title: Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation-specific monoclonal antibodies.
abstract: Normal primary platelet aggregation requires agonist-mediated activation of membrane GPIIb-IIIa, binding of fibrinogen to GPIIb-IIIa, and cellular events after ligand binding. PAC1 monoclonal antibody distinguishes between resting and activated states of GPIIb-IIIa, and other antibodies preferentially recognize GPIIb (PMI-1) or IIIa (anti-LIBS1) after the binding of fibrinogen or fibrinogen-mimetic peptides, such as GRGDSP. Using these antibodies and platelet flow cytometry, we studied two distinct persistent platelet aggregation abnormalities. Platelets from a thrombasthenic variant, which contained near-normal amounts of GPIIb-IIIa, failed to aggregate or bind PAC1 in response to agonists. In addition, GRGDSP, which binds to normal GPIIb-IIIa without prior cell activation, failed to increase the binding of PMI-1 or anti-LIBS1 to the thrombasthenic platelets, suggesting a primary defect in ligand binding. Chromatography of detergent-solubilized platelets on a KYGRGDS affinity column confirmed that the patient's GPIIb-IIIa lacked the fibrinogen binding site. In another patient with myelofibrosis and defective aggregation, PAC1 failed to bind to adenosine diphosphate-stimulated platelets, but did bind when protein kinase C was directly activated with phorbol myristate acetate. Furthermore, the binding of PMI-1 and anti-LIBS1 increased in response to GRGDSP, confirming a defect in agonist-mediated fibrinogen receptor activation rather than in fibrinogen binding or events distal to binding. These studies indicate that this immunochemical approach is useful in classification of clinical abnormalities of platelet aggregation as defects in either (a) fibrinogen receptor activation, (b) fibrinogen binding, or (c) postoccupancy events.
authors.affiliation: Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA 92037.
authors.forename: M H
authors.initials: MH
authors.lastname: Ginsberg
authors.forename: A L
authors.initials: AL
authors.lastname: Frelinger
authors.forename: S C
authors.initials: SC
authors.lastname: Lam
authors.forename: J
authors.initials: J
authors.lastname: Forsyth
authors.forename: R
authors.initials: R
authors.lastname: McMillan
authors.forename: E F
authors.initials: EF
authors.lastname: Plow
authors.forename: S J
authors.initials: SJ
authors.lastname: Shattil
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology*; D001778:Blood Coagulation Disorders / Q000276:immunology / Q000473:pathology*; D001792:Blood Platelets / Q000737:chemistry / Q000276:immunology* / Q000473:pathology; D002462:Cell Membrane / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D005260:Female; D005434:Flow Cytometry; D014644:Genetic Variation / Q000276:immunology; D006801:Humans; D008024:Ligands; D008875:Middle Aged; D010974:Platelet Aggregation / Q000276:immunology*; D010980:Platelet Membrane Glycoproteins / Q000032:analysis / Q000276:immunology*; D055728:Primary Myelofibrosis / Q000276:immunology / Q000473:pathology; D013915:Thrombasthenia / Q000276:immunology / Q000473:pathology
chemical_list: D000911:Antibodies, Monoclonal; D008024:Ligands; D010980:Platelet Membrane Glycoproteins