A Critical Role for the IKKβ/Lipid Rafts Axis in Regulating Platelet Exocytosis, Via the Spatial Regulation of SNARE Proteins

Lipid rafts, which are membrane microdomains that are rich in cholesterol and glycosphingolipids, have been implicated in the regulation of intracellular protein trafficking. Furthermore, we have previously shown that SNAREs mediate granule-plasma membrane fusion during exocytosis in platelets, and that IKKβ regulates SNAREs and granule exocytosis. Based on these considerations, we sought to investigate the regulatory relationship between IKKβ and lipid rafts, by employing the PF4-Cre/IKKβ^flox/flox platelets. We demonstrate that lipid rafts play a critical role in mediating IKKβ^-/--dependent regulation of SNARE proteins and SNARE complexes. Specifically, we found that methyl β-cyclodextrin (MβCD, which disrupts lipid rafts) is able to inhibit platelet aggregation, dense and alpha granule secretion, PS exposure and αIIbβ3 integrin activation. We also observed that MβCD is able to inhibit platelet spreading, clot retraction, as well as 7S complex formation. In terms of co-localization, the SNARE protein SNAP-23 was found to associate with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in control platelets, but not in the IKKβ^-/- platelets. Furthermore, in resting platelets, plasma membrane SNARE proteins such as syntaxin-11 and SNAP-23 or the granule-associated SNARE vesicle-associated membrane protein VAMP-8 were neither present in the microdomains of control platelets, nor in those from IKKβ^-/- mice. Interestingly however, agonist/thrombin stimulation led to the relocation of syntaxin-11 and VAMP-8 into the lipid rafts, and as part of raft-associated phospho-SNAP-23/syntaxin-11/VAMP-8 complexes in control platelets. This seems to be IKKβ dependent, as these associations did not take place in the IKKβ^-/- platelets. Taken together, these data reveal a critical role for the IKKβ/lipid rafts axis in regulating platelet exocytosis, via the spatial regulation of SNARE proteins.