Antiplatelet effects of caffeic acid due to Ca2+ mobilization- inhibition via cAMP-dependent inositol-1,4,5-trisphosphate receptor phosphorylation

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 a2 (txa2)-associated microsomal cyclooxygenase-1 (cox-1) activity in collagen (10 μg/ml)-stimulated platelet aggregation. methods: washed platelets (108/ml) obtained from sprague-dawley rats (6-7 weeks old,male) were preincubated for 3 minutes at 37° in the presence of 2 mm exogenous cacl2 with or without cafa or other materials,stimulated with collagen (10 μg/ml) for 5 minutes,then used to determine the levels of intracellular cytosolic ca2+ ([ca2+]i),txa2,cyclic adenosine monophosphate (camp),cyclic guanosine monophosphate (cgmp),cox-1 activity,vasp and ip3r phosphorylation. results: cafa dose-dependently inhibited collagen-induced platelet aggregation and suppressed the production of txa2,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-ser157,an a-kinase substrate,but not the phosphorylation of vasp-ser239,a g-kinase substrate. we demonstrate that cafa increases camp and subsequently phosphorylates both ip3r and vasp-ser157 through a-kinase activation to inhibit [ca2+]i mobilization and txa2 production via the inhibition of the cox-1 activity. 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.