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von Willebrand factor (VWF)-R1205H variant (Vicenza) results in markedly enhanced VWF clearance in humans that has been shown to be largely macrophage-mediated. However, the biological mechanisms underlying this enhanced clearance remain poorly understood.

•A cluster of 4 lysine residues (K1405, K1406, K1407 and K1408) in the VWF-A1 domain constitutes a critical binding site for macrophage LRP1.•BT200 interaction with the VWF-A1 domain in proximity to this lysine cluster significantly attenuates macrophage LRP1-mediated clearance.

Von Willebrand factor (VWF) contains a number of free thiols, the majority of which are located in its C‐domains, and these have been shown to alter VWF function, However, the impact of free thiols on function following acute exposure of VWF to collagen under high and pathological shear stress has not been determined. VWF free thiols were blocked with N‐ethylmaleimide and flow assays performed under high and pathological shear rates to determine the impact on platelet capture and collagen binding function. Atomic force microscopy (AFM) was used to probe the interaction of VWF with collagen and molecular simulations conducted to determine the effect of free thiols on the flexibility of the VWF‐C4 domain. Blockade of VWF free thiols reduced VWF‐mediated platelet capture to collagen in a shear‐dependent manner, with platelet capture virtually abolished above 5000 s−1and in regions of stenosis in microfluidic channels. Direct visualization of VWF fibers formed under extreme pathological shear rates and analysis of collagen‐bound VWF attributed the effect to altered binding of VWF to collagen. AFM measurements showed that thiol‐blockade reduced the lifetime and strength of the VWF‐collagen bond. Pulling simulations of the VWF‐C4 domain demonstrated that with one or two reduced disulphide bonds the C4 domain has increased flexibility and the propensity to undergo free‐thiol exchange. We conclude that free thiols in the C‐domains of VWF enhance the flexibility of the molecule and enable it to withstand high shear forces following collagen binding, demonstrating a previously unrecognized role for VWF free thiols.

Von Willebrand factor (VWF) contains a number of free thiols, the majority of which are located in its C‐domains, and these have been shown to alter VWF function, However, the impact of free thiols on function following acute exposure of VWF to collagen under high and pathological shear stress has not been determined.

Previous studies have demonstrated that the A1A2A3 domains of von Willebrand factor (VWF) play a key role in regulating macrophage‐mediated clearance in vivo. In particular, the A1‐domain has been shown to modulate interaction with macrophage low‐density lipoprotein receptor‐related protein‐1 (LRP1) clearance receptor. Furthermore, N‐linked glycans within the A2‐domain have been shown to protect VWF against premature LRP1‐mediated clearance. Importantly, however, the specific regions within A1A2A3 that enable macrophage binding have not been defined.

Glycan determinants on von Willebrand factor (VWF) play critical roles in regulating its susceptibility to proteolysis and clearance. Abnormal glycosylation has been shown to cause von Willebrand disease (VWD) in a number of different mouse models. However, because of the significant technical challenges associated with accurate assessment of VWF glycan composition, the importance of carbohydrates in human VWD pathogenesis remains largely unexplored. To address this, we developed a novel lectin-binding panel to enable human VWF glycan characterization. This methodology was then used to study glycan expression in a cohort of 110 patients with low VWF compared with O blood group-matched healthy controls. Interestingly, significant interindividual heterogeneity in VWF glycan expression was seen in the healthy control population. This variation included terminal sialylation and ABO(H) blood group expression on VWF. Importantly, we also observed evidence of aberrant glycosylation in a subgroup of patients with low VWF. In particular, terminal α(2-6)-linked sialylation was reduced in patients with low VWF, with a secondary increase in galactose (Gal) exposure. Furthermore, an inverse correlation between Gal exposure and estimated VWF half-life was observed in those patients with enhanced VWF clearance. Together, these findings support the hypothesis that loss of terminal sialylation contributes to the pathophysiology underpinning low VWF in at least a subgroup of patients by promoting enhanced clearance. In addition, alterations in VWF carbohydrate expression are likely to contribute to quantitative and qualitative variations in VWF levels in the normal population. This trial was registered at www.clinicaltrials.govas #NCT03167320.

Glycan determinants on von Willebrand factor (VWF) play critical roles in regulating its susceptibility to proteolysis and clearance. Abnormal glycosylation has been shown to cause von Willebrand disease (VWD) in a number of different mouse models. However, because of the significant technical challenges associated with accurate assessment of VWF glycan composition, the importance of carbohydrates in human VWD pathogenesis remains largely unexplored. To address this, we developed a novel lectin-binding panel to enable human VWF glycan characterization. This methodology was then used to study glycan expression in a cohort of 110 patients with low VWF compared with O blood group-matched healthy controls. Interestingly, significant interindividual heterogeneity in VWF glycan expression was seen in the healthy control population. This variation included terminal sialylation and ABO(H) blood group expression on VWF. Importantly, we also observed evidence of aberrant glycosylation in a subgroup of patients with low VWF. In particular, terminal a(2-6)-linked sialylation was reduced in patients with low VWF, with a secondary increase in galactose (Gal) exposure. Furthermore, an inverse correlation between Gal exposure and estimated VWF half-life was observed in those patients with enhanced VWF clearance. Together, these findings support the hypothesis that loss of terminal sialylation contributes to the pathophysiology underpinning low VWF in at least a subgroup of patients by promoting enhanced clearance. In addition, alterations in VWF carbohydrate expression are likely to contribute to quantitative and qualitative variations in VWF levels in the normal population. This trial was registered at www.clinicaltrials.gov as #NCT03167320.

Previous studies have shown that loss of terminal sialic acid causes enhanced von Willebrand factor (VWF) clearance through the Ashwell-Morrell receptor (AMR). In this study, we investigated (1) the specific importance of N- vs O-linked sialic acid in protecting against VWF clearance and (2) whether additional receptors contribute to the reduced half-life of hyposialylated VWF. α2-3-linked sialic acid accounts for <20% of total sialic acid and is predominantly expressed on VWF O-glycans. Nevertheless, specific digestion with α2-3 neuraminidase (α2-3Neu-VWF) was sufficient to cause markedly enhanced VWF clearance. Interestingly, in vivo clearance experiments in dual VWF−/−/Asgr1−/− mice demonstrated enhanced clearance of α2-3Neu-VWF even in the absence of the AMR. The macrophage galactose-type lectin (MGL) is a C-type lectin that binds to glycoproteins expressing terminal N-acetylgalactosamine or galactose residues. Importantly, the markedly enhanced clearance of hyposialylated VWF in VWF−/−/Asgr1−/− mice was significantly attenuated in the presence of an anti-MGL inhibitory antibody. Furthermore, dose-dependent binding of human VWF to purified recombinant human MGL was confirmed using surface plasmon resonance. Additionally, plasma VWF:Ag levels were significantly elevated in MGL1−/− mice compared with controls. Collectively, these findings identify MGL as a novel macrophage receptor for VWF that significantly contributes to the clearance of both wild-type and hyposialylated VWF.

Previous studies have shown that loss of terminal sialic acid causes enhanced von Willebrand factor (VWF) clearance through the Ashwell-Morrell receptor (AMR). In this study, we investigated (1) the specific importance of N- vs O-linked sialic acid in protecting against VWF clearance and (2) whether additional receptors contribute to the reduced half-life of hyposialylated VWF. α2-3-linked sialic acid accounts for <20% of total sialic acid and is predominantly expressed on VWF O-glycans. Nevertheless, specific digestion with α2-3 neuraminidase (α2-3Neu-VWF) was sufficient to cause markedly enhanced VWF clearance. Interestingly, in vivo clearance experiments in dual VWF−/−/Asgr1−/−mice demonstrated enhanced clearance of α2-3Neu-VWF even in the absence of the AMR. The macrophage galactose-type lectin (MGL) is a C-type lectin that binds to glycoproteins expressing terminal N-acetylgalactosamine or galactose residues. Importantly, the markedly enhanced clearance of hyposialylated VWF in VWF−/−/Asgr1−/−mice was significantly attenuated in the presence of an anti-MGL inhibitory antibody. Furthermore, dose-dependent binding of human VWF to purified recombinant human MGL was confirmed using surface plasmon resonance. Additionally, plasma VWF:Ag levels were significantly elevated in MGL1−/−mice compared with controls. Collectively, these findings identify MGL as a novel macrophage receptor for VWF that significantly contributes to the clearance of both wild-type and hyposialylated VWF.

Supplemental Digital Content is available in the text.

Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However, the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and A1-A2-A3. Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo.

Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However, the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and A1-A2-A3. Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo.

Supplemental Digital Content is available in the text.

Plasmodium falciparum malaria infection is associated with an early marked increase in plasma von Willebrand factor (VWF) levels, together with a pathological accumulation of hyperreactive ultra-large VWF (UL-VWF) multimers. Given the established critical role of platelets in malaria pathogenesis, these increases in plasma VWF raise the intriguing possibility that VWF may play a direct role in modulating malaria pathogenesis. To address this hypothesis, we used an established murine model of experimental cerebral malaria (ECM), in which wild-type (WT) C57BL/6J mice were infected with Plasmodium berghei ANKA. In keeping with findings in children with P falciparum malaria, acute endothelial cell activation was an early and consistent feature in the murine model of cerebral malaria (CM), resulting in significantly increased plasma VWF levels. Despite the fact that murine plasma ADAMTS13 levels were not significantly reduced, pathological UL-VWF multimers were also observed in murine plasma following P berghei infection. To determine whether VWF plays a role in modulating the pathogenesis of CM in vivo, we further investigated P berghei infection in VWF-/- C57BL/6J mice. Clinical ECM progression was delayed, and overall survival was significantly prolonged in VWF-/- mice compared with WT controls. Despite this protection against ECM, no significant differences in platelet counts or blood parasitemia levels were observed between VWF-/- and WT mice. Interestingly, however, the degree of ECM-associated enhanced blood–brain barrier permeability was significantly attenuated in VWF-/- mice compared with WT controls. Given the significant morbidity and mortality associated with CM, these novel data may have direct translational significance.

Plasmodium falciparummalaria infection is associated with an early marked increase in plasma von Willebrand factor (VWF) levels, together with a pathological accumulation of hyperreactive ultra-large VWF (UL-VWF) multimers. Given the established critical role of platelets in malaria pathogenesis, these increases in plasma VWF raise the intriguing possibility that VWF may play a direct role in modulating malaria pathogenesis. To address this hypothesis, we used an established murine model of experimental cerebral malaria (ECM), in which wild-type (WT) C57BL/6J mice were infected with Plasmodium bergheiANKA. In keeping with findings in children with P falciparummalaria, acute endothelial cell activation was an early and consistent feature in the murine model of cerebral malaria (CM), resulting in significantly increased plasma VWF levels. Despite the fact that murine plasma ADAMTS13 levels were not significantly reduced, pathological UL-VWF multimers were also observed in murine plasma following P bergheiinfection. To determine whether VWF plays a role in modulating the pathogenesis of CM in vivo, we further investigated P bergheiinfection in VWF−/−C57BL/6J mice. Clinical ECM progression was delayed, and overall survival was significantly prolonged in VWF−/−mice compared with WT controls. Despite this protection against ECM, no significant differences in platelet counts or blood parasitemia levels were observed between VWF−/−and WT mice. Interestingly, however, the degree of ECM-associated enhanced blood–brain barrier permeability was significantly attenuated in VWF−/−mice compared with WT controls. Given the significant morbidity and mortality associated with CM, these novel data may have direct translational significance.

ADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. The mechanisms of VWF recognition by ADAMTS13 have yet to be fully resolved. Most studies have focused on the role of exosites within the VWF A2 domain, involved in interaction with the ADAMTS13 spacer domain. In the present study, we expressed different C-terminal domain VWF fragments and evaluated their binding to ADAMTS13 and its truncated mutants, MDTCS and del(TSP5-CUB). Using plate binding assay and surface plasmon resonance, we identified a novel ADAMTS13 binding site (KD ∼ 86 nM) in the region of VWF spanning residues 1874 to 2813, which includes the VWF D4 domain and that interacts with the C-terminal domains of ADAMTS13. We show that the interaction occurs even when VWF is in static conditions, assumed to be globular and where the VWF A2 domain is hidden. We demonstrate that C-terminal VWF fragments, as well as an antibody specifically directed toward the VWF D4 domain, inhibit VWF proteolysis by ADAMTS13 under shear conditions. We propose that this novel VWF C-terminal binding site may participate as the initial step of a multistep interaction ultimately leading to proteolysis of VWF by ADAMTS13.

ADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. The mechanisms of VWF recognition by ADAMTS13 have yet to be fully resolved. Most studies have focused on the role of exosites within the VWF A2 domain, involved in interaction with the ADAMTS13 spacer domain. In the present study, we expressed different C-terminal domain VWF fragments and evaluated their binding to ADAMTS13 and its truncated mutants, MDTCS and del(TSP5-CUB). Using plate binding assay and surface plasmon resonance, we identified a novel ADAMTS13 binding site (KD∼ 86 nM) in the region of VWF spanning residues 1874 to 2813, which includes the VWF D4 domain and that interacts with the C-terminal domains of ADAMTS13. We show that the interaction occurs even when VWF is in static conditions, assumed to be globular and where the VWF A2 domain is hidden. We demonstrate that C-terminal VWF fragments, as well as an antibody specifically directed toward the VWF D4 domain, inhibit VWF proteolysis by ADAMTS13 under shear conditions. We propose that this novel VWF C-terminal binding site may participate as the initial step of a multistep interaction ultimately leading to proteolysis of VWF by ADAMTS13.

We examined the role of N-linked glycan structures of VWF on its interaction with ADAMTS13. PNGase F digestion followed by lectin analysis demonstrated that more than 90% of VWF N-linked glycan chains could be removed from the molecule (PNG-VWF) without disruption of its multimeric structure or its ability to bind to collagen. PNG-VWF had an approximately 4-fold increased affinity for ADAMTS13 compared with control VWF. PNG-VWF was cleaved by ADAMTS13 faster than control VWF and was also proteolysed in the absence of urea. Occupancy of the N-linked glycan sites at N1515 and N1574 and their presentation of ABO(H) blood group sugars were confirmed with an isolated tryptic fragment. Recombinant VWF was mutated to prevent glycosylation at these sites. Mutation of N1515 did not alter ADAMTS13 binding or increase rate of ADAMTS13 proteolysis. Mutation of N1574 increased the susceptibility of VWF to ADAMTS13 proteolysis and allowed cleavage in the absence of urea. Mutation of N1574 in the isolated recombinant VWF-A2 domain also increased binding and ADAMTS13 proteolysis. These data demonstrate that the N-linked glycans of VWF have a modulatory effect on the interaction with ADAMTS13. At least part of this effect is conformational, but steric hindrance may also be important.

We examined the role of N-linked glycan structures of VWF on its interaction with ADAMTS13. PNGase F digestion followed by lectin analysis demonstrated that more than 90% of VWF N-linked glycan chains could be removed from the molecule (PNG-VWF) without disruption of its multimeric structure or its ability to bind to collagen. PNG-VWF had an approximately 4-fold increased affinity for ADAMTS13 compared with control VWF. PNG-VWF was cleaved by ADAMTS13 faster than control VWF and was also proteolysed in the absence of urea. Occupancy of the N-linked glycan sites at N1515 and N1574 and their presentation of ABO(H) blood group sugars were confirmed with an isolated tryptic fragment. Recombinant VWF was mutated to prevent glycosylation at these sites. Mutation of N1515 did not alter ADAMTS13 binding or increase rate of ADAMTS13 proteolysis. Mutation of N1574 increased the susceptibility of VWF to ADAMTS13 proteolysis and allowed cleavage in the absence of urea. Mutation of N1574 in the isolated recombinant VWF-A2 domain also increased binding and ADAMTS13 proteolysis. These data demonstrate that the N-linked glycans of VWF have a modulatory effect on the interaction with ADAMTS13. At least part of this effect is conformational, but steric hindrance may also be important.

O'Donnell: Bayer: Research Funding, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Leo Pharma: Speakers Bureau; Octapharma: Speakers Bureau; CSL Behring: Consultancy; Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Research Funding; Baxter: Research Funding, Speakers Bureau; Shire: Research Funding, Speakers Bureau.

Introduction

No relevant conflicts of interest to declare.

Enhanced plasma clearance of von Willebrand factor (VWF) plays an important role in the etiology of both type 1 and type 2 VWD. Nevertheless, although significant progress has been achieved in understanding the structure and functional properties of VWF, the mechanism(s) responsible for modulating VWF clearance from the plasma remain poorly understood. Accumulating recent data suggests that hepatic and splenic macrophages play key roles in modulating VWF clearance. A number of putative macrophage receptors for VWF have been also been described, including LRP1, β2-integrins and Siglec-5. In addition, it is well recognised that variation in VWF glycan expression significantly influences its clearance rate. In particular, terminal ABO(H) blood group determinants which are predominantly expressed on the N-linked glycans of human VWF significantly modulate its rate of clearance. Critically however, the molecular mechanisms through which specific macrophage receptors interact with particular regions of the complex VWF glycoprotein have not been defined.

No relevant conflicts of interest to declare.

During biosynthesis, Factor VIII (FVIII) undergoes complex post-translational modification including significant glycosylation. Consequently each FVIII molecule can contains 25 N- and 6 O-linked glycans. These carbohydrate structures are of physiological significance. For example, FVIII glycan expression modulates intracellular trafficking and also regulates FVIII clearance by dendritic cells. Nevertheless, the molecular mechanisms through which glycan structures influence FVIII biology remains poorly defined. Interestingly, carbohydrate-binding galectins (Gal) -1 and -3 have recently been reported to bind human VWF. Moreover, these galectin interactions significantly influence VWF function. In this study, based upon similar glycans expression profiles, we hypothesised that galectins might also constitute novel binding partners for human FVIII.

Von Willebrand factor (VWF) is a large plasma glycoprotein that mediates platelet tethering at sites of vascular injury. This function is dependent upon its multimeric size, which is modulated in plasma through proteolysis by ADAMTS13. Terminal ABO blood group sugars presented on the N-linked glycans of VWF alter the susceptibility of VWF to cleavage by ADAMTS13. Two predicted N-linked glycan sites (N1515 & N1574) are in close proximity to the ADAMTS13 cleavage site (Y1605-M1606). However, is it not known whether these predicted sites are occupied, or indeed, present the ABO sugars. In this study, purified plasma VWF was digested with trypsin, and the resulting fragments separated by ion-exchange chromatography. A 55kDa fragment observed under reducing SDS-PAGE was identified as VWF residues 1493–1849 by mass spectrocosopy and N-terminal sequencing. This fragment encompassed the predicted N-linked glycan sites at N1515 & N1574. Incomplete PNGase F digestion produced three protein bands, indicating that both glycosylation sites were occupied. Furthermore, analysis with blood group specific lectins demonstrated presentation of the ABO sugars on these glycan chains. This data suggests that the N1515 and N1574 glycosylation sites and subsequent ABO(H) sugar modification may influence VWF proteolysis. To further elucidate the role of other VWF glycan structures on susceptibility to ADAMTS13 cleavage, purified VWF was treated with neuraminidase to remove terminal sialic acid residues, producing asialo VWF (As-VWF), and PNGase F to remove whole N-linked glycan chains. (Nless-VWF). Lectin analysis demonstrated removal of >95% of terminal sialic residues and >75% whole N-linked glycan chains. Both AsVWF and Nless-VWF bound to type III collagen with similar affinity to wild type VWF (KD 1.9nM, 1.6nM, 1.4nM respectively) and demonstrated similar multimeric composition. Interestingly, As-VWF had decreased susceptibility to ADAMTS13 cleavage, but bound ADAMTS13 with comparable affinity to wild type VWF (KD 11nM & 12nM respectively). Nless-VWF was cleaved faster by ADAMTS13 and bound ADAMTS13 with ~ 4-fold increased affinity (KD 3.4nM). This data demonstrates that the glycan moiety of VWF plays an important role in mediating the interaction with ADAMTS13.

Von Willebrand factor (VWF) is a large plasma glycoprotein that mediates platelet tethering at sites of vascular injury. This function is dependent upon its multimeric size, which is modulated in plasma through proteolysis by ADAMTS13. Terminal ABO blood group sugars presented on the N-linked glycans of VWF alter the susceptibility of VWF to cleavage by ADAMTS13. Two predicted N-linked glycan sites (N1515 & N1574) are in close proximity to the ADAMTS13 cleavage site (Y1605-M1606). However, is it not known whether these predicted sites are occupied, or indeed, present the ABO sugars. In this study, purified plasma VWF was digested with trypsin, and the resulting fragments separated by ion-exchange chromatography. A 55kDa fragment observed under reducing SDS-PAGE was identified as VWF residues 1493–1849 by mass spectrocosopy and N-terminal sequencing. This fragment encompassed the predicted N-linked glycan sites at N1515 & N1574. Incomplete PNGase F digestion produced three protein bands, indicating that both glycosylation sites were occupied. Furthermore, analysis with blood group specific lectins demonstrated presentation of the ABO sugars on these glycan chains. This data suggests that the N1515 and N1574 glycosylation sites and subsequent ABO(H) sugar modification may influence VWF proteolysis. To further elucidate the role of other VWF glycan structures on susceptibility to ADAMTS13 cleavage, purified VWF was treated with neuraminidase to remove terminal sialic acid residues, producing asialo VWF (As-VWF), and PNGase F to remove whole N-linked glycan chains. (Nless-VWF). Lectin analysis demonstrated removal of >95% of terminal sialic residues and >75% whole N-linked glycan chains. Both AsVWF and Nless-VWF bound to type III collagen with similar affinity to wild type VWF (KD1.9nM, 1.6nM, 1.4nM respectively) and demonstrated similar multimeric composition. Interestingly, As-VWF had decreased susceptibility to ADAMTS13 cleavage, but bound ADAMTS13 with comparable affinity to wild type VWF (KD11nM & 12nM respectively). Nless-VWF was cleaved faster by ADAMTS13 and bound ADAMTS13 with ~ 4-fold increased affinity (KD3.4nM).