High Concentrations Of Urea Research Articles

Effect of Urea and Glycerol on the Adsorption of Ribonuclease A at the Air−Water Interface

This study reports on the influence of nonionic cosolvents on the interfacial structure of ribonuclease A (RNase) adsorbed at the air-water interface. We applied X-ray reflectometry to obtain detailed volume fraction profiles of the adsorbed layers and to follow the effect of glycerol and urea on the adsorbate structure as a function of cosolvent concentration. Under all conditions studied, the adsorbed RNase layer maintains its compact shape, and the adsorbed RNase molecules adopt a flat-on orientation at the interface. Both kosmotropic glycerol and chaotropic urea exert profound effects on the adsorbate: The surface excess decreases linearly with glycerol content and is also reduced at low urea concentration. However, at high urea concentration, parts of the adsorbed layer are dehydrated and become exposed to air. The electron density and volume fraction profiles of the adsorbed protein provide clear evidence that these effects are ruled by different mechanisms.

Mechanisms of amphipathic helical peptide denaturation by guanidinium chloride and urea: a molecular dynamics simulation study

Urea and GdmCl are widely used to denature proteins at high concentrations. Here, we used MD simulations to study the denaturation mechanisms of helical peptide in different concentrations of GdmCl and urea. It was found that the helical structure of the peptide in water simulation is disappeared after 5ns while the helicity of the peptide is disappeared after 70ns in 2M urea and 25ns in 1M GdmCl. Surprisingly, this result shows that the helical structure in low concentration of denaturants is remained more with respect to that solvated in water. The present work strongly suggests that urea interact more preferentially to non-polar and aromatic side chains in 2M urea; therefore, hydrophobic residues are in more favorable environment in 2M urea. Our results also reveal that the hydrogen bonds between urea and the backbone is the dominant mechanism by which the peptide is destabilized in high concentration of urea. In 1M and 2M GdmCl, GdmCl molecules tend to engage in transient stacking interactions with aromatics and hydrophobic planar side chains that lead to displacement of water from the hydration surface, providing more favorable environment for them. This shows that accumulation of GdmCl around hydrophobic surfaces in 1M and 2M GdmCl solutions prevents proper solvation of the peptide at the beginning. In high GdmCl concentrations, water solvate the peptide better than 1M and 2M GdmCl. Therefore, our results strongly suggest that hydrogen bonds between water and the peptide are important factors in the destabilization of peptide in GdmCl solutions.

Plasmodium Falciparum-Induced Kidney and Liver Dysfunction in Malaria Patients in Freetown, Sierra Leone

This study was undertaken to investigate the effect of Plasmodium falciparum infection on kidney and liver function parameters in malaria patients in Freetown, Sierra Leone. Blood samples taken from 64 malaria patients and 64 non-malaria volunteers at Abanita and Blue Shield Hospitals, Freetown Sierra Leone between January to April, 2009 were examined. Changes in serum biochemical parameters were analysed using normal range values as baseline. Serum bilirubin, alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations were significantly elevated in falciparum malaria patients compared to their non-malaria counterparts which is an indication of defective liver function. Most of patients with falciparum malaria also have significantly high serum concentrations of urea, creatinine, sodium and potassium showing alteration in kidney function. This study suggests that malaria parasites could be responsible for derangement of kidney and liver functions in patients and could therefore contribute to organ damage in affected individuals if not treated. Keywords: Kidney function, Liver function, Malaria, Plasmodium falciparum

Self‐Association of Unfolded Outer Membrane Proteins

We have investigated self-association propensities of aqueous unfolded (U(AQ)) forms of eight outer membrane proteins (OMPs), OmpA, OmpW, OmpX, PagP, OmpT, OmpLa, FadL, and Omp85. We found that high urea concentrations maintain all of these OMPs as monomers and that OmpA and OmpX remain monomeric upon dilution to 1 M urea. A pH screen showed that basic pH supports the least amount of U(AQ) OMP self-association, consistent with earlier studies showing that basic pH was optimal for better folding efficiencies. The addition of KCl increased U(AQ) OMP self-association, although the magnitudes of the responses were varied. These studies showed that urea can be used to tune the amount of U(AQ) OMP self-association and indicate that the presence of some urea may be useful in optimizing folding conditions because it diminishes aggregation.

Synthesis of yttria nanoparticles using NIPAM/AAc copolymer

PurposeThe purpose of this paper is to evaluate the effect of copolymer and starting material concentrations in homogeneous precipitation synthesis of Yttria nanoparticles and red‐emitting nanophosphors Y2O3:Eu3+. N‐isopropylacrylamide and acrylic acid (NIPAM/AAc) and urea are used.Design/methodology/approachTo optimise synthesis condition of Y2O3:Eu3+ nanophosphor NIPAM/AAc copolymer was used as a modifier and the effect of various concentration of yttrium ions, urea and precipitation time on size, morphology and emission spectra were investigated.FindingsUsing NIPAM/AAc copolymer shows significant improvement on size and dispersion of nanoparticles. It is found that yttrium concentration, varying between 0.006 and 0.03 M, has a profound impact on the average size of particles, which systematically increases from 65 to over 165 nm. The rate of precipitation reaction, however, is shown to be independent of yttrium concentration. In contrast, as urea concentration increases from 0.2 to 5 M, the average particle size exhibits a gradual decrease from 183 to 70 nm. At extremely high urea concentration such as 5 M, a significant level of inter‐particle agglomeration is observed.Originality/valueBased on this paper, the authors have successfully prepared some promising nanophosphors. The nanoparticles are studied by X‐ray diffraction, transmission electronic microscopy, zeta sizer, Infra red and photoluminescence spectroscopy.

Purification of high yields of catalytically active lysyl oxidase directly from Escherichia coli cell culture

Lysyl oxidase is a highly insoluble enzyme requiring high concentrations of urea to solubilize. A method to obtain lysyl oxidase in high yields directly from an Escherichia coli culture without the need for refolding of inclusion bodies has been developed using nutrient rich media. pET21b was used to overexpress the lysyl oxidase enzyme and to introduce a C-terminal 6X histidine tag for purification. Lysyl oxidase yields of 10 mg of active and properly folded enzyme per liter of media have been obtained. Purification was achieved via affinity chromatography using a Ni–NTA column. Copper content was found to be 19%. LTQ cofactor formation in LOX is a self-processing event in the presence of copper. LTQ content was determined to be 24% based on reaction with phenylhydrazine to form a phenylhydrazone adduct. Quantification of this adduct was attained using the previously reported extinction coefficient of 15.4 mM −1cm −1. LTQ presence was also verified by redox cycling. Specific enzymatic activity was measured to be 0.31 U/mg, one of the highest activities reported.

Study of conformational changes in glucoamylase ofAspergillus awamori nakazawa in presence of denaturants through CD-spectroscopy

In present study, changes in the pattern or motifs of secondary structures of glucoamylase from Aspergillus awamori nakazawa in the presence of denaturants such as urea, guanidine-HCl (Gdn-HCl), and at different pH, were studied through CD-spectroscopy. It was observed that in native state glucoamylase entirely comprised of β-sheets. CD-spectra of glucoamylase in the presence of 6 M urea lost its spectral regular characteristic; and ellipticity became zero between 207 and 218 nm. With 0.5 M urea concentration glucoamylase showed approximately double negative ellipticity between 207 and 218 nm than the control, which indicates increase in β-sheets and decrease in random coil contents. But higher concentration of urea (⩾6 M) and Gdn-HCl (⩾3 M) completely unfold the enzyme. At lower concentration (0.1 M) of Gdn-HCl negative peak got shifted from 208 to 219 nm to a very sharp peak at 198 nm with lower intensity than the control. It was also observed that glucoamylase posses higher β-sheet in acidic media than in basic media. Glucoamylase remains active in a broad range of pH (3.0–11.0) and maximum activity was observed at pH 4.5. Activity of glucoamylase does not vary too much between pH 5.5 and 9.0. Conformational changes during wide range of pH were supported with its activity coincided.

Identification of p65-Associated Phosphoproteins by Mass Spectrometry after On-Plate Phosphopeptide Enrichment Using Polymer-oxotitanium Films

Stimuli-induced protein phosphorylation plays a vital role in signal transduction and transcriptional activities in eukaryotic cells. This work aims to develop analysis techniques that rapidly detect stimulus-specific intracellular protein phosphorylation and association, with specific emphasis on identifying phosphoproteins associated with p65, a nuclear regulatory factor. The analytical strategy includes immunoprecipitation of the target protein along with its associated proteins, tryptic digestion directly on the antibody beads, on-plate phosphopeptide enrichment for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) to identify phosphopeptides and phosphorylation sites. Enrichment of the phosphopeptides from the tryptic digest occurs on a polymer-oxotitanium-modified gold wafer (Au-P-oxoTi) and consumes as little as 1 microL of digest solution. The Au-P-oxoTi wafers can capture both mono- and multiphosphorylated peptides from model protein digests containing high concentrations of nonphosphopeptides, urea, and salts. When combined with MALDI-MS/MS, the enrichment technique reveals nine phosphopeptides from p65-associated proteins immunoprecipitated from human acute monocytic leukemia (THP-1) cell nuclear extracts. Semiquantitative MALDI-MS shows that the levels of these proteins increase dramatically after treatment with tumor necrosis factor (TNF)-alpha. Overall, these techniques facilitated the identification of five p65-associated proteins, two of which were not previously reported to interact with p65.

Uremic Thrombocytopathy Is not about Urea

Platelet dysfunction in renal failure is attributable to high levels of small, partly dialyzable molecules known as uremic toxins, hence the term "uremic thrombocytopathy." Although a variety of moieties contribute to platelet dysfunction or abnormal interactions between platelets and the vascular wall, urea remains a potential factor. Here, we studied three family members with familial azotemia, a rare autosomal dominant syndrome characterized by high plasma urea resulting from impaired urinary excretion but normal renal function otherwise. Platelet function, assessed in vitro and by traditional bleeding time, was normal in all individuals. Abnormal platelet function in patients with renal failure is not caused by high concentrations of urea.

Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins.

The goal of this article is to gain a better understanding of the denatured state ensemble (DSE) of proteins through an experimental and computational study of their denaturation by urea. Proteins unfold to different extents in urea and the most hydrophobic proteins have the most compact DSE and contain almost as much secondary structure as folded proteins. Proteins that unfold to the greatest extent near pH 7 still contain substantial amounts of secondary structure. At low pH, the DSE expands due to charge-charge interactions and when the net charge per residue is high, most of the secondary structure is disrupted. The proteins in the DSE appear to contain substantial amounts of polyproline II conformation at high urea concentrations. In all cases considered, including staph nuclease, the extent of unfolding by urea can be accounted for using the data and approach developed in the laboratory of Wayne Bolen (Auton et al., Proc Natl Acad Sci 2007; 104:15317-15323).

Effect of osmotic shock and urea on phosphatidylserine scrambling in thrombocyte cell membranes

Blood passing the renal medulla enters a strongly hypertonic environment challenging functional properties and survival of blood cells. In erythrocytes, exposure to hyperosmotic shock stimulates Ca(2+) entry and ceramide formation with subsequent cell membrane scrambling, an effect partially reversed by high concentrations of Cl(-) or urea. Cell membrane scrambling with phosphatidylserine exposure is part of the procoagulant phenotype of platelets. Coagulation in the hypertonic renal medulla would jeopardize blood flow in the vasa recta. The present study thus explored whether hypertonic environment and urea modify phosphatidylserine exposure of human platelets. FACS analysis was employed to estimate cytosolic Ca(2+) activity with Fluo3 fluorescence, ceramide formation, P-selectin, and glycoprotein IIb/IIIa activation with fluorescent antibodies and phosphatidylserine exposure with annexin V-binding. The spontaneous platelet aggregation was measured by impedance aggregometry. Hyperosmotic shock (addition of 500 mM sucrose or 250 mM NaCl) significantly enhanced cytosolic Ca(2+) activity, ceramide formation, phosphatidylserine exposure, platelet degranulation, and aggregability. Addition of 500 mM urea to isotonic saline did not significantly modify cytosolic Ca(2+) activity, ceramide abundance, or annexin V-binding but significantly blunted the respective effects of hypertonic shock following addition of 500 mM sucrose. In isotonic solutions, both ceramide (20 microM) and Ca(2+) ionophore ionomycin (0.5 microM) increased annexin V-binding, effects again significantly blunted by 500 mM urea. Moreover, oxidative stress by addition of 0.5 mM peroxynitrite increased cytosolic Ca(2+) activity and triggered annexin V-binding, effects again blunted in the presence of 500 mM urea. The observations reveal that hyperosmotic shock and oxidative stress trigger a procoagulant platelet phenotype, an effect blunted by the presence of high urea concentrations.

Tailored Titanium Dioxide Nanomaterials: Anatase Nanoparticles and Brookite Nanorods as Highly Active Photocatalysts

High quality brookite TiO2 nanorods have been obtained by the thermal hydrolysis of commercially available aqueous solutions of titanium bis(ammonium lactate) dihydroxide in the presence of high concentrations of urea (≥6.0 M) as an in situ OH− source. Biphasial anatase/brookite mixtures are obtained at lower urea concentrations. The ratios between anatase and brookite can readily be tailored by the control of the urea concentration. The obtained powders have been characterized by X-ray diffraction, Raman spectroscopy, field emission-scanning electron microscopy, high-resolution transmission electron microscopy, UV−vis diffuse reflectance spectra, and nitrogen adsorption. The photocatalytic activity of pure anatase nanoparticles, of anatase/brookite mixtures, and of pure brookite nanorods has been assessed by hydrogen evolution from aqueous methanol solution as well as by the degradation of dichloroacetic acid (DCA) in aqueous solution. The results indicate that the photocatalytic hydrogen evolution activ...

Human serum albumin binding ibuprofen: A 3D description of the unfolding pathway in urea

Small angle X-ray scattering (SAXS) technique, supported by light scattering measurements and spectroscopic data (circular dichroism and fluorescence) allowed us to restore the 3D structure at low resolution of defatted human serum albumin (HSA) in interaction with ibuprofen. The data were carried out on a set of HSA solutions with urea concentrations between 0.00 and 9.00 M. The Singular Value Decomposition method, applied to the complete SAXS data set allowed us to distinguish three different states in solution. In particular a native conformation N (at 0.00 M urea), an intermediate I1 (at 6.05 M urea) and an unfolded structure U (at 9.00 M urea) were recognized. The low-resolution structures of these states were obtained by exploiting both ab initio and rigid body fitting methods. In particular, for the protein without denaturant, a conformation recently described (Leggio et al., PCCP, 2008, 10, 6741–6750), very similar to the crystallographic heart shape, with only a slight reciprocal movement of the three domains, was confirmed. The I1 structure was instead characterized by only a closed domain (domain III) and finally, the recovered structure of the U state revealed the characteristic feature of a completely open state. A direct comparison with the free HSA pointed out that the presence of the ibuprofen provokes a shift of the equilibrium towards higher urea concentrations without changing the unfolding sequence. The work represents a type of analysis which could be exploited in future investigations on proteins in solution, in the binding of drugs or endogenous compounds and in the pharmacokinetic properties as well as in the study of allosteric effects, cooperation or anticooperation mechanisms.

Functional implications of the three-dimensional architecture of the rat renal inner medulla

A new, region-based mathematical model of the urine concentrating mechanism of the rat renal inner medulla (IM) was used to investigate the significance of transport and structural properties revealed in recent studies that employed immunohistochemical methods combined with three-dimensional computerized reconstruction. The model simulates preferential interactions among tubules and vessels by representing two concentric regions. The inner region, which represents a collecting duct (CD) cluster, contains CDs, some ascending thin limbs (ATLs), and some ascending vasa recta; the outer region, which represents the intercluster region, contains descending thin limbs, descending vasa recta, remaining ATLs, and additional ascending vasa recta. In the upper portion of the IM, the model predicts that interstitial Na(+) and urea concentrations (and osmolality) in the CD clusters differ significantly from those in the intercluster regions: model calculations predict that those CD clusters have higher urea concentrations than the intercluster regions, a finding that is consistent with a concentrating mechanism that depends principally on the mixing of NaCl from ATLs and urea from CDs. In the lower IM, the model predicts that limited or nearly zero water permeability in descending thin limb segments will increase concentrating effectiveness by increasing the rate of solute-free water absorption. The model predicts that high urea permeabilities in the upper portions of ATLs and increased contact areas of longest loop bends with CDs both modestly increase concentrating capability. A surprising finding is that the concentrating capability of this region-based model falls short of the capability of a model IM that has radially homogeneous interstitial fluid at each level but is otherwise analogous to the region-based model.

Ceramide in Suicidal Death of Erythrocytes

The suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage, membrane blebbing and cell membrane phospholipid scrambling resulting in phosphatidylserine exposure at the cell surface. Eryptosis is stimulated in a wide variety of diseases including sepsis, haemolytic uremic syndrome, malaria, sickle-cell anemia, beta-thalassemia, glucose-6-phosphate dehydrogenase (G6PD)-deficiency, phosphate depletion, iron deficiency and Wilson’s disease. Moreover, eryptosis is elicited by osmotic shock, oxidative stress, energy depletion as well as a wide variety of endogenous mediators and xenobiotics. Excessive eryptosis is observed in erythrocytes lacking the cGMP-dependent protein kinase type I (cGKI) or the AMP-activated protein kinase AMPK. Inhibitors of eryptosis include erythropoietin, nitric oxide NO, catecholamines and high concentrations of urea. Eryptosis-triggering diseases and chemicals are partially effective by stimulating the formation of ceramide, which in turn fosters cell membrane scrambling. Accordingly, ceramide-induced eryptosis participates in the pathophysiology of several diseases and contributes to the effects of a large number of xenobiotics. The mechanisms underlying ceramide formation in erythrocytes are, however, still ill defined. In case of osmotic cell shrinkage, ceramide formation is apparently due to activation of phospholipase 2, leading to formation of platelet activating factor PAF and PAF-dependent stimulation of ceramide formation, which possibly involves acid sphingomyelinase. Additional experiments are needed to conclusively define the ceramide-generating enzyme and the ceramide-dependent cellular events eventually leading to suicidal erythrocyte death.

Detection of biomolecules from solutions with high concentration of salts using probe electrospray and nano-electrospray ionization mass spectrometry

Probe electrospray ionization (PESI) is a recently developed ionization technique which uses a solid needle or wire as sampling probe and ESI emitter instead of capillary. PESI is free from the clogging problem and it has high tolerance to salts and urea. We present herein a comparative study of the probe electrospray ionization (PESI) and nano-electrospray ionization (nano-ESI) for the measurement of biomolecules in the sample solutions with high concentrations of salts and urea. Our results show that PESI could provide equivalent ionization performance with nano-ESI, and in certain cases it could be superior to nano-ESI for the samples with high concentration (>100 mM) of salts and urea. Therefore, PESI can be useful for the direct analysis of cell/tissue extracts and protein digestion without or reduced procedures in sample purification.

Catalytic oxygenation of phenols by arthropod hemocyanin, an oxygen carrier protein, from Portunus trituberculatus

The hexamer (Pt-6Hc) of swimming crab Portunus trituberculatus hemocyanin (Pt-Hc) and one of its monomeric subunits (Pt-1Hc) have been purified and converted to an efficient phenol monooxygenase (phenolase) by treatment with urea. To explore the intrinsic chemical reactivity of the dicopper center of Pt-Hc, the spectroscopic features and phenol monooxygenase (phenolase) activity of the isolated proteins have been examined in detail. The oxy-forms involving a (mu-eta(2):eta(2)-peroxo)dicopper(II) species (oxy-Hc) of Pt-6Hc and Pt-1Hc are relatively stable in 0.5 M borate buffer (pH 9.0) even in the presence of a high concentration of urea (3 M) at 25 degrees C. The catalytic activity of monomeric Pt-1Hc in the oxygenation reaction (multi-turnover reaction) of 4-methylphenol to 4-methyl-1,2-dihydroxybenzene (4-methylcatechol) was higher than that of hexameric Pt-6Hc, and its catalytic activity was further accelerated by the addition of urea. Kinetic deuterium isotope effect analysis and Hammett analysis using a series of p-substituted phenol derivatives under anaerobic conditions (single-turnover reaction) have indicated that the monooxygenation reaction of phenols to catechols by the peroxo species of oxy-Hc proceeds via electrophilic aromatic substitution mechanism as in the case of tyrosinase (dinuclear copper monooxygenase). The effect of urea on the redox functions of oxy-Hc is discussed on the basis of spectroscopic analysis and reactivity studies.

PH-dependent urea-induced unfolding of stem bromelain: Unusual stability against urea at neutral pH

Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a function of pH using circular dichroism and fluorescence emission spectroscopy. Urea-induced denaturation studies at pH 4.5 showed that SB unfolds through a two-state mechanism and yields DeltaG (free energy difference between the fully folded and unfolded forms) of approximately 5.0 kcal/mol and C(m) (midpoint of the unfolding transition) of approximately 6.5 M at 25 degrees C. Very high concentration of urea (9.5 M) provides unusual stability to the protein with no more structural loss and transition to a completely unfolded state.

Molecular coin slots for urea

Membrane-bound protein channels that allow only urea to pass through are vital to the kidney's ability to conserve water. Crystal structures show that the channels select urea molecules by passing them through thin slots. The osmotic gradient necessary to facilitate water resorption in the kidney is maintained by high concentrations of urea. The free diffusion of urea across the cell membrane is slow, so highly conserved urea transporters have evolved to facilitate selective permeation of urea. Here Levin et al. report the X-ray crystal structure of a bacterial homologue of the urea transporter, from Desulfovibrio vulgaris. The transporter contains a pore with a selectivity filter that can accommodate multiple dehydrated urea molecules in single file, revealing that the transporter operates by a channel-like mechanism.

Terminal α2-6 Linked Sialic Acid Expression On VWF Specifically Enhances Proteolysis by ADAMTS13.

Abstract 30VWF is a large plasma sialoglycoprotein that mediates platelet tethering at sites of vascular injury. VWF function is dependent upon VWF multimeric composition, which is regulated in plasma by ADAMTS13. ABO(H) blood group determinants expressed on VWF N-linked glycans significantly influence susceptibility to ADAMTS13 proteolysis. In this study, we investigated whether terminal sialic acid residues expressed on the N- and O-linked glycans of VWF may also regulate proteolysis by ADAMTS13. VWF was initially purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF sialylation was modified using specific exoglycosidases and quantified by lectin-binding ELISA. The rate of glycosidase-treated VWF proteolysis by ADAMTS13 was determined by incubation with recombinant ADAMTS13 and subsequent measurement of residual VWF collagen binding activity.Complete VWF deglycosylation has been shown to enhance the rate of proteolysis by ADAMTS13. In contrast, enzymatic desialylation of VWF by α2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired the rate of ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to only 50±14% by ADAMTS13, compared to 11±7% for untreated VWF (p<0.01) at the same time point. Despite this, Neu-VWF exhibited increased susceptibility to proteases other than ADAMTS13 (trypsin, chymotrypsin and cathepsin B; all p<0.05). VWF sialylation is therefore a specific enhancer of ADAMTS13-mediated proteolysis. Consequently, quantification and molecular distribution of VWF sialylation was examined by sequential digestion and HPLC analysis. Total sialic acid expression on pdVWF was 167nmol/mg, of which the majority (133.4nmol/mg or 80.1%) was present on its N-linked glycan chains. Interestingly, despite the resistance to ADAMTS13 proteolysis observed upon complete desialylation, digestion of pdVWF with α2-3 neuraminidase to remove predominantly O-linked sialic acid did not influence the rate of ADAMTS13 proteolysis.Previous studies have demonstrated that VWF expressing different blood groups exhibit altered rates of proteolysis by ADAMTS13 (O ≥ B > A ≥ AB). Since α2-6 linked sialic acid and ABO(H) determinants are both expressed as terminal antigens on VWF N-linked glycans, the effect of desialylation upon blood group-specific VWF proteolysis by ADAMTS13 was determined. As expected, untreated group O VWF was cleaved significantly faster than group AB-VWF (p<0.05). However, the ability of ABO blood group to regulate ADAMTS13 proteolysis was completely ablated upon VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. This indicates that VWF sialylation constitutes a more important determinant of susceptibility to ADAMTS13 proteolysis than ABO(H) expression.Sialic acid can mediate protein-protein interactions through either conformational and/or charge-mediated mechanisms. Despite this, sodium metaperiodate treatment of pdVWF to remove sialic acid anionic charge did not influence the rate of proteolysis by ADAMTS13. In contrast, the ability of sialic acid to specifically enhance ADAMTS13 proteolysis of VWF was significantly attenuated at high urea concentrations (≥2M), supporting the hypothesis that VWF sialylation enhances proteolysis by ADAMTS13 by promoting a ADAMTS13-specific permissive conformation.These novel data demonstrate that although sialic acid protects VWF against proteolysis by serine and cysteine proteases, it also specifically enhances susceptibility to proteolysis by ADAMTS13. Moreover, the magnitude of this sialic acid-specific effect on VWF proteolysis by ADAMTS13 is more marked than that attributable to N-linked ABO(H) blood group antigen expression. Therefore, quantitative variation in VWF sialylation represents a key regulator of VWF multimeric composition, and as such, is likely to be of clear patho-physiological significance. Disclosures:No relevant conflicts of interest to declare.