Structural differences in active site-labeled thrombin complexes with hirudin isoinhibitors.

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Hirudin, a potent thrombin inhibitor from medicinal leeches, exists in recombinant forms with comparable efficacy to isolated versions.
• Understanding the precise binding interactions of hirudin variants with different thrombin forms is crucial for developing targeted anticoagulants.

Purpose of the Study:

• To employ sensitive spectroscopic techniques to differentiate the binding of two recombinant hirudins, HV1 and HV2-Lys 47, to various human thrombin active sites.
• To investigate how sequence differences in hirudin isoinhibitors influence their interaction with specific thrombin structures.

Main Methods:

• Utilized active site-directed covalent reporter groups, including fluorosulfonylphenyl nitroxide spin labels, dansyl fluoride, and p-nitrophenylanthranilate.
• Applied spectroscopic methods to monitor and quantify the binding of recombinant hirudins (HV1, HV2-Lys 47) to labeled human alpha-, epsilon-, and zeta-thrombins.

Main Results:

• Observed greater immobilization of nitroxide spin labels in thrombin complexes with HV2-Lys 47 compared to HV1.
• Demonstrated that dansyl and anthraniloyl fluorophore moieties detected distinct binding differences between HV1 and HV2-Lys 47.
• Identified differential interactions, particularly with thrombin's loop 145-150, correlating with epsilon- and zeta-thrombin cleavage sites.

Conclusions:

• Spectroscopic methods effectively distinguish binding nuances between recombinant hirudin variants HV1 and HV2-Lys 47.
• Sequence variations, specifically at residues 24 and 47 affecting charge, likely account for the observed differences in hirudin-thrombin interactions.