The C-terminal sequences of the ?57.5 chain of human fibrinogen constitute a plasmin sensitive epitope that is exposed in crosslinked fibrin

The ? chain of human plasma fibrinogen is heterogeneous with three forms differing in length at the C-terminus. Alternative RNA splicing produces two ? chain mRNAs encoding ?50 and ?57.5 polypeptides, while fibrinogen ?55 is produced by post-translational modification of the ?57.5 chain. The composition of purified variant ? chain fibrinogens, which comprise 10% to 13% total plasma fibrinogen, is predominantly heterodimeric (Aa, Bß, ?50/?55 or Aa, Bß, ?50/?57.5), whereas the composition of purified fibrinogen with the major form of the ? chain is homodimeric (Aa, Bß, ?50/?50). These ? chain variations interrupt sequences that mediate platelet-fibrinogen interactions. Therefore, the structure and function of ?57.5 C-terminal sequences were investigated using synthetic peptides and a specific monoclonal antibody (MoAb), L2B. The L2B epitope was localized and included ?57.5 chain residues 409-412 (Arg-Pro-Glu-His), as determined by differential enzyme-linked immunosorbent assay (ELISA) reactivity with a His-412 deleted synthetic peptide and by Western blot analysis of plasmin cleaved fibrinogen ?57.5. L2B had no effect on adenosine diphosphate (ADP)-induced platelet aggregation supported by either fibrinogen ?50 or ?57.5. High concentrations (0.5 to 1 mmol/L) of synthetic peptide ?57.5_408-416 only weakly inhibited ADP-induced platelet aggregation supported by either fibrinogen ?50 or ?57.5. Binding of fibrinogen ?50 (IC₅₀ = 780 µmol/L) or ?57.5 (IC₅₀ = 650 µmol/L) to ADP-stimulated platelets was weakly inhibited, and MoAb L2B failed to inhibit fibrinogen ?57.5 binding. Peptide ?57.5_408-416 failed to dissociate platelet-bound fibrinogens. These data indicate that the ?_408-416 sequence of fibrinogen ?55 or ?57.5 alone is unlikely to bind to the platelet fibrinogen receptor, glycoprotein IIb-IIIa (GPIIb-IIIa), in support of platelet aggregation under physiologic conditions. The sequence recognized by L2B does not resemble known GPIIb-IIIa binding site peptide sequences [Arg-Gly-Asp-Ser (RGDS) or ?50_400-411] as determined by competitive inhibition ELISA comparing these binding site synthetic peptides with ?57.5_408-416. This epitope is available for binding MoAb L2B in ?55 or ?57.5 chain dimers and binds to all ?57.5₄₀₈-₄₁₆ epitopes equally in non-crosslinked and factor XIIIa crosslinked fibrin clots. The availability of the L2B epitope in crosslinked fibrin indicates that it is exposed in the fibrin clot, which is in agreement with the predicted secondary structure of the ?57.5 chain C-terminus.