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摘要
Aim: In this study, we investigated the effects of caffeic acid (CAFA), a phenolic acid, on Ca2+-antagonistic cyclic nucleotides associated with the phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) and vasodilator-stimulated phosphoprotein (VASP) and the thromboxane A(2) (TXA(2))-associated microsomal cyclooxygenase-1 (COX-1) activity in collagen (10 mu g/mL)-stimulated platelet aggregation. %26lt;br%26gt;Methods: Washed platelets (10(8)/mL) obtained from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 minutes at 37 degrees C in the presence of 2 mM exogenous CaCl2 with or without CAFA or other materials, stimulated with collagen (10 mu g/mL) for 5 minutes, then used to determine the levels of intracellular cytosolic Ca2+ ([Ca2+] i), TXA(2), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), COX-1 activity, VASP and IP3R phosphorylation. %26lt;br%26gt;Results: CAFA dose-dependently inhibited collagen-induced platelet aggregation and suppressed the production of TXA(2), an aggregation-inducing autacoid associated with the strong inhibition of COX-1 in platelet microsomes exhibiting cytochrome C reductase activity. CAFA dose-dependently inhibited collagen-elevated [Ca2+]i mobilization, which was increased by a cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS, but not a cGMP-dependent protein kinase (G-kinase) inhibitor, Rp-8-Br-cGMPS. In addition, CAFA significantly increased the formation of cAMP and cGMP, intracellular Ca2+-antagonists that function as aggregation-inhibiting molecules. CAFA increased IP3R (320 kDa) phosphorylation, indicating the inhibition of IP3-mediated Ca2+ release from internal stores (i. e. the dense tubular system) via the IP3R on collagen-activated platelets. Furthermore, CAFA-induced IP3R phosphorylation was strongly inhibited by an A-kinase inhibitor, Rp-8-Br-cAMPS, but only mildly inhibited by a G-kinase inhibitor, Rp-8-Br-cGMPS. These results suggest that the inhibition of [Ca2+] i mobilization by CAFA is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CAFA elevated the phosphorylation of VASP-Ser(157), an A-kinase substrate, but not the phosphorylation of VASP-Ser(239), a G-kinase substrate. We demonstrate that CAFA increases cAMP and subsequently phosphorylates both IP3R and VASP-Ser(157) through A-kinase activation to inhibit [Ca2+] i mobilization and TXA(2) production via the inhibition of the COX-1 activity. %26lt;br%26gt;Conclusions: These results strongly indicate that CAFA is a potent beneficial compound that elevates the level of cAMP-dependent protein phosphorylation in collagen-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.
- 出版日期2014
