Lactotransferrin binding to its platelet receptor inhibits platelet aggregation

Abstract

A fluorescent lactotransferrin probe was prepared by coupling 5-( { [2-(carbhydrazino)methyl]-thio) acety1)amino fluorescein to aldehyde groups that were produced by a mild periodic-acid oxidation of the glycan moieties of lactotransferrin. In this manner, the receptor-binding site of the lactotransferrin remains active in contrast to the binding site of the lactotransferrin derivatized with fluorescein isothiocyanate. The fluorescent probe allowed us to characterize, by flow cytometry, the binding of lactotransferrin to non-activated human platelets. The putative lactotransferrin platelet receptor was purified and its immunological and physico-chemical properties were found to be very similar to those of the receptor previously isolated from activated human lymphocytes. Lactotransferrin inhibits ADP-induced platelet aggregation at concentrations down to 5 nM, which can be reached in the plasma after leukocyte degranulation. Inhibition of platelet aggregation was also observed with the N-terminal fragment of lactotransferrin (residues 3 -281 ; 50% inhibition = 2 pM) and with CFQWQRNMRKVRGPPVSC synthetic octodecapeptide (residues 20 -37 ; 50% iahibition = 20 pM) corresponding to one of the two external loops (residues 28-34 and 39-42) where we recently located the receptor-binding site. The activity (50% inhibition = 500 pM) of the tetrapeptide KRDS (residues 39-42), which has already been described, was at least 25-times and 16000-times lower than the activity of the octodecapeptide and of the lactotransferrin molecules, respectively. Finally, the inhibition was demonstrated to be mediated by a mechanism which requires the binding of lactotransferrin to its putative receptor and not to platelet glycoprotein IIb-IIIa.Lactotransfemn (also called lactoferrin), discovered by Montreuil et al. in human milk [l, 21 was also found in human granulocytes [3] and numerous epithelial secretions [4, 51. Major progress has been made in the determination of its glycan [6] and peptide [7,8] sequences, three-dimensional structure and the understanding of iron capture [9, 101. In contrast, little is known about either its biological activity or target cells. Lactotransferrin has been proposed to be involved in iron absorption [2, 11, 121, growth-factor activity [13, 141, antibacterial activity [15, 161 and immunological processes [17], but none were definitively established. The nature of the target cells was investigated and binding sites were described for enterocytes [ l l , 12, 18, 191, liver cells [20, 213 and leukocytes [22]. However, no platelet lactotransferrin receptor was found [22] cyte-membrane receptor [23 -251. It has been recently shown that the KRDS tetrapeptide, which corresponds to loop 39-42 of lactotransferrin molecule and is thus an integral part of the lactotransferrin receptor binding site, inhibits platelet aggregation [26 -281. We have therefore investigated the presence of the lactotransferrin receptor on non-activated human platelets. In order to achieve this and because the fluorescein-isothiocyanat...

A Cell-penetrating Peptide Derived from Human Lactoferrin with Conformation-dependent Uptake Efficiency

“…In contrast to our peptide, binding to these receptors was therefore saturable. Quite remarkably, regarding binding to the lymphocyte lactoferrin receptor and the platelet lactoferrin receptor, evidence has been obtained for the involvement of the loop domain corresponding to the lactoferrin-derived CPP (49,50). Competition experiments with our peptide for the binding of lactoferrin may thus help to better define ligand binding domains for other types of lactoferrin receptors.…”

Candidacidal Activities of Human Lactoferrin Peptides Derived from the N Terminus

“…The exact mechanism by which hLF peptides exert their bactericidal activity is not known. A number of antimicrobial peptides have been shown to bind cell wall components of gram-positive and gram-negative bacteria (19) and C. albicans (15), and lactoferrin receptors on a variety of cells have been described (17,30,36,37). Previous studies have shown that the cytotoxic activity of human neutrophil defensins l to 3 against C. albicans (29), bacteria (41), and tumor cells (31) is energy dependent.…”

“…hLF (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) in killing C. albicans, since it was necessary to use 10 times more hLF(21-31) than hLF(1-11) for a similar level of candidacidal activity. Next, to find out which N-terminal amino acids of hLF are essential in the candidacidal activity of the peptide comprising the first cationic domain, we compared the candidacidal activities of hLF(1-11) and fragments thereof.…”

Lactoferrin Inhibits Platelet Production from Human Megakaryocytes in Vitro

“…It has been reported that LF receptors are expressed on intestine, 1) monocytes, 2) osteoblasts, 3) PHAactivated lymphocytes, 4) PLTs, 5) and megakaryocytic cells. 6) LF levels in the serum of healthy donors are in the range of 2 to 7 mg/ml.…”

“…9,10) To date, it has been reported that LF is involved in a variety of biological functions, such as cell growth and differentiation, [11][12][13] modulation of the inflammatory response, 14) anti-viral effects, 15) and regulation of myelopoiesis. 16,17) LF showed inhibitory effects on PLT aggregation by binding to the LF receptor which exists on the surface of PLTs, 5) but it is unclear whether LF can act on MKs though the LF receptor on the MKs. 6) Therefore, we assume that LF may contribute to differentiation of MKs as well as being involved in cell proliferation and differentiation.…”