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Characterisation of GEA 3175 on human platelets: comparison with S-nitroso-N-acetyl-D,L-penicillamine
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
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.

Place, publisher, year, edition, pages
2004. Vol. 496, no 1-3, 1-9 p.
Keyword [en]
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: urn:nbn:se:liu:diva-20473DOI: 10.1016/j.ejphar.2004.06.002PubMedID: 15288569OAI: oai:DiVA.org:liu-20473DiVA: diva2:234626
Available from: 2009-09-09 Created: 2009-09-09 Last updated: 2017-12-13Bibliographically 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
2. Pharmacological evaluation of the NO/cGMP signalling system
Open this publication in new window or tab >>Pharmacological evaluation of the NO/cGMP signalling system
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Platelet activation and inhibition are tightly balanced processes, regulated by various endogenous molecules. In this regard, the endothelium plays a key role in mediating inhibition of platelets by releasing nitric oxide (NO) and cAMP-elevating prostaglandins.

The present study put emphasis on drugs that activate directly or modulate the NO/cGMP signalling pathway.

The specific aims were i) to compare two different NO-containing compounds; namely the S-nitrosothiol SNAP and the oxatriazole derivative GEA 3175; ii) to evaluate the mechanism of drug action of the nitrosoamine dephostatin; iii) to investigate cross-talk mechanisms between the NO/cGMP and the cAMP signalling pathways. These studies were perfom1ed using human blood platelets and iliac arteries obtained from pigs.

The present data revealed that SNAP but not GEA 3175 released detectable amounts of NO. Despite that, both compounds elevated cGMP, inhibited rises in [Ca2+]i and stimulated phosphorylation of vasodilator stimulated phosphoprotein (VASP). Moreover, the results showed that NO/cGMP-induced inhibition of Ca2+ responses, but not NO/cGMP-mediated V ASP phosphorylation, was rapidly desensitised. The results showed that an unstable NO-donor more effectively induced desensitisation.

Dephostatin, originally characterised as a protein tyrosine phosphatase (PTP) inhibitor, was found to modulate the NO/cGMP signalling in a complex way. More specifically, dephostatin is an effective NO-scavenger and surprisingly, it also serves as a source of NO and thereby induces cGMPmediated vasorelaxation. In platelets, dephostatin abolished NO/cGMP-mediated inhibition of cytosolic Ca2+, but augmented NO/cGMP-induced VASP phosphorylation.

cGMP-induced inhibition of type 3 phosphodiesterases (PDE3) enhanced adenosine-mediated inhibition of platelets. This effect was of main importance for the suppression of several platelet responses. However, inhibition of Ca2+ influx was another cGMP-specific mechanism contributing to a powerful inhibition of the platelets.

In conclusion: The present results show that release of NO from drug molecules is not a prerequisite for NO/cGMP-mediated cellular and intracellular responses. On the contrary, drug stability in terms of NO-release appears to be crucial in platelet desensitisation to NO. Therefore it is important to gain more knowledge about the NO/cGMP-signalling pathway regarding future drug design of NO-containing drugs. The findings presented in this thesis suggest that dephostatin can prove to be a very useful tool in this area of research.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2005. 82 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 919
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-31869 (URN)17696 (Local ID)91-85297-56-8 (ISBN)17696 (Archive number)17696 (OAI)
Public defence
2005-11-24, Elsa Brändströmssalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-01Bibliographically approved

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Asplund Persson, Anna KPalmér, LouiseGunnarsson, PeterGrenegård, Magnus

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