A monoclonal antibody directed against a granule membrane glycoprotein (GMP-140/PADGEM, P-selectin, CD62P) inhibits ristocetin-induced platelet aggregation

Br J Haematol. 1996 Feb;92(2):442-51. doi: 10.1046/j.1365-2141.1996.d01-1485.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / pharmacology*
  • Cell Adhesion
  • Dose-Response Relationship, Drug
  • Electrophoresis, Gel, Two-Dimensional
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Neutrophils / physiology
  • P-Selectin / immunology*
  • Platelet Activation
  • Platelet Aggregation / drug effects*
  • Ristocetin / pharmacology*
  • Thrombasthenia / blood*
  • Thrombin / pharmacology

Substances

  • Antibodies, Monoclonal
  • P-Selectin
  • Ristocetin
  • Thrombin