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 gamma' chain (gamma'408-427). Comparison of the gamma' amino acid sequence (VRPEHPAETEYDSLYPEDDL) with other protein sequences known to bind to thrombin exosites such as those in GPIbalpha, 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 gamma' 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 gamma' peptides was prepared for evaluation of their inhibition of 125I-labeled PPACK-thrombin binding to fibrin. gamma'414-427 was as effective an inhibitor as gamma'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 gamma'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 gamma' 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.
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Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee 53201-2178, USA. dmeh@bcsew.edu
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D000595:Amino Acid Sequence; D001667:Binding, Competitive; D005337:Fibrin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization; D013917:Thrombin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000494:pharmacology
D010446:Peptide Fragments; C005662:tyrosine O-sulfate; D014443:Tyrosine; D005337:Fibrin; D013917:Thrombin