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α1-acid glycoprotein (AGP)-induced platelet shape change involvesthe Rho/Rho kinase signalling pathway
Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
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. Vol. 102, no 4, 694-703 p.
Keyword [en]
shape change, Rho-kinase, Platelets, α1-acid glycoprotein
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-20474DOI: 10.1160/TH09-03-0156ISI: 000271039400013OAI: oai:DiVA.org:liu-20474DiVA: diva2:234628
Available from: 2009-09-09 Created: 2009-09-09 Last updated: 2014-01-13Bibliographically approved
In thesis
1. α1-acid glycoprotein modulates the function of human neutrophils and platelets
Open this publication in new window or tab >>α1-acid glycoprotein modulates the function of human neutrophils and platelets
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:nbn:se:liu:diva-20476 (URN)978-91-7393-614-9 (ISBN)
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

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Gunnarsson, PeterLevander, LouisePåhlsson, PeterGrenegård, Magnus

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