Human platelet glycoprotein IIIb binds to thrombospondin fragments bearing the C-terminal region, and/or the type I repeats (CSVTCG motif), but not to the N-terminal heparin-binding region.
Major blood membrane platelet glycoprotein IIIb (GPIIIb), also termed GPIV or CD365, has been identified as a receptor for thrombospondin (TSP), collagen and Plasmodium falciparum-infected erythrocytes. The aim of the present study was to identify region(s) of TSP involved in binding of GPIIIb. Proteolytic fragments of TSP (M(r) 140 kDa, 120-18 kDa and 27 kDa on SDS/PAGE under reducing conditions) were purified by f.p.l.c. and identified by N-terminal gas-phase sequencing, e.l.i.s.a. and Western blots using monoclonal antibodies directed against defined domains of TSP. The 140 kDa and 120-18 kDa fragments (C-terminal region), but not the 27 kDa fragment (N-terminal region), were shown to bind to GPIIIb by using e.l.i.s.a. and affinity-chromatography systems. TSP binding to a GPIIIb-affinity column was Ca(2+)-dependent and reduced by 45% in the presence of EDTA. Moreover, TSP was only partially eluted with EDTA from a Ca(2+)-equilibrated GPIIIb column. A fragment of 68 kDa, obtained by further digestion of the 140 kDa fragment, bound to the GPIIIb-Sepharose affinity column. This fragment, or stalk-like region, bears the TSP type I repeats that show sequence similarity to regions on properdin, Plasmodium falciparum proteins and antistasin. Peptides (CSVTCG or SVTCGGGV) representing these repeats bound isolated GPIIIb in a Ca(2+)-independent way, but did not completely inhibit the GPIIIb and TSP interaction. These studies indicate that GPIIIb binds to the TSP via the C-terminal region and/or the CSVTCG motif, but not to the N-terminal region. Interaction between GPIIIb and the TSP C-terminal region or the CSVTCG motif is respectively Ca(2+)-dependent and -independent.
Catimel
B
B
INSERM U331, Faculté de Médecine Alexis Carrel, Institut Pasteur de Lyon, France.
Leung
L
L
el Ghissasi
H
H
Mercier
N
N
McGregor
J
J
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018955:CD36 Antigens; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid / Q000502:physiology; D012685:Sepharose; D019699:Thrombospondins
D015703:Antigens, CD; D018955:CD36 Antigens; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D019699:Thrombospondins; D006493:Heparin; D012685:Sepharose; D002918:Chymotrypsin