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The nitrosoamine dephostatin interacts with nitric oxide/cGMP signalling and modulates cytosolic calcium responses in human platelets
Linköpings universitet, Institutionen för medicin och vård, Farmakologi. Linköpings universitet, Hälsouniversitetet.
Linköpings universitet, Institutionen för medicin och vård, Farmakologi. Linköpings universitet, Hälsouniversitetet.
Linköpings universitet, Institutionen för molekylär och klinisk medicin, Medicinsk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
Linköpings universitet, Institutionen för medicin och vård, Farmakologi. Linköpings universitet, Hälsouniversitetet.
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Abstract [en]

1 The effects of the nitrosoamine dephostatin on cytosolic Ca2+ and nitric oxide (NO)/cGMP signallings in human platelets were investigated.

2 Dephostatin has been characterised as a protein tyrosine phosphatase (PTP) inhibitor, and may on that account affect platelet responses. However, western blot analysis revealed that dephostatin (1-100 µM) did not increase tyrosine-specific protein phosphorylation.

3 Dephostatin dose-dependently (0.003-3 µM) inhibited thrombin-, thrombin-receptor activating peptide-, and ADP-stimulated rises in cytosolic free Ca2+ concentration, [Ca2+]i. in fura-2-loaded platelets. Surprisingly, higher doses of dephostatin (10-30 µM) augmented thrombin-triggered Ca2+ response. This dual action of dephostatin mainly involved modulation of Ca2+ influx.

4 The results revealed that dephostatin antagonised NO/cGMP- and prostaglandin E1/cAMP-mediated inhibition of [Ca2+]1. The action of the thrornbin-neutralising peptide hirudin was, however, unaffected.

5 Dephostatin did not affect basal or NO-mediated rises in platelet cGMP content. On the other hand, dephostatin alone directly increased the phosphorylation of ser239 on vasodilator-stimulated phosphoprotein (VASP) and markedly enhanced NO/cGMP-dependent VASP phosphorylation.

6 Cell functional studies revealed that dephostatin amplified NO-induced inhibition of platelet aggregation. Opposite to that, dephostatin diminished the inhibitory action of NO on phosphatidylserine exposure.

7 In conclusion, the results revealed that dephostatin affects a wide range of mechanisms involved in Ca2+ homeostasis in platelets. These effects are apparently not due to inhibition of PTPs. However, enhancement of VASP phosphorylation represents another important molecular mechanism of dephostatin. Considering NO signalling, the results indicate that dephostatin may be an excellent tool when elucidating the relative importance of inhibition of Ca2+ versus VASP phosphorylation.

Nyckelord [en]
calcium, cGMP, dephostatin, nitric oxide, platelets, vasodilator-stimulated phosphoprotein
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:liu:diva-82119OAI: oai:DiVA.org:liu-82119DiVA, id: diva2:557946
Tillgänglig från: 2012-10-01 Skapad: 2012-10-01 Senast uppdaterad: 2012-10-01Bibliografiskt granskad
Ingår i avhandling
1. Pharmacological evaluation of the NO/cGMP signalling system
Öppna denna publikation i ny flik eller fönster >>Pharmacological evaluation of the NO/cGMP signalling system
2005 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Linköping: Linköping University Electronic Press, 2005. s. 82
Serie
Linköping University Medical Dissertations, ISSN 0345-0082 ; 919
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
urn:nbn:se:liu:diva-31869 (URN)17696 (Lokalt ID)91-85297-56-8 (ISBN)17696 (Arkivnummer)17696 (OAI)
Disputation
2005-11-24, Elsa Brändströmssalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Svenska)
Opponent
Tillgänglig från: 2009-10-09 Skapad: 2009-10-09 Senast uppdaterad: 2012-10-01Bibliografiskt granskad

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Asplund Persson, AnnaLarsson, JennyGrenegård, Magnus

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