| 1. |
- Balogh, Istvan, et al.
(författare)
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Analysis of Gas6 in Human Platelets and Plasma.
- 2005
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1524-4636. ; 25:6, s. 1280-1286
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Tidskriftsartikel (refereegranskat)abstract
- Objective - Gas6 is a member of the vitamin K-dependent protein family. Gas6- deficient mice were found to be resistant to thrombosis because of defective platelet function. Mouse Gas6 was demonstrated to be present in platelets and found to be involved in platelet aggregation. The aim of this study was to investigate the presence of Gas6 in human platelets and plasma and determine its role in platelet function. Methods and Results - The presence of Gas6 in human platelets and plasma was analyzed using sensitive immunologic methods. Mass spectrometry and ELISA were used to identify and quantify Gas6 in plasma. Gas6 was demonstrated to be present in human plasma, at a concentration determined to be 13 to 23 ng/mL (0.16 to 0.28 nM). Furthermore, plasma Gas6 levels were found to be lower in patients administered with warfarin. However, Gas6 was undetectable in human platelets. Conclusions - This is the first report to identify and quantify Gas6 in human plasma. However, Gas6 protein was not detected in human platelets, suggesting that any potential platelet-specific function could be because of Gas6 from the circulation. These findings open up new directions regarding the role of Gas6 in normal and pathophysiological situations such as inflammation, autoimmune disease, thrombosis and arteriosclerosis.
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| 2. |
- Ekman, Carl, et al.
(författare)
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Gas6 is complexed to soluble tyrosine kinase receptor Axl in human blood.
- 2010
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Ingår i: Journal of Thrombosis and Haemostasis. - : Elsevier BV. - 1538-7933 .- 1538-7836. ; 8:4, s. 838-844
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Tidskriftsartikel (refereegranskat)abstract
- Summary Background: The vitamin K-dependent Gas6 protein (product of growth arrest specific gene 6) binds to, and activates the TAM receptor tyrosine kinases Tyro3, Axl, and Mer. Gas6 and the TAM receptors have been suggested to be important for primary platelet functions, but Gas6 cannot be found in human platelets. However, Gas6 is present in human plasma at a concentration of around 0.2 nM, which is 1,000-fold lower than that of the homologous protein S. The Axl and Mer receptors can be cleaved close to the cell membrane, yielding soluble molecules consisting of the extracellular parts of the receptors. Objective: To investigate if soluble Axl (sAxl) is present in human serum and plasma and if Gas6 circulates in complex with sAxl. Methods: We expressed recombinant sAxl, raised antibodies, developed and validated an ELISA for Axl. Serum and plasma were analyzed using ELISAs for Gas6, Axl, and sAxl-Gas6 complexes. Serum was gel filtered and fractions analyzed by the different ELISAs to determine if Gas6 in serum is free or complexed. Immunoprecipitation was used to investigate binding between Gas6 and sAxl in serum. Results: sAxl is present in serum and plasma at around 0.6 nM and all Gas6 is bound to sAxl. No complexes between Gas6 and the soluble forms of Mer and Tyro3 could be detected, indicating that sAxl is the physiological binder of Gas6 in human serum. Conclusions: Gas6 in circulation is bound to sAxl suggesting circulating Gas6 to be inhibited and incapable of stimulating the TAM receptors.
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| 3. |
- Hafizi, Sassan, et al.
(författare)
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The Ran binding protein RanBPM interacts with Axl and Sky receptor tyrosine kinases.
- 2005
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Ingår i: International Journal of Biochemistry & Cell Biology. - : Elsevier BV. - 1878-5875 .- 1357-2725. ; 37:Jun 16, s. 2344-2356
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Tidskriftsartikel (refereegranskat)abstract
- Axl belongs to a particular subfamily of transmembrane receptor tyrosine kinases, the biological ligand for which is the growth/survival factor Gas6. However, little is known about the molecular mechanisms for Axl activation and signal transduction. We have previously identified a novel interaction between the intracellular domain of Axl and Ran binding protein in microtubule organising centre (RanBPM). In the present study, we investigated further the nature of the RanBPM interaction with Axl. A wide distribution of RanBPM mRNA expression in human tissues and various human cancer cell lines was detected. The strength of interaction of both proteins in yeast was comparable to that with the other Axl-binding proteins phosphatidylinositol 3-kinase and Grb2. A truncated version of RanBPM with the SPRY-LisH domain region omitted failed to interact with Axl in yeast. RanBPM was also found to interact in yeast with the Axl homologue, Sky/Tyro3. The interaction between Axl intracellular domain and RanBPM was reproduced in coimmunoprecipitation experiments in both cell-free and mammalian cell systems. Furthermore, coimmunoprecipitation revealed endogenous Axl and RanBPM to interact in several mammalian cell lines in a constitutive manner. Stimulation of COS cells with Gas6 caused increased Axl tyrosine phosphorylation although appeared not to influence the RanBPM-Axl association. In conclusion, we have identified and characterised a novel interaction between RanBPM and the related receptor tyrosine kinases, Axl and Sky. This novel insight into the signalling interactions of Axl and Sky may shed further light on their suspected roles in tumourigenesis, inflammation as well as other cell proliferative diseases.
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| 4. |
- Stenhoff, Jonas
(författare)
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Structural and Functional Studies of Gas6
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The vitamin K-dependent and ubiquitously expressed 82 kDa glycoprotein product of the growth-arrest-specific-gene 6, Gas6, shows 44% sequence identity to the anticoagulant protein, protein S. Both proteins share further the same domain organization. A short loop region, 4 epidermal growth factor-like modules and an SHBG-like region follow the N-terminal Gla-domain, which confers membrane-binding capability. The Gas6 SHBG interacts with the receptor tyrosine kinases of the Tyro3 subfamily, Axl, Tyro3 and Mer. As a ligand to these receptors Gas6 is implicated in mediating for example growth, survival, migration, clearance of apoptotic cells and platelet response amplification. The investigations described in this thesis were aimed at shedding further light on the structure and function of these relatively recently discovered proteins. In study I, purified Gas6 was added to Gas6 deficient mouse cardiac fibroblasts, which responded by mitogenesis and increased survival. Employing a sensitive ELISA, in study II, Gas6 could be detected in human circulation (? 18 ng/mL) but not in human platelets. In study III it's shown that most of the Gas6 present in human circulation likely is complex bound to soluble extracellular domains of Axl. We further show that Gas6 ability to interact with its receptors relies on calcium. Study IV shows the high tendency of Gas6 to form multimers during purification. The multimers, which fail to interact with Axl, show slow association but high affinity binding towards bilayers containing phosphatidylserine. By contrast the monomers display fast association, low affinity binding and preference for bilayers containing phosphatidylethanolamine in addition to phosphatidylserine.
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