Regional variation in accessibility of automated external defibrillators in British Columbia.

OBJECTIVES: Bystander-applied Automated External Defibrillators (AED) improve outcomes for out-of-hospital cardiac arrest. AED placement is often driven by private enterprise or non-for-profit agencies, which may result in inequitable access. We sought to compare AED availability between four regions in British Columbia (BC). METHODS: We identified AEDs (confirmed to be operational) and emergency medical system (EMS)-treated out-of-hospital cardiac arrests (OHCA) from provincial registries. We compared AED availability between BC's four most populous regions. The primary outcome was the total regional weekly accessible AED-hours per 100,000 population. We also examined: AEDs per 100,000 population and per km2, the ratio of AEDs to OHCA, and the distance from each OHCA to the closest AED. RESULTS: From provincial registries, we included 879 AEDs from BC's four most populous regions, where 9333 EMS-treated OHCA occurred over a 5-year period. The most common AED location types were stores, public community centres, and office buildings. Ten percent of AEDs were accessible for all hours. Weekly accessible AED-hours/100,000 population in the four regions were: 3845, 1734, 1594, and 1299. AEDs/100,000 population ranged from 22 to 48, and AEDs/km2 ranged from 0.0048 to 0.20. The number of OHCAs per AED per year ranged from 1.1 to 2.8. The median OHCA-to-closest AED distance ranged from 503 (IQR 244, 947) to 925 (IQR 455, 1501) metres. The regional mean accessibility of individual AEDs ranged between 59 and 79 h per week. CONCLUSION: BC's four most populous regions demonstrate substantial variability in AED accessibility. Further benefit could be derived from AEDs if placed in locations accessible all hours. Our data may encourage community planning efforts to use data-based strategies to systematically place AEDs in optimal locations with strategies to maximize accessibility.

ABSTRAIT: OBJECTIFS: Les défibrillateurs externes automatisés (DEA) appliqués par les témoins améliorent les résultats en cas d'arrêt cardiaque hors hôpital. Le placement des DEA est souvent dirigé par une entreprise privée ou des organismes sans but lucratif, ce qui peut entraîner un accès inéquitable. Nous avons cherché à comparer la disponibilité des DEA entre quatre régions de la Colombie-Britannique. MéTHODES: Nous avons identifié les DEA (dont la mise en service a été confirmée) et les SMU (système médical d'urgence) traités par arrêt cardiaque hors hôpital (AHC) dans les registres provinciaux. Nous avons comparé la disponibilité des DEA entre les quatre régions les plus peuplées de la Colombie-Britannique. Le résultat principal était le nombre total d'heures de DEA accessibles hebdomadaires par région pour 100000 habitants. Nous avons également examiné : les DEA par 100000 habitants et par km2, le rapport entre les DEA et l'AHCA, et la distance entre chaque AHCA et le DEA le plus proche. RéSULTATS: À partir des registres provinciaux, nous avons inclus 879 DEA des quatre régions les plus peuplées de la Colombie-Britannique, où 9333 OHCA traités par les SMU se sont produits sur une période de 5 ans. Les types de DEA les plus courants étaient les magasins, les centres communautaires publics et les immeubles de bureaux. Dix pour cent des DEA étaient accessibles toutes les heures. La population hebdomadaire accessible en heures AED/100000 habitants dans les quatre régions était de 3845, 1734, 1594 et 1299. Le nombre de DEA/100 000 habitants variait de 22 à 48, et le nombre de DEA/km2 variait de 0,018 à 0,018. Le nombre de CASO par DEA par année variait de 1,1 à 2,8. La distance médiane entre le DEA OHCA et le DEA le plus proche variait de 503 mètres (IQR 244, 947) à 925 mètres (IQR 455, 1501). L'accessibilité moyenne régionale des DEA individuels variait entre 59 et 79 heures par semaine. CONCLUSION: Les quatre régions les plus populeuses de la Colombie-Britannique présentent une variabilité importante de l'accessibilité aux DEA. D'autres avantages pourraient découler des DEA s'ils sont placés dans des endroits accessibles toutes les heures. Nos données peuvent encourager les efforts de planification communautaire à utiliser des stratégies fondées sur les données pour placer systématiquement les DEA dans des endroits optimaux avec des stratégies pour maximiser l'accessibilité.

Utilization and cost-effectiveness of school and community center AED deployment models in Canadian cities.

BACKGROUND: The optimal locations and cost-effectiveness of placing automated external defibrillators (AEDs) for out-of-hospital cardiac arrest (OHCAs) in urban residential neighbourhoods are unclear. METHODS: We used prospectively collected data from 2016 to 2018 from the British Columbia OHCA Registry to examine the utilization and cost-effectiveness of hypothetical AED deployment in municipalities with a population of over 100 000. We geo-plotted OHCA events using seven hypothetical deployment models where AEDs were placed at the exteriors of public schools and community centers and fetched by bystanders. We calculated the "radius of effectiveness" around each AED within which it could be retrieved and applied to an individual prior to EMS arrival, comparing automobile and pedestrian-based retrieval modes. For each deployment model, we estimated the number of OHCAs within the "radius of effectiveness". RESULTS: We included 4017 OHCAs from ten urban municipalities. The estimated radius of effectiveness around each AED was 625 m for automobile and 240 m for pedestrian retrieval. With AEDs placed outside each school and community center, 2567 (64%) and 605 (15%) of OHCAs fell within the radii of effectiveness for automobile and pedestrian retrieval, respectively. For each AED, there was an average of 1.20-2.66 and 0.25-0.61 in-range OHCAs per year for automobile retrieval and pedestrian retrieval, respectively, depending on the deployment model. All of our proposed surpassed the cost-effectiveness threshold of 0.125 OHCA/AED/year provided > 5.3-11.6% in-range AEDs were brought-to-scene. CONCLUSIONS: The systematic deployment of AEDs at schools and community centers in urban neighbourhoods may result in increased application and be a cost-effective public health intervention.

Incremental gains in response time with varying base location types for drone-delivered automated external defibrillators.

INTRODUCTION: Drone-delivered automated external defibrillators (AEDs) may reduce delays to defibrillation for out-of-hospital cardiac arrests (OHCAs). We sought to determine how integration of drones and selection of drone bases between emergency service stations (i.e., paramedic, fire, police) would affect 9-1-1 call-to-arrival intervals. METHODS: We identified all treated OHCAs in southern Vancouver Island, British Columbia, Canada from Jan. 2014 to Dec. 2020. We developed mathematical models to select 1-5 optimal drone base locations from each of: paramedic stations, fire stations, police stations, or an unrestricted grid-based set of points to minimize drone travel time to OHCAs. We evaluated models on the estimated first response interval assuming that drones were integrated with existing OHCA response. We compared median response intervals with historical response, as well as across drone base locations. RESULTS: A total of 1610 OHCAs were included in the study with a historical median response interval of 6.4 minutes (IQR 5.0-8.6). All drone-integrated response systems significantly reduced the median response interval to 4.2-5.4 minutes (all P < 0.001), with grid-based stations using 5 drones resulting in the lowest response interval (4.2 minutes). Median response times between drone base location types differed by 6-16 seconds, all comparisons of which were statistically significant (all P < 0.02). CONCLUSION: Integrating drone-delivered AEDs into OHCA response may reduce first response intervals, even with a small quantity of drones. Implementing drone response with only one emergency service resulted in similar response metrics regardless of the emergency service hosting the drone base and was competitive with unrestricted drone base locations.

Preparation of heparin/heparan sulfate oligosaccharides with internal N-unsubstituted glucosamine residues for functional studies.

The rare N-unsubstituted glucosamine (GlcNH (3)(+)) residues in heparan sulfate (HS) have important biological and pathophysiological roles. However, it is difficult to prepare naturally-occuring, GlcNH(3)(+)-containing oligosaccharides from HS because of their low abundance, as well as the inherent problems in both excising and identifying them. Therefore, the ability to chemically generate a series of structurally-defined oligosaccharides containing GlcNH(3)(+) residues would greatly contribute to investigating their natural role in HS. In this study, a series of heparin/HS oligosaccharides, from dp6 up to dp16 in length that possess internal GlcNH(3)(+) residues were prepared by a combination of chemical modification and heparinase I digestion. Purification and structural analysis of the major species derived from the octa- to dodeca-saccharide size fractions indicated the introduction of between 1 and 3 internal GlcNH(3)(+) residues per oligosaccharide. In addition, a GlcNH(3)(+) residue was selectively introduced into an internal position in a tetrasaccharide species by direct chemical modification. This selectivity has potential as an alternative procedure for preparing internally-modified oligosaccharides of various lengths. The utility of such oligosaccharides was demonstrated by a comparison of the binding of three different tetrasaccharide species containing 0, 1 and 2 free amino groups to the NK1 truncated variant of hepatocyte growth factor/scatter factor.

Decrease in emergency medical services utilization during early stages of the COVID-19 pandemic in British Columbia.

OBJECTIVE: To date in the COVID-19 pandemic, there has been a decrease in patients accessing emergency health services, (EHS) but research has been conducted in areas with a very high incidence of COVID-19. In an area with a low COVID-19 incidence, we estimate changes in EHS use. METHODS: We compared EHS encounters in British Columbia from March 15 (the date of school and business closures) to May 15, 2020, when compared to the same period in 2019. We categorized EHS encounters into 18 presenting complaints and prespecified critical care complaints including major trauma, cardiac arrest, stroke, and ST-elevation myocardial infarction. We analyzed by descriptive methods. RESULTS: Comparing 2019 to 2020, total EHS encounters decreased from 83,925 (incidence rate 834 per 100,000 person-months) to 71,611 (incidence rate 701 per 100,000 person-months) for a decrease of 133 per 100,000 person-months (95% CI 126-141). The top 18 codes had a significant decrease in every category except respiratory and anxiety. Encounters for critically ill patients decreased significantly overall from 3019 to 2753 (incidence rate difference 3.1 per 100,000 person-months, 95% CI 1.6-4.5), including stroke, trauma, and STEMI, but the incidence of OHCA appeared stable. CONCLUSION: In a single province with a low incidence of COVID-19, there was a 15% reduction in overall EHS use and a 9% reduction in critical illness. EHS planners will need to match patient need with available resources.

RéSUMé: OBJECTIFS: Jusqu'à présent dans la pandémie de Covid-19, il y a eu une diminution du nombre de patients ayant accès aux services de santé d'urgence, mais des recherches ont été menées dans des zones à très forte incidence de Covid-19. Dans une zone à faible incidence de Covid-19, nous estimons les changements dans l'utilisation des services de santé d'urgence. MéTHODES: Nous avons comparé les cas des services de santé d'urgence en Colombie-Britannique du 15 mars (date de fermeture des écoles et des entreprises) au 15 mai 2020, par rapport à la même période en 2019. Nous avons classé les cas des services de santé d'urgence en 18 plaintes de présentation et des plaintes de soins intensifs pré-spécifiées, y compris un traumatisme majeur, un arrêt cardiaque, un accident vasculaire cérébral et un infarctus du myocarde avec élévation du segment ST. Nous avons analysé par des méthodes descriptives. RéSULTATS : En comparant 2019 à 2020, le nombre total des cas des services de santé d'urgence est passé de 83 925 (taux d'incidence de 834 pour 100 000 personnes-mois) à 71 611 (taux d'incidence de 701 pour 100 000 personnes-mois) pour une diminution de 133 pour 100 000 personnes-mois (IC à 95 % 126 à 141). Les 18 codes principaux ont connu une diminution significative dans toutes les catégories, sauf respiratoire et anxiété. Les cas chez les patients gravement malades ont globalement diminué de manière significative de 3 019 à 2 753 (différence de taux d'incidence de 3,1 pour 100 000 personnes-mois, IC à 95 % de 1,6 à 4,5), y compris les accidents vasculaires cérébraux, les traumatismes et les STEMI, mais l'incidence des arrêts cardiaque hors hôpital semble stable. CONCLUSIONS: Dans une seule province avec une faible incidence de Covid-19, il y a eu une réduction de 15 % de l'utilisation globale des services de santé d'urgence et une réduction de 9 % des maladies graves. Les organisateurs des services de santé d'urgence devront faire correspondre les besoins des patients avec les ressources disponibles.

Interaction of human ß-defensin 2 (HBD2) with glycosaminoglycans.

Human ß-defensin 2 (HBD2) is a member of the defensin family of antimicrobial peptides that plays important roles in the innate and adaptive immune system of both vertebrates and invertebrates. In addition to their direct bactericidal action, defensins are also involved in chemotaxis and Toll-like receptor activation. In analogy to chemokine/glycosaminoglycan (GAG) interactions, GAG-defensin complexes are likely to play an important role in chemotaxis and in presenting defensins to their receptors. Using a gel mobility shift assay, we found that HBD2 bound to a range of GAGs including heparin/heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate. We used NMR spectroscopy of (15)N-labeled HBD2 to map the binding sites for two GAG model compounds, a heparin/HS pentasaccharide (fondaparinux sodium; FX) and enzymatically prepared DS hexasaccharide (DSdp6). We identified a number of basic amino acids that form a common ligand binding site, which indicated that these interactions are predominantly electrostatic. The dissociation constant of the [DSdp6-HBD2] complex was determined by NMR spectroscopy to be 5 ± 5 µM. Binding of FX could not be quantified because of slow exchange on the NMR chemical shift time scale. FX was found to induce HBD2 dimerization as evidenced by the analysis of diffusion coefficients, (15)N relaxation, and nESI-MS measurements. The formation of FX-bridged HBD2 dimers exhibited features of a cooperative binding mechanism. In contrast, the complex with DSdp6 was found to be mostly monomeric.

Characterization and binding activity of the chondroitin/dermatan sulfate chain from Endocan, a soluble endothelial proteoglycan.

Endocan is a recently identified soluble chondroitin/dermatan sulfate (CS/DS) proteoglycan. Synthesized by endothelial cells, it has been found to be over-expressed in the vasculature surrounding a number of tumors, and by promoting growth factor mitogenic activities, hepatocyte growth factor/scatter factor (HGF/SF) in particular, it supports cellular proliferation. In this work, we characterized the glycosaminoglycan (GAG) chain of Endocan, purified either from the naturally producing human umbilical vein endothelial cells (HUVEC) or from a recombinant over-expression system in human embryonic kidney cells (HEK). Compositional analysis using different chondroitinases as well as nuclear magnetic resonance studies revealed that the GAG chains from both sources share many characteristics, with the exception of size (15 and 40 kDa, respectively, for HUVEC and HEK-293 cells). The DS-specific, IdoA-containing disaccharides contribute 30% of the chain (15% of which are 2-O-sulfated) and are mostly clustered in tetra- (35%), hexa- (12%), and octa- (5%) saccharide domains. Highly sulfated D, E, and B disaccharide units (HexA2S-GalNAc6S, HexA-GalNAc4S6S, and HexA2S-GalNAc4S) were also detected in significant amounts in both chains and may account for the HGF/SF-binding activity of the CS/DS. This work establishes that HEK-293 cells can be engineered to provide a valuable source of Endocan with authentic CS/DS chains, enabling the purification of sufficient amounts for structural and/or binding analysis and providing a possible model of Endocan CS/DS chain organization.

Lysine and arginine side chains in glycosaminoglycan-protein complexes investigated by NMR, cross-linking, and mass spectrometry: a case study of the factor H-heparin interaction.

We have used the interaction between module 7 of complement factor H (CFH approximately 7) and a fully sulfated heparin tetrasaccharide to exemplify a new approach for studying contributions of basic side chains to the formation of glycosaminoglycan (GAG)-protein complexes. We first employed HISQC and H(2)CN NMR experiments to monitor the side-chain resonances of lysines and arginines in (15)N, (13)C-labeled protein during titrations with a fully sulfated heparin tetrasaccharide under physiological conditions. Under identical conditions and using (15)N-labeled protein, we then cross-linked tetrasaccharide to CFH approximately 7 and confirmed the 1:1 stoichiometry by FT-ICR-MS. We subsequently characterized this covalent protein-GAG conjugate by NMR and further MS techniques. MALDI-TOF MS identified protein fragments obtained via trypsin digestion or chemical fragmentation, yielding information concerning the site of GAG attachment. Combining MS and NMR data allowed us to identify the side chain of K405 as the point of attachment of the cross-linked heparin oligosaccharide to CFH approximately 7. On the basis of the analysis of NMR and MS data of the noncovalent and cross-linked CFH approximately 7-tetrasaccharide complexes, we conclude that the K446 side chain is not essential for binding the tetrasaccharide, despite the large chemical shift perturbations of its backbone amide (15)N and (1)H resonances during titrations. We show that R444 provides the most important charge-charge interaction within a C-terminal heparin-binding subsite of CFH approximately 7 whereas side chains of R404, K405, and K388 are the predominant contributors to an N-terminal binding subsite located in the immediate vicinity of residue 402, which is implicated in age-related macular degeneration (AMD).

Residual dipolar coupling investigation of a heparin tetrasaccharide confirms the limited effect of flexibility of the iduronic acid on the molecular shape of heparin.

The solution conformation of a fully sulfated heparin-derived tetrasaccharide, I, was studied in the presence of a 4-fold excess of Ca(2+). Proton-proton and proton-carbon residual dipolar couplings (RDCs) were measured in a neutral aligning medium. The order parameters of two rigid hexosamine rings of I were determined separately using singular value decomposition and ab initio structures of disaccharide fragments of I. The order parameters were very similar implying that a common order tensor can be used to analyze the structure of I. Using one order tensor, RDCs of both hexosamine rings were used as restraints in molecular dynamics simulations. RDCs of the inner iduronic acid were calculated for every point of the molecular dynamics trajectory. The fitting of the calculated RDCs of the two forms of the iduronic acid to the experimental values yielded a population of (1)C(4) and (2)S(o) conformers of iduronic acid that agreed well with the analysis based on proton-proton scalar coupling constants. The glycosidic linkage torsion angles in RDC-restrained molecular dynamics (MD) structures of I are consistent with the interglycosidic three-bond proton-carbon coupling constants. These structures also show that the shape of heparin is not affected dramatically by the conformational flexibility of the iduronic acid ring. This is in line with conclusions of previous studies based on MD simulations and the analysis of (1)H-(1)H NOEs. Our work therefore demonstrates the effectiveness of RDCs in the conformational analysis of glycosaminoglycans.

A simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples.

Sulfated glycosaminoglycans regulate the biological functions of a wide variety of proteins, primarily through high affinity interactions mediated by specific sugar sequences or patterns/densities of sulfation. Disaccharide analysis of such glycosaminoglycans yields important diagnostic and comparative structural information on sulfate patterning. When applied to specific oligosaccharides it can also make a vital contribution to sequence elucidation. Standard UV detection of lyase-generated disaccharides resolved by HPLC can lack sufficient sensitivity and be compromised by contaminating UV signals, when dealing with scarce tissue- or cell culture-derived material. Various methods exist for improved detection, but usually involve additional HPLC hardware and often necessitate different procedures for analyzing different glycosaminoglycans. We describe a simple procedure, requiring only standard HPLC instrumentation, involving prederivatization of disaccharides with 2-aminoacridone with no cleanup of samples, followed by a separation by reverse-phase HPLC that is sensitive to as little as approximately 100 pg (approximately 10(-13) mol) of an individual disaccharide, thereby allowing analyses of >10 ng of total glycosaminoglycan. Importantly, separate analysis of both HS/heparin and CS/DS species within a mixed glycosaminoglycan pool can be performed using the same procedure on a single column. We demonstrate its applicability in dealing with small quantities of material derived from rat liver (where we demonstrate a high abundance of the unusual CS-E species within the CS/DS pool) and MDCK cells (which revealed a HS species of relatively low N-sulfation, but high O-sulfation). This simplified method should find a widespread utility for analyzing glycosaminoglycans from limited animal and cell culture samples.

Investigating antimalarial drug interactions of emetine dihydrochloride hydrate using CalcuSyn-based interactivity calculations.

The widespread introduction of artemisinin-based combination therapy has contributed to recent reductions in malaria mortality. Combination therapies have a range of advantages, including synergism, toxicity reduction, and delaying the onset of resistance acquisition. Unfortunately, antimalarial combination therapy is limited by the depleting repertoire of effective drugs with distinct target pathways. To fast-track antimalarial drug discovery, we have previously employed drug-repositioning to identify the anti-amoebic drug, emetine dihydrochloride hydrate, as a potential candidate for repositioned use against malaria. Despite its 1000-fold increase in in vitro antimalarial potency (ED50 47 nM) compared with its anti-amoebic potency (ED50 26-32 uM), practical use of the compound has been limited by dose-dependent toxicity (emesis and cardiotoxicity). Identification of a synergistic partner drug would present an opportunity for dose-reduction, thus increasing the therapeutic window. The lack of reliable and standardised methodology to enable the in vitro definition of synergistic potential for antimalarials is a major drawback. Here we use isobologram and combination-index data generated by CalcuSyn software analyses (Biosoft v2.1) to define drug interactivity in an objective, automated manner. The method, based on the median effect principle proposed by Chou and Talalay, was initially validated for antimalarial application using the known synergistic combination (atovaquone-proguanil). The combination was used to further understand the relationship between SYBR Green viability and cytocidal versus cytostatic effects of drugs at higher levels of inhibition. We report here the use of the optimised Chou Talalay method to define synergistic antimalarial drug interactivity between emetine dihydrochloride hydrate and atovaquone. The novel findings present a potential route to harness the nanomolar antimalarial efficacy of this affordable natural product.

Quantitative and qualitative alterations of heparan sulfate in fibrogenic liver diseases and hepatocellular cancer.

Heparan sulfate (HS), due to its ability to interact with a multitude of HS-binding factors, is involved in a variety of physiological and pathological processes. Remarkably diverse fine structure of HS, shaped by non-exhaustive enzymatic modifications, influences the interaction of HS with its partners. Here we characterized the HS profile of normal human and rat liver, as well as alterations of HS related to liver fibrogenesis and carcinogenesis, by using sulfation-specific antibodies. The HS immunopattern was compared with the immunolocalization of selected HS proteoglycans. HS samples from normal liver and hepatocellular carcinoma (HCC) were subjected to disaccharide analysis. Expression changes of nine HS-modifying enzymes in human fibrogenic diseases and HCC were measured by quantitative RT-PCR. Increased abundance and altered immunolocalization of HS was paralleled by elevated mRNA levels of HS-modifying enzymes in the diseased liver. The strong immunoreactivity of the normal liver for 3-O-sulfated epitope further increased with disease, along with upregulation of 3-OST-1. Modest 6-O-undersulfation of HCC HS is probably explained by Sulf overexpression. Our results may prompt further investigation of the role of highly 3-O-sulfated and partially 6-O-desulfated HS in pathological processes such as hepatitis virus entry and aberrant growth factor signaling in fibrogenic liver diseases and HCC.

The binding properties of minimal oligosaccharides reveal a common heparan sulfate/dermatan sulfate-binding site in hepatocyte growth factor/scatter factor that can accommodate a wide variety of sulfation patterns.

Heparan sulfate (HS)/heparin and dermatan sulfate (DS) both bind with high affinity to hepatocyte growth factor/scatter factor (HGF/SF) and function as necessary co-factors in vitro. How both these two structurally distinct glycosaminoglycans (GAGs) are recognized has remained unclear. We have now reconciled this issue using a panel of minimal tri- and tetrasaccharide sequences of variable but well defined sulfation patterns in combination with further development of the gel mobility shift assay to allow simultaneous comparisons of relative protein affinities/selectivities for different oligosaccharides. From this approach it would seem that a minimum binding sequence is a disulfated trisaccharide comprised of an internal iduronate flanked by monosulfated hexosamine residues and that additional sulfation further enhances affinity. However, the similarity in recognition of HS/heparin and DS seems to arise primarily from a lack of any apparent positional requirement for sulfation. Thus, isomers of HS/heparin tetrasaccharides containing only two sulfates irrespective of whether they are purely N-, 2-O-, or 6-O-sulfates bind with equivalent apparent affinity as a disulfated DS tetrasaccharide. In addition, the NMR chemical shifts induced in NK1 (the truncated variant of HGF/SF comprised of the N-terminal and first Kringle domains) by titration with either heparin or DS oligosaccharides strongly indicate that both bind to essentially the same site. Together, these observations reveal an unexpected degree of flexibility in the GAG-HGF/SF interface, allowing a single binding site in the protein to accommodate iduronate-containing sequences of variable sulfation pattern and/or density from different GAGs.

Interactions of hepatocyte growth factor/scatter factor with various glycosaminoglycans reveal an important interplay between the presence of iduronate and sulfate density.

Hepatocyte growth factor/scatter factor (HGF/SF) has a cofactor requirement for heparan sulfate (HS) and dermatan sulfate (DS) in the optimal activation of its signaling receptor MET. However, these two glycosaminoglycans (GAGs) have different sugar backbones and sulfation patterns, with only the presence of iduronate in common. The structural basis for GAG recognition and activation is thus very unclear. We have clarified this by testing a wide array of natural and modified GAGs for both protein binding and activation. Comparisons between Ascidia nigra (2,6-O-sulfated) and mammalian (mainly 4-O-sulfated) DS species, as well as between a panel of specifically desulfated heparins, revealed that no specific sulfate isomer, in either GAG, is vital for interaction and activity. Moreover, different GAGs of similar sulfate density had comparable properties, although affinity and potency notably increase with increasing sulfate density. The weaker interaction with CS-E, compared with DS, shows that GlcA-containing polymers can bind, if highly sulfated, but emphasizes the importance of the flexible IdoA ring. Our data indicate that the preferred binding sites in DS in vivo will be comprised of disulfated, IdoA(2S)-containing motifs. In HS, clustering of N-/2-O-/6-O-sulfation in S-domains will lead to strong reactivity, although binding can also be mediated by the transition zones where sulfates are mainly at the N- and 6-O- positions. GAG recognition of HGF/SF thus appears to be primarily driven by electrostatic interactions and exhibits an interesting interplay between requirements for iduronate and sulfate density that may reflect in part a preference for particular sugar chain conformations.

Novel heparin/heparan sulfate mimics as inhibitors of HGF/SF-induced MET activation.

The synthesis of simple, non-sugar glycosaminoglycan (GAG) mimics has been achieved and the analogues evaluated for their ability to inhibit the activation of the MET receptor by hepatocyte growth factor/scatter factor (HGF/SF).

Structure shows that a glycosaminoglycan and protein recognition site in factor H is perturbed by age-related macular degeneration-linked single nucleotide polymorphism.

A common single nucleotide polymorphism in the factor H gene predisposes to age-related macular degeneration. Factor H blocks the alternative pathway of complement on self-surfaces bearing specific polyanions, including the glycosaminoglycan chains of proteoglycans. Factor H also binds C-reactive protein, potentially contributing to noninflammatory apoptotic processes. The at risk sequence contains His (rather than Tyr) at position 402 (384 in the mature protein), in the seventh of the 20 complement control protein (CCP) modules (CCP7) of factor H. We expressed both His(402) and Tyr(402) variants of CCP7, CCP7,8, and CCP6-8. We determined structures of His(402) and Tyr(402) CCP7 and showed them to be nearly identical. The side chains of His/Tyr(402) have similar, solvent-exposed orientations far from interfaces with CCP6 and -8. Tyr(402) CCP7 bound significantly more tightly than His(402) CCP7 to a heparin affinity column as well as to defined-length sulfated heparin oligosaccharides employed in gel mobility shift assays. This observation is consistent with the position of the 402 side chain on the edge of one of two glycosaminoglycan-binding surface patches on CCP7 that we inferred on the basis of chemical shift perturbation studies with a sulfated heparin tetrasaccharide. According to surface plasmon resonance measurements, Tyr(402) CCP6-8 binds significantly more tightly than His(402) CCP6-8 to immobilized C-reactive protein. The data support a causal link between H402Y and age-related macular degeneration in which variation at position 402 modulates the response of factor H to age-related changes in the glycosaminoglycan composition and apoptotic activity of the macula.

Conformation of glycosaminoglycans by ion mobility mass spectrometry and molecular modelling.

We have performed conformational analyses of heparin-derived oligosaccharide ions in the gas phase using a combination of ion-mobility mass spectrometry and molecular modelling. Negative mode electrospray ionisation was used to generate singly (disaccharide, [C12H15NO19S3Na3]-) and doubly charged (tetrasaccharides, [C24H30N2O38S6Na6]2- and [C24H31N2O35S5Na5]2-) ions containing three and six Na+ ions, respectively. Good agreement was obtained between the experimental and theoretical cross sections. The latter were obtained using modelled structures generated by the AMBER-based force field. Analysis of the conformations of the oligosaccharide ions shows that sodium cations play a major role in stabilizing these ions in the gas phase. This was seen in the formation of oligomers of the disaccharide ion and "compact" structures of tetrasaccharide ions. Interestingly, the gas phase conformations of the three tetrasaccharide ions with different primary structures were significantly different.

The mode of action of heparan and dermatan sulfates in the regulation of hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor, in addition to binding to its specific signal-transducing receptor, Met, also interacts with both heparan and dermatan sulfates with high affinity. We have investigated the comparative role of these two glycosaminoglycans in the activation of Met by hepatocyte growth factor/scatter factor. Using glycosaminoglycan-deficient CHO pgsA-745 cells we have shown that growth factor activity is critically dependent upon glycosaminoglycans, and that heparan sulfate and dermatan sulfate are equally potent as co-receptors. Cross-linked 1:1 conjugates of growth factor and either heparan or dermatan sulfate do not dimerize under physiological conditions and are biologically active. This implies that a ternary signaling complex with Met forms in vivo. Native Met isolated from CHO pgsA-745 cells shows only very weak intrinsic affinity for heparin in vitro. Also, a heparin-derived hexasaccharide, which is the minimal size for high affinity binding to the growth factor alone, is sufficient to induce biological activity. Together these observations imply that the role of these glycosaminoglycan may be primarily to effect a conformational change in hepatocyte growth factor/scatter factor, rather than to induce a necessary growth factor dimerization, or to stabilize a ternary complex by additionally interacting with Met.

Syndecan-1 and -4 synthesized simultaneously by mouse mammary gland epithelial cells bear heparan sulfate chains that are apparently structurally indistinguishable.

Many of the biological functions attributed to cell surface heparan sulfate (HS) proteoglycans, including the Syndecan family, are elicited through the interaction of their HS chains with soluble extracellular molecules. Tightly controlled, cell-specific sulfation and epimerization of HS precursors endows these chains with highly sulfated, iduronate-rich regions, which are major determinants of cytokine and matrix-protein binding and which are interspersed by N-acetylated, poorly sulfated regions. Until this study, there have been no comprehensive structural comparisons made on HS chains decorating simultaneously expressed, but different, syndecan core proteins. In this paper we demonstrate that the HS chains on affinity-purified syndecan-1 and -4 from murine mammary gland cells are essentially identical by a number of parameters. Size determination, disaccharide analyses, enzymatic and chemical scission methods, and affinity co-electrophoresis all failed to reveal any significant differences in fine structure, domain organization, or ligand-binding properties of these HS species. These findings lead us to suggest that the imposition of the fine structure onto HS occurs independently of the core protein to which it is attached and that these core proteins, in addition to the HS chains, may play a pivotal role in the various biological functions ascribed to these macromolecules.

The interactions of hepatocyte growth factor/scatter factor and its NK1 and NK2 variants with glycosaminoglycans using a modified gel mobility shift assay. Elucidation of the minimal size of binding and activatory oligosaccharides.

Full-length hepatocyte growth factor/scatter factor interacts with both heparan and dermatan sulfates and is critically dependent upon them as cofactors for activation of the tyrosine kinase receptor Met. Two C-terminally truncated variants (NK1 and NK2) of this growth factor also occur naturally. Their glycosaminoglycan binding properties are not clear. We have undertaken a comparative study of the heparan/dermatan sulfate binding characteristics of all three proteins. This has entailed the development of a modified gel mobility shift assay, utilizing fluorescence end-tagged oligosaccharides, that is also widely applicable to the analysis of many glycosaminoglycan-protein interactions. Using this we have shown that all three hepatocyte growth factor/scatter factor variants share identical heparan/dermatan sulfate binding properties and that both glycosaminoglycans occupy the same binding site. The minimal size of the oligosaccharide that binds with high affinity in all cases is a tetrasaccharide from heparan sulfate but a hexasaccharide from dermatan sulfate. These findings demonstrate that functional glycosaminoglycan binding is restricted to a binding site situated solely within the small N-terminal domain. The same minimal size fractions are also able to promote hepatocyte growth factor/scatter factor-mediated activation of Met and consequent downstream signaling in the glycosaminoglycan-deficient Chinese hamster ovary pgsA-745 cells. A covalent complex of heparan sulfate tetrasaccharide with monovalent growth factor is also active. The binding and activity of tetrasaccharides put constraints upon the possible interactions and molecular geometry within the ternary signaling complex.