Lactotransferrin binding to its platelet receptor inhibits platelet aggregation.
A fluorescent lactotransferrin probe was prepared by coupling 5-(([2-(carbhydrazino)methyl]-thio)acetyl)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 microM) and with CFQWQRNMRKVRGPPVSC synthetic octodecapeptide (residues 20-37; 50% inhibition = 20 microM) 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 microM) 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.
Laboratoire de Chimie Biologique, Centre National de la Recherche Scientifique no 111, Université des Sciences et Technologies de Lille France.
B
B
Leveugle
J
J
Mazurier
D
D
Legrand
C
C
Mazurier
J
J
Montreuil
G
G
Spik
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D007781:Lactoferrin / Q000378:metabolism / Q000494:pharmacology*; D037102:Lectins / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism*
D037102:Lectins; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; C038340:lactoferrin receptors; D000244:Adenosine Diphosphate; D007781:Lactoferrin