Determination of the putative binding site for fibronectin on platelet glycoprotein IIb-IIIa complex through a hydropathic complementarity approach.
We have applied the principle of complementary hydropathy to the prediction of the binding site for fibronectin (FN) and for the alpha-chain of fibrinogen in the platelet receptor complex glycoprotein (GP) IIb-IIIa. Since both ligands bind to it through their respective RGDS (Arg-Gly-Asp-Ser) domains and since both have been cloned, we were able to deduce the amino acid sequence of the binding site from the nucleotide sequence coding for RGDS in both proteins. The deduced peptides were very similar. Antibodies raised against a synthetic peptide WTVPTA (Trp-Thr-Val-Pro-Thr-Ala) deduced from the cloned rat FN RGDS domain block ADP-mediated platelet aggregation; this block can be overcome by additional fibrinogen. In Western blots of whole cell platelet extracts run under reducing conditions, this antibody binds to a 108-kDa band. It also binds to affinity-purified GP IIIa. Furthermore, it reacts strongly with GP IIIa immunoprecipitated by a commercially available anti-GP IIb-IIIa monoclonal antibody. Binding of affinity-purified GP IIb-IIIa complex to fibronectin is inhibited by the 110-kDa FN fragment. Similar inhibitions can be effected by WTVPTA (Trp-Thr-Val-Pro-Thr-Ala) and GAVSTA (Gly-Ala-Val-Ser-Thr-Ala) predicted from the rat and human fibronectin nucleotide sequences, respectively. GAGSTA (Gly-Ala-Gly-Ser-Thr-Ala) and GARSTA (Gly-Ala-Arg-Ser-Thr-Ala) related to the human peptide but with discrepant hydropathies are noninhibitory.
Pasqualini
R
R
Ludwig Institute for Cancer Research, Faculdade de Medicina, Universidade de São Paulo, Brazil.
Chamone
D F
DF
Brentani
R R
RR
D000328:Adult; D000595:Amino Acid Sequence; D000954:Antigens, Surface / Q000235:genetics / Q000302:isolation & purification; D001792:Blood Platelets / Q000378:metabolism*; D015815:Cell Adhesion Molecules; D015342:DNA Probes; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000235:genetics / Q000302:isolation & purification*
D000954:Antigens, Surface; D015815:Cell Adhesion Molecules; D015342:DNA Probes; D005353:Fibronectins; D009842:Oligopeptides; D010980:Platelet Membrane Glycoproteins; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic; C046617:arginyl-glycyl-aspartyl-serine