A monoclonal antibody directed against a granule membrane glycoprotein (GMP-140/PADGEM, P-selectin, CD62P) inhibits ristocetin-induced platelet aggregation.
P-selectin (also called CD62, GMP-140, PADGEM, CD62P) is a recently described member of a family of vascular adhesion receptors expressed by activated platelets and endothelial cells that are involved in leucocyte cell adhesion. The aim of this study was to characterize a new monoclonal antibody (LYP7) directed against activated human blood platelets that inhibits ristocetin-induced platelet aggregation. Immunoadsorbent affinity chromatography and immunoprecipitation studies showed that LYP7 (IgG1) bound a surface-labelled glycoprotein (GP) which changed its apparent molecular mass (M(r)) on reduction from 138 kD (situated below GPIIb) to 148 kD (above GPIIb alpha). LYP7 and S12, a monoclonal antibody directed against P-selectin immunoprecipitated the same band. Using ELISA assay, purified P-selectin was shown to bind LYP7 and S12 monoclonal antibodies. Binding sites of 125I-labelled LYP7, which was greatly increased on thrombin-stimulated (2 U/ml) washed platelets (10825 +/- 2886, mean +/- SD) Kd = 1.5 +/- 0.5 nM) compared to resting platelets (2801 +/- 1278, mean +/- SD) (Kd = 1.5 +/- 0.6 nM), was found to be normal on thrombin-stimulated platelets taken from a patient with grey platelet syndrome or a patient with Glanzmann thrombasthenia. LYP7 (IgG1, F(ab')2 or Fab fragments) inhibited ristocetin-induced platelet aggregation of platelets in a dose-dependent fashion without affecting the binding of von Willebrand (vWf) factor. However, agglutination of formaldehyde-fixed platelets induced by ristocetin was not affected by monoclonal antibody LYP7. In addition, the binding of thrombin-activated platelets to neutrophils was inhibited by monoclonal antibody LYP7. These results strongly suggest that P-selectin, by promoting cell-cell contact, may play an active role in platelet-platelet interactions.
Boukerche
H
H
INSERM U331, Faculty of Medicine Alexis Carrel, Lyon, France.
Ruchaud-Sparagano
M H
MH
Rouen
C
C
Brochier
J
J
Kaplan
C
C
McGregor
J L
JL
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D002448:Cell Adhesion; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009504:Neutrophils / Q000502:physiology; D019007:P-Selectin / Q000276:immunology*; D015539:Platelet Activation; D010974:Platelet Aggregation / Q000187:drug effects*; D012310:Ristocetin / Q000494:pharmacology*; D013915:Thrombasthenia / Q000097:blood*; D013917:Thrombin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D019007:P-Selectin; D012310:Ristocetin; D013917:Thrombin