Abstract
BackgroundHirudin is an anti-coagulation protein produced by the salivary glands of the medicinal leech Hirudomedicinalis. It is a powerful and specific thrombin inhibitor. The novel recombinant hirudin, RGD-hirudin, which contains an RGD motif, competitively inhibits the binding of fibrinogen to GPIIb/IIIa on platelets, thus inhibiting platelet aggregation while maintaining its anticoagulant activity.ResultsRecombinant RGD-hirudin and six mutant variants (Y3A, S50A, Q53A, D55A, E57A and I59A), designed based on molecular simulations, were expressed in Pichia pastoris. The proteins were refolded and purified to homogeneity as monomers by gel filtration and anion exchange chromatography. The anti-thrombin activity of the six mutants and RGD-hirudin was tested. Further, we evaluated the binding of the mutant variants and RGD-hirudin to thrombin using BIAcore surface plasmon resonance analysis (SPR). Kinetics and affinity constants showed that the KD values of all six mutant proteins were higher than that of RGD-hirudin.ConclusionsThese findings contribute to a novel understanding of the interaction between RGD-hirudin and thrombin.
Highlights
Hirudin is an anti-coagulation protein produced by the salivary glands of the medicinal leech Hirudomedicinalis
It blocks the thrombin-mediated conversion of fibrinogen to fibrin during clot formation, but unlike heparin, it is a direct thrombin inhibitor (DTI) [1] that is not inactivated by platelet factor 4 (PF4) [2,3]
A previous study has shown that the inhibitor is a small peptide (65 amino acids, 7 kDa) that binds to active thrombin and irreversibly inactivates it [4]
Summary
Hirudin is an anti-coagulation protein produced by the salivary glands of the medicinal leech Hirudomedicinalis It is a powerful and specific thrombin inhibitor. Asp was mutated to Gln, Glu was mutated to Pro, and Glu was added These changes improve the hydrophobicity of the protein and allow the recombinant RGD-hirudin to interact more effectively with the fibrinogen recognition exosite of thrombin, resulting in a specific activity of 12,000 ATU/mg [15]. Given these changes, we hypothesized that the interaction between RGD-hirudin and thrombin would be similar to that between wild-type hirudin and thrombin. Anti-platelet aggregation activity was tested by classic turbidity assays
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