The role of lipid rafts in GPVI-mediated platelet activation

Lipid rafts are microdomains within the plasma membrane containing high concentrations of cholesterol and saturated lipids and are believed to regulate signaling in a wide range of cells, including platelets. The GPVI receptor reportedly associates with the lipid rafts in both resting and activated platelets. It has been suggested that there is a reduction in GPVI signaling in raft-disrupted platelets following activation with low to moderate concentrations of GPVI agonist. Since secreted adenosine 5'-diphosphate (ADP) potentiates GPVI-induced platelet aggregation, we investigated whether the decrease in GPVI signaling found in platelets with disrupted rafts was due to the loss of agonist potentiation by ADP. We show that lipid raft disruption decreases aggregation induced by only low concentrations of convulxin, but this decrease is eliminated in the presence of ADP antagonists. Our data indicates that lipid rafts do not play a direct role in proximal GPVI signaling responses induced by convulxin or collagen. Interestingly, however, raft disruption directly reduced GPVI signaling induced by CRP in the presence and absence of ADP feedback. We hypothesize that these variable results are due to the inherent differences among the GPVI agonists.; Conversely, we found the integrity of lipid rafts was essential for agonist-induced dense granule secretion. Platelets with disrupted rafts consistently had inhibited serotonin and ATP release compared to untreated platelets stimulated with agonist in the presence of ADP and fibrinogen antagonists. Furthermore, this inhibition was apparent at a range of GPVI agonist concentrations. Not only was GPVI-induced secretion affected when lipid rafts were disrupted, but PAR-induced secretion was inhibited as well. This suggests that lipid rafts are involved in a fundamental step of platelet exocytosis that is mediated by various agonists that signal through distinct pathways. Our data reveals that a component of the universal secretory machinery---the SNARE protein SNAP-23---is affected by raft disruption. We detected SNAP-23 in the lipid rafts of resting control platelets but this association was prevented in MbetaCD-treated platelets. We also found that the phosphorylation of an unidentified 18 kDa, SNARE-associated protein is reduced following raft disruption. Therefore, we conclude that lipid rafts have a function in SNARE-mediated platelet exocytosis.