title: Structural requirements of platelet chemokines for neutrophil activation.
abstract: Using recombinantly expressed proteins and synthetic peptides, we examined the structural/functional features of the platelet chemokines, neutrophil-activating peptide-2 (NAP-2) and platelet factor 4 (PF4); that were important in their activation of neutrophils. Previous studies with the chemokine interleukin-8 (IL-8) had shown that the N-terminal region preceding the first cysteine residue was critical in defining neutrophil-activating properties. We examined whether NAP-2 and PF4 had similar structural requirements. In the Ale-glu-leu-arg (AELR) N-terminus of NAP-2, substitution of E or R abolished Ca2+ mobilization and elastase secretion. Unlike the parent molecule PF4, AELR/PF4, the hybrid formed by replacing the N-terminal sequence of PF4 before the first cysteine residue with the homologous sequence of NAP-2, stimulated Ca2+ mobilization and elastase secretion. Furthermore, the effect of amino acid substitutions in the ELR motif differed from those seen with NAP-2 in that conserved substitutions of E or R in NAP-2 abolished activity, but only reduced neutrophil activation in the hybrid. These studies show that just as with IL-8, the N-termini of NAP-2 and PF4 are critical for high-level neutrophil-activating function. Desensitization studies provided information on receptor binding. NAP-2, which binds almost exclusively to the type 2 IL-8 receptor (IL-8R), did not desensitize neutrophils to activation by IL-8 because IL-8 could bind to and activate via both type 1 and 2 IL-8R. AELR/PF4 appears to bind to both types of receptors because it desensitized neutrophils to NAP-2 activation; but was not desensitized by NAP-2, and because it desensitized to and was desensitized by IL-8. Thus, although NAP-2 and AELR/PF4 share approximately 60% amino acid homology, they have different receptor affinities. Studies were performed to define the role of the C-termini of these platelet chemokines in receptor binding. Heparin and a monoclonal antibody specific for the heparin-binding domain of PF4 both inhibited Ca2+ mobilization and elastase release, further suggesting that the C-terminus of these chemokines is important in receptor binding. Synthetic NAP-2(51-70) failed to mobilize Ca2+, whereas PF4(47-70) and PF4(58-70) induced Ca2+ mobilization and secretion of elastase at high concentrations. Pertussis toxin inhibited neutrophil activation by 40% to 50%, establishing a role for G-protein-coupled receptors such as the IL-8Rs in activation by the PF4 C-terminal peptides.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Physiology, Temple University School of Medicine, Philadelphia, PA.
authors.forename: Z
authors.initials: Z
authors.lastname: Yan
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.forename: J C
authors.initials: JC
authors.lastname: Holt
authors.forename: G J
authors.initials: GJ
authors.lastname: Stewart
authors.forename: S
authors.initials: S
authors.lastname: Niewiarowski
authors.forename: M
authors.initials: M
authors.lastname: Poncz
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001792:Blood Platelets / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018375:Neutrophil Activation; D010196:Pancreatic Elastase / Q000378:metabolism; D010455:Peptides / Q000502:physiology*; D037342:Pertussis Toxin; D010978:Platelet Factor 4 / Q000502:physiology*; D018123:Receptors, Interleukin / Q000502:physiology; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D001620:beta-Thromboglobulin
chemical_list: D017931:DNA Primers; C061615:PPBP protein, human; D010455:Peptides; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D001620:beta-Thromboglobulin; D010978:Platelet Factor 4; C009862:connective tissue-activating peptide; D006493:Heparin; D037342:Pertussis Toxin; D010196:Pancreatic Elastase; D002118:Calcium