Ca2+-dependent binding of a synthetic Arg-Gly-Asp (RGD) peptide to a single site on the purified platelet glycoprotein IIb-IIIa complex.
The platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a member of the integrin receptor family that recognizes adhesive proteins containing the Arg-Gly-Asp (RGD) sequence. In the present study the binding characteristics of the synthetic hexapeptide Tyr-Asn-Arg-Gly-Asp-Ser (YNRGDS, a sequence present in the fibrinogen alpha-chain at position 570-575) to purified GP IIb-IIIa were determined by equilibrium dialysis. The binding of 125I-YNRGDS to GP IIb-IIIa was specific, saturable, and reversible. The apparent dissociation constant was 1.0 +/- 0.2 microM, and the maximal binding capacity was 0.92 +/- 0.02 mol of 125I-YNRGDS/mol of GP IIb-IIIa, indicating that GP IIb-IIIa contains a single binding site for RGD peptides. The binding of 125I-YNRGDS to purified GP IIb-IIIa showed many of the characteristics of fibrinogen binding to activated platelets: the binding was inhibited by fibrinogen, by the monoclonal antibody A2A9, and by the dodecapeptide from the C terminus of the fibrinogen gamma-chain. In addition, the binding of 125I-YNRGDS to GP IIb-IIIa was divalent cation-dependent. Our data suggest that two divalent cation binding sites must be occupied for YNRGDS to bind: one site is specific for calcium and is saturated at 1 microM free Ca2+, whereas the other site is less specific and reaches saturation at millimolar concentrations of either Ca2+ or Mg2+. The results of the present study support the hypothesis that the RGD domains within the adhesive proteins are responsible for their binding to GP IIb-IIIa.
Steiner
B
B
Pharmaceutical Research Department, F. Hoffmann-La Roche and Co., Basel, Switzerland.
Cousot
D
D
Trzeciak
A
A
Gillessen
D
D
Hadváry
P
P
D000595:Amino Acid Sequence; D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes; D008274:Magnesium / Q000502:physiology; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D007457:Iodine Radioisotopes; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen; C046617:arginyl-glycyl-aspartyl-serine; D008274:Magnesium; D002118:Calcium