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Differential proteomic analysis of platelets suggested possible signal cascades network in platelets treated with salvianolic acid B.

Ma C, Yao Y, Yue QX, Zhou XW, Yang PY, Wu WY, Guan SH, Jiang BH, Yang M, Liu X, Guo DA - PLoS ONE (2011)

Bottom Line: Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C.The regulation of SB on protein levels was confirmed by Western blotting.The signal cascades network induced by SB after its binding with integrin α2β1 was predicted.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People's Republic of China.

ABSTRACT

Background: Salvianolic acid B (SB) is an active component isolated from Danshen, a traditional Chinese medicine widely used for the treatment of cardiovascular disorders. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin α2β1. But, the signal cascades in platelets after SB binding are still not clear.

Methodology/principal findings: In the present study, a differential proteomic analysis (two-dimensional electrophoresis) was conducted to check the protein expression profiles of rat platelets with or without treatment of SB. Proteins altered in level after SB exposure were identified by MALDI-TOF MS/MS. Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. The regulation of SB on protein levels was confirmed by Western blotting. The signal cascades network induced by SB after its binding with integrin α2β1 was predicted. To certify the predicted network, binding affinity of SB to integrin α2β1 was checked in vitro and ex vivo in platelets. Furthermore, the effects of SB on protein levels of hsp70, coronin-1B and intracellular levels of Ca²+ and reactive oxygen species (ROS) were checked with or without pre-treatment of platelets using antibody against integrin α2β1. Electron microscopy study confirmed that SB affected cytoskeleton structure of platelets.

Conclusions/significance: Integrin α2β1 might be one of the direct target proteins of SB in platelets. The signal cascades network of SB after binding with integrin α2β1 might include regulation of intracellular Ca²+ level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets.

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Effect of SB treatment on the shape of platelets.A, Representative result of scanning electron microscopy analysis. (a), Magnification 1000X, scale bar = 20 µm. (b), Higher magnification views of the boxed fields in (a), magnification 6000X, scale bar = 5 µm. (c) Higher magnification views of the boxed fields in (b), magnification 12000X, scale bar = 2 µm. B, The percentage of platelets with spindle-like shape in total platelets (result from 10 fields of view of 3 different experiments). *Significant difference from the control group at P<0.05.
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pone-0014692-g006: Effect of SB treatment on the shape of platelets.A, Representative result of scanning electron microscopy analysis. (a), Magnification 1000X, scale bar = 20 µm. (b), Higher magnification views of the boxed fields in (a), magnification 6000X, scale bar = 5 µm. (c) Higher magnification views of the boxed fields in (b), magnification 12000X, scale bar = 2 µm. B, The percentage of platelets with spindle-like shape in total platelets (result from 10 fields of view of 3 different experiments). *Significant difference from the control group at P<0.05.

Mentions: Since the signal network suggested that SB might affect cytoskeleton structure and SB caused decrease in cell size as showed in flow cytometry analysis, further study using electron microscopic examination was conducted to check the effect of SB on platelets. As shown in A of Fig. 6, SB did induce change of cell shape in platelets. The control platelets was round or oval discoid while part of the platelets change to more slim shape like spindle-shaped after 100 µM SB treatment. Statistical analysis of the percentage of platelets with spindle-like shape in total platelets (result from 10 fields of view of 3 different experiments) indicated that the change in the shape of platelets after SB treatment was significant (B of Fig. 6).


Differential proteomic analysis of platelets suggested possible signal cascades network in platelets treated with salvianolic acid B.

Ma C, Yao Y, Yue QX, Zhou XW, Yang PY, Wu WY, Guan SH, Jiang BH, Yang M, Liu X, Guo DA - PLoS ONE (2011)

Effect of SB treatment on the shape of platelets.A, Representative result of scanning electron microscopy analysis. (a), Magnification 1000X, scale bar = 20 µm. (b), Higher magnification views of the boxed fields in (a), magnification 6000X, scale bar = 5 µm. (c) Higher magnification views of the boxed fields in (b), magnification 12000X, scale bar = 2 µm. B, The percentage of platelets with spindle-like shape in total platelets (result from 10 fields of view of 3 different experiments). *Significant difference from the control group at P<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040754&req=5

pone-0014692-g006: Effect of SB treatment on the shape of platelets.A, Representative result of scanning electron microscopy analysis. (a), Magnification 1000X, scale bar = 20 µm. (b), Higher magnification views of the boxed fields in (a), magnification 6000X, scale bar = 5 µm. (c) Higher magnification views of the boxed fields in (b), magnification 12000X, scale bar = 2 µm. B, The percentage of platelets with spindle-like shape in total platelets (result from 10 fields of view of 3 different experiments). *Significant difference from the control group at P<0.05.
Mentions: Since the signal network suggested that SB might affect cytoskeleton structure and SB caused decrease in cell size as showed in flow cytometry analysis, further study using electron microscopic examination was conducted to check the effect of SB on platelets. As shown in A of Fig. 6, SB did induce change of cell shape in platelets. The control platelets was round or oval discoid while part of the platelets change to more slim shape like spindle-shaped after 100 µM SB treatment. Statistical analysis of the percentage of platelets with spindle-like shape in total platelets (result from 10 fields of view of 3 different experiments) indicated that the change in the shape of platelets after SB treatment was significant (B of Fig. 6).

Bottom Line: Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C.The regulation of SB on protein levels was confirmed by Western blotting.The signal cascades network induced by SB after its binding with integrin α2β1 was predicted.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People's Republic of China.

ABSTRACT

Background: Salvianolic acid B (SB) is an active component isolated from Danshen, a traditional Chinese medicine widely used for the treatment of cardiovascular disorders. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin α2β1. But, the signal cascades in platelets after SB binding are still not clear.

Methodology/principal findings: In the present study, a differential proteomic analysis (two-dimensional electrophoresis) was conducted to check the protein expression profiles of rat platelets with or without treatment of SB. Proteins altered in level after SB exposure were identified by MALDI-TOF MS/MS. Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. The regulation of SB on protein levels was confirmed by Western blotting. The signal cascades network induced by SB after its binding with integrin α2β1 was predicted. To certify the predicted network, binding affinity of SB to integrin α2β1 was checked in vitro and ex vivo in platelets. Furthermore, the effects of SB on protein levels of hsp70, coronin-1B and intracellular levels of Ca²+ and reactive oxygen species (ROS) were checked with or without pre-treatment of platelets using antibody against integrin α2β1. Electron microscopy study confirmed that SB affected cytoskeleton structure of platelets.

Conclusions/significance: Integrin α2β1 might be one of the direct target proteins of SB in platelets. The signal cascades network of SB after binding with integrin α2β1 might include regulation of intracellular Ca²+ level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets.

Show MeSH
Related in: MedlinePlus