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Inhibitory effect of caffeic acid on ADP-induced thrombus formation and platelet activation involves mitogen-activated protein kinases.

Lu Y, Li Q, Liu YY, Sun K, Fan JY, Wang CS, Han JY - Sci Rep (2015)

Bottom Line: In this study, we investigated the antithrombotic effect of CA in mouse cerebral arterioles and venules using intravital microscopy.Our results demonstrated that CA (1.25-5 mg/kg) significantly inhibited thrombus formation in vivo.Additionally, CA attenuated p38, ERK, and JNK activation, and enhanced cAMP levels.

View Article: PubMed Central - PubMed

Affiliation: Department of gynaecology, Beijing Royal Integrative Medicine Hospital, Beijing, China.

ABSTRACT
Caffeic acid (CA), one of the active constituents of Radix Salvia miltiorrhizae, exhibits antioxidant and anti-inflammatory activities. However, few studies have assessed the ability of CA to inhibit platelet mediated thrombus generation in vivo. In this study, we investigated the antithrombotic effect of CA in mouse cerebral arterioles and venules using intravital microscopy. The antiplatelet activity of CA in ADP stimulated mouse platelets in vitro was also examined in attempt to explore the underlying mechanism. Our results demonstrated that CA (1.25-5 mg/kg) significantly inhibited thrombus formation in vivo. In vitro, CA (25-100 μM) inhibited ADP-induced platelet aggregation, P-selectin expression, ATP release, Ca(2+) mobilization, and integrin αIIbβ3 activation. Additionally, CA attenuated p38, ERK, and JNK activation, and enhanced cAMP levels. Taken together, these data provide evidence for the inhibition of CA on platelet-mediated thrombosis in vivo, which is, at least partly, mediated by interference in phosphorylation of ERK, p38, and JNK leading to elevation of cAMP and down-regulation of P-selectin expression and αIIbβ3 activation. These results suggest that CA may have potential for the treatment of aberrant platelet activation-related diseases.

No MeSH data available.


Related in: MedlinePlus

CA inhibits ADP-induced conformational changes in platelet integrin αIIbβ3.Washed platelets were preincubated with CA or vehicle at 37 °C for 20 minutes followed by addition of fluorescent anti-integrin αIIbβ3 monoclonal antibody (JON/A), and stimulated with ADP for 20 min. Positive αIIbβ3 expression was detected by flow cytometry. (A) Result from a typical experiment. (B) Quantitative evaluation of the data from at least four independent experiments. Data are expressed as means ± SEM. *p < 0.05 vs control group; #p < 0.05 vs ADP group.
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f8: CA inhibits ADP-induced conformational changes in platelet integrin αIIbβ3.Washed platelets were preincubated with CA or vehicle at 37 °C for 20 minutes followed by addition of fluorescent anti-integrin αIIbβ3 monoclonal antibody (JON/A), and stimulated with ADP for 20 min. Positive αIIbβ3 expression was detected by flow cytometry. (A) Result from a typical experiment. (B) Quantitative evaluation of the data from at least four independent experiments. Data are expressed as means ± SEM. *p < 0.05 vs control group; #p < 0.05 vs ADP group.

Mentions: The interaction of agonists with platelet receptors triggers inside-out signals that result in conformational changes in the αIIbβ3 integrin, leading to the exposure of neoepitopes and facilitating fibrinogen binding. One of the newly exposed epitopes is recognized by antibody JON/A and the binding of JON/A to the platelet surface is used as a measure of αIIbβ3 integrin activation. Figure 8A shows the fluorescence signal of PE-conjugated JON/A bound to the activated αIIbβ3 receptor of platelets in the presence of various concentration of CA. ADP (20 μM) stimulation evoked a significantly greater αIIbβ3 activation in platelets compared with control group. On the other hand, CA pretreatment markedly suppressed ADP-induced activation of αIIbβ3 being statistically significant at 100 μM (Fig. 8B). This result suggests that CA pretreatment hinders the conformational changes in αIIbβ3 that are required to reveal the binding site for fibrinogen and, most likely, post-ligand occupancy events such as changes in the shape and spreading of the platelets (Supplemental Fig. 3).


Inhibitory effect of caffeic acid on ADP-induced thrombus formation and platelet activation involves mitogen-activated protein kinases.

Lu Y, Li Q, Liu YY, Sun K, Fan JY, Wang CS, Han JY - Sci Rep (2015)

CA inhibits ADP-induced conformational changes in platelet integrin αIIbβ3.Washed platelets were preincubated with CA or vehicle at 37 °C for 20 minutes followed by addition of fluorescent anti-integrin αIIbβ3 monoclonal antibody (JON/A), and stimulated with ADP for 20 min. Positive αIIbβ3 expression was detected by flow cytometry. (A) Result from a typical experiment. (B) Quantitative evaluation of the data from at least four independent experiments. Data are expressed as means ± SEM. *p < 0.05 vs control group; #p < 0.05 vs ADP group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561902&req=5

f8: CA inhibits ADP-induced conformational changes in platelet integrin αIIbβ3.Washed platelets were preincubated with CA or vehicle at 37 °C for 20 minutes followed by addition of fluorescent anti-integrin αIIbβ3 monoclonal antibody (JON/A), and stimulated with ADP for 20 min. Positive αIIbβ3 expression was detected by flow cytometry. (A) Result from a typical experiment. (B) Quantitative evaluation of the data from at least four independent experiments. Data are expressed as means ± SEM. *p < 0.05 vs control group; #p < 0.05 vs ADP group.
Mentions: The interaction of agonists with platelet receptors triggers inside-out signals that result in conformational changes in the αIIbβ3 integrin, leading to the exposure of neoepitopes and facilitating fibrinogen binding. One of the newly exposed epitopes is recognized by antibody JON/A and the binding of JON/A to the platelet surface is used as a measure of αIIbβ3 integrin activation. Figure 8A shows the fluorescence signal of PE-conjugated JON/A bound to the activated αIIbβ3 receptor of platelets in the presence of various concentration of CA. ADP (20 μM) stimulation evoked a significantly greater αIIbβ3 activation in platelets compared with control group. On the other hand, CA pretreatment markedly suppressed ADP-induced activation of αIIbβ3 being statistically significant at 100 μM (Fig. 8B). This result suggests that CA pretreatment hinders the conformational changes in αIIbβ3 that are required to reveal the binding site for fibrinogen and, most likely, post-ligand occupancy events such as changes in the shape and spreading of the platelets (Supplemental Fig. 3).

Bottom Line: In this study, we investigated the antithrombotic effect of CA in mouse cerebral arterioles and venules using intravital microscopy.Our results demonstrated that CA (1.25-5 mg/kg) significantly inhibited thrombus formation in vivo.Additionally, CA attenuated p38, ERK, and JNK activation, and enhanced cAMP levels.

View Article: PubMed Central - PubMed

Affiliation: Department of gynaecology, Beijing Royal Integrative Medicine Hospital, Beijing, China.

ABSTRACT
Caffeic acid (CA), one of the active constituents of Radix Salvia miltiorrhizae, exhibits antioxidant and anti-inflammatory activities. However, few studies have assessed the ability of CA to inhibit platelet mediated thrombus generation in vivo. In this study, we investigated the antithrombotic effect of CA in mouse cerebral arterioles and venules using intravital microscopy. The antiplatelet activity of CA in ADP stimulated mouse platelets in vitro was also examined in attempt to explore the underlying mechanism. Our results demonstrated that CA (1.25-5 mg/kg) significantly inhibited thrombus formation in vivo. In vitro, CA (25-100 μM) inhibited ADP-induced platelet aggregation, P-selectin expression, ATP release, Ca(2+) mobilization, and integrin αIIbβ3 activation. Additionally, CA attenuated p38, ERK, and JNK activation, and enhanced cAMP levels. Taken together, these data provide evidence for the inhibition of CA on platelet-mediated thrombosis in vivo, which is, at least partly, mediated by interference in phosphorylation of ERK, p38, and JNK leading to elevation of cAMP and down-regulation of P-selectin expression and αIIbβ3 activation. These results suggest that CA may have potential for the treatment of aberrant platelet activation-related diseases.

No MeSH data available.


Related in: MedlinePlus