The platelet-fibrinogen interaction. Evidence for proximity of the A alpha chain of fibrinogen to platelet membrane glycoproteins IIb/III

Eur J Biochem. 1984 Feb 15;139(1):5-11. doi: 10.1111/j.1432-1033.1984.tb07968.x.

Abstract

Fibrinogen participates in platelet aggregation via specific inducible receptors on the cell surface. We have used a photoactivable bifunctional reagent, N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate, SANAH, to derivatize 125I-labeled-fibrinogen (125I-Fg) and crosslink it to ADP-stimulated platelets. Binding experiments established that 125I-Fg and 125I-Fg-SANAH interacted with platelets with the same kinetics and affinity as unlabeled fibrinogen. After photoactivation of the platelet-bound 125I-Fg-SANAH, polyacrylamide gel electrophoresis under reducing conditions revealed formation of a high molecular weight covalent complex with coordinate loss of the A alpha chain. 125I-Fg-SANAH missing the extreme carboxy-terminal region of the A alpha chain failed to crosslink to the platelets under similar conditions. Crosslinked 125I-Fg-SANAH was extracted from the cells in 1% Triton X-100, and immunoprecipitation with antibodies specific for platelet membrane glycoproteins was used to identify components of the complex. With antibodies to the glycoprotein IIb/III complex (anti-GP IIb/III), 40 +/- 9% of the extracted 125I-Fg-SANAH was immunoprecipitated. Omission of photoactivation, platelets, or ADP from the reaction or use of unmodified 125I-Fg resulted in less than 5% immunoprecipitation by the anti-GP IIb/III. As controls for specificity, anti-(glycoprotein Ib) or anti-IgG immunoprecipitated less than 5% of the extracted 125I-Fg-SANAH. Under similar conditions, 45% of the GP IIb/III from surface-labeled platelets was recovered in the anti-GP IIb/III immunoprecipitate. These results indicate that the A alpha chain of fibrinogen comes in close proximity to GP IIb/III when the molecule is bound to its platelet receptor.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Azides
  • Binding Sites
  • Blood Platelets / metabolism*
  • Chemical Precipitation
  • Cross-Linking Reagents
  • Electrophoresis, Polyacrylamide Gel
  • Fibrinogen / metabolism*
  • Glycoproteins / blood*
  • Humans
  • Immunochemistry
  • Kinetics
  • Membrane Proteins / blood*
  • Photochemistry
  • Platelet Aggregation
  • Platelet Membrane Glycoproteins
  • Protein Binding

Substances

  • Azides
  • Cross-Linking Reagents
  • Glycoproteins
  • Membrane Proteins
  • Platelet Membrane Glycoproteins
  • N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate
  • Fibrinogen