Adhesive Surface Determines Raft Composition in Platelets Adhering Under Flow

Abstract number: P2090

van Lier¹ M, Verhoef¹ S, Lee² F, Farndale RW² , Gorter¹ G, Ohno-Iwashita³ Y, Akkerman¹ JWN, Heijnen¹ HFG

¹¹Laboratory for Thrombosis and Haemostasis, Department of Haematology, UMCU, and Institute of Biomembranes, Utrecht, the Netherlands ¹¹Laboratory for Thrombosis and Haemostasis, Department of Haematology, UMCU, and Institute of Biomembranes, Utrecht, the Netherlands ²²Department of Biochemistry, University of Cambridge, Cambridge, UK ³³Department of Protein Biochemistry, Tokyo Metropolian Institute of Gerontology, Tokyo, Japan

Adhesion to von Willebrand factor (vWf) induces platelet spreading, whereas adhesion to collagen induces aggregation. Here we report that cholesterol-rich domains (CRDs) play a crucial role in the signaling in adhered platelets. CRDs were characterized by confocal microscopy using biotinylated perfringolysin-O (BCQ), a q-toxin that binds selectively to membrane cholesterol. CRD distribution was compared with the localization of glycoprotein Ib (GPIb), the vWF receptor, glycoprotein VI (GPVI), a collagen receptor, and tyrosine phosphorylated proteins, using appropriate antibodies. CRDs were isolated from non-stimulated and adhered platelets using 1% CHAPS, followed by sucrose gradient fractionation. Following adhesion at a shear rate of 800 s^-1, CRDs concentrate in filopodia of platelets adhering to both surfaces. Clustered CRDs contain both GPIb and GPVI. GPIb distribution is enriched in filopodia on vWF-adhering platelets and random on collagen-adhering platelets. Tyrosine phosphorylated proteins show a punctated staining in filopodia upon adhesion to vWf. Also in aggregates formed on collagen, clusters of tyrosine-phosphorylated proteins are found. Electron microscopical analysis shows co-clusters of GPVI and LAT, and GPVI and SLP-76 on a collagen surface. Biochemical analysis of CRDs shows a 2.8 ± 0.5 fold enrichment of GPIb (but not GPVI) upon adhesion to vWf, and a 3.3 ± 0.3 fold enrichment of GPVI (but not GPIb) upon adhesion to collagen. Cholesterol-depletion impairs the spreading on vWf, the aggregate formation on collagen, and the localization of GPIb in filopodia on vWF. The initial attachment and the formation of filopodia remain unchanged. These data show that CRDs are dynamic structures and adapt their localization and composition to the adhesive substrate to ensure optimal signal generation.

Acknowledgements:  Supported by NHF grant 2001B171.