The amino acid sequence in fibrin responsible for high affinity thrombin binding

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 g amma' chain (gamma '408-427). Comparison of the gamma' amino acid sequence (VRPEHPAETEYDSLYPEDDL) with other protein sequences known to bind to thromb in exosites such as those in GPIb alpha, the platelet thrombin receptor, th rombomodulin, and hirudin suggests no homology or consensus sequences, but Glu and Asp enrichment are common to all. Tyrosine sulfation in these seque nces enhances thrombin exosite binding, but this has not been uniformly inv estigated. The fibrinogen gamma' chain mass determined by electrospray ioni zation mass spectrometry, was 50,549 Da, a value 151 Da greater than predic ted from its amino acid/carbohydrate sequence. Since each sulfate group inc reases mass by 80 Da, this indicates that both tyrosines at 418 and 422 are sulfated. A series of overlapping gamma' peptides was prepared for evaluat ion of their inhibition of I-125-labeled PPACK-thrombin binding to fibrin, gamma '414-427 was as effective an inhibitor as gamma '408-427 and its bind ing affinity was dependent on all carboxy-terminal residues. Mono Tyr-sulfa ted peptides were prepared by substituting non-sulfatable Phe for Tyr at ga mma' 418 or 422. Sulfation at either Tyr residue increased binding competit ion compared with non-sulfated peptides, but was less effective than doubly sulfated peptides, which had 4 to 8-fold greater affinity. The reverse gam ma' 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.