Usefulness of Lung Ultrasound as a Method for Early Diagnosis of Interstitial Lung Disease in Patients with Rheumatoid Arthritis.

Purpose: To determine the value of lung ultrasound (LUS) compared to high-resolution computed tomography (HRCT) in the early diagnosis of interstitial lung disease (ILD) in patients with rheumatoid arthritis (RA). Patients and Methods: An observational prospective study was performed. Were included patients with respiratory symptoms or/and, patients with crackles in auscultation during medical consultation. All patients underwent to chest X-rays, LUS, HRCT,and respiratory function tests. Results: A total of 192 patients with RA were included. Mean disease duration was 16.8 ± 11.1 years. 72% were positive for rheumatoid factor or anti-citrullinated antibodies. Of the total number of subjects, 54.7% had respiratory symptoms. The other patients did not have respiratory symptoms, but they did have had crackles on pulmonary auscultation. B lines > 11.5 on the ROC curve predicted ILD (AUC 0.63; CI 95%: 0.55-0.71; p < 0.003). A DLCO value of <7.13 significantly predicted the presence of ILD (AUC 0.61; 95% CI: 0.52-0.70; p < 0.028). Conclusion: The findings of this study suggest that LUS is a valuable tool for the early diagnosis of ILD in patients with RA, and together with DLCO, can adequately predict the presence of ILD in this population. LUS also helps to determine which patients with respiratory symptoms and signs suggestive for ILD are undergo to HRCT.

Evaluation of Phenol-Substituted Diphyllin Derivatives as Selective Antagonists for Ebola Virus Entry.

Ebola virus (EBOV) is an aggressive filoviral pathogen that can induce severe hemorrhagic fever in humans with up to 90% fatality rate. To date, there are no clinically effective small-molecule drugs for postexposure therapies to treat filoviral infections. EBOV cellular entry and infection involve uptake via macropinocytosis, navigation through the endocytic pathway, and pH-dependent escape into the cytoplasm. We report the inhibition of EBOV cell entry via selective inhibition of vacuolar (V)-ATPase by a new series of phenol-substituted derivatives of the natural product scaffold diphyllin. In cells challenged with Ebola virus, the diphyllin derivatives inhibit viral entry dependent upon structural variations to low nanomolar potencies. Mechanistically, the diphyllin derivatives had no effect on uptake and colocalization of viral particles with endocytic marker LAMP1 but directly modulated endosomal pH. The most potent effects were reversible exhibiting higher selectivity than bafilomycin or the parent diphyllin. Unlike general lysosomotrophic agents, the diphyllin derivatives showed no major disruptions of endocytic populations or morphology when examined with Rab5 and LAMP1 markers. The dilated vacuole phenotype induced by apilimod treatment or in constitutively active Rab5 mutant Q79L-expressing cells was both blocked and reversed by the diphyllin derivatives. The results are consistent with the action of the diphyllin scaffold as a selective pH-dependent viral entry block in late endosomes. Overall, the compounds show improved selectivity and minimal cytotoxicity relative to classical endosomal acidification blocking agents.

Design of an algorithm for the diagnostic approach of patients with joint pain.

BACKGROUND: Rheumatic diseases are a reason for frequent consultation with primary care doctors. Unfortunately, there is a high percentage of misdiagnosis. OBJECTIVE: To design an algorithm to be used by primary care physicians to improve the diagnostic approach of the patient with joint pain, and thus improve the diagnostic capacity in four rheumatic diseases. METHODS: Based on the information obtained from a literature review, we identified the main symptoms, signs, and paraclinical tests related to the diagnosis of rheumatoid arthritis, spondyloarthritis with peripheral involvement, systemic lupus erythematosus with joint involvement, and osteoarthritis. We conducted 3 consultations with a group of expert rheumatologists, using the Delphi technique, to design a diagnostic algorithm that has as a starting point "joint pain" as a common symptom for the four diseases. RESULTS: Thirty-nine rheumatologists from 18 countries of Ibero-America participated in the Delphi exercise. In the first consultation, we presented 94 items to the experts (35 symptoms, 31 signs, and 28 paraclinical tests) candidates to be part of the algorithm; 74 items (25 symptoms, 27 signs, and 22 paraclinical tests) were chosen. In the second consultation, the decision nodes of the algorithm were chosen, and in the third, its final structure was defined. The Delphi exercise lasted 8 months; 100% of the experts participated in the three consultations. CONCLUSION: We present an algorithm designed through an international consensus of experts, in which Delphi methodology was used, to support primary care physicians in the clinical approach to patients with joint pain. Key Points • We developed an algorithm with the participation of rheumatologists from 18 countries of Ibero-America, which gives a global vision of the clinical context of the patient with joint pain. • We integrated four rheumatic diseases into one tool with one common symptom: joint pain. It is a novel tool, as it is the first algorithm that will support the primary care physician in the consideration of four different rheumatic diseases. • It will improve the correct diagnosis and reduce the number of paraclinical tests requested by primary care physicians, in the management of patients with joint pain. This point was verified in a recently published study in the journal Rheumatology International (reference number 31).

A patients' view of OA: the Global Osteoarthritis Patient Perception Survey (GOAPPS), a pilot study.

BACKGROUND: Globally, osteoarthritis (OA) is the third condition associated with disability. There is still poor treatment in OA but science holds the key to finding better treatments and a cure. It is essential to learn what's important to patients from them to implement the most effective OA management. The OA Patients Task Force, conducted the Global OA Patient Perception Survey (GOAPPS)-the first global survey made by patients to analize the quality of life (QoL) & patient perceptions of care. The goal was to collect data on OA patients' perception of OA to understand patients' needs and expectations to improve OA management. METHODS: Observational, cross-sectional study by online survey data collection from six countries, translated into three languages. The questionnaire was comprised of 3 sections: patient demographics and clinical symptomology characteristics; relationship with physicians: perception of attention, treatment, and information provided; and OA impact on daily activity and QoL. The results of the survey were evaluated using the Limited Data Set. The survey results were analyzed using descriptive statistics to characterize the patients' answers. Additionally, Cronbach's alpha was calculated to determine internal consistency validity. RESULTS: A total of 1512 surveys were completed in 6 countries. 84.2% of respondents reported pain/tenderness and 91.1% experienced limitations to physical activities. 42.3% of patients were not satisfied with their current OA treatment. 86% had comorbidities, especially hypertension, and obesity. 51.3 and 78% would like access to additional drug or additional non-drug/non-surgical treatments respectively. 48.2% of patients perceived their QoL to be affected by OA. The Cronbach's alpha was 0.61. CONCLUSIONS: OA has a significant impact on patients' daily activities and their desire to play an active role in managing this disease. Patients are seeking additional treatments, especially no pharmacological/no surgical treatments stressing the need for investing in clinical research, implementing OA preventive measures, and managing interventions to improve the healthcare value chain in OA.

High-Throughput, High-Resolution Interferometric Light Microscopy of Biological Nanoparticles.

Label-free, visible light microscopy is an indispensable tool for studying biological nanoparticles (BNPs). However, conventional imaging techniques have two major challenges: (i) weak contrast due to low-refractive-index difference with the surrounding medium and exceptionally small size and (ii) limited spatial resolution. Advances in interferometric microscopy have overcome the weak contrast limitation and enabled direct detection of BNPs, yet lateral resolution remains as a challenge in studying BNP morphology. Here, we introduce a wide-field interferometric microscopy technique augmented by computational imaging to demonstrate a 2-fold lateral resolution improvement over a large field-of-view (>100 × 100 µm2), enabling simultaneous imaging of more than 104 BNPs at a resolution of ∼150 nm without any labels or sample preparation. We present a rigorous vectorial-optics-based forward model establishing the relationship between the intensity images captured under partially coherent asymmetric illumination and the complex permittivity distribution of nanoparticles. We demonstrate high-throughput morphological visualization of a diverse population of Ebola virus-like particles and a structurally distinct Ebola vaccine candidate. Our approach offers a low-cost and robust label-free imaging platform for high-throughput and high-resolution characterization of a broad size range of BNPs.

Global management of patients with knee osteoarthritis begins with quality of life assessment: a systematic review.

BACKGROUND: Knee osteoarthritis (KOA) is a prevalent form of chronic joint disease associated with functional restrictions and pain. Activity limitations negatively impact social connectedness and psychological well-being, reducing the quality of life (QoL) of patients. The purpose of this review is to summarize the existing information on QoL in KOA patients and share the reported individual factors, which may influence it. METHODS: We conducted a systematic review examining the literature up to JAN/2017 available at MEDLINE, EMBASE, Cochrane, and PsycINFO using KOA and QOL related keywords. Inclusion criteria were QOL compared to at least one demographic factor (e.g., age, gender), lifestyle factor (e.g., functional independence), or comorbidity factor (e.g., diabetes, obesity) and a control group. Analytical methods were not considered as part of the original design. RESULTS: A total of 610 articles were reviewed, of which 62 met inclusion criteria. Instruments used to measure QoL included: SF-36, EQ-5D, KOOS, WHOQOL, HAS, AIMS, NHP and JKOM. All studies reported worse QoL in KOA patients when compared to a control group. When females were compared to males, females reported worse QOL. Obesity as well as lower level of physical activity were reported with lower QoL scores. Knee self-management programs delivered by healthcare professionals improved QoL in patients with KOA. Educational level and higher total mindfulness were reported to improve QoL whereas poverty, psychological distress, depression and lacking familial relationships reduce it. Surgical KOA interventions resulted in good to excellent outcomes generally; although, results varied by age, weight, and depression. CONCLUSION: KOA has a substantial impact on QoL. In KOA patients, QoL is also influenced by specific individual factors including gender, body weight, physical activity, mental health, and education. Importantly, education and management programs designed to support KOA patients report improved QoL. QoL data is a valuable tool providing health care professionals with a better comprehension of KOA disease to aid implementation of the most effective management plan.

Pan-American League of Associations for Rheumatology-Central American, Caribbean and Andean Rheumatology Association Consensus-Conference Endorsements and Recommendations on the Diagnosis and Treatment of Chikungunya-Related Inflammatory Arthropathies in Latin America.

BACKGROUND/OBJECTIVE: Although mortality rates related with chikungunya (CHIK) outbreaks in Latin America's (LA's) dengue-endemic rural and new urban regions are low, dealing with symptoms and sequelae can both produce a significant burden of disease and diminish quality of life-from many months to years-after the acute phase of the infection, with a significant impact on public and individual health.The aim of this work was to establish Pan-American League of Associations for Rheumatology-Central American, Caribbean and Andean Rheumatology Association (ACCAR) consensus-conference endorsements and recommendations on the diagnosis and treatment of CHIK-related inflammatory arthropathies transmitted by Aedes aegypti and Aedes albopictus in LA. METHODS: Based on the Consensus Development Conference format, a panel of ACCAR rheumatologist voting members (n = 10) took part in this Pan-American League of Associations for Rheumatology initiative. Experts voted from a previous content analysis of the medical literature on CHIK, 4 subsequent topic conferences, and a workshop. Consensus represents the majority agreement (≥80%) achieved for each recommendation. RESULTS: The experts' panel reached 4 overarching principles: (1) CHIK virus (CHIKV) is a re-emergent virus transmitted by 2 species of mosquitoes: A. aegypti and A. albopictus; (2) CHIKV caused massive outbreaks in LA; (3) chronic CHIKV infection produces an inflammatory joint disease that, in some cases, can last for several months to years, and (4) currently, there are no vaccines or antivirals licensed for CHIKV infections. RECOMMENDATIONS: Pan-American League of Associations for Rheumatology-ACCAR achieved 13 endorsements and recommendations on CHIK categorized in 3 groups: (1) epidemiology and clinical manifestations, (2) diagnosis, and (3) treatment, representing the consensus agreement from the panel's members.

Reliability of OMERACT ultrasound elementary lesions in gout: results from a multicenter exercise.

The aim of this study was to evaluate the reliability of the outcome measures in rheumatology (OMERACT) definitions for ultrasound (US) elementary lesions in gout through an image reading exercise. Images from patients with gout (static images and videos) were collected. As an initial step, we carried out a image reading exercise within the experts of the Pan-American League of Associations for Rheumatology (PANLAR) US Study Group (n = 16). The following step consisted in a web-based exercise with the participation of larger number of sonographers (n = 63) from different centers. Images were rated evaluating the presence/absence of any US elementary lesion. Inter- and intra-reader reliabilities were analyzed using kappa coefficients. Participants were stratified according to their level of experience. In the first exercise, inter-reader kappa values were 0.45 for aggregates, 0.57 for tophus, 0.69 for erosions, and 0.90 for double contour (DC). Intra-reader kappa values were 0.86, 0.76, 0.80, and 0.90, respectively. The web-based exercise showed inter-reader kappa values for aggregates, tophus, erosions, and DC of 0.42, 0.49, 0.69, and 0.79, respectively. The intra-reader kappa values were 0.62, 0.69, 0.77, and 0.85, respectively. Reliability was not influenced by the sonographer's level of experience. The reliability of the new OMERACT US definitions for elementary lesions in gout ranged from moderate to excellent, depending on the type of lesion.

Regulation of glycosaminoglycan biogenesis is critical for sensitive-period-dependent vocal ontogeny.

In the brain, the extracellular matrix (ECM) plays a central role during neural development and thus modulates critical-period regulated behavioral ontogeny. The major components of the ECM are glycosaminoglycans (GAGs) including chondroitin sulfate (CS). However, the specific roles of GAGs in behavioral development are largely unknown. It has been shown that xylosides affect the biological functions of GAGs through modulating GAG biosynthesis. Particularly, xylosides affect GAG biosynthesis through priming of GAG chains (priming activity), competing with endogenous core proteins that carry GAG initiation sites (decoy activity), or both. Using birdsong as our model, we investigated, for the first time, how xyloside-mediated modulation of GAG biogenesis affects song development. Xylosides infused into motor cortex of juvenile birds alter song development by specifically affecting ontogeny of the stereotyped sequence rather than the acoustic structure of syllables. Further analyses reveal that observed changes can be attributed to the priming activity rather than the decoy activity of xylosides. Collectively, these results suggest that regulation of GAG biogenesis through chemical biology approaches may allow promising therapeutic interventions of critical-period-dependent central nervous system plasticity. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1401-1412, 2017.

The 2017 EULAR standardised procedures for ultrasound imaging in rheumatology.

BACKGROUND: In 2001, the European League Against Rheumatism developed and disseminated the first guidelines for musculoskeletal (MS) ultrasound (US) in rheumatology. Fifteen years later, the dramatic expansion of new data on MSUS in the literature coupled with technological developments in US imaging has necessitated an update of these guidelines. OBJECTIVES: To update the existing MSUS guidelines in rheumatology as well as to extend their scope to other anatomic structures relevant for rheumatology. METHODS: The project consisted of the following steps: (1) a systematic literature review of MSUS evaluable structures; (2) a Delphi survey among rheumatologist and radiologist experts in MSUS to select MS and non-MS anatomic structures evaluable by US that are relevant to rheumatology, to select abnormalities evaluable by US and to prioritise these pathologies for rheumatology and (3) a nominal group technique to achieve consensus on the US scanning procedures and to produce an electronic illustrated manual (ie, App of these procedures). RESULTS: Structures from nine MS and non-MS areas (ie, shoulder, elbow, wrist and hand, hip, knee, ankle and foot, peripheral nerves, salivary glands and vessels) were selected for MSUS in rheumatic and musculoskeletal diseases (RMD) and their detailed scanning procedures (ie, patient position, probe placement, scanning method and bony/other landmarks) were used to produce the App. In addition, US evaluable abnormalities present in RMD for each anatomic structure and their relevance for rheumatology were agreed on by the MSUS experts. CONCLUSIONS: This task force has produced a consensus-based comprehensive and practical framework on standardised procedures for MSUS imaging in rheumatology.

A glycan-based approach to therapeutic angiogenesis.

Angiogenesis, the sprouting of new blood vessels from existing vasculature, involves multiple complex biological processes, and it is an essential step for hemostasis, tissue healing and regeneration. Angiogenesis stimulants can ameliorate human disease conditions including limb ischemia, chronic wounds, heart disease, and stroke. The current strategies to improve the bioavailability of pro-angiogenic growth factors, including VEGF and FGF2, have remained largely unsuccessful. This study demonstrates that small molecules, termed click-xylosides, can promote angiogenesis in the in vitro matrigel tube formation assay and the ex ovo chick chorioallantoic membrane assay, depending on their aglycone moieties. Xyloside treatment enhances network connectivity and cell survivability, thereby, maintaining the network structures on matrigel culture for an extended period of time. These effects were achieved via the secreted xyloside-primed glycosaminoglycans (GAG) chains that in part, act through an ERK1/2 mediated signaling pathway. Through the remodeling of GAGs in the extracellular matrix of endothelial cells, the glycan approach, involving xylosides, offers great potential to effectively promote therapeutic angiogenesis.

PANLAR Consensus Recommendations for the Management in Osteoarthritis of Hand, Hip, and Knee.

OBJECTIVE: The objective of this consensus is to update the recommendations for the treatment of hand, hip, and knee osteoarthritis (OA) by agreeing on key propositions relating to the management of hand, hip, and knee OA, by identifying and critically appraising research evidence for the effectiveness of the treatments and by generating recommendations based on a combination of the available evidence and expert opinion of 18 countries of America. METHODS: Recommendations were developed by a group of 48 specialists of rheumatologists, members of other medical disciplines (orthopedics and physiatrists), and three patients, one for each location of OA. A systematic review of existing articles, meta-analyses, and guidelines for the management of hand, hip, and knee OA published between 2008 and January 2014 was undertaken. The scores for Level of Evidence and Grade of Recommendation were proposed and fully consented within the committee based on The American Heart Association Evidence-Based Scoring System. The level of agreement was established through a variation of Delphi technique. RESULTS: Both "strong" and "conditional" recommendations are given for management of hand, hip, and knee OA and nonpharmacological, pharmacological, and surgical modalities of treatment are presented according to the different levels of agreement. CONCLUSIONS: These recommendations are based on the consensus of clinical experts from a wide range of disciplines taking available evidence into account while balancing the benefits and risks of nonpharmacological, pharmacological, and surgical treatment modalities, and incorporating their preferences and values. Different backgrounds in terms of patient education or drug availability in different countries were not evaluated but will be important.

How is the ultrasound in rheumatology used, implemented, and applied in Latin American centers? Results from a multicenter study.

This study aimed to perform an overview of how ultrasound (US) is being used, implemented, and applied in rheumatologic centers in Latin America (LA). A retrospective, multicenter 1-year experience study was undertaken. Eighteen centers from eight countries were involved. The following information were collected: demographic data, indication to perform an US examination, physician that required the examination, and the anatomical region required for the examination. A total of 7167 patients underwent an US examination. The request for US examinations came most frequently from their own institution (5981 (83.45 %)) than from external referral (1186 (16.55 %)). The services that more frequently requested an US examination were rheumatology 5154 (71.91 %), followed by orthopedic 1016 (14.18 %), and rehabilitation 375 (5.23 %). The most frequently scanned area was the shoulder in 1908 cases (26.62 %), followed by hand 1754 (24.47 %), knee 1518 (21.18 %), ankle 574 (8.01 %), and wrist 394 (5.50 %). Osteoarthritis was the most common disease assessed (2279 patients (31.8 %)), followed by rheumatoid arthritis (2125 patients (29.65 %)), psoriatic arthritis (869 patients (12.1 %)), painful shoulder syndrome (545 (7.6 %)), connective tissue disorders (systemic sclerosis 339 (4.7 %), polymyositis/dermatomyositis 107 (1.4 %), Sjögren's syndrome 60 (0.8 %), and systemic lupus erythematosus 57 (0.8 %)). US evaluation was more frequently requested for diagnostic purposes (3981 (55.5 %)) compared to follow-up studies (2649 (36.9 %)), research protocols (339 (4.73 %)), and invasive guided procedures (198 (2.76 %)). US registered increasing applications in rheumatology and highlighted its positive impact in daily clinical practice. US increases the accuracy of the musculoskeletal clinical examination, influence the diagnosis, and the disease management.

Osteoarthritis in Latin America: Study of Demographic and Clinical Characteristics in 3040 Patients.

BACKGROUND: Latin America is a heterogeneous region made up of different populations, cultures, latitudes, altitudes, and immigrants from different areas and ethnic groups. OBJECTIVE: The purpose of this study is to describe the clinical and demographic profile of patients with osteoarthritis (OA) evaluated by a selected group of rheumatologists in 13 Latin American countries. METHODS: A descriptive, observational, cross-sectional study was conducted in 13 Latin American countries of patients with symptomatic OA. Data were collected over a 3-month period using an ad hoc questionnaire to evaluate the clinical and demographic features of OA seen by rheumatologists. RESULTS: Among the 3040 patients, their average age was 62.5 years, and female-to-male ratio was 4.8:1. Patients with body mass index of greater than 30 kg/m or obesity was found in 38.2%. Approximately 88% had primary OA. Joints with OA were as follows: knee 31.2%, hand 9.5%, hand and knee 22.9%, proximal and distal interphalangeal joints (erosive OA) 6.5%, axial 6.6%, and hip 1.3%. Approximately 88.5% had radiographic severity of grade 2 or 3 on Kellgren-Lawrence scale (0-4). Nonsteroidal anti-inflammatory drugs were the predominant OA treatment included in combinations with glucosamine sulfate/chondroitin and viscosupplementation. Associated comorbidities included hypertension (39%), obesity (36.3%), diabetes mellitus (12%), and without comorbidity (12.7%). CONCLUSIONS: This is 1 of the largest population studies that evaluated the characteristics of OA in 3040 patients evaluated by rheumatologists in 13 Latin American countries. This study provides important data for each Latin American country to develop new health care planning in management of OA.

Chemoenzymatically prepared heparan sulfate containing rare 2-O-sulfonated glucuronic acid residues.

The structural diversity of natural sulfated glycosaminoglycans (GAGs) presents major promise for discovery of chemical biology tools or therapeutic agents. Yet, few GAGs have been identified so far to exhibit this promise. We reasoned that a simple approach to identify such GAGs is to explore sequences containing rare residues, for example, 2-O-sulfonated glucuronic acid (GlcAp2S). Genetic algorithm-based computational docking and filtering suggested that GlcAp2S containing heparan sulfate (HS) may exhibit highly selective recognition of antithrombin, a key plasma clot regulator. HS containing only GlcAp2S and 2-N-sulfonated glucosamine residues, labeled as HS2S2S, was chemoenzymatically synthesized in just two steps and was found to preferentially bind antithrombin over heparin cofactor II, a closely related serpin. Likewise, HS2S2S directly inhibited thrombin but not factor Xa, a closely related protease. The results show that a HS containing rare GlcAp2S residues exhibits the unusual property of selective antithrombin activation and direct thrombin inhibition. More importantly, HS2S2S is also the first molecule to activate antithrombin nearly as well as the heparin pentasaccharide although being completely devoid of the critical 3-O-sulfonate group. Thus, this work shows that novel functions and mechanisms may be uncovered by studying rare GAG residues/sequences.

Enzymatic synthesis of heparan sulfate and heparin.

Heparan sulfate (HS) polysaccharide chains have been shown to orchestrate distinct biological functions in several systems. Study of HS structure-function relations is, however, hampered due to the lack of availability of HS in sufficient quantities as well as the molecular heterogeneity of naturally occurring HS. Enzymatic synthesis of HS is an attractive alternative to the use of naturally occurring HS, as it reduces molecular heterogeneity, or a long and daunting chemical synthesis of HS. Heparosan, produced by E. coli K5 bacteria, has a structure similar to the unmodified HS backbone structure and can be used as a precursor in the enzymatic synthesis of HS-like polysaccharides. Here, we describe an enzymatic approach to synthesize several specifically sulfated HS polysaccharides for biological studies using the heparosan backbone and a combination of recombinant biosynthetic enzymes such as C5-epimerase and sulfotransferases.

Synthesis and biomedical applications of xylosides.

Xylosides modulate the biosynthesis of sulfated glycosaminoglycans (GAGs) in various cell types. A new class of xylosides called "click-xylosides" has been synthesized for their biostability, ease of chemical synthesis, and tunable sulfated GAG biogenesis in vitro and in vivo. These click-xylosides have several therapeutic and biomedical applications in the regulation of angiogenesis, tumor inhibition, and regeneration. This protocol focuses on the synthesis of click-xylosides, their cellular priming activities, and biomedical applications.

Determinants of versican-V1 proteoglycan processing by the metalloproteinase ADAMTS5.

Proteolysis of the Glu(441)-Ala(442) bond in the glycosaminoglycan (GAG) ß domain of the versican-V1 variant by a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif (ADAMTS) proteases is required for proper embryo morphogenesis. However, the processing mechanism and the possibility of additional ADAMTS-cleaved processing sites are unknown. We demonstrate here that if Glu(441) is mutated, ADAMTS5 cleaves inefficiently at a proximate upstream site but normally does not cleave elsewhere within the GAGß domain. Chondroitin sulfate (CS) modification of versican is a prerequisite for cleavage at the Glu(441)-Ala(442) site, as demonstrated by reduced processing of CS-deficient or chondroitinase ABC-treated versican-V1. Site-directed mutagenesis identified the N-terminal CS attachment sites Ser(507) and Ser(525) as essential for processing of the Glu(441)-Ala(442) bond by ADAMTS5. A construct including only these two GAG chains, but not downstream GAG attachment sites, was cleaved efficiently. Therefore, CS chain attachment to Ser(507) and Ser(525) is necessary and sufficient for versican proteolysis by ADAMTS5. Mutagenesis of Glu(441) and an antibody to a peptide spanning Thr(432)-Gly(445) (i.e. containing the scissile bond) reduced versican-V1 processing. ADAMTS5 lacking the C-terminal ancillary domain did not cleave versican, and an ADAMTS5 ancillary domain construct bound versican-V1 via the CS chains. We conclude that docking of ADAMTS5 with two N-terminal GAG chains of versican-V1 via its ancillary domain is required for versican processing at Glu(441)-Ala(442). V1 proteolysis by ADAMTS1 demonstrated a similar requirement for the N-terminal GAG chains and Glu(441). Therefore, versican cleavage can be inhibited substantially by mutation of Glu(441), Ser(507), and Ser(525) or by an antibody to the region of the scissile bond.

A serotonin-induced N-glycan switch regulates platelet aggregation.

Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associated with platelet aggregation. Mass spectrometry of total surface glycoproteins on platelets isolated from wild-type mice infused for 24 hours with saline or 5-HT revealed that the content of glycoproteins on platelets from 5-HT-infused mice switched from predominantly N-acetyl-neuraminic acid (Neu5Ac) to N-glycolyl-neuraminic acid (Neu5Gc). Cytidine monophosphate-N-acetylneuraminate hydroxylase (CMAH) synthesizes Neu5Gc from Neu5Ac. Up-regulation of Neu5Gc content on the platelet surface resulted from an increase in the catalytic function, not expression, of CMAH in platelets of 5-HT-infused mice. The highest level of Neu5Gc was observed in platelets of 5-HT-infused, 5-HT transporter-knock out mice, suggesting that the surface delineated 5-HT receptor on platelets may promote CMAH catalytic activity. These new findings link elevated levels of plasma 5-HT to altered platelet N-glycan content, a previously unrecognized abnormality that may favor platelet aggregation.