The platelet glycoprotein Ib-IX-V complex anchors lipid rafts to the membrane skeleton: implications for activation-dependent cytoskeletal translocation of signaling molecules.

BACKGROUND

The glycoprotein (GP) Ib-IX-V complex attaches platelets to areas of endothelial damage by binding von Willebrand factor (VWF), an interaction that transmits intracellular activation signals. These signals require that the complex associates with both lipid rafts and the membrane cytoskeleton, but it is not clear whether the same GPIb-IX-V subpopulation associates with both structures.

OBJECTIVES

To determine which subpopulation of GPIb-IX-V associates with lipid rafts, and the consequences of that interaction.

METHODS

We analyzed the content of proteins (particularly the GPIb-IX-V complex) and lipids in rafts from detergent lysates of platelets before and after removal of the actin cytoskeleton alone or both the actin cytoskeleton and membrane skeleton (by successive centrifugations of 15,800 x g and 100,000 x g).

RESULTS

In unstimulated platelets, little raft-associated GPIb-IX-V sedimented with the actin skeleton; most was removed by sedimentation of the membrane skeleton. The Src family kinase Lyn followed the same pattern. In VWF-activated platelets, almost all of the GPIb-IX-V complex and Lyn in rafts sedimented with the actin cytoskeleton, consistent with a previously described crosslinking of the membrane and actin skeletal structures following platelet activation. Disruption of the GPIbalpha-filamin linkage with N-ethylmaleimide prevented depletion of raft-associated GPIb-IX-V by skeletal sedimentation. Not all raft-associated proteins and lipids followed this pattern.

CONCLUSION

These results suggest that the raft association and cytoskeletal linkage of the GPIb-IX-V complex are interrelated, and both are required for optimal receptor function, perhaps because raft association attracts signaling proteins and membrane skeletal association allows these proteins to move en masse to new locations.