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α1-acid glycoprotein modulates the function of human neutrophils and platelets
Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Arts and Sciences.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The acute-phase protein α1-acid glycoprotein (AGP; orosomucoid) was initially identified andcharacterised in the 1950s. The normal plasma concentration is around 0.5-1 mg/ml butduring inflammation the concentration increase several fold and the carbohydrate compositionof the protein changes. AGP is a highly glycosylated protein with 45 % of the molecularweight consisting of glycans. These glycans are believed to be of importance for the functionof the protein. However, the precise physiological role of AGP is still unclear.

The present thesis reveals that AGP at physiological concentration induce calcium elevationin human neutrophils and platelets. In neutrophils this response was enhanced several fold ifsurface L-selectin was pre-engaged. Our results showed that this L-selectin-mediatedamplification was abolished if the neutrophils were pre-treated with Src or phosphoinositide3-kinase (PI3K) inhibitors. AGP alone did not induce production of reactive oxygen species(ROS) in neutrophils. However, if the neutrophils were activated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) a subsequent addition of AGP caused aprominent ROS response. Moreover, both the calcium rise and the ROS response weredepending on sialic acid residues on AGP. In the case of calcium elevation we defined thereceptor as sialic-acid-binding immunoglobulin-like lectin (Siglec)-5 on the neutrophil.

In platelets, AGP induced a Rho-kinase dependent phosphorylation of myosin phosphatasetarget subunit-1 (MYPT1) and a minor calcium response. This resulted in a prominent plateletshape change (i.e. spherical shape and granule centralization) recorded as change in lighttransmission and by differential interference contrast (DIC) microscopy. The shape changecaused by AGP was strongly suppressed by inhibition of Rho-kinase and abolished by Rhokinaseinhibition combined with chelation of intracellular calcium. No other manifestations ofplatelet activation like aggregation or secretion were registered. Opposite to neutrophils theeffect of AGP on platelets was not mediated by an interaction between sialic acid and siglecmolecules. However, the results indicated that AGP may bind to a collagen/thrombospondin-1surface receptor. Endogenous inhibitors like nitric oxide (NO) and adenosine abolished theAGP-induced platelet shape change. The antagonizing action of NO on shape change causedby AGP was long acting. In comparison, other aspects of agonist-induced platelet activation(e.g. intracellular calcium elevations) are only transiently suppressed by NO. This indicatesthat endothelium-derived NO may play a crucial role to counter balance the effect of AGP in vivo.

Take together the results in this thesis reveal that AGP can initiate intracellular signalling andmodulate functional responses in neutrophils and platelets.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 65 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1135
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-20476ISBN: 978-91-7393-614-9 (print)OAI: oai:DiVA.org:liu-20476DiVA: diva2:234640
Public defence
2009-09-04, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-09-09 Created: 2009-09-09 Last updated: 2012-02-03Bibliographically approved
List of papers
1. The acute-phase protein alpha 1-acid glycoprotein (AGP) induces rises in cytosolic Ca2+ in neutrophil granulocytes via sialic acid binding immunoglobulin-like lectins (siglecs)
Open this publication in new window or tab >>The acute-phase protein alpha 1-acid glycoprotein (AGP) induces rises in cytosolic Ca2+ in neutrophil granulocytes via sialic acid binding immunoglobulin-like lectins (siglecs)
2007 (English)In: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, ISSN 1530-6860, Vol. 21, no 14, 4059-4069 p.Article in journal (Refereed) Published
Abstract [en]

We studied whether the acute-phase protein alpha1-acid glycoprotein (AGP) induces rises in [Ca2+]i in neutrophils and sought to identify the corresponding AGP receptor (or receptors). We found that AGP elicited a minimal rise in [Ca2+]i in Fura-2-loaded neutrophils, and this response was markedly enhanced by pretreatment with anti-L-selectin antibodies. (The EC50 value of the AGP-induced Ca2+ response was 9 microg/ml.) Activation of phospholipase-C, Src tyrosine kinases, and PI3 kinases proved to be essential for the AGP-mediated increase in [Ca2+]i, whereas the p38 MAPK and SYK signaling pathways were not involved. Furthermore, antibodies against sialic acid binding, immunoglobulin-like lectin 5 (Siglec-5) and oligosaccharide 3'-sialyl-lactose both antagonized the AGP-induced response and caused an immediate increase in [Ca2+]i in anti-L-selectin-treated neutrophils, which indicates a signaling capacity of Siglec-5. We used modified forms of AGP (treated with mild periodate or neuraminidase) to establish the importance of sialic acid residues. The modified forms of AGP caused a much smaller rise in [Ca2+]i than did unaltered AGP. Affinity chromatography confirmed that unchanged AGP, but not neuraminidase-treated AGP, bound to Siglec-5. Our report provides the first evidence for a signaling capacity by AGP through a defined receptor. Pre-engagement of L-selectin significantly enhanced this signaling capacity.

Keyword
Orosomucoid, plasma protein, calcium signaling, carbohydrate, L-selectin, phagocyte
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20262 (URN)10.1096/fj.07-8534com (DOI)17675532 (PubMedID)
Available from: 2009-09-01 Created: 2009-09-01 Last updated: 2009-09-09Bibliographically approved
2. Sialic Acid Dependent and Independent Effects of alpha 1-Acid Glycoprotein (AGP) on Human Platelets
Open this publication in new window or tab >>Sialic Acid Dependent and Independent Effects of alpha 1-Acid Glycoprotein (AGP) on Human Platelets
2008 (English)In: 2008 Meeting of the Society for Glycobiology, 2008, Vol. 18, no 11, 990-990 p.Conference paper, Published paper (Other academic)
Abstract [en]

Objective: We have recently shown that terminal sialic acid residues are essential for α1-acidglycoprotein (AGP)-induced Ca2+ mobilization in neutrophils. The aim of the present studywas to establish the importance of sialic acid-residues on AGP in modulating humanneutrophil functions, with emphasis on the generation of reactive oxygen species (ROS).Material and methods: ROS were measured by luminol-enhanced chemiluminescence inisolated human neutrophils.

Results: We found that AGP did not provoke ROS generation in resting or L-selectin presensitizedneutrophils. Moreover, AGP did not affect the N-formyl-methionyl-leucylphenylalanine(fMLP)-induced ROS generation but it slightly suppressed opsonized zymosaninducedresponses. However, when the neutrophils were pre-stimulated with fMLP, thefollowing addition of AGP provoked a marked ROS response. Dose-response studies and timestudies revealed that the ROS generating capacity of AGP was maximal at a concentration of0.05 mg/ml and when given 3-10 min after addition of fMLP. A desialylated form of AGP orpre-treatment of neutrophils with 3’- and 6’-sialyllactose caused a substantial lower ROSresponse in neutrophils pre-stimulated with fMLP.

Conclusions: Our data show that AGP can stimulate a second ROS response in fMLP preactivatedneutrophils and that terminal sialic acid residues on AGP play a crucial role in thisregard.

Keyword
α1-acid glycoprotein, neutrophils, reactive oxygen species, sialic acid
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16353 (URN)
Available from: 2009-01-16 Created: 2009-01-16 Last updated: 2009-09-09
3. Characterisation of GEA 3175 on human platelets: comparison with S-nitroso-N-acetyl-D,L-penicillamine
Open this publication in new window or tab >>Characterisation of GEA 3175 on human platelets: comparison with S-nitroso-N-acetyl-D,L-penicillamine
2004 (English)In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 496, no 1-3, 1-9 p.Article in journal (Refereed) Published
Abstract [en]

By comparing the effect of two nitric oxide (NO)-containing compounds, we found that S-nitroso-N-acetyl-d,l-penicillamine (SNAP), but not GEA 3175 (1,2,3,4-Oxatriazolium,3-(3-chloro-2-metylphenyl)-5-[[(4-methylphenyl)sulfonyl]amino]-, hydroxide inner salt), released NO. Despite this, both drugs elevated cyclic guanosine 3′,5′-monophosphate (cGMP) levels in human platelets. However, SNAP was more effective after short exposure times (5 and 20 s). The compounds also inhibited thrombin-induced rises in cytosolic Ca2+. Time studies revealed that the action of SNAP rapidly declined by increasing the length of incubation (from 5 s to 30 min). This desensibilisation phenomenon mainly involved the release of Ca2+ from intracellular stores. In comparison, GEA 3175-induced inhibition of cytosolic Ca2+ signalling was much more long-lasting. The soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reversed the effect of GEA 3175 on cytosolic Ca2+. Consequently, this inhibition depends solely on the increase in cGMP. In summary, differences between GEA 3175 and SNAP were observed in NO releasing, cGMP elevating and Ca2+ suppressive properties.

Keyword
NO (Nitric Oxide), cGMP (cyclic guanosine 3′, 5′-monophosphate), Calcium, Aggregation, Platelet, SNAP (S-nitroso-N-acetyl-d, l-penicillamine), GEA 3175
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20473 (URN)10.1016/j.ejphar.2004.06.002 (DOI)15288569 (PubMedID)
Available from: 2009-09-09 Created: 2009-09-09 Last updated: 2017-12-13Bibliographically approved
4. α1-acid glycoprotein (AGP)-induced platelet shape change involvesthe Rho/Rho kinase signalling pathway
Open this publication in new window or tab >>α1-acid glycoprotein (AGP)-induced platelet shape change involvesthe Rho/Rho kinase signalling pathway
2009 (English)In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 102, no 4, 694-703 p.Article in journal (Refereed) Published
Abstract [en]

α1-acid glycoprotein (AGP) is an acute-phase protein that contributes to inflammation processes. The role of AGP in platelet activation and thrombosis is, however, largely unknown. Therefore, we thoroughly investigated the effects of AGP on human platelets. Platelets were isolated from healthy volunteers and subsequently exposed to AGP. Platelet responses were monitored as change in light transmission, intracellular calcium concentration, light microscopy and protein phosphorylation by Western blot. We found that AGP induced platelet shape change independently of a second release of adenine nucleotides or thromboxane A2, and that effect was abolished by endotheliumderived platelet inhibitors such as nitric oxide (NO) and adenosine. Furthermore, AGP triggered a minor calcium response and a pronounced Rho/Rho-kinase-dependent increase in Thr696 phosphorylation of myosin phosphatase target subunit 1 (MYPT1). Moreover, the Rho/Rho-kinase inhibitor Y-27632 significantly decreased the AGP-induced shape change. The results also showed that the AGP-elicited shape change was antagonised by pretreatment with low doses of collagen and thrombospondin- 1. Our results describe a novel mechanism by which AGP stimulates platelet shape change via activation of the Rho/Rhokinase signalling pathway. Physiological important platelet inhibitors, such as NO, completely counterbalance the effect of AGP. Hence, the present study indicates that AGP directly contributes to platelet activation, which in turn might have an impact in physiological haemostasis and/or pathological thrombosis.

Place, publisher, year, edition, pages
Stuttgart, Germany: Schattauer Gmbh, 2009
Keyword
shape change, Rho-kinase, Platelets, α1-acid glycoprotein
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20474 (URN)10.1160/TH09-03-0156 (DOI)000271039400013 ()
Available from: 2009-09-09 Created: 2009-09-09 Last updated: 2017-12-13Bibliographically approved

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