitating factors are different, although, in both forms, a severe ADAMTS13 deficiency characterizes their physiopathology. This review is aimed at exploring the possible factors responsible for the different clinical and pathological features occurring in hereditary and immune-mediated TTPs.

ADAMTS13 inhibition to treat acquired von Willebrand syndrome during mechanical circulatory support device implantation

BackgroundAcquired von Willebrand syndrome (aVWS) is common in patients with mechanical circulatory support (MCS) devices. In these patients, the high shear stress in the device leads to increased shear‐induced proteolysis of von Willebrand factor (VWF) by A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, number 13 (ADAMTS13). As a result, the high molecular weight (HMW) VWF multimers are lost, leading to a decreased VWF function and impaired hemostasis that could explain the bleeding complications that are frequently observed in these patients. To counteract this abnormal VWF degradation by ADAMTS13, we developed a novel targeted therapy, using an anti‐ADAMTS13 monoclonal antibody (mAb) that inhibits the shear‐induced proteolysis of VWF by ADAMTS13. MethodsHuman or bovine blood was circulated through in vitro MCS device systems with either inhibitory anti‐ADAMTS13 mAb 3H9 or 17C7 (20 μg/ml) or control anti‐ADAMTS13 mAb 5C11 or phosphate buffered saline (PBS). VWF multimers and function (collagen binding activity) were determined at different time points. Next, Impella pumps were implanted in calves and the calves were injected with PBS and subsequently treated with mAb 17C7. VWF, ADAMTS13, and blood parameters were determined. ResultsWe demonstrated that blocking ADAMTS13 could prevent the loss of HMW VWF multimers in in vitro MCS device systems. Importantly, our antibody could reverse aVWS in a preclinical Impella‐induced aVWS calf model. ConclusionHence, inhibition of ADAMTS13 could become a novel therapeutic strategy to manage aVWS in MCS device patients.

In vitro study of red blood cell and VWF damage in mechanical circulatory support devices based on blood-shearing platform.

Blood damage induced by mechanical circulatory support devices (MCSDs) remains a significant challenge to optimal clinical care. Although researchers have been conducting in vitro studies, the major determinant of blood damage is still unclear. An optimized capillary tube blood-shearing platform with custom designed parts was constructed to investigate the influence of two flow-dependent parameters (shear stress and exposure time) on the shear-induced damage of red blood cells and von Willebrand factor (VWF). Blood samples under different high shear stress and instantaneous exposure time were obtained by changing the flow rate and the length of capillary tube. Plasma free hemoglobin assay and immunoblotting of VWF were then performed on the sheared blood samples. The quantitative correlation between the hemolysis index and the two flow-dependent parameters was found following the power law mathematical model under the flow condition with high shear stress and instantaneous exposure time. The degradation of high molecular weight VWF was not obvious under high shear stress factor. However, the degradation of high molecular weight VWF was found as the result of the accumulation over exposure time under non-physiological shear stress, which was consistent with the different mechanism of VWF damage comparing to red blood cell damage. Compared to peak shear stress, exposure time has a greater effect on both red blood cell and VWF damage. To improve the hemocompatibility of MCSDs, it is more important to avoid regions of slow blood flow with non-physiological shear stress under laminar flow conditions.

Acquired von Willebrand Syndrome and Desmopressin Resistance During Venovenous Extracorporeal Membrane Oxygenation in Patients With COVID-19: A Prospective Observational Study.

OBJECTIVES:Although COVID-19 is associated with high von Willebrand factor (vWF) parameters promoting thrombosis, venovenous extracorporeal membrane oxygenation (vvECMO) is associated with the development of acquired von Willebrand syndrome (AVWS) promoting bleeding. This study was designed to assess both the incidence and severity of AVWS in COVID-19 patients undergoing vvECMO, and the benefit of comprehensive vWF analyses.DESIGN:Prospective observational study.SETTING:ICU at a tertiary-care center.PATIENTS:Twenty-seven consecutive COVID-19 patients with acute respiratory distress syndrome (ARDS) requiring vvECMO.MEASUREMENTS AND MAIN RESULTS:Comprehensive vWF analyses (including sodium dodecyl-sulfate polyacrylamide gel electrophoresis) were performed before, during, and after vvECMO. In a subgroup of 12 patients with AVWS, effectiveness of treatment with desmopressin was assessed. The patients’ mean age was 53 years (range, 23–73), 70% were male, and all had various comorbidities. Following markedly elevated vwf antigen (vWF: Ag; mean, 546% (sd, 282]), vWF collagen binding capacity (mean, 469% [sd, 271]), vWF activity (vWF:A; mean, 383% [sd, 132]), and factor VIII activity (mean, 302% [sd, 106]), and only borderline decreases in high-molecular-weight (HMW) vWF multimers before vvECMO, all of these variables decreased and HMW vWF multimers became undetectable within hours following initiation of vvECMO. All variables fully recovered within 3–38 hours after discontinuation of vvECMO. During vvECMO, decreases in the vWF:A/vWF:Ag ratio correlated with absent HMW vWF multimers. Desmopressin did not affect vWF parameters.CONCLUSIONS:In patients with COVID-19-associated ARDS, AVWS developed soon after initiation of vvECMO. The vWF:A/vWF:Ag ratio was a suitable screening test for AVWS. As desmopressin was ineffective, bleeding during vvECMO-associated AVWS should preferably be treated with concentrates containing vWF.

TLT-1 Regulates Thrombus Growth Under Non-Inflammatory Conditions

BackgroundThrombus formation is a complex, dynamic and multistep process, based on two crucial steps: platelet adhesion and platelet aggregation that both involve the large multimeric plasma glycoprotein Von Willebrand Factor (VWF). VWF binding to the GPIb/X/V complex initiates platelet adhesion to the vessel wall at high shear stress and triggers platelet activation resulting in the generation of thrombin and activation of integrin αIIbβ3 on the platelet surface. This activation of αIIbβ3 in turn leads to outside-in signalling and promotes binding of αIIbβ3 to fibrinogen and VWF, mediating thrombus growth. Trigging receptor expressed on myeloid cells like transcript-1 (TLT-1) is a transmembrane receptor, which is targeted to α-granules of platelets and megakaryocytes. Thrombin-induced platelet activation rapidly presents TLT-1 on the platelet surface and releases a soluble form (sTLT-1) into the circulation. To date the only known ligand for TLT-1 is fibrinogen and TLT-1 has been implicated in the regulation of inflammation-associated thrombosis. Interestingly, a putative interaction of VWF with TLT-1 was indicated by a screen with known platelet receptors.AimWe aimed to evaluate the effect of TLT-1/VWF interaction on platelet aggregation and thrombus formation.MethodsRecombinant TLT-1 and VWF were purified and the interaction between TLT-1 and VWF was analyzed by surface plasmon resonance. Static interaction was confirmed by an ELISA based binding assay. Flow assays assessed TLT-1 dependent thrombus formation in vitro. The effects of TLT-1 knockout on thrombus formation in vivo were examined via intravital microscopy of the flow restricted inferior vena cava (IVC) and imaging of platelet attachment and fibrin formation over 6 hours. Furthermore, thrombus formation and resolution was followed by high resolution ultrasound imaging after stenosis induction for 28 days. Integrin aIIbb3 activation was analysed by flow cytometry using the JonA antibody in murine platelet rich plasma.ResultsVWF bound to soluble TLT-1 with high affinity in a calcium dependent manner (K D = 1.9 nM). The binding site on VWF was mapped to the A3D4 domains and high molecular weight VWF multimers had the greatest affinity for TLT-1. Moreover, HEK293 cells transfected with TLT-1 bound to VWF and VWF strings formed specifically on TLT-1 expressing cells, confirming the interaction between the two proteins. VWF inhibited the binding of fibrinogen to TLT-1, suggesting that VWF is a preferred binding partner of TLT-1.Human platelets exhibited increased TLT-1 surface expression after TRAP-6 induced platelet activation and TLT-1 was detected throughout thrombi formed under flow. Furthermore, a TLT-1 blocking antibody inhibited the interaction of TLT-1 with VWF and reduced platelet capture to type I collagen under shear stress. Ex vivo perfusion of blood from TLT-1 knock out mice over type I collagen also resulted in reduced thrombus formation compared to blood from wild-type mice. TLT-1 knock-out platelets were activated by thrombin similar to wild-type controls, based on P-selectin expression in platelet rich plasma. However, activation of integrin αIIbβ3 determined by JonA staining was reduced in the absence of TLT-1. This phenotype of reduced integrin αIIbβ3 activation on P-selectin positive platelets was phenocopied by the thrombin platelet response in platelet rich plasma from VWF -/- mice, but not GPIbα-deficient mice, indicating that the TLT-1-VWF interaction on platelets directly influences integrin αIIbβ3 activation. Significantly, thrombus formation was markedly reduced in TLT-1 knockout mice in the IVC model in vivo in comparison to wild-type mice.ConclusionsThis study demonstrates that TLT-1 is a novel platelet ligand for VWF, and that TLT-1 may preferentially bind VWF over fibrinogen. We propose a TLT-1/VWF dependent integrin αIIbβ3 activation mechanism which plays a pivotal role in thrombus formation under non-inflammatory and potentially inflammatory conditions. DisclosuresRuf: ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company; ARCA bioscience: Consultancy, Patents & Royalties.

The influence of surface roughness on the damage of von Willebrand Factor under shear flow condition.

Despite technological advances in mechanical circulatory support devices to treat end-stage heart failure, blood damage induced by non-physiological shear stress in operation often triggered clinical hemocompatibility complications. The loss of high molecular weight von Willebrand Factor (HMW-VWF) has been considered as an essential cause of gastrointestinal bleeding. In addition to the mechanics factors, interface factors may also affect blood damage, especially the surface characteristics. In this study, the effect of surface roughness on VWF damage under flow condition was investigated. A roller pump circulation experimental platform with a roughness embedded sample chamber was constructed to provide blood shearing flow condition. VWF molecular weight analysis, VWF antigen (VWF-Ag) concentration assay, and VWF ristocetin cofactor activity (VWF-Rico) assay were performed on the sheared blood samples. These variables are the main functional indicators of VWF. It was found that the surface roughness induced VWF damage is mainly caused by the loss of HMW-VWF rather than reducing the total amount of VWF. The threshold value of surface roughness for a rapid increase in the degradation of HMW-VWF under low flow rate was obtained between Ra 0.4 and 0.6 μm, which was smaller than the threshold for hemolysis. Our findings indicated that VWF is more sensitive to the interface factor of surface roughness than red blood cells, thus has a higher requirement for blood pump design. It could provide reference for the material design and processing in developing mechanical circulatory support devices.

ADAMTS13 regulation of VWF multimer distribution in severe COVID‐19

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

Quantitative Analysis of Von Willebrand Factor (VWF) Multimers in Von Willebrand Disease (VWD) Patients Recruited through the Zimmerman Program for the Molecular and Clinical Biology of VWD

Quantitative Analysis of Von Willebrand Factor (VWF) Multimers in Von Willebrand Disease (VWD) Patients Recruited through the Zimmerman Program for the Molecular and Clinical Biology of VWD

Ultra-Low Von Willebrand Factor (VWF) Adsorption Under Shear with a Polymer Coating for Blood-Contacting Devices

Introduction. Many human diseases are treated with blood-contacting medical devices. However, they are often compromised because of blood proteins that accumulate on their surfaces. Such accumulation activates both platelets and blood coagulation, and patients supported by blood-contacting devices are at high risk of both bleeding and clotting. Blood passing through medical devices is often subjected to high shear stresses (especially near surfaces) and flow acceleration, conditions that we have shown promote rapid deposition of the large plasma glycoprotein von Willebrand factor (VWF) onto uncoated surfaces. Additionally, acquired Von Willebrand syndrome (AVWS) - a bleeding disorder caused by deficiency of high molecular weight VWF multimers - has been described in patients supported with ECMOs (extracorporeal membrane oxygenators) and LVADs (left ventricular assist devices). Blood in these patients experiences high shear stress when passing through the blood pump in ECMOs and LVADs. Thus, the adsorption of VWF to artificial surfaces under shear is likely to initiate platelet binding and the adsorption of other plasma proteins, including clotting proteins. Here, we show that polymers generated from the sulfobetaine methacrylate (SBMA) when coated onto surfaces are able to render these surfaces resistant to VWF binding (< 5 ng/cm2) under both static conditions and under shear stress.Preliminary results. We synthesized SBMA polymers (pSBMA) from SBMA according to methods described in the literature. Previously, we tested various coating conditions for pSBMA to achieve the lowest VWF adsorption. Under optimal conditions, VWF adsorption to pSBMA-coated polypropylene tubes was 4% of that to uncoated tubes (< 2.6 ng/cm2) after the tubes were exposed to a VWF solution (5 µg/mL) under shear applied with a vortexer (3000 rpm at RT for 90 min). In this work, we prepared a microfluidic device of polydimethylsiloxane that contained a tortuous flow channel to mimic shear stress conditions of blood vessels and tested the ability of pSBMA to prevent VWF deposition and platelet binding under different shear stress conditions.Methods. Purified plasma VWF (2.5 µg/mL) was perfused through the uncoated and pSBMA-coated devices for 1 h with a gravity-driven pump. To image the VWF structure, 5% BSA was perfused through the system to block nonspecific protein adsorption. Then FITC-labeled anti-human VWF antibody was perfused into the chamber system. After incubation at room temperature for 1 h, PBS was introduced to wash the system. The devices were fixed with 3.7% formaldehyde, incubated, and washed with PBS. We also perfused 1 mL of citrated human whole blood containing platelets stained with calcein AM through the devices at 0.05 mL/min with syringe pump to image platelet binding.Results. Under both conditions, VWF and platelet binding to the pSBMA-coated device was much lower than to the uncoated device. On the uncoated device, VWF formed aggregates which could not be washed away. By quantifying fluorescence intensity, we found that most VWF adsorbed along curves compared to the straight regions, the inlet, or the outlet of the chamber, consistent with shear stress simulation results that indicate that VWF deposits in the regions of highest shear and elongational flow. When whole blood was perfused through the chamber, the platelets bound to the entire device surface, with the inlet and outlet having relatively higher levels. In contrast, in the pSBMA-coated device, VWF bound loosely and could be removed almost completely with washing. We then perfused whole blood through uncoated or pSBMA-coated chambers without VWF pre-perfusion. Unlike the findings with VWF pre-perfusion, there was no apparent platelet binding in either device, indicating that the high level of platelet binding on the uncoated devices required time for VWF to deposit on the surface.Conclusions. PSBMA effectively prevented VWF adsorption under shear in two types of devices, and was stable during storage. We expect this coating, or a variation of it to significantly enhance the lifetime of blood-contacting devices and reduce both costs and medical complications of their use. DisclosuresNo relevant conflicts of interest to declare.

Characterization of a Nanobody That Binds Von Willebrand Factor and Prevents Von Willebrand Factor Cleavage By Plasmin

Introduction: Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adherence to exposed subendothelial collagen at sites of vascular injury. To prevent spontaneous platelet aggregation, accumulation of high molecular weight vWF multimers is prevented by ADAMTS13-mediated cleavage in the vWF A2 domain. Moreover, emerging data demonstrates plasmin-mediated cleavage of vWF in its open conformation in the A1-A2 linker, suggesting a role for plasmin in regulation of vWF multimer composition as a back up for ADAMTS13.Aim: We recently identified a llama-derived anti-vWF nanobody recognizing vWF in a conformation sensitive to plasmin cleavage. Our aim was to characterize this nanobody in terms of binding to- and interference with vWF functionality.Methods and results: The nanobody was produced by immunization of llamas with wildtype vWF, followed by preparation of cDNA from isolated peripheral blood lymphocyte-derived RNA and selection of the vWF A1 domain specific nanobody via phage display. To study recognition of vWF by the nanobody and possible modulating factors we developed an ELISA assay based on a nanobody-coated microtiter plate. Plasmin pre-treatment of normal pooled plasma (NPP) significantly increased nanobody recognition of vWF, but only after subjecting the plasma to shear by vortexing. Ristocetin also increased recognition by the nanobody; however, addition of plasmin to ristocetin-opened vWF in NPP did not further enhance nanobody binding. This suggests that, in contrast to ristocetin, vortexing was not sufficient to fully expose all vWF A1 domains, but the remaining hidden A1 domains were exposed by plasmin cleavage, allowing additional vWF to bind to the nanobody.Ellipsometry was used to assess competition of the nanobody and plasmin binding to vWF. Polyclonal anti-human vWF antibodies were coated to silicon slides, followed by addition of vWF concentrate (Haemate) or recombinant vWF R1306W, plasmin and the nanobody in varying order. We found that plasmin binds much more efficiently to R1306W vWF than to globular vWF. If the nanobody was bound to the R1306W vWF first, plasmin could not bind anymore. Conversely, after plasmin binding, only a small proportion of the nanobody still recognizes the plasmin-sensitive conformation of vWF, suggesting plasmin occupied the majority but not all binding sites and a possible difference in affinity for this conformation of vWF.A flow-cytometry based platelet-activation assay was performed to study the effect of plasmin on the ability of vWF in its plasmin-sensitive conformation to bind to washed platelets. Fluorescent detection of vWF binding was mediated by FITC-labeled monoclonal anti-vWF antibodies or biotin-conjugated labeled nanobody in combination with streptavidin-FITC. Addition of plasmin increased ristocetin-modified vWF binding to platelets by 3.7-fold. This effect of plasmin was abrogated in the presence of heparin, consistent with work by Brophy et al. (2017) showing that heparin prevents cleavage of vWF by plasmin. Hence, it appears that plasmin-cleaved vWF has increased binding affinity to platelets. Of note, a similar pattern in ristocetin-activated vWF to platelet binding upon addition of plasmin (and heparin) was detected by the nanobody. This demonstrates that the nanobody does not interfere with platelet binding of vWF. Moreover, binding of plasmin-cleaved vWF resulted in reduced platelet activation, as determined from P-selectin expression.Discussion: In conclusion, we have identified an anti-vWF nanobody capable of blocking plasmin binding to- and subsequent cleavage of vWF. This nanobody is unable to bind globular vWF in solution or vWF immobilized by polyclonal anti-vWF antibodies, but efficiently interacts with shear- and ristocetin-activated vWF and recombinant vWF comprising the type 2B R1306W mutation. The nanobody epitope on vWF is expected to overlap with- or lie in close proximity to the plasmin binding site in the A1-A2 linker, possibly resulting in steric hindrance. Current studies focus on providing more direct evidence of the vWF binding site of the nanobody. From a clinical perspective, the sensitivity of R1306W vWF to plasmin-mediated cleavage may explain the bleeding diathesis in VWD type 2B patients with this mutation. DisclosuresNo relevant conflicts of interest to declare.

Mathematical models for shear-induced blood damage based on vortex platform.

Non-physiological shear stress in Ventricular Assist Device (VAD) is considered to be an important trigger of blood damage, which has become the biggest shackle for clinical application. The researches on blood damage in literature were limited to qualitative but did not make much quantitative analysis. The purpose of this study was to investigate the quantitative influence of two flow-dependent parameters: shear stress (rotational speed) and exposure time on the shear-induced damage of red blood cells and von Willebrand Factor (vWF). A vortex blood-shearing platform was constructed to conduct in vitro experiments. Free hemoglobin assay and vWF molecular weight analysis were then performed on the sheared blood samples. MATLAB was used for regression fitting of original experimental data. The quantitative correlations between the hemolysis index, the degradation of high molecular weight vWF and the two flow-dependent parameters were found both following the power law model. The mathematic models indicated that the sensitivity of blood damage on red blood cells and vWF to exposure time was both greater than that of shear stress. Besides, the damage of vWF was more serious than that of red blood cells at the same flow condition. The models could be used to predict blood damage in blood-contacting medical devices, especially for the slow even stagnant blood flow regions in VAD, thus may provide useful guidance for VAD development and improvement. It also indicated that the vortex platform can be used to study the law of blood damage for the simple structure and easy operation.

Efficacy and safety of a recombinant Von Willebrand Factor treatment in patients with inherited Von Willebrand Disease requiring surgical procedures.

Von Willebrand Disease is a common inherited haemorrhagic disorder due to a deficiency of Von Willebrand Factor (VWF). In case of surgical procedures in patients who are not responsive or have contraindications to desmopressin, replacement therapy with VWF concentrates is indicated. Until recently, only plasma-derived VWF concentrates were available. A new recombinant VWF (rVWF) concentrate that contains no Factor VIII (FVIII) but a high amount of high molecular weight VWF multimers has been available in France since 2018. Describe real-world experience of using rVWF in surgical procedures. Sixty-three surgeries for 55 patients were retrospectively analysed in 7 French haemostasis centres. During minor surgeries, the median (range) number of infusions was 1 (1-8) with a preoperative loading dose of 35 (19-56) rVWF IU/kg and a total median dose of 37.5IU (12-288). During major surgeries, the median (range) number of infusions was only 3 (1-14) with a median preoperative loading dose of 36IU (12-51) rVWF IU/kg, and a total median dose of 108IU (22-340) rVWF IU/kg. The overall clinical efficacy was qualified as excellent/good in 61 of the procedures (97%), moderate in 1 (1.5%) and poor in 1 (1.5%). There was no accumulation of VWF or FVIII during postoperative monitoring. No thromboembolic events, anti-VWF antibodies or adverse events were reported. This French 'real-world' experience shows that a few infusions and low doses of rVWF provided effective prevention of bleeding in major and minor surgeries in inherited VWD, with no clinically significant safety concerns.

Correcting dominant‐negative von Willebrand disease

Correcting dominant‐negative von Willebrand disease

Semiautomatic VWF Multimer Densitometric Analysis: Validation of the Clinical Accuracy and Clinical Implications in Von Willebrand Disease

Semiautomatic VWF Multimer Densitometric Analysis: Validation of the Clinical Accuracy and Clinical Implications in Von Willebrand Disease

Inhibition of adamts13: a novel therapy to treat mechanical circulatory support-induced acquired von willebrand syndrome

Abstract Introduction Bleeding is the most frequent adverse event in patients with continuous flow mechanical circulatory support (CF-MCS) and has been linked to the occurrence of acquired von Willebrand syndrome (aVWS). MCS devices cause an increased shear-induced proteolysis of von Willebrand factor (VWF) by ADAMTS13, leading to aVWS. Hence, specifically blocking ADAMTS13 might be an efficient way to rescue the loss of HMW VWF multimers in CF-MCS patients. Purpose To investigate if blocking ADAMTS13, using an in-house developed inhibitory anti-ADAMTS13 monoclonal antibody (mAb), prevents the loss of high molecular weight (HMW) VWF multimers in in vitro CF-MCS systems and to determine the efficacy of this therapy in a CF-MCS calf model. Methods Human blood was perfused through in vitro CF-MCS systems (Heartmate II and Impella CP, axial flow heart pumps) in the presence of the inhibitory or control mAb (20 μg/mL). Bovine blood was perfused through an in vitro Impella 5.5 system with the inhibitory mAb (20 μg/mL) or PBS. Next, Impella 5.5 pumps were implanted in calves. One dose of the inhibitory mAb (600 μg/kg) or PBS was injected eight days after Impella implantation. Plasma samples were analysed for VWF multimers, VWF antigen (VWF:Ag) and VWF collagen binding activity (VWF:CB). Results A time-dependent decrease in HMW VWF multimers was observed in both in vitro CF-MCS systems in the presence of the control mAb, leading to a 70% reduction of HMW VWF multimers, 180 minutes (min) after blood perfusion (p=0.01 for HM II and p=0.0003 for Impella). This was also reflected by a severely decreased VWF:CB/VWF:Ag ratio (0.59±0.11 and 0.52±0.10 at 180 min versus 1.00±0.06 and 1.07±0.09 before perfusion, for the HM II (p=0.03) and Impella (p=0.001) respectively). Interestingly, blocking ADAMTS13 using the inhibitory mAb prevented the loss of HMW VWF multimers in both systems (p=0.50 for the HM II and p=0.06 for the Impella, 180 min after the start of perfusion). The preservation of HMW VWF multimers was also reflected by normal VWF:CB/VWF:Ag ratios (0.92±0.16 and 0.97±0.11 at 180 min versus 0.93±0.09 and 1.19±0.12 before perfusion for the HM II (p=0.75) and Impella (p=0.06) respectively). Blocking bovine ADAMTS13 using the inhibitory mAb could prevent the loss of HMW VWF multimers in the in vitro Impella 5.5 system, showing that the calf is a good preclinical animal model to study the in vivo effect of this novel therapy. Impella implantation in the calves led to a decrease in HMW VWF multimers (Figure 1A and B). Hence, this animal model represents the VWF laboratory features of MCS-induced aVWS. Moreover, the loss of HMW VWF multimers after pump implantation could be rescued after injection of the inhibitory mAb (Figure 1A and B). Conclusion Blocking ADAMTS13 rescues MCS-induced VWF proteolysis in calves. Hence, inhibiting ADAMTS13 function could become a promising therapeutic strategy to rescue aVWS-induced bleeding in MCS patients. Figure 1. Impella calf model Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Fund for Scientific Research Flanders

Von Willebrand factor increases in experimental cerebral malaria but is not essential for late‐stage pathogenesis in mice

BackgroundCerebral malaria (CM) is the most severe complication of malaria. Endothelial activation, cytokine release, and vascular obstruction are essential hallmarks of CM. Clinical studies have suggested a link between von Willebrand factor (VWF) and malaria pathology. ObjectivesTo investigate the contribution of VWF in the pathogenesis of experimental cerebral malaria (ECM). MethodsBoth Vwf+/+ and Vwf−/− mice were infected with Plasmodium berghei ANKA (PbANKA) to induce ECM. Alterations of plasma VWF and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), platelet count, neurological features, and accumulation of platelets and leukocytes in the brain were examined following infection. ResultsPlasma VWF levels significantly increased upon PbANKA infection in Vwf+/+ animals. While ADAMTS13 activity was not affected, high molecular weight VWF multimers disappeared at the end‐stage ECM, possibly due to an ongoing hypercoagulability. Although the number of reticulocytes, a preferential target for the parasites, was increased in Vwf−/− mice compared to Vwf+/+ mice early after infection, parasitemia levels did not markedly differ between the two groups. Interestingly, Vwf−/− mice manifested overall clinical ECM features similar to those observed in Vwf+/+ animals. At day 8.5 post‐infection, however, clinical ECM features in Vwf−/− mice were slightly more beneficial than in Vwf+/+ animals. Despite these minor differences, overall survival was not different between Vwf−/− and Vwf+/+ mice. Similarly, PbANKA‐induced thrombocytopenia, leukocyte, and platelet accumulations in the brain were not altered by the absence of VWF. ConclusionsOur study suggests that increased VWF concentration is a hallmark of ECM. However, VWF does not have a major influence in modulating late‐stage ECM pathogenesis.