Summary
Binding of the synthetic peptide AAKDSDWPEASDEDWNYKAPSGAR, a fibrinogen α27–50 analog,
to thrombin was studied by inhibition assays and affinity chromatography. Peptide
α27–50 corresponds to a segment of human fibrinogen downstream from the thrombin cleavage
site, with cysteine residues at positions 28, 36, 45 and 49 replaced by alanine. The
peptide inhibited clotting of fibrinogen with an inhibition constant of 190–400 μM.
Cleavage of fibrinopeptides A and B was inhibited by the peptide and the peptide was
competitive with fibrinogen for thrombin. Inhibition of the small substrate tosyl-Gly-Pro-Arg-p-nitroaniline
was not observed indicating that the peptide did not block the active site of the
enzyme. Peptide α27–50 that was covalently linked to Sepharose bound active siteinhibited
thrombin at low ionic strength and was eluted at higher salt concentration. The peptide
was not cleaved on overnight exposure to thrombin as determined by reverse phase HPLC.
In summary, the peptide bound to, but was not a substrate for thrombin. These results
suggest that this region of fibrinogen contributes to binding of thrombin.
