Synergistic functions of protein phosphorylation and calcium mobilization in platelet activation

When human platelets were stimulated by synthetic diacylglycerol such as 1-oleoyl-2-acetyl-glycerol, which was a potent activator in vitro of Ca²⁺ -activated, phospholipid-dependent protein kinase (protein kinase a protein having M(r) ~ 40,000 (40-kilodalton protein) was rapidly phosphorylated, just as it was by natural extracellular messengers such as thrombin. Fingerprint analysis appeared to indicate that protein kinase C was indeed responsible for this 40-kilodalton protein phosphorylation in intact platelets. Under these conditions, neither inositol phospholipid breakdown nor endogenous diacylglycerol formation was observed, indicating that the synthetic diacylglycerol intercalated into the membrane and directly activated protein kinase C without interaction with cell surface receptors. During this process, the diacylglycerol was converted in situ to the corresponding phosphatidate, 1-oleoyl-2-acetyl-glyceryl-3-phosphoric acid. Experiments with the synthetic diacylglycerol and Ca²⁺ ionophore A23187 suggested that the protein phosphorylation catalyzed by protein kinase C was a prerequisite requirement for the release of serotonin, and that the receptor-linked protein phosphorylation and Ca²⁺ mobilization acted synergistically to elicit the full physiological cellular response.