NOVEL FACTOR XA AND PLASMA KALLIKREIN INHIBITORY-ACTIVITIES OF THE 2ND KUNITZ-TYPE INHIBITORY DOMAIN OF URINARY TRYPSIN-INHIBITOR

Urinary trypsin inhibitor is a glycoprotein with a structure in which two Kunitz-type inhibitory domains are linked in a row. We isolated tw o genes encoding the 70 amino acid sequence from the 78th amino acid ( Thr) to the C-terminal and the 68 amino acid sequence from the 80th (A la) to the C-terminal of human urinary trypsin inhibitor, both which c orrespond to the second Kunitz-type inhibitory domain, and then constr ucted expression plasmids by ligating it to the E. coli alkaline phosp hatase signal peptide gene. These plasmids under the control of the tr yptophan promoter expressed the second domain in E. coli strain JE5505 which lacks the membrane lipoprotein. The recombinant second domain p urified from the culture supernatant of the transformant inhibited try psin, plasmin, leukocyte elastase and chymotrypsin which are known to be inhibited by urinary trypsin inhibitor. In addition it inhibited bl ood coagulation factor Xa and plasma kallikrein in a concentration dep endent and competitive manner, and significantly prolonged the plasma- based activated partial thromboplastin time (APTT). The truncated natu ral counterpart obtained by a limited degradation of human urinary try psin inhibitor also revealed the identical inhibitory activities.