Immunoregulatory activity of peptides related to platelet factor 4.

Abstract

Platelet factor 4 (PF4), a secreted platelet protein, alleviates concanavalin A-induced immunosuppression in mice. We now rind that activity also resides in (i) the C-terminal tridecapeptide of PF4 (P13S), (ii) an analog of this in which arginine replaces the lysine residues and in which the last two amino acids are absent, (iii) the C-terminal 18 amino acids of low-affmity platelet factor 4, which is very similar to P13S, and (iv) peptide fragments of P13S that contain only 5-9 amino acids. P13S treated with fluorescamine to derivatize the free amino groups retained immunoregulatory activity but did not bind to heparin-agarose. The N-terminal and middle portions of PF4, polylysine, protamine, and three unrelated peptides were inactive in this assay.Platelet factor 4 (PF4), a 7800-kDa secretable platelet protein with anti-heparin and chemotactic activity (1-3), alleviates immunosuppression induced in mice by injection of concanavalin A (Con A) or y-irradiated reticulum sarcoma cells (4, 5). Earlier results (5) showed that it was active at 0.2 ,ug per mouse and secretable from as few as 2 x 107 human platelets. Recent results have shown activity at 0.05 gg.Recombinant PF4 with additional amino acids at each end of the molecule and with aspartic acid instead of asparagine at position 47 is also active (6). We now report studies on the immunoregulatory activity of fragments of PF4. MATERIALS AND METHODSSource of Peptides. The amino acid sequences of the PF4 molecule and other peptides studied are shown in Fig. 1. T. Maione (Repligen, Cambridge, MA) donated recombinant preparations of (i) PF4 (rPF4) identical to the native molecule, (ii) the C-terminal 41 amino acids (designated C41), (iii) the N-terminal 29 amino acids (designated N29) of PF4, and (iv) synthetic P13S, the C-terminal 13 amino acids of PF4. Low-affinity PF4 (LA-PF4) was prepared from extracts of fresh platelets and was separated from PF4, ,8-thromboglobulin, and platelet basic protein by chromatography on CMSephadex (7). Peptides H18, H22, and H24 were derived from reduced S-pyridylethyl-modified native PF4 (900 ug) by digestion with 60 ,ug of protease isolated from Staphylococcus aureus V8 (Pierce) at 370C for 3 hr in 0.05 M sodium phosphate (pH 7.8). The digest was fractionated on a Vydac wide-pore C18 reverse-phase column (The Separations Group, Hesperia, CA) equilibrated with 0.1% trifluoroacetic acid and was eluted with a gradient of 0-55% acetonitrile in the same solvent. Identity of the collected peaks was established by automated N-terminal sequencing.The C-terminal octadecapeptide of LA-PF4, designated P18D, and a second preparation of P13S were synthesized by Peninsula Laboratories and were provided by Stefan Niewiarowski (Temple University). Peptides unrelated to l 2 3 4 5

Platelet Factor 4: Production, Structure, and Physiologic and Immunologic Action

“…The immunoregulatory effects of mouse PF4, native and recombinant human PF4 (rPF4), and synthetic C-terminal peptides of human PF4 have been described by our group (18,(95)(96)(97)(98)(99)(100)(101)(102) and others (46,103).…”

Biological activity of the growth factor-induced cytokine N51: structure-function analysis using N51/Interleukin-8 chimeric molecules.

“…Alternatively, the ot-helical tail has been proposed to significantly contribute to the binding with the IL-8 receptor(s) and to confer ligand specificity (8,9). This possibility is based on the observations that a C-terminal tridecapeptide of PF4 has both chemotactic and immunoregulatory activities (43). This observation is in contrast with the finding that IL-8 truncated of its C-terminal at helix and onward into the , sheet remained active (7).…”

Binding to heparan sulfate or heparin enhances neutrophil responses to interleukin 8.

“…Chemokines are cationic and bind heparin. The interaction with heparin and related glycosaminoglycans (GAGs), however, has been thoroughly studied for platelet factor 4 only (4)(5)(6)(7)(8).…”

“…It has been demonstrated that platelet factor 4 binds to GAGs through its C-terminal a-helix (5)(6)(7), and this led to the suggestion that IL-8 interacts with heparin and heparan sulfate at the same site (15). In this paper, we show that the capacity of IL-8 to bind heparin decreases markedly with progressive shortening of the a-helix, and we present evidence for a functional role of heparan sulfate and heparin as enhancers of IL-8 activity.…”