Recombinant
tissue factor pathway inhibitor (rTFPI) has been expressed in four mammalian expression systems using human SK
hepatoma, mouse C127, baby hamster kidney (BHK), and Chinese hamster ovary (CHO) cells as hosts. On
sodium dodecyl sulfate polyacrylamide gel electrophoresis, the immunoaffinity purified rTFPIs all show broad bands and the mean molecular weight of SK
hepatoma and C127 rTFPIs (M(r) approximately 38,000) appear larger than those of BHK and CHO rTFPIs (M(r) approximately 35,000). All these
proteins inhibit
factor Xa and appear to bind
factor Xa with 1:1 stoichiometry. The ability of these
proteins to inhibit
tissue factor-induced coagulation in plasma was examined using a prothrombin time assay. The relative activities of SK rTFPI:C127 rTFPI:BHK rTFPI:CHO rTFPI were found to be 28:15:2.1:1. By Western blot using specific
antisera against the amino- and carboxy-termini of
TFPI as probes, it is found that all the immunoaffinity purified rTFPIs possess approximately equal amounts of the amino terminus, but the C127 and BHK rTFPIs are deficient in carboxy terminus and the CHO rTFPI is essentially devoid of this region of the
protein.
Mono S chromatography allowed separation of the full-length and the truncated molecules with high and low
anticoagulant activities, respectively. The above results suggest that proteolysis of the carboxy terminus of
TFPI occurs to different extent when
TFPI is expressed in different cells and that the carboxy terminal region of the
TFPI molecule is important for the inhibition of
tissue factor-induced coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)