Cloning and expression of rat coagulation factor VII.
Smaller and widely available animals such as rats are commonly used to evaluate antithrombotic drug candidates in vivo. However, the isolation and purification of FVII from rats and other species is very challenging because they are present in extremely low levels in plasma (approximately 10 nM). Furthermore, purification of FVII from other coagulation factors present in the plasma such as prothrombin, factor IX and factor X can often be very challenging and labor-intensive. To facilitate studies on the role of the extrinsic pathway of coagulation in rats, a full-length cDNA-encoding rat factor VII was isolated using polymerase-mediated DNA amplification using a rat liver cDNA library. The cDNA codes for a 41-residue signal/propeptide region, followed by a 405-residue mature protein consisting of the light chain with gamma-carboxy glutamic acid (gla) including epidermal growth factor domains (EGF) and the heavy chain with the serine protease catalytic domain. Rat factor VII cDNA was transfected into human embryonic kidney 293 cells and several cell lines that constitutively express rat factor VII were established. The media from the stable lines expressing recombinant rat FVII were rapidly screened for functional activity and were found to normalize clotting time of FVII-depleted human plasma. The supernatants were also functionally active in the presence of tissue factor in chromogenic assays by measuring FVIIa activation using a tripeptide chromogenic substrate and in a two-stage, coupled assay measuring the generation of FXa. Recombinant rat FVII may be an important new tool in the development of novel antithrombotic drugs.
Seetharam
Shobha
S
Cardiovacular Department, The Bristol Myers Squibb Company, P.O. Box 400, Experimental Station, Wilmington, DE 19880, USA. shoram2@yahoo.com
Murphy
Kathleen
K
Atkins
Charity
C
Feuerstein
Giora
G
D000818:Animals; D001483:Base Sequence; D001780:Blood Coagulation Tests; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D018076:DNA, Complementary; D005167:Factor VII / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D014162:Transfection
D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D011994:Recombinant Proteins; D005167:Factor VII