Fine structural and functional consequences of deglycosylation of the platelet adhesion receptor GPIb-IX (CD 42b).
To investigate the role of the glycosylation of the platelet receptor glycoprotein Ib (GPIb, CD 42b), platelets and purified GPIb were deglycosylated by neuraminidase, O- and N-glycosidases. N-deglycosylation and neuraminic-acid cleavage had little effect on ristocetin and botrocetin-induced platelet agglutination. However, O-deglycosylation reduced the response by approximately 50%, and total deglycosylation (the combination of all three glycosidases) fully abolished the response to ristocetin. Interestingly, binding of von Willebrand Factor (vWF) to purified GPIb in the presence of ristocetin and botrocetin in a standardized microtiter plate assay was not altered by partial or even by total deglycosylation. Electron microscopy indicated that the normally stretched approximately 50 nm long molecule was approximately 32 nm after N-deglycosylation, approximately 20 nm after O-deglycosylation, and reduced in a approximately 15 nm long collapse by total deglycosylation. These results suggest that deglycosylation has major structural impacts on GPIb, strongly impairing platelet-vWF interactions; however, vWF binding to isolated GPIb remains unaffected.
Moshfegh
K
K
Department of Medicine, University Hospital of Bern, Switzerland.
Lengweiler
S
S
Häner
M
M
Aebi
U
U
Steiner
B
B
Beer
J H
JH
D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology; D002240:Carbohydrate Sequence; D003435:Crotalid Venoms / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D009439:Neuraminidase / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D012310:Ristocetin / Q000494:pharmacology; D014841:von Willebrand Factor / Q000378:metabolism
D003435:Crotalid Venoms; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D012310:Ristocetin; C030961:botrocetin; D006026:Glycoside Hydrolases; D009439:Neuraminidase