title: Dynamic redistribution of major platelet surface receptors after contact-induced platelet activation and spreading. An immunoelectron microscopy study.
abstract: The authors used an immunogold labeling procedure to investigate the redistribution of platelet receptors and their ligands on the surface of contact-activated adherent platelets before and after thrombin stimulation. During the initial stage of platelet adhesion, a typical segregation of receptors occurred. Gold particles identifying glycoprotein (GP) Ib (CD42b) and GPIIb-IIIa (CD41a) remained distributed over the entire platelet surface, whereas gold particles identifying GPIa-IIa (CDw 49b) and GPIV (CD36) were found essentially overlying the granulomere; p24 (CD9) was present at the peripheral platelet rim and over the cell body. An increased labeling of GPIIb-IIIa, GPIV and p24 was also observed on pseudopods, with GPIIb-IIIa and GPIV concentrated at the enlarged extremities and at sites of contact between two platelets, whereas GPIb was absent from pseudopods. After thrombin stimulation of adherent platelets, GPIb underwent a relocation to the cell center, in contrast to GPIIb-IIIa which still remained randomly distributed over the cell body. To investigate whether ligand distribution paralleled this receptor segregation, platelet released von Willebrand factor (vWF), fibrinogen (Fg) and thrombospondin (TSP) were visualized. During the early stages of platelet activation, surface labeling for all three adhesive proteins was minimal and almost undetectable. Occasionally, intragranular Fg and vWF was accessible to gold-coupled antibodies, with vWF exhibiting the typical eccentric alpha-granular localization. At later stages of activation and especially after thrombin stimulation, no surface labeling for vWF was observed, whereas immunogold particles identifying vWF were still present inside enlarged clear vacuoles. In contrast, labeling of Fg and TSP was increased over the granulomere and extended to the cell periphery and the pseudopods, but was absent from the hyalomere, despite the presence of GPIIb-IIIa molecules. Double labeling experiments showed colocalization of Fg and TSP, GPIV and TSP, as well as Fg and GPIIb-IIIa, although no typical coclustering of GPIIb-IIIa and GPIV or GPIIb-IIIa and p24 was apparent. Our results further suggest that 1) on surface activated adherent platelets, not all GPIIb-IIIa molecules become competent to bind Fg, 2) GPIa-IIa is not anchored to the platelet membrane skeleton, and 3) during the early stage of platelet activation, a communication exists between the alpha granules and the platelet surface.
authors.affiliation: INSERM U91, Hôpital Henri Mondor, Créteil, France.
authors.forename: N
authors.initials: N
authors.lastname: Kieffer
authors.forename: J
authors.initials: J
authors.lastname: Guichard
authors.forename: J
authors.initials: J
authors.lastname: Breton-Gorius
mesh_terms: D015703:Antigens, CD / Q000032:analysis / Q000502:physiology; D001792:Blood Platelets / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure*; D002450:Cell Communication / Q000502:physiology*; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D005340:Fibrinogen / Q000032:analysis; D006046:Gold; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins; D016253:Microscopy, Immunoelectron / Q000379:methods; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000032:analysis* / Q000502:physiology; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000502:physiology; D060245:Tetraspanin 29; D013917:Thrombin / Q000494:pharmacology; D019699:Thrombospondins; D014841:von Willebrand Factor / Q000032:analysis
chemical_list: D015703:Antigens, CD; C554869:CD9 protein, human; D008024:Ligands; D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D060245:Tetraspanin 29; D019699:Thrombospondins; D014841:von Willebrand Factor; D006046:Gold; D005340:Fibrinogen; D013917:Thrombin