liu.seSearch for publications in DiVA
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Direct Evidence for Thioredoxin in Platelets: Studies on its release into human plasma
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0001-5082-6423
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Thioredoxin is a multifunctional protein involved in protecting cells against oxidative stress, and it acts as a co-cytokine and chemotactic factor during an immune response. Mechanisms behind thioredoxin release and transport in the blood are unknown, although the presence of thioredoxin in extracellular fluids is indisputable. We investigated the role of circulatory thioredoxin. We have previously found that release of selenoprotein thioredoxin reductase to the blood is induced by oxidative stress, and thioredoxin reductase is present in plasma (Söderberg, Sabaf, and Rosén, Cancer Res. 60, 2281; 2000). Both thioredoxin and protein disulfide isomerase are substrates for thioredoxin reductase, and both are exposed on the surface oflymphocytes and monocytes. Plasma thioredoxin level is elevated by oxidative stress, a condition often seen in burn patients and HIV carriers. Three of our present findings demonstrate that platelets contain thioredoxin and that circulatory thioredoxin is transported primarily in platelets in healthy blood donors. First, thioredoxin, but not thioredoxin reductase was detected in platelets by deconvolution fluorescence microscopy. Second, plasma thioredoxin concentration was sensitive to thrombocytolysis: a significant decrease in thioredoxin was seen in plasma samples (n = 20) pretreated with the platelet degranulation inhibitors theophylline, adenosine, dipyridamol, acetylsalicylic acid, and apyrase (thioredoxin decreased from 28 down to 8 ng/ml; p < 0.0001). Release of thioredoxin from platelets was induced by the thioloxidant diamide but not by platelet degranulation caused by thrombin, a thrombin peptide (SFLLRN) and collagen, ADP, or PMA-ionophore. Third, in thrombocytopenic and thrombocytemic patients, plasma levels of thioredoxin, but not ß- thromboglobulin, were strongly correlated with platelet numbers (correlation coefficient, r = 0.7). Summarizing, we found direct evidence that thioredoxin is present in platelets and is liberated by oxidative stress (diamide). This suggests that platelets are essential for prompt delivery of this cellular reducing agent to sites of injury, where it can activate coagulation factors and subsequently reduce or balance reactive oxygen species released by inflammatory macrophages.

National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-79886OAI: oai:DiVA.org:liu-79886DiVA: diva2:544474
Available from: 2012-08-14 Created: 2012-08-14 Last updated: 2017-09-22Bibliographically approved
In thesis
1. Thioredoxin system in normal and transformed human cells
Open this publication in new window or tab >>Thioredoxin system in normal and transformed human cells
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Thioredoxin (Trx) and thioredoxin reductase (TrxR), together with NADPH, constitute the Trx system, a major antioxidant entity that helps maintain a reducing environment within living cells. Trx designates a family of proteins that are related on the basis of structure and function. Human full-length Trx is a 12 kDa redox-active protein that contains the evolutionarily conserved active site sequence Cys-Gly Pro-Cys. Truncated Trx is a shorter, 10 kDa form of human Trx that shows complete homology to the N-terminal 80 or 84 amino acids of 12 kDa Trx. Truncated Trx displays no reducing activity, even though it contains an intact active site. Human TrxR is a homodimeric, FAD-containing, selenoprotein that reduces oxidized Trx back to the enzymatically active form by consuming NADPH. Expression of human Trx and TrxR is induced by a variety of stressors. The Trx system functions directly and indirectly in important biological features such as DNA synthesis, gene expression, co-cytokine activity, chemokine properties, scavenging reactive oxygen intermediates, apoptosis, and growth regulation.

Aims: The overall objective of this research was to examine the regulation and biological role of the Trx system in normal and transformed cells, and a possible connection with tumorigenesis. The specific aims were as follows: i) to study the presence and function of full-length and truncated Trx in normal and transformed human cells; ii) to assess the expression and function of TrxR; iii) to investigate the functional importance of truncated Trx; iv) to examine the regulatory mechanisms behind secretion of Trx, truncated Trx, and TrxR; v) to study full-length secreted Trx in human plasma.

Methods: Using Köhler and Milstein hybridoma technology, monoclonal antibodies against full-length and truncated Trx, as well as TrxR, were developed and applied in various immunological methods to detect the proteins in and secreted from normal and transformed human cells.

Results: Using selective monoclonal antibodies distinct intracellular localization of full-length and truncated Trx was shown. The latter was present in lower amounts, primarily associated with the plasma membrane; only small amounts of the 12 kDa were present on the plasma membrane. Moreover, we found that human TrxR was secreted from normal and transformed cells via a Golgi-dependent pathway. Using sensitive EUSPOT, TrxR release was quantified at the single-cell level and found to be inducible. TrxR was detected in plasma from healthy blood donors, and this protein was overexpressed in transformed cells, compared to their normal counterparts. Truncated 10 kDa Trx was present in and secreted from normal and transformed cells. Treatment of normal peripheral blood monocytes with LPS, IL-1, IFN-γ, PMA, ionomycin, and the thiol oxidant diarnide induced secretion of Trx. Trx was found within platelets, and liberation of this Trx was induced by diamide, implying a novel mechanism for release of Trx.

Conclusions: Monoclonal antibodies were generated against full-length and truncated Trx as well as TrxR and proved to be useful tools for investigating these proteins. Full-length and truncated Trx were found to have different cellular functions, suggesting that C-temrinal truncation regulates the activity of Trx. Secretion of TrxR and the presence of thls enzyme in plasma imply that the Trx system has both intracellular and extracellular functions. The presence of Trx in platelets indicates participation of the protein in coagulation and possibly also in the effects of platelets that maintain the reducing capacity of human plasma.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2000. 86 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 642
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25707 (URN)10083 (Local ID)91-7219-743-9 (ISBN)10083 (Archive number)10083 (OAI)
Public defence
2000-10-13, Berzeliussalen, Hälsouniversitetet, Linköping, 09:30 (Swedish)
Opponent
Supervisors
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-09-22Bibliographically approved

Open Access in DiVA

No full text

Authority records BETA

Lindahl, Tomas L.Rosén, Anders

Search in DiVA

By author/editor
Lindahl, Tomas L.Rosén, Anders
By organisation
Cell biologyFaculty of Health SciencesDepartment of Clinical Chemistry
Medical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 385 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf