Characterization of 1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine (AGEPC)-induced protein phosphorylation in rabbit platelets: Inhibitory effects of AGEPC analogs

Abstract

1- O-Alkyl-2-acetyl- sn-glycero-3-phosphocholine (AGEPC) induced phosphorylation of two proteins having molecular masses of approximately 20- and 40-kDa in washed rabbit platelets in a concentration- and time-dependent manner. Sequential stimulation with AGEPC did not induce additional protein phosphorylation, supporting the concept of desensitization of the AGEPC receptors responsible for biological activity. AGEPC analogs 1- O-octadecyl-2-acetyl- sn-glycero-3-phosphoric acid-6′-trimethylammonium hexyl ester and 1- O-octadecyl-2-acetyl- sn-glycero-3-phosphoric acid-10′-trimethylammonium decyl ester (U66985 and U66982), containing polar head groups with methylene chain lengths of C6 and C10, did not cause protein phosphorylation, but they did inhibit the AGEPC-induced events. Thus protein phosphorylation is closely associated with the receptor-mediated stimulation of platelets and is a useful indicator of the signaling process initiated through the receptors. Other synthetic analogs of AGEPC such as rac-3-( N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate and 1-( N-n-pentadecylcarbamoyloxy)-2-methoxy- rac-glycero-3-phosphocholine (CV3988 and U68043) were also shown to be inhibitors of the AGEPC-induced protein phosphorylation. Inhibition by these analogs was specific for AGEPC since there was no observed effect of thrombin, ADP, 12- O-tetradecanoyl-phorbol 13-acetate (TPA) and arachidonic acid-induced changes. The extent of inhibition was dependent on the concentration of AGEPC and its analogs and did not change with time after the addition of AGEPC. In platelets incubated with AGEPC analogs before and simultaneously with the addition of AGEPC, protein phosphorylation was prevented; however, addition of AGEPC to platelets shortly before the addition of these analogs showed a high response. In experiments where platelets were previously incubated with AGEPC analogs and washed with buffer containing 0.5% bovine serum albumin, AGEPC-induced protein phosphorylation was recovered to a level of 80%. These observations support the conclusion that AGEPC stimulates platelets through its specific receptor, and that the AGEPC analogs bind to the AGEPC receptor and block that pathway sensitive to AGEPC stimulation but not because of the desensitization of its receptor. On the other hand, in platelets where phosphorylation of the 40-kDa protein was induced by a 2-min preincubation with 3 × 10 −10 M TPA, 5 × 10 −10 M AGEPC-induced serotonin release decreased by 51% compared to a control value. But the IC 50 value for U66985 (2 × 10 −8 M) with 5 × 10 −10 M AGEPC then was not affected by the pretreatment with TPA prior to the addition of U66985. These observations showed that the activation of protein kinase C pathway suppressed the AGEPC-induced secretion reaction but did not affect the mode of inhibitory action by the antagonists.