Stabilization of platelet-fibrinogen interactions is an integral property of the glycoprotein IIb-IIIa complex.
Fibrinogen binding to platelets is multiphasic and culminates in the stabilization of platelet-fibrinogen interactions characterized by the resistance of bound fibrinogen to dissociation by ethylenediaminetetraacetic acid (EDTA) or excess unlabeled fibrinogen. Controversy exists, however, with regard to the exclusive role of the glycoprotein IIb-IIIa (GPIIb-IIIa) complex in this process. Thus the reversibility of fibrinogen binding to purified GPIIb-IIIa and GPIIb-IIIa activated by a monoclonal antibody (D3) on otherwise resting platelets was examined. GPIIb-IIIa was isolated by affinity chromatography on concanavalin A followed by gel filtration on Sephacryl S-300 and immobilized directly on plastic microtiter wells or immunocaptured by immobilized anti-GPIIb or GPIIIa antibodies. The extent of GPIIb-IIIa deposition, 0.14 to 0.27 pmol/well, was determined by using a monoclonal, anti-GPIIb-IIIa antibody (10E5). Maximum fibrinogen binding occurred after 60 minutes at 22 degrees C in the presence of 300 micrograms/ml fibrinogen, when 0.014 to 0.030 pmol fibrinogen bound per well. Assuming a 1:1 relationship between fibrinogen binding and GPIIb-IIIa occupancy, these data suggest that approximately 10% to 20% of immobilized GPIIb-IIIa was in an active confirmation. After 60 minutes, 65% +/- 13% of bound fibrinogen was resistant to dissociation by excess unlabeled fibrinogen, and 53% +/- 24% failed to dissociate with 10 mmol/L EDTA. Fibrinogen fragment D1 also bound irreversibly to immobilized GPIIb-IIIa (52% +/- 18%).(ABSTRACT TRUNCATED AT 250 WORDS)
Peerschke
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EI
Department of Pathology, SUNY at Stony Brook 11794-7300.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000527:Alprostadil / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001792:Blood Platelets / Q000502:physiology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004347:Drug Interactions; D004355:Drug Stability; D004492:Edetic Acid / Q000494:pharmacology; D005340:Fibrinogen / Q000502:physiology*; D006801:Humans; D010980:Platelet Membrane Glycoproteins / Q000032:analysis / Q000276:immunology / Q000502:physiology*; D013997:Time Factors
D000911:Antibodies, Monoclonal; D010980:Platelet Membrane Glycoproteins; D000244:Adenosine Diphosphate; D005340:Fibrinogen; D004492:Edetic Acid; D000527:Alprostadil