Signaling Mediated by Glycoprotein Ib-IX-V/VWF Interaction Mainly Takes Place in GEMs In Human Platelets

Abstract number: P2086

Asazuma¹ N, Kaneko¹ M, Satoh¹ K, Suzuki-Inoue¹ K, Inoue¹ O, Handa² M, Berndt³ MC, Ozaki¹ Y

¹¹Department of Clinical and Laboratory Medicine, University of Yamanashi, Yamanashi, Japan ¹¹Department of Clinical and Laboratory Medicine, University of Yamanashi, Yamanashi, Japan ²²Department of Transfusion Medicine and Cell Therapy, Keio University School of Medicine, Tokyo, Japan ³³Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia

The platelet glycoprotein (GP) Ib-IX-V, which binds von Willebrand factor (VWF), plays important roles in platelet adhesion and subsequent thrombus formation at sites of vascular injury, particularly under high shear stress. Recent studies have revealed that VWF/GPIb-IX-V signals through a set of signaling molecules, Src, PI-3Kinase, FcR g-chain, Syk and PLC g2. It is now recognized that there are particular membrane regions known as glycosphingolipid-enriched microdomains (GEMs, also known as rafts) in platelet membranes. GEMs are rich in cholesterol and glycosphingolipids, and appear to provide platforms for interactions between many of the signaling molecules, particularly those related to tyrosine phosphorylation. In this study, we investigated the role of GEMs in VWF/GPIb-IX-V signaling, utilizing biochemical isolation of GEMs by sucrose density ultra-centrifugation in the presence of 0.1% Triton X-100, and the cholesterol-depleting reagent, methyl b-cyclodextrin (MbCD). Isolation of GEMs and non-GEMs in resting platelets revealed that 10 to 13% of GPIb-IX-V localized in GEMs in agreement with a previous report (Shrimpton, JEM 2002), and GEM-associated-GPIb-IX-V increased in a time-dependent manner after platelet activation induced by VWF/GPIb-IX-V interaction. Association of GPIb-IX-V with FcR g-chain was observed in GEMs and only the FcR g-chain present in GEMs underwent tyrosine phosphorylated in VWF-botrocetin-stimulated platelets, which was completely inhibited by 15 mM MbCD treatment. Upon VWF/GPIb-IX-V interaction, Src associated with GPIb-IX-V located in GEMs but not with those in non-GEMs, and GPIb-IX-V-associated Src was the activated form of Src with 416 tyrosine residue phosphorylated. While PLC g2 was present both in GEMs and non-GEMs, only PLC g2 located in GEMs underwent tyrosine phosphorylation upon VWF/GPIb-IX-V interaction, and this was completed abrogated by 15 mM MbCD treatment. While the mechanism by which GPIb-IX-V is recruited to GEMs remains to be investigated, our findings suggest that the GPIb-IX-V signal transduction pathway mainly takes place in GEMs, and that an intact GEM structure is required for GPIb-IX-V signaling.