Structure-function and refolding studies of the thrombin-specific inhibitor hirudin

Area of Science:

• Biochemistry
• Protein Engineering
• Pharmacology

Background:

• Hirudin is a potent thrombin inhibitor.
• Understanding hirudin's structure-activity relationship is crucial for developing anticoagulants.

Purpose of the Study:

• To develop a novel expression and purification system for recombinant desulfo-hirudin (HV-1).
• To characterize the activity, refolding properties, and inhibition mechanism of hirudin.
• To identify key functional regions of hirudin responsible for thrombin inhibition.

Main Methods:

• Recombinant expression and purification of desulfo-hirudin (HV-1).
• Assays for specific activity and inhibition constant (Ki) against human alpha-thrombin.
• Analytical gel filtration for multimerization studies.
• Refolding studies at high concentration and alkaline pH.
• Thrombin inhibition assays using synthetic hirudin peptide fragments.

Main Results:

• High specific activity (10,000 antithrombin units/mg) and low Ki (0.2 pM) for recombinant HV-1.
• Rapid refolding of reduced and denatured hirudin at high concentration ( > 50 mg/ml) and pH 10.
• Evidence of hirudin multimerization at neutral pH, forming a 1:1 complex with thrombin.
• Peptide fragments 42-65 and 51-65 effectively inhibited thrombin, indicating key inhibitory regions.

Conclusions:

• Hirudin possesses unusual structural and refolding properties.
• Thrombin inhibition involves noncovalent interactions with multiple sites on thrombin.
• The Lys-47 region contributes to binding but is not essential for inhibition.