A role for glycosphingolipid‐enriched microdomains in platelet glycoprotein Ib‐mediated platelet activation

Summary. Background: Glycoprotein (GP) Ib, a platelet von Willebrand factor (VWF) receptor, plays a crucial role in thrombosis and hemostasis. As recent reports have suggested that GPIb partially locates in a particular region, designated as glycosphingolipid-enriched microdomains (GEMs), we hypothesized that GEMs play a central role in GPIb-mediated platelet activation. Methods: Platelets were stimulated by VWF/botrocetin to activate platelets through GPIb. GEMs and non-GEMs were isolated by sucrose density gradient ultracentrifugation and the location of signaling molecules characterized. The role of GEMs-mediated signaling in platelet behavior was tested by platelet aggregation and by platelet interaction with immobilized VWF under flow conditions when GEMs were disrupted by methyl-β-cyclodextrin (MβCD). Results: GPIb was partially translocated to GEMs upon VWF/botrocetin stimulation. Immunoprecipitation of GPIb in GEMs and non-GEMs revealed that the tyrosine kinases, Src and Lyn, were associated with GPIb only in GEMs after GPIb-stimulation, and not in non-GEMs. Activation of PLCγ2 was more intense in GEMs than non-GEMs. Disruption of GEMs by MβCD strongly inhibited tyrosine phosphorylation of Syk and PLCγ2. Functional studies revealed that stable adhesion of platelets to a VWF-coated surface under flow was impaired by GEM disruption by MβCD. Conclusion: The combined results suggest that GEMs play an important role in GPIb-mediated platelet activation.