REGULATION OF PLATELET GLYCOPROTEIN IIB/IIIA (INTEGRIN ALPHA(IIB)BETA(3)) FUNCTION VIA THE THROMBIN RECEPTOR

Binding sites on glycoprotein (GP) IIb/IIIa exposed by 0.5 unit/ml alpha-thrombin are insensitive to prostaglandin I-2 (PGI(2)), in contrast with sites exposed by ADP or platelet-activating factor. Here we show that the thrombin receptor agonist peptide (TRAP) (SFLLRN; 15 mu M) opens almost the same number of GPIIb/IIIa molecules as 0.5 unit/ml alpha-thrombin (64840+/-8920 compared with 81050+/-6030 molecules of fibronectin bound/platelet), but these sites rapidly close on addition of PGI(2). To investigate whether alpha-thrombin and TRAP initiate different signalling pathways, we measured phospholipase C (PLC)-mediated control of GPIIb/IIIa and its sensitivity to cyclic AMP. Optimal concentrations of alpha-thrombin and TRAP activated PLC maximally, but TRAP induced only about 50% protein kinase C (PKC) activation after 10 min stimulation compared with alpha-thrombin. These concentrations also suppressed PGI,-induced cyclic AMP accumulation, with alpha-thrombin inducing complete inhibition and TRAP about 10% less. Direct activation of PKC by phorbol 12-myristate 13-acetate confirmed earlier observations that PGI(2)-induced cyclic AMP accumulation is partly inhibited via PKC. Applying different concentrations of alpha-thrombin, TRAP or a combination of alpha-thrombin and the thrombin receptor inhibitory peptide (TRIP) (Mpr-F-Cha-Cha-RKPNDK-NH2; 800 mu M) (Mpr, 3-mercaptopropionic acid; Cha, cyclohexylalanine), we show that the different means of stimulating the thrombin receptor all suppressed PGI(2)-induced cyclic AMP accumulation via (i) activation of PKC and (ii) activation of the heterotrimeric G-protein, G(i). We conclude that complete inhibition of cyclic AMP accumulation requires activation of both PKC and G(i), as observed with 0.5 unit/ml alpha-thrombin. Although TRAP almost fully exposes GPIIb/IIIa, its activation of PKC is incomplete, enabling PGI(2) to raise cyclic AMP concentration from 1.4+/-0.7 to 4.1+/-1.3 nmol/ 10(11) platelets (P < 0.005) which is sufficient to close exposed GPIIb/IIIa molecules.