Section 7
Chapter 6,737

The platelet fibrinogen interaction evidence for proximity of the a alpha chain of fibrinogen to platelet membrane glyco proteins iib glyco protein iii

Marguerie, G.A.; Thomas Maison, N.; Ginsberg, M.H.; Plow, E.F.

European Journal of Biochemistry 139(1): 5-12


ISSN/ISBN: 0014-2956
Accession: 006736241

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Fibrinogen participates in [human] platelet aggregation via specific inducible receptors on the cell surface. A photoactivable bifunctional reagent, N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate, SANAH, was used 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 MW 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 < 5% immunoprecipitation by the anti-GP IIb/III. As controls for specificity, anti-(glycoprotein Ib) or anti-IgG immunoprecipitated < 5% of the extracted 125I-Fg-SANAH. Under similar conditions, 45% of the GPIIb/III from surface-labeled platelets was recovered in the anti-GP IIB/III immunoprecipitated. The A.alpha. chain of fibrinogen apparently comes in close proximity to GP IIb/III when the molecule is bound to its platelet receptor.

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