Endogenous platelet fibrinogen: its modulation after surface expression is related to size‐selective access to and conformational changes in the bound fibrinogen

Abstract

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.

An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function.

“…Interestingly, both ticlopidine and clopidogrel also neutralize ADP-induced inhibition of PGE1-activated adenylate cyclase (39,40 Binding of mAbs to activation-dependent epitopes on GP Ilb-IIIa or bound fibrinogen was then performed as a measure of platelet activation in whole blood. The mAbs included PAC-1, which binds close to a fibrinogen-binding site on GP fibHIIa (see reference 44), AP-6 which binds to a determinant within the /3(211-221) sequence (18), and two anti-RIBS mAbs (F26 and 9F9) that recognize epitopes on fibrinogen after it has bound to GP fib-Ila (20,21,45 …”

“…F26 was provided by Dr. H. Gralnick (National Institutes of Health, Bethesda, MD) and 9F9 by Dr. A. Budzynski (Temple University, Philadelphia, PA). They are anti-RIBS (receptor induced binding site) mAbs which recognize determinants expressed when fibrinogen is attached to the GP IIb-IIIa receptor, but not when it is in the soluble phase (20,21). They were used as purified IgG, F26 at 5 Mg/ml and 9F9 at 20 MLg/ml.…”

Expression of Markers of Platelet Activation and the Interpatient Variation in Response to Abciximab

“…The epitope for AP-6 is a ligand-induced binding site (LIBS) exposed on GP IIb-IIIa complexes after agonist-induced fibrinogen binding. 22 The anti-receptor-induced binding site (RIBS) IgG murine mAb, F26, recognizes receptor-bound fibrinogen on platelets 23 and was provided by Dr H. Gralnick (Bethesda, Md). AP-2 (from Dr Kunicki) is an IgG mAb that reacts with a complex-dependent determinant common to unactivated and activated GP IIb-IIIa.…”

Autoimmune thrombocytopenic purpura (AITP) and acquired thrombasthenia due to autoantibodies to GP IIb–IIIa in a patient with an unusual platelet membrane glycoprotein composition

“…AP2 (anti-GP IIb-IIIa) and AP6 (an IgM recognizing an anti-LIBS [''ligand-induced binding site''] on GP IIIa) were generously made available by Dr. Thomas Kunicki (Scripps Research Institute, La Jolla, CA) [25,26]. F26 (recognizing an anti-RIBS [''receptorinduced binding site''] on fibrinogen) was kindly provided by Professor Harvey Gralnick (NIH, Washington, DC) [27]. PAC-1 (an IgM recognizing activated but unoccupied GP IIb-IIIa complexes) was purchased from the University of Pennsylvania, Philadelphia [28].…”