Off-target effect of the Epac agonist 8-pCPT-2'-O-Me-cAMP on P2Y12 receptors in blood platelets

Abstract

The primary target of the cAMP analogue 8-pCPT-2'-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2'-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2'-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2'O-Me-cAMP binding into the purinergic platelet receptor P2Y12 revealed that the analogue docks similar to the P2Y12 antagonist 2MeSAMP. The 8-pCPT-2'-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2'-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2'-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y12 receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2'-O-Me-cAMP did not affect platelet activation at similar concentrations.

Keywords: (Rp)-adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer; (β-phenyl-1), N(2)-etheno-8-bromoguanosine-3′,5′-cyclic monophosphate; 2-methylthio-adenosine diphosphate; 2-methylthio-adenosine monophosphate; 2MeSADP; 2MeSAMP; 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole-3′,5′-cyclic monophosphorothioate, Sp-isomer; 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate; 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer; 8-Br-PET-cGMP; 8-bromoadenosineadenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer; 8-pCPT-2′-O-Me-cAMP; ADP; Blood platelets; CP/CPK; Epac; P2Y(12) receptor; PI3K; PKA; PKG; PMA; Rp-8-Br-cAMPS; Rp-cAMPS; Sp-5, 6-DCL-cBIMPS; Sp-8-pCPT-2′-O-Me-cAMPS; Thromboxane; TxA(2); adenosine diphosphate; cAMP; cAMP-activated protein kinase; cGMP-activated protein kinase; creatine phosphate/creatine phosphokinase; cyclic adenosine monophosphate; exchange factor directly activated by cAMP; phorbol 12-myristate 13-acetate; phosphatidyl-inositol-3 kinase; thromboxane receptor A(2).