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Thrombin inhibition by covalently bound hirudin
R K Ito1, M D Phaneuf, F W LoGerfo
1New England Deaconess Hospital/Harvard Medical School, Department of Surgery, Boston.
Summary
Recombinant hirudin (rHir) covalently bound to biomaterials retains significant thrombin inhibition. This immobilization technique localizes anticoagulant activity, with only a ten-fold decrease in potency.
Area of Science:
- Biochemistry
- Biomaterials Science
- Pharmacology
Background:
- Hirudin is a potent natural thrombin inhibitor.
- Recombinant and synthesized hirudin are increasingly available.
- Covalent immobilization of hirudin offers potential for localized anticoagulant therapy.
Purpose of the Study:
- To investigate the efficacy of covalently immobilized recombinant hirudin (rHir).
- To assess the impact of immobilization on rHir's antithrombin activity.
- To evaluate rHir's potential for surface localization of anticoagulant effects.
Main Methods:
- Derivatization of rHir using heterobifunctional crosslinking reagents.
- Formation of covalent crosslinks between rHir and albumin to create conjugates.
- Immobilization of rHir onto Dacron prosthetic graft patches.
- Assays to measure alpha-thrombin inhibition and platelet aggregation.
Main Results:
- Both derivatized rHir and conjugates showed a ten-fold decrease in alpha-thrombin inhibition compared to rHir alone.
- Immobilized rHir on Dacron grafts inhibited alpha-thrombin in solution at an 8:1 molar ratio.
- The ED50 for rHir in inhibiting platelet aggregation was 0.12 x 10(-6) microM.
- The conjugate ED50 showed an eleven-fold loss of activity (1.37 x 10(-6) microM).
Conclusions:
- Covalent immobilization of rHir results in a manageable ten-fold loss of inhibitory activity.
- This technique effectively localizes antithrombin activity to surfaces or soluble carriers.
- Immobilized rHir holds promise for targeted anticoagulant applications.