The platelet-fibrinogen interaction. Evidence for proximity of the A alpha chain of fibrinogen to platelet membrane glycoproteins IIb/III.
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.
D001386:Azides; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D011232:Chemical Precipitation; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism*; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D007120:Immunochemistry; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood*; D010777:Photochemistry; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins; D011485:Protein Binding
D001386:Azides; D003432:Cross-Linking Reagents; D006023:Glycoproteins; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; C037733:N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate; D005340:Fibrinogen