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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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Prediction and certification of signal network of SB after binding with integrin α2β1.A. Predicted signal network of SB. The red dot illustrates integrin α2β1 while the blue dots are un-direct target-related proteins found in the proteomic study. The intermediate partners are shown in grey. B. Revised signal network of SB. C. Real time binding affinity measurement of SB to the integrin α2-I protein in vitro using Biacore 3000. Representative sensorgrams were obtained from injections of SB at concentrations of 16.8, 24, 34.3, 49, 70 and 100 µM (curves from bottom to top) over the immobilized integrin α2-I surface. D. Influence of SB treatment on binding between integrin α2β1 on platelets and its specific FITC-conjugated antibody. Both representative flow cytometry analysis result and quantitative results were shown. E. Western blotting analysis of protein levels of coronin-1 B, hsp70 and p-hsp70 in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. F. Flow cytometry analysis result of intracellular Ca(2+) level in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both respresentative flow cytometry analysis result and quantitative results were shown. G. Flow cytometry analysis result of intracellular ROS level in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both representative flow cytometry analysis result and quantitative results were shown. H. Flow cytometry analysis result of cell size of platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both representative flow cytometry analysis result and quantitative results (FSC values) were shown. *Significant difference from the control group at P<0.05, **Significant difference from the control group at P<0.01, #Significant difference from the antibody treatment group at P<0.05.
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pone-0014692-g005: Prediction and certification of signal network of SB after binding with integrin α2β1.A. Predicted signal network of SB. The red dot illustrates integrin α2β1 while the blue dots are un-direct target-related proteins found in the proteomic study. The intermediate partners are shown in grey. B. Revised signal network of SB. C. Real time binding affinity measurement of SB to the integrin α2-I protein in vitro using Biacore 3000. Representative sensorgrams were obtained from injections of SB at concentrations of 16.8, 24, 34.3, 49, 70 and 100 µM (curves from bottom to top) over the immobilized integrin α2-I surface. D. Influence of SB treatment on binding between integrin α2β1 on platelets and its specific FITC-conjugated antibody. Both representative flow cytometry analysis result and quantitative results were shown. E. Western blotting analysis of protein levels of coronin-1 B, hsp70 and p-hsp70 in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. F. Flow cytometry analysis result of intracellular Ca(2+) level in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both respresentative flow cytometry analysis result and quantitative results were shown. G. Flow cytometry analysis result of intracellular ROS level in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both representative flow cytometry analysis result and quantitative results were shown. H. Flow cytometry analysis result of cell size of platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both representative flow cytometry analysis result and quantitative results (FSC values) were shown. *Significant difference from the control group at P<0.05, **Significant difference from the control group at P<0.01, #Significant difference from the antibody treatment group at P<0.05.

Mentions: In our previous proteomic study, the effect of a mixture of salvianolic acids on the protein expression profile of platelets was checked and 12 differentially expressed proteins were found [11]. By comparing the 12 proteins found in the previous study with the 20 proteins found in the present study, we could identify 6 proteins that appeared in the both studies. They were heat shock-related 70 kDa protein 2 (heat shock protein 2), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. Interestingly, these 6 proteins were closely related to integrin α2β1, the possible direct target protein of SB identified in our previous study [9]. The possible signal cascades network in platelets after binding of SB to integrin α2β1 was predicted (A of Fig. 5). After binding to integrin α2β1, SB might induce regulation of its un-direct target-related protein through direct activation (heat shock protein 2), regulation of Ca(2+) level (copine I), ROS level (peroxiredoxin-2) and reorganization of cytoskeleton structure (LIM domain protein CLP-36, coronin-1 B and cytoplasmic dynein intermediate chain 2C).


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)

Prediction and certification of signal network of SB after binding with integrin α2β1.A. Predicted signal network of SB. The red dot illustrates integrin α2β1 while the blue dots are un-direct target-related proteins found in the proteomic study. The intermediate partners are shown in grey. B. Revised signal network of SB. C. Real time binding affinity measurement of SB to the integrin α2-I protein in vitro using Biacore 3000. Representative sensorgrams were obtained from injections of SB at concentrations of 16.8, 24, 34.3, 49, 70 and 100 µM (curves from bottom to top) over the immobilized integrin α2-I surface. D. Influence of SB treatment on binding between integrin α2β1 on platelets and its specific FITC-conjugated antibody. Both representative flow cytometry analysis result and quantitative results were shown. E. Western blotting analysis of protein levels of coronin-1 B, hsp70 and p-hsp70 in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. F. Flow cytometry analysis result of intracellular Ca(2+) level in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both respresentative flow cytometry analysis result and quantitative results were shown. G. Flow cytometry analysis result of intracellular ROS level in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both representative flow cytometry analysis result and quantitative results were shown. H. Flow cytometry analysis result of cell size of platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both representative flow cytometry analysis result and quantitative results (FSC values) were shown. *Significant difference from the control group at P<0.05, **Significant difference from the control group at P<0.01, #Significant difference from the antibody treatment group at P<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040754&req=5

pone-0014692-g005: Prediction and certification of signal network of SB after binding with integrin α2β1.A. Predicted signal network of SB. The red dot illustrates integrin α2β1 while the blue dots are un-direct target-related proteins found in the proteomic study. The intermediate partners are shown in grey. B. Revised signal network of SB. C. Real time binding affinity measurement of SB to the integrin α2-I protein in vitro using Biacore 3000. Representative sensorgrams were obtained from injections of SB at concentrations of 16.8, 24, 34.3, 49, 70 and 100 µM (curves from bottom to top) over the immobilized integrin α2-I surface. D. Influence of SB treatment on binding between integrin α2β1 on platelets and its specific FITC-conjugated antibody. Both representative flow cytometry analysis result and quantitative results were shown. E. Western blotting analysis of protein levels of coronin-1 B, hsp70 and p-hsp70 in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. F. Flow cytometry analysis result of intracellular Ca(2+) level in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both respresentative flow cytometry analysis result and quantitative results were shown. G. Flow cytometry analysis result of intracellular ROS level in platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both representative flow cytometry analysis result and quantitative results were shown. H. Flow cytometry analysis result of cell size of platelets under SB treatment with or without pretreatment of antibody against integrin α2β1. Both representative flow cytometry analysis result and quantitative results (FSC values) were shown. *Significant difference from the control group at P<0.05, **Significant difference from the control group at P<0.01, #Significant difference from the antibody treatment group at P<0.05.
Mentions: In our previous proteomic study, the effect of a mixture of salvianolic acids on the protein expression profile of platelets was checked and 12 differentially expressed proteins were found [11]. By comparing the 12 proteins found in the previous study with the 20 proteins found in the present study, we could identify 6 proteins that appeared in the both studies. They were heat shock-related 70 kDa protein 2 (heat shock protein 2), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. Interestingly, these 6 proteins were closely related to integrin α2β1, the possible direct target protein of SB identified in our previous study [9]. The possible signal cascades network in platelets after binding of SB to integrin α2β1 was predicted (A of Fig. 5). After binding to integrin α2β1, SB might induce regulation of its un-direct target-related protein through direct activation (heat shock protein 2), regulation of Ca(2+) level (copine I), ROS level (peroxiredoxin-2) and reorganization of cytoskeleton structure (LIM domain protein CLP-36, coronin-1 B and cytoplasmic dynein intermediate chain 2C).

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