title: Characterization of an anti-thrombospondin monoclonal antibody (P8) that inhibits human blood platelet functions. Normal binding of P8 to thrombin-activated Glanzmann thrombasthenic platelets.
abstract: Stimulated human blood platelets release thrombospondin, an alpha-granule glycoprotein of 450 kDa. The aim of this work was to characterize an anti-thrombospondin monoclonal antibody (P8) in order to study the role of thrombospondin in platelet functions. The presence of thrombospondin receptor sites on resting and thrombin-stimulated platelets of three Glanzmann's thrombasthenia patients and normal donors was investigated using the P8 monoclonal antibody. Monoclonal antibody P8 was extensively characterized using ELISA, immunoprecipitation, immunoadsorbent affinity chromatography combined with tryptic peptide map analysis and crossed immunoelectrophoretic techniques. Labelled P8 bound strongly to thrombin-stimulated normal platelets (n = 14917 +/- 420, mean +/- SD) (Kd = 9.2 +/- 3.0 nM) and poorly to resting platelets (n = 2697 +/- 1278) (Kd = 24.8 +/- 18.6 nM). Moreover, the number of binding sites for P8 on thrombin-stimulated platelets from three Glanzmann's thrombasthenia patients, lacking the IIb-IIIa glycoprotein complex, were found similar to normal samples. F(ab')2 fragments of P8 inhibited aggregation of, and reduced secretion from, washed platelets stimulated by low concentrations of thrombin (0.05-0.06 U/ml) and collagen (0.5-0.6 microgram/ml). F(ab')2 fragments of P8 inhibited thrombin-induced platelet aggregation, but did not reduce fibrinogen binding (n) nor affect its dissociation constant (Kd). Inhibition of platelet aggregation by P8 suggests that thrombospondin plays an active role in promoting platelet aggregation, at low concentrations of thrombin and collagen. Normal binding of P8 to thrombin-stimulated Glanzmann thrombasthenic platelets indicates the presence of a thrombospondin receptor on the platelet surface distinct from the GPIIb-IIIa complex.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale Unite 63, Faculté de Médecine Alexis Carrel, Université Claude, Bernard, Lyon, France.
authors.forename: H
authors.initials: H
authors.lastname: Boukerche
authors.forename: J L
authors.initials: JL
authors.lastname: McGregor
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001791:Blood Platelet Disorders / Q000503:physiopathology*; D001792:Blood Platelets / Q000502:physiology*; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005340:Fibrinogen / Q000378:metabolism; D006023:Glycoproteins / Q000276:immunology* / Q000502:physiology; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000502:physiology; D013915:Thrombasthenia / Q000503:physiopathology*; D013917:Thrombin / Q000494:pharmacology; D019699:Thrombospondins
chemical_list: D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D010980:Platelet Membrane Glycoproteins; D019699:Thrombospondins; D005340:Fibrinogen; D013917:Thrombin