Mechanisms of agonist synergism in human isolated platelets

The synergism between various platelet agonists which results in an aggregation response has been described frequently in vitro, however there are no satisfactory explanations for the mechanisms controlling this response. In vivo, platelets are exposed to many aggregating stimuli at low concentrations at an injury site and synergism between such stimuli would enable an intensive aggregation and release response to occur. This study has investigated the intracellular mechanisms associated with agonist synergism in human isolated platelets using the following agonist combinations; adrenaline and thrombin, adrenaline and LDL. The action of LDL alone in activating platelets was also studied. Subthreshold concentrations of adrenaline (5-20 μM), thrombin (0.006-0.02 U/ml) and LDL (0.1-0.2 mg protein/ml), which individually did not cause an aggregation response were combined in pairs to produce a synergistic aggregation response, which was not inhibited by the presence of aspirin. There was no hydrolysis of phosphatidylinositol 4,5, bisphosphate or production of inositol trisphosphate associated with the synergism. Using quin 2, no increases in intracellular calcium concentration ([Ca2+]i ) were detected during the synergistic aggregation responses, however small [([Ca2+]i increases were detected using aequorin. No release or metabolism of arachidonic acid from membrane phospholipids was observed. In agreement with the calcium results, there was no phosphorylation of the 20 kDa protein. However, despite no significant diacylglycerol production, the 47 kDa protein was clearly phosphorylated following synergism between both the agonist combinations. Whether the phosphorylation of the 47 kDa protein is important in the initiation of a synergistic response remains to be determined and the presence of other pathways must be considered. Activation of platelets with 2.75 mg protein/ml LDL was accompanied by a slow increase in inositol trisphosphate production and intracellular calcium levels. Low LDL concentrations (<0.25 mg protein/ml) did not stimulate either aggregation or any intracellular mechanism