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-beta-cyclodextrin (MbetaCD).


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 PLCgamma2 was more intense in GEMs than non-GEMs. Disruption of GEMs by MbetaCD strongly inhibited tyrosine phosphorylation of Syk and PLCgamma2. Functional studies revealed that stable adhesion of platelets to a VWF-coated surface under flow was impaired by GEM disruption by MbetaCD.


CONCLUSIONS
The combined results suggest that GEMs play an important role in GPIb-mediated platelet activation.
Jin
W
W
Department of Laboratory and Clinical Medicine, University of Yamanashi, Yamanashi, Japan.
Inoue
O
O
Tamura
N
N
Suzuki-Inoue
K
K
Satoh
K
K
Berndt
M C
MC
Handa
M
M
Goto
S
S
Ozaki
Y
Y
D002448:Cell Adhesion; D006028:Glycosphingolipids / Q000378:metabolism*; D006801:Humans; D015539:Platelet Activation / Q000502:physiology*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000502:physiology*; D011485:Protein Binding; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism
D006028:Glycosphingolipids; D019038:Platelet Glycoprotein GPIb-IX Complex; D019061:src-Family Kinases