INHIBITION OF THROMBIN BY HIRUDIN GENETICALLY FUSED TO WILD-TYPE OR MUTANT ANTITHROMBIN

Recombinant fusion proteins consisting of hirudin variant 3 (HV3) fuse d at its C-terminus to either of two forms of mature rabbit antithromb in (AT) were generated in COS-1 cells. HV3 fused to wild-type AT was d esignated HAT, while a similar chimeric protein in which the P12 resid ue of AT was mutated from Ala to Thr, was designated HAT(H) for the Ha milton (A382T) mutation. Addition of the HV3 domain resulted in a decr eased mobility of both HAT and HAT(H) relative to COS-derived AT (68 k Da versus 60 kDa). Both proteins had a greatly increased ability to in hibit thrombin in amidolytic activity assays, relative to recombinant AT. Addition of heparin to these reactions was without effect. Incubat ion of conditioned media containing recombinant AT with I-125-labelled thrombin resulted in the formation of SDS-stable AT-IIa complexes; no such complexes were detected in identical reactions containing either HV3-AT fusion protein. The two proteins did not differ significantly in their ability to compete for the binding of I-125-labelled thrombin to immobilized HV1 (CGP 39393). Both proteins were found to bind to h eparin-Sepharose, but less tightly than unfused AT. This property was demonstrated by the peak elution of the fusion proteins at 0.65 M NaCl , as compared to that of COS-derived AT at 1.05 M NaCl. We conclude th at the fusion proteins inhibit thrombin with similar affinity to unfus ed hirudin via their hirudin and not their antithrombin domains. The h eparin-binding capability of these proteins may indicate the acquisiti on of vessel wall binding capacity by these novel forms of recombinant hirudin. Copyright (C) 1996 Elsevier Science Ltd