title: Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18.
abstract: Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence-activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins "activation reporter." When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP- or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.
authors.affiliation: G. Bizzozero Laboratory of Platelet and Leukocyte Pharmacology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Evangelista
authors.forename: S
authors.initials: S
authors.lastname: Manarini
authors.forename: S
authors.initials: S
authors.lastname: Rotondo
authors.forename: N
authors.initials: N
authors.lastname: Martelli
authors.forename: R
authors.initials: R
authors.lastname: Polischuk
authors.forename: J L
authors.initials: JL
authors.lastname: McGregor
authors.forename: G
authors.initials: G
authors.lastname: de Gaetano
authors.forename: C
authors.initials: C
authors.lastname: Cerletti
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001792:Blood Platelets / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D018821:CD18 Antigens / Q000276:immunology / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D046911:Macromolecular Substances; D016177:Macrophage-1 Antigen / Q000502:physiology*; D008274:Magnesium / Q000502:physiology; D008345:Manganese / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019007:P-Selectin / Q000276:immunology / Q000502:physiology*; D015539:Platelet Activation; D011487:Protein Conformation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D018821:CD18 Antigens; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D016177:Macrophage-1 Antigen; D019007:P-Selectin; D008345:Manganese; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011505:Protein-Tyrosine Kinases; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium