Anne Louise Sørensen

Dual roles for hepatic lectin receptors in the clearance of chilled platelets.

Rapid chilling causes glycoprotein-Ib (GPIb) receptors to cluster on blood platelets. Hepatic macrophage β2 integrin binding to β-N-acetylglucosamine (β-GlcNAc) residues in the clusters leads to rapid clearance of acutely chilled platelets after transfusion. Although capping the β-GlcNAc moieties by galactosylation prevents clearance of short-term–cooled platelets, this strategy is ineffective after prolonged refrigeration. We report here that prolonged refrigeration increased the density and concentration of exposed galactose residues on platelets such that hepatocytes, through Ashwell-Morell receptor binding, become increasingly involved in platelet removal. Macrophages rapidly removed a large fraction of transfused platelets independent of their storage conditions. With prolonged platelet chilling, hepatocyte-dependent clearance further diminishes platelet recovery and survival after transfusion. Inhibition of chilled platelet clearance by both β2 integrin and Ashwell-Morell receptors may afford a potentially simple method for storing platelets in the cold.

Role of sialic acid for platelet life span: exposure of β-galactose results in the rapid clearance of platelets from the circulation by asialoglycoprotein receptor–expressing liver macrophages and hepatocytes

Although surface sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet circulation lifetime is not fully clarified. We show that thrombocytopenia in mice deficient in the St3gal4 sialyltransferase gene (St3Gal-IV(-/-) mice) is caused by the recognition of terminal galactose residues exposed on the platelet surface in the absence of sialylation. This results in accelerated platelet clearance by asialoglycoprotein receptor-expressing scavenger cells, a mechanism that was recently shown to induce thrombocytopenia during Streptococcus pneumoniae sepsis. We now identify platelet GPIbalpha as a major counterreceptor on ST3Gal-IV(-/-) platelets for asialoglycoprotein receptors. Moreover, we report data that establish the importance of sialylation of the von Willebrand factor in its function.

Leishmania major surface protease Gp63 interferes with the function of human monocytes and neutrophils in vitro

In the present study the effect of Leishmania major surface protease Gp63 on the chemotaxis and oxidative burst response of human peripheral blood monocytes and neutrophils was investigated. It was shown that prior incubation of cells with Gp63 inhibited chemotaxis of neutrophils but not monocytes towards the chemotactic peptide f‐met‐leu‐phe. On the other hand, chemotaxis of both neutrophils and monocytes towards zymosan‐activated serum containing C5a was inhibited by Gp63. Monocyte and neutrophil chemiluminescence response to opsonized zymosan was reduced by preincubation of the cells with Gp63 in a concentration‐dependent manner. Notably, monocytes were inhibited to a much greater degree than neutrophils by a given concentration of Gp63, and they were also inhibited at much lower concentrations of the protease. The inhibitory effect of Gp63 on chemotaxis and chemiluminescence was completely abolished by heat inactivation of the protease at 70 °C for 15 min. Neither neutrophil nor monocyte chemiluminescence was inhibited by Gp63 when cells were stimulated with PMA. Our data suggest that the major surface protease Gp63 might play an important role in the initial stages of Leishmania/macrophage interactions and the intracellular survival of the parasite.

The origin and function of platelet glycosyltransferases

Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and antiangiogenic factors throughout the vascular system. Although they are anucleated cells that lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartments, past studies have shown that platelets have glycosyltransferase activities. In the present study, we show that members of 3 distinct glycosyltransferase families are found within and on the surface of platelets. Immunocytology and flow cytometry results indicated that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results in the release of soluble glycosyltransferase activities and that platelets contain sufficient levels of sugar nucleotides for detection of glycosylation of exogenously added substrates. Therefore, the results of the present study show that blood platelets are a rich source of both glycosyltransferases and donor sugar substrates that can be released to function in the extracellular space. This platelet-glycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biologic functions.

Carbohydrate clearance receptors in transfusion medicine.

BACKGROUND Complex carbohydrates play important functions for circulation of proteins and cells. They provide protective shields and refraction from non-specific interactions with negative charges from sialic acids to enhance circulatory half-life. For recombinant protein therapeutics carbohydrates are especially important to enhance size and reduce glomerular filtration loss. Carbohydrates are, however, also ligands for a large number of carbohydrate-binding lectins exposed to the circulatory system that serve as scavenger receptors for the innate immune system, or have more specific roles in targeting of glycoproteins and cells. SCOPE OF REVIEW Here we provide an overview of the common lectin receptors that play roles for circulating glycoproteins and cells, and present a discussion of ways to engineer glycosylation of recombinant biologics and cells to improve therapeutic effects. MAJOR CONCLUSIONS While the pharmaceutical industry has learned how to exploit carbohydrates to improve pharmacokinetic properties of recombinant therapeutics, our understanding of how to improve cell-based therapies by manipulation of complex carbohydrates is still at its infancy. Progress with the latter has recently been achieved with cold-stored platelets, where exposure of uncapped glycans lead to rapid clearance from circulation by several lectin-mediated pathways. GENERAL SIGNIFICANCE Understanding lectin-mediated clearance pathways is essential for progress in development of biological pharmaceuticals.

Glycans and glycosylation of platelets : current concepts and implications for transfusion

Purpose of reviewPlatelet products are currently stored at room temperature, because refrigeration causes their rapid clearance from the circulation upon transfusion. Glycans have recently been emphasized as important determinants for the clearance of refrigerated platelets. The present review addresses the current knowledge of platelet glycans and the potential of glycosylation for improving platelet storage. Recent findingsRemoval of refrigerated platelets from the circulation is partly mediated by recognition of clustered β-N-acetylglucosamine on platelet surface glycoproteins by the αMβ2 hepatic lectin receptor. Capping the exposed β-N-acetylglucosamine residues by enzymatic galactosylation restored the circulation of short-term chilled murine platelets, introducing a novel method that allows for cold storage of platelet. Recent studies have, however, shown that galactosylation is not sufficient to restore circulation of long-term refrigerated platelets. Additional data indicate that differential carbohydrate-mediated mechanisms may exist for clearance of short-term and long-term cold-stored platelets. SummaryRoom temperature storage of platelet products increases the risk of transfusion-mediated sepsis and accelerates platelet deterioration, limiting platelet shelf life. Recent evidence suggests that glycoengineering of platelets might allow for their cold storage, significantly improving the quality of platelet products.

Se NMR spectroscopy as a sensitive probe for Hammett σ constants.

Herein we showcase the use of a combination of (1)H, (13)C, and (77)Se NMR spectroscopy as a sensitive tool for correlation analysis. A series of substituted O-aryl selenocarbamates [ArOC(Se)N(CH3)2] and Se-aryl selenocarbamates [ArSeC(O)N(CH3)2] have been investigated by means of (1)H, (13)C, and (77)Se NMR spectroscopy. We have determined the (1)H, (13)C, and (77)Se chemical shift values as well as both one- and two-bond heteronuclear (13)C-(77)Se coupling constants, and the changes in both the chemical shift values and the coupling constants were found to obey linear free energy relationships with Hammetts σ(p) and σ(p)(-) constants. For the eight studied O-aryl selenocarbamates, we observe linear free energy correlations with two of the (13)C and (77)Se chemical shift values and as well as one (13)C-(77)Se coupling constant. With the five examples of Se-aryl selenocarbamates, linear correlations are observed with three different (13)C-(77)Se coupling constants. The strong internal consistency validates the use of (77)Se NMR spectroscopy for correlation analysis.

Long-term visual outcome in a Danish population of patients with idiopathic intracranial hypertension

Idiopathic intracranial hypertension (IIH) is characterized by raised intracranial pressure (ICP), normal cerebrospinal composition and exclusion of alternative causes to increased ICP. The aim of this study was to evaluate long‐term visual outcome in a Danish population of IIH patients.

Noninvasive measurement of reepithelialization and microvascularity of suction-blister wounds with benchmarking to histology: Suction-blister wound healing model in humans

We explored use of the suction‐blister wound model in the assessment of not only epidermal regeneration but also pain, the microvascular response and bacteriology. The effects of topical zinc sulfate were studied to articulate the methodologies in this double‐blind trial. One epidermal suction blister (10 mm) was induced on each buttock in 30 healthy volunteers (15 females:15 males) and deroofed on day 0. The wounds were randomized to daily treatment with 1.4% zinc sulfate shower gel (n = 20), placebo (n = 20) or control (n = 20). Digital photography coupled with planimetry, transepidermal water loss (TEWL) measurement and optical coherence tomography (OCT) was benchmarked to the gold standard of histology of 60 full‐thickness wound biopsies on day 4. Pain increased after application of the shower gels. Microvessel density, determined from OCT images, increased from day 0 to day 2 in the three groups but increased more with the placebo than with the zinc shower gel (p = 0.003) or the control treatment (p = 0.002) and correlated (rS = 0.313, p = 0.015) with the inflammatory response on day 4, as determined by histology. Coagulase‐negative staphylococci were more common in wounds compared with skin (p = 0.002) and was reduced (p = 0.030) with zinc sulfate treatment. Planimetric analysis of digital wound images was not biased (p = 0.234) compared with histology, and TEWL measurements showed no correlation (rS = 0.052, p = 0.691) with epithelialization. Neoepidermal formation, determined by histology, did not differ (p = 0.290) among the groups. Zinc sulfate reduced (p = 0.031) the release of lactate dehydrogenase from cultured gel‐treated keratinocytes isolated from the blister roofs. Therefore, combination of the standardized suction‐blister wound model with noninvasive planimetry and OCT is a useful tool for assessing wound therapies. Zinc sulfate transiently dampened inflammation and reduced bacterial growth.