Isolation and Characterization of Novel Blood Coagulation Factor Xa (FXa) Inhibitor (R-020) and its Variants

Urinary trypsin inhibitor (UTI) isolated from human urine is an acidic glycoprotein with molecular weight of 67,000 and is known to inhibit trypsin, plasmin, leucocyte elastase, α-chymotrypsin and other enzymes. UTI is composed of two Kunitz-type domains and the second domain is responsible for its antitryptic activity. In order to determine the detailed inhibitory spectrum of the second Kunitz-type domain of UTI (R-020) against various proteases, I cloned two types of R-020 cDNAs: one encodes the 70-amino acid sequence from the 78th amino acid (Thr) to the C-terminal (R-020 TN70) and the other encodes the 68-amino acid sequence from the 80th (Ala) to the C-terminal (R-020, AN68). Their products are secreted into the cultured supernatant of E.coli JE5505 transformants. R-020 TN70 and R-020 AN68 purified from the cultured supernatant inhibited not only human trypsin, human plasmin, human leucocyte elastase and humanα-chymotrypsin which were already known to be inhibited by UTI but also the human blood coagulation factor Xa and plasma kallikrein. Furthermore, to increase the FXa inhibitory activity I constructed a model of the structure of the R-020 TN70-FXa complex with reference to that of bovine pancreatic trypsin inhibitor-bovine trypsin complex and investigated the binding state of R--20 TN70 and FXa. Based on this information, one amino acid of R-020 TN70 and AN68 was substituted with recombinant DNA technology. The inhibitory effect of the product on FXa was increased in four variants after single amino acid substitution. Four other variants with two amino acids substitution modified by combining the above mentioned single amino acid substitution showed an enhanced inhibitory effect on FXa in comparison with those with single amino acid substitution. Because the electrostatic interaction within these R-020 variants-FXa complexes seemed stronger, this increase in their inhibitory effect on FXa was speculated to be a consequence of more potent binding between the enzyme and the inhibitors