Inhibition of Sialic Acid Loss Greatly Improves Survival of Refrigerated Platelets

Abstract Abstract 1133 Platelets have the shortest shelf life of all major blood components and are the most difficult to store, a fact that complicates platelet transfusion practices. Platelet refrigeration could slow bacterial growth and possibly retard the loss of platelet function following storage. However, in contrast to other blood components, platelets do not tolerate refrigeration and are rapidly cleared from the circulation. We demonstrated that two distinct pathways recognizing GPIba remove refrigerated platelets in recipient's livers: 1) αMβ2 integrins (Mac-1) on hepatic resident macrophages (Kupffer cells) selectively recognize irreversibly clustered b-N-acetylglucosamine (β-GlcNAc)–terminated glycans on GPIbα, and 2) hepatic Asialoglycoprotein (Asg) receptors (Ashwell Morell receptors) recognize desialylated GPIba. We here investigated the mechanism of sialic acid loss during refrigeration. We show, that when refrigerated platelets are rewarmed, they secrete active sialidases, including the lysosomal sialidase Neu1 that remove sialic acid from platelet receptors, specifically from GPIbα. Platelets also express Neu3 on their surfaces, however Neu3 expression appears to be unaffected by platelet refrigeration. Importantly, the recovery and circulation of refrigerated platelets is greatly improved by storage in the presence of the competitive sialidase inhibitor N-Acetylneuraminic Acid, 2,3-Dehydro-2-deoxy-Sodium Salt (DANA). Desialylated von Willebrand receptor (vWfR) complex is also a target for metalloproteinases (MMPs), as GPIbα and GPV are cleaved from the surface of refrigerated platelets. Receptor shedding is inhibited by the metalloproteinase inhibitor GM6001 and does not occur in ADAM17ΔZn/ΔZn platelets expressing inactive ADAM17. Critically, desialylation in the absence of metalloproteinase-mediated receptor shedding is sufficient to induce the rapid clearance of platelets from circulation. Desialylation of platelet vWfR therefore triggers platelet clearance, and primes GPIbα and GPV for metalloproteinase-dependent cleavage. We conclude that desialylation of platelets is caused by increased surface sialidase activity following refrigeration and desialylation of glycoproteins, specifically of GPIbα, promotes receptor cleavage by MMPs. Disclosures: Liu: Velicomedical, Inc: Employment.

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Desialylation accelerates platelet clearance after refrigeration and initiates GPIbα metalloproteinase-mediated cleavage in mice

Blood ◽

10.1182/blood-2011-05-355628 ◽

2012 ◽

Vol 119 (5) ◽

pp. 1263-1273 ◽

Cited By ~ 105

Author(s):

A. J. Gerard Jansen ◽

Emma C. Josefsson ◽

Viktoria Rumjantseva ◽

Qiyong Peter Liu ◽

Hervé Falet ◽

...

Keyword(s):

Sialic Acid ◽

Von Willebrand Factor ◽

Rapid Clearance ◽

Receptor Shedding ◽

Von Willebrand ◽

Willebrand Factor ◽

Lysosomal Sialidase

AbstractWhen refrigerated platelets are rewarmed, they secrete active sialidases, including the lysosomal sialidase Neu1, and express surface Neu3 that remove sialic acid from platelet von Willebrand factor receptor (VWFR), specifically the GPIbα subunit. The recovery and circulation of refrigerated platelets is greatly improved by storage in the presence of inhibitors of sialidases. Desialylated VWFR is also a target for metalloproteinases (MPs), because GPIbα and GPV are cleaved from the surface of refrigerated platelets. Receptor shedding is inhibited by the MP inhibitor GM6001 and does not occur in Adam17ΔZn/ΔZn platelets expressing inactive ADAM17. Critically, desialylation in the absence of MP-mediated receptor shedding is sufficient to cause the rapid clearance of platelets from circulation. Desialylation of platelet VWFR therefore triggers platelet clearance and primes GPIbα and GPV for MP-dependent cleavage.

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Sialic Acid Loss Regulates Platelet Survival and Integrity

Blood ◽

10.1182/blood.v122.21.3504.3504 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3504-3504

Author(s):

Renata Grozovsky ◽

Gerard Jansen ◽

Karin M. Hoffmeister

Keyword(s):

Flow Cytometry ◽

Sialic Acid ◽

Conflicts Of Interest ◽

Surface Expression ◽

Sialidase Activity ◽

Acetylneuraminic Acid ◽

Platelet Survival ◽

Sialidase Inhibitor ◽

Glycan Degradation

Abstract It becomes increasingly apparent that, besides the intrinsic apoptotic machinery, surface glycan modifications regulate platelet survival. Platelets with reduced α2,3-linked sialic acid during sepsis due to S. pneumoniae infection, after cold storage, or in mice lacking the sialyltransferase ST3GalIV are cleared by the hepatic Ashwell-Morell receptor (AMR, ASGPR1/2). Platelet survival in Asgr2-/- mice was increased by ∼35% when compared to that of WT mice, which results in a ∼50% increase in circulating platelet counts, despite a loss of surface sialic acid. We reasoned that sialidase activity increases on the surface of circulating platelets as they age, a process that would facilitate sialic acid hydrolysis and removal from the circulation. To test this hypothesis, we directly injected the sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) into WT mice and determined endogenous platelet circulatory times. Platelet survival was prolonged by ∼30% (T1/2 of 62.0 ± 2.7 h) in DANA-treated mice, compared to that of mock-treated mice (T1/2 of 47.5 ± 4.3 h). DANA injections decreased terminal sialic acid loss on circulating platelets by ∼40% by day 2, compared to control platelets, as evidenced by binding of RCA-I lectin that specifically recognizes terminal β1-4 galactose moieties exposed by sialic acid removal. Freshly isolated, resting platelets from Asgr2-/- mice (AMR-platelets) were significantly smaller in size (22%) and had increased sialidase Neu1 (∼5 fold), but not Neu3 surface expression, when compared to WT platelets or St3gal4-/- platelets, as measured by flow cytometry. We next investigated if AMR-platelets age/deteriorate faster upon in vitro storage. Platelets were isolated from WT, Asgr2-/- and St3gal4-/- mice and stored for 24hrs at room temperature, and sialidase expression (Neu1 and Neu3) as well as microvesiculation were measured by flow cytometry. Although significant Neu1 and Neu3 surface expression increase was measured on platelets from all phenotype after storage, Neu1 and Neu3 surface expression was significantly higher in AMR-platelets (∼2 and 4 fold, respectively) when compared to WT and St3gal4-/- platelets. AMR-platelets, but not St3gal4-/- platelets microvesiculated upon storage, consistent with a faster deterioration of aged AMR-platelets. We next injected into WT and Asgr2-/- mice the BH3 mimetic, ABT-737, which binds and inhibits the pro-apoptotic Bcl-2, Bcl-xL and Bcl-w. After injection of ABT-737, platelets in the Asgr2-/- mouse were cleared more efficiently (∼20%) from the circulation when compared to those in WT mice. Collectively, our data show that blood borne sialidases contribute to loss of sialic acid during circulation to regulate platelet survival. Our data also suggest that platelet glycan degradation, i.e. sialic acid loss, may trigger the intrinsic apoptotic machinery in platelets, linking glycan degradation and intrinsic apoptotic machinery in the clearance mechanisms regulating platelet survival. Disclosures: No relevant conflicts of interest to declare.

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Autoantibody Mediated Desialylation Impairs Human Thrombopoiesis and Platelet Life Span

Blood ◽

10.1182/blood-2019-131725 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2346-2346

Author(s):

Irene Marini ◽

Jan Zlamal ◽

Lisann Pelzel ◽

Wolfgang Bethge ◽

Christoph Faul ◽

...

Keyword(s):

Sialic Acid ◽

Life Span ◽

Conflicts Of Interest ◽

Lectin Binding ◽

Cell Interaction ◽

Bleeding Tendency ◽

Sialidase Inhibitor ◽

Von Willebrand ◽

The Impact

Background: The low platelet count in autoimmune thrombocytopenia (ITP) is caused by enhanced destruction of opsonised platelets in the spleen upon binding of the anti-platelet autoantibodies (AAbs) to the glycoproteins (GPs) express on PLT's surface. Data from animal model suggested that desialylation may contribute to PLT destruction in ITP. However, accumulating evidence suggests that reduction of PLT generation from megakaryocytes (MKs) in bone morrow is also responsible thrombocytopenia in ITP. Based on these considerations, we hypothesized that AAb-mediated desialylation of the GPs expressed on PLT and MKs may interfere with PLT formation and life span. Methods: Sera from 100 ITP patients were investigated in this study. AAb-induced desialylation was detected using a lectin binding assay (LBA) by flow cytometry (FC). To investigate the impact of desialylation on the life-span of human PLTs, the NSG mouse model was used. PLTs and MKs functions were assessed after AAb treatment using proplatelet formation test and adhesion assays on different surfaces. Results: Sera from 35/100 (35%) ITP patients induced cleavage of sialic acid from PLT surface. Injection of desialylating AAbs in vivo resulted in accelerated clearance of human PLTs which was significantly reduced by a specific sialidase inhibitor that prevents desialylation on the PLT surface (survival after 5h: 29%, range 22-40% vs. 48%, range 41-53%, p=0.014, respectively). Desialylating AAbs caused a significant reduction in PLT adhesion to fibrinogen and von Willebrand factor (mean of % adherent PLTs compared to control IgG: 34±6%, p=0.004 and 26±2%, p=0.001, respectively). Interestingly, PLT adhesion was recovered in the presence of a sialidase inhibitor (mean of % adherent PLTs: 86±6%, p=0.001 and 67±10, p=0.020, respectively). IgG fractions from 7/10 (70%) ITP-sera were able to cleave sialic acid and induce exposure of ß-galactose residues on CD34+-derived MKs. Desialylating AAbs induced lower ability to form proplatelet extensions compared to control IgG, which was significantly increased in the presence of the sialidase inhibitor (mean of % proplatelet forming MKs: 42±11% vs. 90±9%, p=0.032, respectively). Conclusion: Our findings show that AAbs from a subgroup of ITP patients are not only able to cleave sialic acid on surface of human PLTs, but also on MKs leading to accelerate PLT destruction and impaired thrombopoiesis, respectively. In addition, we observed that AAb-mediated receptor desialyation interferes with cell interaction with extracellular matrix proteins leading to impaired PLT adhesion, MK differentiation and thrombopoiesis. These novel findings highlight the multiple effects of AAbs in ITP and add to the existing evidence that ITP is rather a group of disorders sharing common characteristics, namely loss of immune tolerance toward PLT and MK antigens and increased bleeding tendency. Disclosures No relevant conflicts of interest to declare.

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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

Blood ◽

10.1182/blood-2009-01-199414 ◽

2009 ◽

Vol 114 (8) ◽

pp. 1645-1654 ◽

Cited By ~ 114

Author(s):

Anne Louise Sørensen ◽

Viktoria Rumjantseva ◽

Sara Nayeb-Hashemi ◽

Henrik Clausen ◽

John H. Hartwig ◽

...

Keyword(s):

Sialic Acid ◽

Blood Cells ◽

Asialoglycoprotein Receptor ◽

Platelet Surface ◽

Terminal Galactose ◽

Report Data ◽

Rapid Clearance ◽

Von Willebrand ◽

Willebrand Factor

AbstractAlthough 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 GPIbα 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.

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Role of sialidase in glycoprotein utilization by Tannerella forsythia

Microbiology ◽

10.1099/mic.0.052498-0 ◽

2011 ◽

Vol 157 (11) ◽

pp. 3195-3202 ◽

Cited By ~ 39

Author(s):

Sumita Roy ◽

Kiyonobu Honma ◽

C. W. Ian Douglas ◽

Ashu Sharma ◽

Graham P. Stafford

Keyword(s):

Sialic Acid ◽

Tannerella Forsythia ◽

Similar Reduction ◽

Biofilm Growth ◽

Sialidase Activity ◽

Sialidase Inhibitor ◽

Initial Adhesion ◽

Coated Surfaces

The major bacterial pathogens associated with periodontitis include Tannerella forsythia. We previously discovered that sialic acid stimulates biofilm growth of T. forsythia, and that sialidase activity is key to utilization of sialoconjugate sugars and is involved in host–pathogen interactions in vitro. The aim of this work was to assess the influence of the NanH sialidase on initial biofilm adhesion and growth in experiments where the only source of sialic acid was sialoglycoproteins or human oral secretions. After showing that T. forsythia can utilize sialoglycoproteins for biofilm growth, we showed that growth and initial adhesion with sialylated mucin and fetuin were inhibited two- to threefold by the sialidase inhibitor oseltamivir. A similar reduction (three- to fourfold) was observed with a nanH mutant compared with the wild-type. Importantly, these data were replicated using clinically relevant serum and saliva samples as substrates. In addition, the ability of the nanH mutant to form biofilms on glycoprotein-coated surfaces could be restored by the addition of purified NanH, which we show is able to cleave sialic acid from the model glycoprotein fetuin and, much less efficiently, 9-O-acetylated bovine submaxillary mucin. These data show for the first time that glycoprotein-associated sialic acid is likely to be a key in vivo nutrient source for T. forsythia when growing in a biofilm, and suggest that sialidase inhibitors might be useful adjuncts in periodontal therapy.

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An Orthologue of Bacteroides fragilis NanH Is the Principal Sialidase in Tannerella forsythia

Journal of Bacteriology ◽

10.1128/jb.01618-08 ◽

2009 ◽

Vol 191 (11) ◽

pp. 3623-3628 ◽

Cited By ~ 33

Author(s):

Hayley Thompson ◽

Karen A. Homer ◽

Susmitha Rao ◽

Veronica Booth ◽

Arthur H. F. Hosie

Keyword(s):

Sialic Acid ◽

Biochemical Characterization ◽

Bacteroides Fragilis ◽

Periodontal Pathogen ◽

Putative Virulence Factor ◽

Tannerella Forsythia ◽

Sialidase Activity ◽

Acetylneuraminic Acid ◽

Ph Range

ABSTRACT Sialidase activity is a putative virulence factor of the anaerobic periodontal pathogen Tannerella forsythia, but it is uncertain which genes encode this activity. Characterization of a putative sialidase, SiaHI, by others, indicated that this protein alone may not be responsible for all of the sialidase activity. We describe a second sialidase in T. forsythia (TF0035), an orthologue of Bacteroides fragilis NanH, and its expression in Escherichia coli. Sialidase activity of the expressed NanH was confirmed by using 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid as a substrate. Biochemical characterization of the recombinant T. forsythia NanH indicated that it was active over a broad pH range, with optimum activity at pH 5.5. This enzyme has high affinity for 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (Km of 32.9 ± 10.3 μM) and rapidly releases 4-methylumbelliferone (V max of 170.8 ± 11.8 nmol of 4-methylumbelliferone min−1 mg of protein−1). E. coli lysates containing recombinant T. forsythia NanH cleave sialic acid from a range of substrates, with a preference for α2-3 glycosidic linkages. The genes adjacent to nanH encode proteins apparently involved in the metabolism of sialic acid, indicating that the NanH sialidase is likely to be involved in nutrient acquisition.

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Desialylation by Edwardsiella tarda is the initial step in the regulation of its invasiveness

Biochemical Journal ◽

10.1042/bcj20190367 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3183-3196

Author(s):

Linh Khanh Vo ◽

Toshiharu Tsuzuki ◽

Yuko Kamada-Futagami ◽

Petros Kingstone Chigwechokha ◽

Akinobu Honda ◽

...

Keyword(s):

Sialic Acid ◽

Mrna Level ◽

Initial Step ◽

Host Cells ◽

Edwardsiella Tarda ◽

Economic Losses ◽

Mrna Levels ◽

Sialidase Activity ◽

Sialidase Inhibitor ◽

The Relationship

Abstract Edwardsiella tarda is a gram-negative bacterium causing significant economic losses to aquaculture. E. tarda possesses NanA sialidase which removes sialic acids from α2–3 sialo-glycoprotein of host cells. However, the relationship between NanA sialidase activity and E. tarda invasiveness remains poorly understood. Furthermore, the pathway of sialic acid metabolism in E. tarda remains to be elucidated. We studied sialidase activity in several E. tarda strains and found that the pathogenic strains exhibited higher sialidase activity and greater up-regulation of the NanA mRNA level than non-pathogenic strain. Pathogenic strains also showed higher rates of infection in GAKS cells, and the infection was drastically suppressed by sialidase inhibitor. Additionally, NanA gene overexpression significantly increased infection and treatment of E. tarda with free sialic acid enhanced the rate of infection in GAKS cells. Sialic acid treatment enhanced mRNA levels of two N-acetylneuraminate lyases and one N-acetylneuraminate cytidylyltransferase. E. tarda uses sialic acid as a carbon source for growth via N-acetylneuraminate lyases. The strains with high N-acetylneuraminate cytidylyltransferase level showed greater sialylation of the lipopolysaccharides and glycoproteins. Our study establishes the significance of desialylation by E. tarda sialidase in the regulation of its invasiveness.

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The Hepatic Asialoglycoprotein Receptor Regulates Platelet Homeostasis

Blood ◽

10.1182/blood.v116.21.2025.2025 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2025-2025

Author(s):

Renata Grozovsky ◽

Gerard Jansen ◽

Karin M. Hoffmeister

Keyword(s):

Sialic Acid ◽

Blood Loss ◽

Human Body ◽

Conflicts Of Interest ◽

Wild Type ◽

Acetylneuraminic Acid ◽

Massive Blood Loss ◽

Platelet Survival ◽

Sialidase Inhibitor

Abstract Abstract 2025 The human body produces and removes more than a 100 billion of platelets every day. The mechanisms responsible for platelet homeostasis are subject to speculation since the 1950's. The most popular hypothesis to date has been antibody-mediated clearance, platelet consumption due to massive blood loss and an internal “senescence timer”. We and others have recently demonstrated that sialic acid deficient platelets due to external triggers such as sepsis or chilling are cleared by hepatic asialoglycoprotein receptors (ASGPR) independently of macrophages. Here, we investigated whether loss of sialic acid mediates platelet clearance in vivo. We show that 1) Injection of the specific sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) lengthened the survival of biotinylated platelets by ∼50% (T1/2 of 72h), compared to mock treated (PBS injected) control mice (T1/2 of 49h); 2) Similarly, biotinylated platelet survival in ASGPR-null mice was prolonged by ∼ 50% (T1/2 of 74h) compared to platelet survival in wild type (WT) mice (T1/2 of 48h); 3) ASGPR-null mice have significantly increased platelet counts, compared to WT (p=0.0004) and platelets isolated from ASGPR-null mice are ∼15% smaller than WT (p=0.03); 4) Platelets isolated from ASGPR-null mice showed significant increased in b-galactose exposure (∼50% increase, i.e. decrease of sialic acid), compared to WT, as evidenced by binding of the b-galactose specific lectin (RCA-I). These data show that the ASGPR not only removes desialylated platelets due to sepsis or chilling, but also regulates platelet homeostasis. Disclosures: No relevant conflicts of interest to declare.

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The fate of orally administered sialic acid: First insights from patients with N-acetylneuraminic acid synthase deficiency and control subjects

Molecular Genetics and Metabolism Reports ◽

10.1016/j.ymgmr.2021.100777 ◽

2021 ◽

Vol 28 ◽

pp. 100777

Author(s):

Christel Tran ◽

Licia Turolla ◽

Diana Ballhausen ◽

Sandrine Cornaz Buros ◽

Tony Teav ◽

...

Keyword(s):

Sialic Acid ◽

Acetylneuraminic Acid ◽

Control Subjects ◽

And Control

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Enhancement of elastin expression by transdermal administration of sialidase isozyme Neu2

Scientific Reports ◽

10.1038/s41598-021-82820-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Akira Minami ◽

Yuka Fujita ◽

Jun Goto ◽

Ayano Iuchi ◽

Kosei Fujita ◽

...

Keyword(s):

Sialic Acid ◽

Skin Diseases ◽

Lower Layer ◽

Elastic Fiber ◽

Sialic Acid Residue ◽

Elastic Fibers ◽

Transdermal Administration ◽

Sialidase Activity ◽

Fiber Assembly ◽

Senescence Accelerated Mice

AbstractReduction of elastin in the skin causes various skin diseases as well as wrinkles and sagging with aging. Sialidase is a hydrolase that cleaves a sialic acid residue from sialoglycoconjugate. Cleavage of sialic acid from microfibrils by the sialidase isozyme Neu1 facilitates elastic fiber assembly. In the present study, we showed that a lower layer of the dermis and muscle showed relatively intense sialidase activity. The sialidase activity in the skin decreased with aging. Choline and geranate (CAGE), one of the ionic liquids, can deliver the sialidase subcutaneously while maintaining the enzymatic activity. The elastin level in the dermis was increased by applying sialidase from Arthrobacter ureafaciens (AUSA) with CAGE on the skin for 5 days in rats and senescence-accelerated mice prone 1 and 8. Sialidase activity in the dermis was considered to be mainly due to Neu2 based on the expression level of sialidase isozyme mRNA. Transdermal administration of Neu2 with CAGE also increased the level of elastin in the dermis. Therefore, not only Neu1 but also Neu2 would be involved in elastic fiber assembly. Transdermal administration of sialidase is expected to be useful for improvement of wrinkles and skin disorders due to the loss of elastic fibers.

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