STRUCTURAL REQUIREMENTS OF PLATELET CHEMOKINES FOR NEUTROPHIL ACTIVATION

Using recombinantly expressed proteins and synthetic peptides, we exam ined the structural/functional features of the platelet chemokines, ne utrophil-activating peptide-2 (NAP-2) and platelet factor 4 (PF4), tha t 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 n eutrophil-activating properties. We examined whether NAP-2 and PF4 had similar structural requirements. In the Ale-glu-leu-arg (AELR) N-term inus of NAP-2, substitution of E or R abolished Ca2+ mobilization and elastase secretion. Unlike the parent molecule PF4, AELR/PF4, the hybr id formed by replacing the N-terminal sequence of PF4 before the first cysteine residue with the homologous sequence of NAP-2, stimulated Ca 2+ mobilization and elastase secretion. Furthermore, the effect of ami no acid substitutions in the ELR motif differed from those seen with N AP-2 in that conserved substitutions of E or R in NAP-2 abolished acti vity, but only reduced neutrophil activation in the hybrid. These stud ies show that just as with IL-8, the N-termini of NAP-2 and PF4 are cr itical for high-level neutrophil-activating function. Desensitization studies provided information on receptor binding. NAP-2, which binds a lmost exclusively to the type 2 IL-8 receptor (IL-8R), did not desensi tize 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 activ ation, 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 sha re approximate to 60% amino acid homology, they have different recepto r affinities. Studies were performed to define the role of the C-termi ni of these platelet chemokines in receptor binding. Heparin and a mon oclonal antibody specific for the heparin-binding domain of PF4 both i nhibited Ca2+ mobilization and elastase release, further suggesting th at the C-terminus of these chemokines is important in receptor binding . Synthetic NAP-2(51-70) failed to mobilize Ca2+, whereas PF47-70 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. Calcium mobil ization studies using HL60 cells recombinantly expressing the type 2 I L-8R show that at least part of the activation by the PF4 C-terminal p eptide is by way this receptor. Thus, our studies show that both the N -terminus immediately preceding the first cysteine residue and the C-t erminus of these chemokines influence their neutrophil activation prop erties. (C) 1994 by The American Society of Hematology.