Abstract
We have established a solid phase binding assay using biotinylated fibrinogen as a ligand. Biotinylation did not alter its binding capacity and affinity to solid phase GPIIb/IIIa. Biotinylated fibrinogen binding, as measured by color development of enzymatic reaction, was specific, time-dependent, reversible, saturable and divalent cation-dependent. Unlabeled fibrinogen and three peptide fragments of fibrinogen, RGDV, RGDS and HHLGGAKQAGDV (gamma-chain 400-411) competitively inhibited the binding with IC50 values of 3.7, 49, 155 and 715nM, respectively. The apparent dissociation constant (Kd) of biotinylated fibrinogen was determined to be 0.33nM.
This simple and high-flux solid phase binding assay can be useful to directly detect the binding between fibrinogen and GPIIb/IIIa.
