Construction and in vitro testing of a novel fab-hirudin-based fusion protein that targets fibrin and inhibits thrombin in a factor xa-dependent manner.
Fibrin targeting of the thrombin inhibitor hirudin via chemical coupling is effective in vitro and in vivo. However, since chemical coupling has limitations, a recombinant approach was taken to improve the fibrin-targeting ability of hirudin. Additionally, to activate hirudin selectively at the target area and thereby limit side effects in an in vivo setting, the authors aimed to construct an inactive precursor molecule that is converted into an active thrombin inhibitor only upon cleavage by factor Xa. Using PCR, the coding region for hirudin was fused to parts of the genomic DNA of the IgG heavy chain that was cloned from the antifibrin antibody-producing hybridoma cell line 59D8. Additionally, a factor Xa recognition site was introduced between the antibody and the hirudin sequence. The fusion construct was then transfected into a heavy-chain loss variant of the hybridoma cell line 59D8. After selection of stable hybridoma clones, the expressed fusion protein was evaluated for its molecular size (57 kd) and its binding ability to the fibrin-specific peptide Bbeta 15-22. The cleavage of the fusion protein by factor Xa was demonstrated by HPLC. The recombinant anticoagulant revealed antithrombin activity only after cleavage by factor Xa. Thus, the newly designed hirudin fusion protein revealed the anticipated functions in vitro. Further experiments are needed to prove whether this precursor anticoagulant allows a highly clot-specific and efficient thrombin inhibition in vivo.
Peter
Karlheinz
K
Department of Cardiology and Angiology, Internal Medicine III, University of Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany. peterkh@medizin.ukl.uni-freiburg.de
Gupta
Amitabh
A
Nordt
Thomas
T
Bauer
Simone
S
Runge
Marschall S
MS
Bode
Christoph
C
D000911:Antibodies, Monoclonal; D000925:Anticoagulants / Q000138:chemical synthesis* / Q000494:pharmacology; D000991:Antithrombins / Q000138:chemical synthesis / Q000494:pharmacology; D002460:Cell Line; D015951:Factor Xa / Q000235:genetics; D005337:Fibrin / Q000037:antagonists & inhibitors*; D006629:Hirudins / Q000235:genetics / Q000494:pharmacology*; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000494:pharmacology*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors*; D014158:Transcription, Genetic; D014162:Transfection
D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D000991:Antithrombins; D006629:Hirudins; D007140:Immunoglobulin Fab Fragments; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005337:Fibrin; D013917:Thrombin; D015951:Factor Xa