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 moi eties of lactotransferrin. In this manner, the receptor-binding site o f the lactotransferrin remains active in contrast to the binding site of the lactotransferrin derivatized with fluorescein isothiocyanate. T he fluorescent probe allowed us to characterize, by flow cytometry, th e binding of lactotransferrin to non-activated human platelets. The pu tative lactotransferrin platelet receptor was purified and its immunol ogical and physico-chemical properties were found to be very similar t o those of the receptor previously isolated from activated human lymph ocytes. 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 o bserved with the N-terminal fragment of lactotransferrin (residues 3-2 81 ; 50% inhibition = 2 muM) and with CFQWQRNMRKVRGPPVSC synthetic oct odecapeptide (residues 20-37; 50% inhibition = 20 muM) corresponding t o one of the two external loops (residues 28-34 and 39-42) where we re cently located the receptor-binding site. The activity (50% inhibition = 500 muM) of the tetrapeptide KRDS (residues 39-42), which has alrea dy been described, was at least 25-times and 16000-times lower than th e activity of the octodecapeptide and of the lactotransferrin molecule s, respectively. Finally, the inhibition was demonstrated to be mediat ed by a mechanism which requires the binding of lactotransferrin to it s putative receptor and not to platelet glycoprotein IIb-IIIa.