Summary
Human fibrin has a low affinity thrombin binding site in its E domain and a high affinity
binding site in the carboxy-terminal region of its variant ’ chain (’408-427). Comparison
of the ’ amino acid sequence (VRPEHPAETEYDSLYPEDDL) with other protein sequences known
to bind to thrombin exosites such as those in GPIb , the platelet thrombin receptor,
thrombomodulin, and hirudin suggests no homology or consensus sequences, but Glu and
Asp enrichment are common to all. Tyrosine sulfation in these sequences enhances thrombin
exosite binding, but this has not been uniformly investigated. The fibrinogen ’ chain
mass determined by electrospray ionization mass spectrometry, was 50,549 Da, a value
151 Da greater than predicted from its amino acid/carbohydrate sequence. Since each
sulfate group increases mass by 80 Da, this indicates that both tyrosines at 418 and
422 are sulfated. A series of overlapping ’ peptides was prepared for evaluation of
their inhibition of 125I-labeled PPACK-thrombin binding to fibrin. ’414-427 was as effective an inhibitor
as ’408-427 and its binding affinity was dependent on all carboxy-terminal residues.
Mono Tyr-sulfated peptides were prepared by substituting non-sulfatable Phe for Tyr
at ’ 418 or 422. Sulfation at either Tyr residue increased binding competition compared
with non-sulfated peptides, but was less effective than doubly sulfated peptides,
which had 4 to 8-fold greater affinity. The reverse ’ peptide or the forward sequence
with repositioned Tyr residues did not compete well for thrombin binding, indicating
that the positions of charged residues are important for thrombin binding affinity
Keywords
Fibrin - thrombin - exosite binding - tyrosine-sulfation
