Inhibition of thrombin by hirudin genetically fused to wild-type or mutant antithrombin.
Recombinant fusion proteins consisting of hirudin variant 3 (HV3) fused at its C-terminus to either of two forms of mature rabbit antithrombin (AT) were generated in COS-I cells. HV3 fused to wild-type AT was designated HAT, while a similar chimeric protein in which the P12 residue of AT was mutated from Ala to Thr, was designated HAT(H) for the Hamilton (A382T) mutation. Addition of the HV3 domain resulted in a decreased mobility of both HAT and HAT(H) relative to COS-derived AT (68 kDa versus 60 kDa). Both proteins had a greatly increased ability to inhibit thrombin in amidolytic activity assays, relative to recombinant AT. Addition of heparin to these reactions was without effect. Incubation of conditioned media containing recombinant AT with 125I-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 125I-labelled thrombin to immobilized HV1 (CGP 39393). Both proteins were found to bind to heparin-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 that the fusion proteins inhibit thrombin with similar affinity to unfused hirudin via their hirudin and not their antithrombin domains. The heparin-binding capability of these proteins may indicate the acquisition of vessel wall binding capacity by these novel forms of recombinant hirudin.
Syed
S
S
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Kelly
K E
KE
Sheffield
W P
WP
D000818:Animals; D000990:Antithrombin III / Q000235:genetics / Q000494:pharmacology*; D019556:COS Cells; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D006629:Hirudins / Q000235:genetics / Q000494:pharmacology*; D009154:Mutation; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors*
D005343:Fibrinolytic Agents; D006629:Hirudins; D011993:Recombinant Fusion Proteins; D000990:Antithrombin III; D013917:Thrombin