Point mutations modifying the thrombin inhibition kinetics and antithrombotic activity in vivo of recombinant hirudin.
The hirudin variant HV2 was modified by in vitro site-specific mutagenesis of HV2 cDNA to generate HV2(Asn-47----Lys), HV2(Asn-47----Arg) and HV2(Lys-35----Thr, Asn-47----Lys). Residues 35 and 47 are positioned respectively within the finger and prothrombin-like domains of hirudin, both of which have been suggested as thrombin binding sites. The modified polypeptides were synthesized in Saccharomyces cerevisiae using a secretion vector and purified from culture supernatants. By analysis of the human alpha-thrombin:hirudin inhibition reaction in steady-state conditions it was shown that the dissociation constants for HV2(Lys-47) and HV2(Arg-47) were 5- to 14-fold lower than for unmodified HV2, whereas mutation of Lys-35 did not significantly alter the inhibition kinetics. Furthermore, HV2(Lys-47), whose sequence is identical to a natural hirudin variant, displayed enhanced anti-thrombotic activity in vivo, having a 100-fold lower ED50 compared to HV2 in the rabbit Wessler venous thrombosis model. These results support a role for the prothrombin-like domain in thrombin binding and, moreover, demonstrate that in vivo antithrombotic efficiency correlates with the dissociation constant of the inhibition reaction.
Degryse
E
E
Transgène SA, Strasbourg, France.
Acker
M
M
Defreyn
G
G
Bernat
A
A
Maffrand
J P
JP
Roitsch
C
C
Courtney
M
M
D000595:Amino Acid Sequence; D000818:Animals; D006629:Hirudins / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors*
D006629:Hirudins; D011994:Recombinant Proteins; D013917:Thrombin