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

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Human fibrin possesses distinct thrombin binding sites: a low-affinity site in the E domain and a high-affinity site in the gamma' chain (gamma'408-427).
• Comparison with other thrombin-binding proteins reveals no sequence homology but a common enrichment of Glu and Asp residues.

Purpose of the Study:

• To investigate the role of tyrosine sulfation in the gamma' chain's carboxy-terminal region in enhancing thrombin exosite binding.
• To determine the affinity and binding characteristics of gamma' chain peptides with varying sulfation patterns.

Main Methods:

• Electrospray ionization mass spectrometry (ESI-MS) to determine the mass of the fibrinogen gamma' chain.
• Synthesis and evaluation of overlapping gamma' peptides, including mono- and di-tyrosine sulfated variants, to assess inhibition of 125I-labeled PPACK-thrombin binding.

Main Results:

• ESI-MS confirmed the fibrinogen gamma' chain mass, indicating dual tyrosine sulfation at positions 418 and 422.
• Doubly tyrosine-sulfated gamma' peptides exhibited 4-to-8-fold greater affinity for thrombin binding compared to non-sulfated or mono-sulfated peptides.
• Peptide binding affinity was dependent on the carboxy-terminal residues and their specific positions.

Conclusions:

• Tyrosine sulfation significantly enhances the binding affinity of the fibrinogen gamma' chain to thrombin's exosite.
• The precise positioning of charged residues, particularly sulfated tyrosines, is critical for high-affinity thrombin binding to fibrin.