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

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.