Decreased accessibility of platelet-bound fibrinogen to antibody and enzyme probes.
The binding of fibrinogen to platelets is a multiphasic process leading to apparently nonreversible associations between fibrinogen and stimulated platelets. To further investigate changes in platelet-fibrinogen interactions, the present study examined the accessibility of platelet-bound fibrinogen and its GPIIb-IIIa receptor to antibody and enzyme probes as a function of time after platelet stimulation with adenosine diphosphate (ADP). Whereas only minimal changes in fibrinogen and 10E5 binding were observed within 60 minutes after platelet stimulation and equilibrium fibrinogen binding, the binding of polyclonal antifibrinogen antibodies decreased significantly (75% +/- 13%, mean +/- SD, n = 9). Similar decreases were noted with rabbit antifibrinogen Fab and F(ab')2 fragments. In addition, plasmin (32 mU/mL) added to platelets five minutes compared with 60 minutes after equilibrium fibrinogen binding dissociated 52% +/- 12% compared with 33% +/- 7% of platelet-bound fibrinogen in five minutes, and 83% +/- 15% compared with 66% +/- 14% of bound fibrinogen in 15 minutes. No difference in plasmin cleavage products was observed, however, by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE). Complete fibrinogen dissociation occurred 30 minutes after plasmin addition, confirming that fibrinogen was not internalized. In contrast, dissociation of platelet-bound fibrinogen by chymotrypsin was less affected by time after equilibrium fibrinogen binding, and minimal changes in antifibrinogen antibody recognition and plasmin-induced dissociation of fibrinogen bound to stimulated but glutaraldehyde-fixed platelets were observed. The data suggest that ADP-induced fibrinogen binding to fresh platelets is accompanied by progressive rearrangements of fibrinogen on the platelet surface.
Peerschke
E I
EI
Department of Pathology, SUNY, Stony Brook 11794.
D000906:Antibodies; D001666:Binding Sites, Antibody; D001792:Blood Platelets / Q000378:metabolism*; D002918:Chymotrypsin; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism; D005340:Fibrinogen / Q000276:immunology / Q000378:metabolism*; D005341:Fibrinolysin; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007700:Kinetics; D015335:Molecular Probes; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism
D000906:Antibodies; D005338:Fibrin Fibrinogen Degradation Products; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D015335:Molecular Probes; D010980:Platelet Membrane Glycoproteins; D005340:Fibrinogen; D002918:Chymotrypsin; D005341:Fibrinolysin