Endogenous platelet fibrinogen: its modulation after surface expression is related to size-selective access to and conformational changes in the bound fibrinogen.
Platelet stimulation results in the release of endogenous platelet fibrinogen which binds to the platelet surface. Previous studies have demonstrated that plasma fibrinogen bound to activated platelets becomes inaccessible to a variety of probes. We have studied endogenous platelet fibrinogen binding to activated platelets by employing an immunopurified polyclonal anti-fibrinogen antibody and F26, a monoclonal anti-fibrinogen antibody, which recognizes fibrinogen only when it is bound to a surface. Employing the Ig or F(ab')2 of the poly- or monoclonal antibody we found a marked decrease of fibrinogen accessibility 30-60 min after platelet activation. In contrast, platelet-bound fibrinogen remains accessible to the Fab fragment of F26 at a constant level for 30 min and increases at 60 min. The reduction of the polyclonal Fab fragment binding at 30 and 60 min is similar to the F26 Ig. These results indicate that the decreased accessibility of bound fibrinogen is related to two mechanisms; (1) that the access route to fibrinogen in size selective for the antibody probes and only small antibody probes, e.g. Fab fragments, can gain access to fibrinogen and (2) fibrinogen undergoes a conformational change(s) after binding which exposes at least one neo-epitope in the D domain of fibrinogen and which may decrease or mask the reactivity of other fibrinogen domains. Only the F26 Fab probe has full access to and identifies fibrinogen present on the platelet surface 60 min after stimulation.
Gralnick
H R
HR
Hematology Service, National Institutes of Health, Bethesda, Maryland 20892.
Williams
S B
SB
McKeown
L
L
Shafer
B
B
Connaghan
G D
GD
Hansmann
K
K
Vail
M
M
Magruder
L
L
D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions / Q000276:immunology; D001792:Blood Platelets / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005340:Fibrinogen / Q000276:immunology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D015539:Platelet Activation / Q000276:immunology; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D005340:Fibrinogen