ACTIVITY OF SECRETED KUNITZ DOMAIN-1 VARIANTS OF TISSUE FACTOR PATHWAY INHIBITOR

Tissue factor pathway inhibitors (TFPI and TFPI-2) are Kunitz domain-t ype serine protease inhibitors which inhibit factor VIIa/tissue factor (VIIa/TF) complexes in a factor Xa-dependent manner. The VIIa/TF and Xa inhibitory activity has been localized to the first two Kunitz doma ins, respectively. Unlike TFPI, TFPI-2 has been reported to exhibit si gnificant Xa-independent VIIa/TF inhibitory activity, perhaps due to a n arginine at the P-1 residue in the first Kunitz domain of TFPI-2 as opposed to a lysine at the comparable residue in TFPI. Two domain TFPI variants, differing in the first Kunitz domain but containing the sec ond Kunitz domain of TFPI, were constructed and secreted by Saccharomy ces cerevisiae in order to test the possibility that a TFPI first Kuni tz domain with a P-1 lysine to arginine change or a hybrid containing the TFPI-2 first Kunitz domain may represent more potent VIIa/TF inhib itors. When yeast supernatants were analyzed for specific activity in the Xa-dependent inhibition of VIIa/TF, neither variant was as active as the truncated TFPI.