Abstract
BackgroundSalvianolic 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 FindingsIn 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 Ca(2+) 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/SignificanceIntegrin 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 Ca(2+) level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets.
Highlights
Salvianolic acid B (SB) is one of the main components of Danshen, a popularly used traditional Chinese medicine for patients with coronary artery disease in both China and other countries including the U.S [1,2,3,4]
Comparing with our previous proteomic study using a mixture of salvianolic acids [11], the results suggested that the most important target-related proteins of SB might be heat shock-related 70 kDa protein 2, LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C
Fifteen upregulated protein spots and five down-regulated protein spots was found as indicated by the arrowed spots in A of Fig. 2 and by the expanded plots in B of Fig. 2
Summary
Salvianolic acid B (SB) is one of the main components of Danshen (root of Salvia miltiorrhiza), a popularly used traditional Chinese medicine for patients with coronary artery disease in both China and other countries including the U.S [1,2,3,4]. The mechanism of Danshen is still not clear but its inhibitive effect on platelet adhesion and aggregation might be one of the important bases for its cardiovascular effects. Our previous study showed that salvianolic acids isolated from Salvia miltiorrhiza could inhibit ADPinduced platelet aggregation of rat platelets both in vitro and ex vivo [10]. Our cooperation study result suggested that SB might inhibit platelet adhesion by a mechanism involving the integrin a2b1 (collagen receptor a2b1) [9]. Comparing with our previous proteomic study using a mixture of salvianolic acids [11], the results suggested that the most important target-related proteins of SB might be heat shock-related 70 kDa protein 2, LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin a2b1. The signal cascades in platelets after SB binding are still not clear
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