High Molecular Weight Von Willebrand Factor Multimers Research Articles

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).

A noncanonical splicing variant c.875-5 T > G in von Willebrand factor causes in-frame exon skipping and type 2A von Willebrand disease

IntroductionA novel variant involving noncanonical splicing acceptor site (c.875-5 T > G) in propeptide coding region of von Willebrand factor (VWF) was identified in a patient with type 2A von Willebrand disease (VWD), who co-inherited with a null variant (p.Tyr271*) and presented characteristic discrepancy of plasma level of VWF antigen and activity, and a selective reduction of both intermediate-molecular-weight (IMWMs) and high-molecular-weight VWF multimers (HMWMs). Materials and methodsVWF mRNA transcripts obtained from peripheral leukocytes and platelets of the patients were investigated to analyze the consequence of c.875-5 T > G on splicing. The impact of the variant on expression and multimer assembly was further analyzed by in vitro expression studies in AtT-20 cells. The intracellular processing of VWF mutant and the Weibel-Palade bodies (WPBs) formation was evaluated by immunofluorescence staining and electron microscopy. ResultsThe mRNA transcript analysis revealed that c.875-5 T > G variant led to exon 8 skipping and an in-frame deletion of 41 amino acids in the D1 domain of VWF (p.Ser292_Glu333delinsLys), yielding a truncated propeptide. Consistent with the patient's laboratory manifestations, the AtT-20 cells transfected with mutant secreted less VWF, with the VWF antigen level in conditioned medium 47 % of wild-type. A slight retention in the endoplasmic reticulum was observed for the mutant. Almost complete loss of IMWMs and HMWMs in the medium and impaired WPBs formation in the cell, indicating truncated VWF propeptide lost its chaperon-like function for VWF multimerization and tubular storage. ConclusionsThe VWF splicing site variant (c.875-5 T > G) causes propeptide truncation, severely compromising VWF multimer assembly and tubular storage.

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

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.

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.

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.

Primary Hemostasis Defect Due to Acquired Von Willebrand Disease and Platelet Activation During Extracorporeal Life Support. In Vitro Correction by Purified VWF

<h3>Purpose</h3> Acquired von Willebrand disease (aVWD) is not uncommon during long-term mechanical circulatory support. However, its development during short-term support is unknown. We aim at investigating the development of aVWD in patients under extracorporeal life support (ECLS) and its potential treatment. <h3>Methods</h3> Patients who received ECLS in our center since June 2020 were included (25). Primary hemostasis was evaluated by: VWF antigen (VWF:Ag) and activity (VWF:GPIbM) measurement (immunoturbidimetry), VWF multimers analysis (agarose-gels and immunoblotting), platelet aggregation by ristocetin (turbidimetry), platelet functional analysis (PFA-200) -collagen/epinephrine (Col/EPI) and collagen/ADP (Col/ADP) cartridges-, and platelet activation (flow cytometry analysis of P-selectin and Lisosomal antigen). Studies were performed the day after implant and 3 days after explant. T-TAS was applied in bleeding patients, using samples before and after in vitro addition of purified VWF. <h3>Results</h3> At day 1 day after implant, all patients showed increased VWF:Ag. In 15 (60%) there was an altered ratio (<0.7) between VWF:GPIbM and VWF:Ag, with loss of VWF high molecular weight multimers (HMWM). In all cases, prolonged occlusion times at the PFA with both cartridges were observed. A significant increased expression of platelet-activation antigens was detected in 5 patients (11% and 24% for P-Selectin and Lysosomal antigen, respectively, vs. control platelets). In bleeding patients, addition of purified VWF to blood samples reduced significantly (p<0.05) the occlusion time (-384s) and increased the AUW (370%) at T-TAS. All patients that survived to explant (16) showed normalization of VWF:GPIbM/VWF:Ag ratio (>0.7) and multimers, with reduction of PFA-200 values. <h3>Conclusion</h3> ECLS caused primary hemostasis alterations, leading to aVWD and platelet activation, independently of platelet counts. Hemostatic efficiency in bleeding patients can be corrected by purified VWF, as derived from the in vitro evidence. The primary hemostasis alteration observed was solved early after support removal.

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.

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.

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.

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.

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

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.

Circulating Von Willebrand factor and high molecular weight multimers as markers of endothelial injury predict COVID-19 in-hospital mortality.

BackgroundCoronavirus disease 2019 (COVID-19) is a respiratory disease associated with endotheliitis and microthrombosis.ObjectivesTo correlate endothelial dysfunction to in-hospital mortality in a bi-centric cohort of COVID-19 adult patients.MethodsConsecutive ambulatory and hospitalized patients with laboratory-confirmed COVID-19 were enrolled. A panel of endothelial biomarkers and von Willebrand factor (VWF) multimers were measured in each patient ≤ 48 h following admission.ResultsStudy enrolled 208 COVID-19 patients of whom 23 were mild outpatients and 189 patients hospitalized after admission. Most of endothelial biomarkers tested were found increased in the 89 critical patients transferred to intensive care unit. However, only von Willebrand factor antigen (VWF:Ag) scaled according to clinical severity, with levels significantly higher in critical patients (median 507%, IQR 428–596) compared to non-critical patients (288%, 230–350, p < 0.0001) or COVID-19 outpatients (144%, 133–198, p = 0.007). Moreover, VWF high molecular weight multimers (HMWM) were significantly higher in critical patients (median ratio 1.18, IQR 0.86–1.09) compared to non-critical patients (0.96, 1.04–1.39, p < 0.001). Among all endothelial biomarkers measured, ROC curve analysis identified a VWF:Ag cut-off of 423% as the best predictor for in-hospital mortality. The accuracy of VWF:Ag was further confirmed in a Kaplan–Meier estimator analysis and a Cox proportional Hazard model adjusted on age, BMI, C-reactive protein and d-dimer levels.ConclusionVWF:Ag is a relevant predictive factor for in-hospital mortality in COVID-19 patients. More than a biomarker, we hypothesize that VWF, including excess of HMWM forms, drives microthrombosis in COVID-19.

Correcting dominant‐negative von Willebrand disease

Correcting dominant‐negative von Willebrand disease

Use of a Von Willebrand Factor/Coagulation Factor VIII Complex for Treatment of Refractory Inherited Platelet Disorders

Inherited platelet disorders are recognized as an important cause of mild to severe bleeding in both children and adults. Patients with platelet disorders may present with mucocutaneous bleeding, gastrointestinal bleeding, menorrhagia, postsurgical, and/or excessive bleeding from traumatic injury. Delta storage pool deficiencies (delta-SPD) are among the most frequent platelet disorders, characterized by dysfunctional dense platelet granules. Bernard Soulier syndrome (BSS) is an autosomal recessive platelet disorder caused by mutations in various polypeptides in the GpIb/IX/V complex, which is the principal receptor for von Willebrand factor (VWF). Treatment of platelet disorders is mainly supportive. Normal hemostasis requires VWF and factor VIII (FVIII) to support platelet adhesion and aggregation at sites of vascular injury. von Willebrand factor is a large multimeric glycoprotein present in human plasma as a series of polymers called multimers. Molecular weights for multimers ranges from 500 kDa for the dimer to over 10,000 kDa for the high molecular weight multimers (HMWM) forming the largest known protein present in human plasma. Each multimeric subunit of VWF has binding sites for the receptor GpIb on nonactivated platelets and the receptor GpIIb/IIIa to facilitate platelet adhesion and platelet aggregation, respectively, making the VWF HMWM important for normal platelet function. Desmopressin (DDAVP), which is known to stimulate the release of VWF and FVIII, is commonly used for treatment of platelet disorders. Potentiation of platelet aggregation at high shear rate may be one mechanism by which DDAVP shortens the prolonged bleeding time of patients with congenital platelet defects. For severe bleeding, platelet transfusion may be required, but patients may develop isoantibodies, rendering this therapy ineffective. For this reason, it may be prudent to reserve platelet transfusion in this patient population for emergent situations, such as trauma. Other patients and/or clinical situations may require recombinant active factor VII (rFVIIa), but this therapy is very costly and not always effective and/or available. Antifibrinolytics may also be used but are not always effective. In four (4) patients with platelet disorders (delta-SPD [n=3]; BSS [n=1]), common supportive therapies were not effective, tolerable, and/or available. It was postulated that off-label infusions of a cost-effective von Willebrand factor/coagulation factor VIII (VWF/FVIII) complex (Wilate, Octapharma SA) might be of benefit in these refractory patients (Table 1). The mechanism of action of DDAVP treatment efficacy relies on the release of existing, stored, functional VWF. In refractory patients with suboptimal VWF functionality, it was reasoned that infusion of exogenous, functional VWF and FVIII could potentially encourage platelet adhesion and aggregation. All refractory patients studied were treated successfully with the VWF/FVIII complex with positive clinical outcomes. As mentioned, the adhesive activity of VWF depends on the size of its multimers, and HMWM are the most effective in supporting interaction with collagen and platelet receptors and in facilitating wound healing under conditions of shear stress in the human vascular system. The VWF/FVIII complex utilized in these patients is known to have minimal amounts of the plasma metalloproteinase ADAMTS13. The HMWM of VWF are, under normal conditions, cleaved by ADAMTS13 to smaller, less adhesive multimers. During the manufacturing process, if the ADAMTS13 is not filtered out of the product almost entirely, the VWF in the vial may become highly proteolyzed. Therefore, a reduction or lack of HMWM resulting from inclusion of ADAMTS13 in the manufactured product is believed to reduce product functionality. Multimeric analysis of the VWF/FVIII complex has shown that it exhibits a physiological triplet structure which resembles normal plasma. In addition, the product has a high safety profile and tolerability as protein impurities are eliminated in the manufacturing process. In summary, the use of a VWF/FVIII complex in four (4) patients with inherited platelet disorders, who were refractory to conventional treatments, provided beneficial, cost-effective clinical outcomes with resolution of bleeding. Disclosures Federizo: Octapharma: Consultancy, Honoraria, Other: Publication support, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; American Thrombosis and Hemostasis Netowrk: Research Funding; Aptevo: Consultancy, Speakers Bureau; National Hemophilia Foundation: Consultancy, Honoraria. OffLabel Disclosure: von Willebrand/FVIII concentrate is currently approved for the treatment of Hemophilia A and von Willebrand. This abstract will review the off-label use of this medication in the treatment of inherited platelet dysfunction.

A Novel VWD Variant Identified in a Multi-Generational Canadian Ojibway Kindred with a Type 2B VWD Phenotypic Expression

Introduction: Type 2 VWD is caused by variants in the von Willebrand factor (VWF) gene leading to impaired function. Distinction between subtypes of Type 2 has traditionally relied on a panel of assays including VWF antigen (VWF:Ag), VWF ristocetin-cofactor activity (VWF:RCo), VWF multimers, and Factor VIII (FVIII:C). In type 2A, there is either impaired synthesis or accelerated degradation of the more adhesive VWF high molecular weight multimers (HMWMs). In type 2B, enhanced VWF-platelet interactions result in binding and removal of the HMWMs, impairing platelet adhesion. Type 2B VWD can manifest spontaneous platelet agglutination and thrombocytopenia. The inheritance patterns of Type 2A and 2B VWD are generally described to be autosomal dominant and fully penetrant. We identified a multigenerational Canadian First Nations Ojibway kindred with VWD harboring a missense variant in exon 28 (A&amp;gt;T transversion at nucleotide 4898), leading to a single amino acid substitution (p.Asn1633Ile). Some individuals in the kindred are heterozygous, and some homozygous, for the causative variant. Objectives: The objective of this case series are to describe the genetic basis and pattern of inheritance of this variant of VWD, and to characterize the laboratory hemostasis characteristics in the affected individuals. Methods: A retrospective case review was conducted on all affected individuals in the kindred known or suspected to harbour this variant. There were 20 individuals seen at our centre over 32 years who were included in this study. We collected clinical and laboratory data to characterize the bleeding phenotype. Results: Sixteen of the 20 individuals in the study were known or inferred to be heterozygous, and 4 were homozygous. We were able to examine laboratory hemostasis in ten individuals in the study (Table 1). The platelet count, FVIII:C and VWF:Ag levels were within or slightly above reference range for all individuals. In the heterozygous individuals, VWF:RCo was mildly reduced, VWF multimer testing showed loss of HMWMs, and ristocetin-induced platelet aggregation (RIPA) testing normal aggregation to a higher dose (1.25 mg/ml), a pattern consistent with mild Type 2A VWD (Table 1). In homozygous individuals, VWF:RCo was markedly reduced, and while RIPA was minimal with low-dose ristocetin, aggregation to the higher dose was normal despite the very low ristocetin cofactor activity in the plasma. The PFA-100 (collagen-epinephrine) closure times were markedly prolonged in the homozygous subjects and in one heterozygous subject, exceeding 300 seconds. Discussion/Conclusion: This novel VWF variant confers a laboratory phenotype consistent with type 2A VWD in the heterozygotes. Homozygotes have more severely impaired hemostatic function. The conventional hallmark of type 2B VWD (hyperresponsiveness to low-dose ristocetin) was not seen, but the fact that full aggregation was obtained in homozygotes despite ristocetin cofactor activities of only 5-6% in the plasma suggests some degree of hyperaggregability, and hence a phenotype more in keeping with Type 2B than 2A. In the future, it would be interesting to see how this missense variant affects GP1b binding to VWF, as RIPA and VWF:RCo are not entirely comparable assays. Disclosures No relevant conflicts of interest to declare.