Covalent Linkage of Streptokinase to Recombinant Hirudin: A Novel Thrombolytic Agent with Antithrombotic Properties

 

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Summary

In a continuing effort to create an agent which has both thrombolytic and antithrombotic properties, streptokinase (SK) was covalently bound to the potent antithrombin agent recombinant hirudin (rHir). Linkage of SK to ¹²⁵I-rHir was accomplished via heterobifunctional crosslinkers in an average molar ratio of 1:1. The ¹²⁵I-rHir-SK complex was purified from starting components by anion exchange and gel filtration chromatography. The major band containing covalently bound ¹²⁵I-rHir had a molecular weight of 53 kDa as determined by SDS-PAGE and autoradiography. Biologic activity of each component was then assayed utilizing the chromogenic substrate for each compound. Complex bound ¹²⁵I-rHir exhibited a 1.2 fold decrease in thrombin inhibition when compared to concentrations of, ²⁵I-rHir greater than 3.13 nM. Complex bound ⁱ²⁵I-SK, replacing the ¹²⁵I label on rHir, displayed a 7.9-fold loss in plasminogen activation when compared to ^l25I-SK. These chromogenic assay results were not adversely altered in the presence of the converse compound’s substrate. The ¹²⁵I-SK-rHir complex (examined at various concentrations) also demonstrated a 0. 17- to 17-fold greater affinity for thrombin immobilized onto Sepha- rose beads as compared to ¹²⁵I-SK. These findings indicate the rHir-SK complex maintained both thrombolytic and antithrombin properties while also obtaining affinity for immobilized thrombin.

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