title: Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton.
abstract: Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet-fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.
authors.affiliation: Department of Pathology, State University of New York, Stony Brook 11794.
authors.forename: E I
authors.initials: EI
authors.lastname: Peerschke
mesh_terms: D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015153:Blotting, Western; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D017830:Octoxynol; D009842:Oligopeptides / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000502:physiology; D011092:Polyethylene Glycols / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D011092:Polyethylene Glycols; D005340:Fibrinogen; D017830:Octoxynol; D004492:Edetic Acid; C046617:arginyl-glycyl-aspartyl-serine