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A soluble tissue factor mutant is a selective anticoagulant and
antithrombotic agent
RF Kelley, CJ Refino, MP O'Connell, N Modi, P Sehl, D Lowe, C Pater and S Bunting
Department of Protein Engineering, Genentech, Inc., South San Francisco, CA
94080, USA.
One approach to developing safer and more efficacious agents for the
treatment of thrombotic disease involves the design and testing of
inhibitors that block specific steps in the coagulation cascade. We
describe here the development of a mutant of human tissue factor (TF) as a
specific antagonist of the extrinsic pathway of blood coagulation and the
testing of this mutant in a rabbit model of arterial thrombosis. Alanine
substitutions of Lys residues 165 and 166 in human TF have been shown
previously to diminish the cofactor function of TF in support of factor X
(FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations
have been incorporated into soluble TF (sTF; residues 1-219) to generate
the molecule "hTFAA." hTFAA binds FVIIa with kinetics and affinity
equivalent to wild-type sTF, but the hTFAA x FVIIa complex shows a 34-fold
reduction in catalytic efficiency for FX activation relative to the
activity measured for sTF x FVIIa. hTFAA inhibits the activation of FX
catalyzed by the complex formed between FVIIa and relipidated TF(1-243).
hTFAA prolongs prothrombin time (PT) determined with human plasma and
relipidated TF(1-243) or membrane bound TF, and has no effect on activated
partial thromboplastin time, but is 70-fold less potent as an inhibitor of
PT with rabbit plasma. The rabbit homologue of this mutant ("rTFAA") was
produced and shown to have greater potency with rabbit plasma. Both hTFAA
and rTFAA display an antithrombotic effect in a rabbit model of arterial
thrombosis with rTFAA giving full efficacy at a lower dose than hTFAA.
Compared to heparin doses of equal antithrombotic potential, hTFAA and
rTFAA cause less bleeding as judged by measurements of the cuticle bleeding
time. These results indicate that TF x FVIIa is a good target for the
development of new anticoagulant drugs for the treatment of thrombotic
disease.
Volume 89,
Issue 9,
pp. 3219-3227,
05/01/1997
Copyright © 1997 by The American Society of Hematology
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