THE CARBOXY-TERMINUS OF TISSUE FACTOR PATHWAY INHIBITOR IS REQUIRED FOR INTERACTING WITH HEPATOMA-CELLS IN-VITRO AND IN-VIVO

Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that directly inhibits coagulation Factor Xa and al so inhibits tissue factor-initiated coagulation, Normal human plasma T FPI exists both as the full-length molecule and as variably carboxy-te rminal truncated forms, We reported recently that the low density lipo protein receptor-related protein mediates the cellular degradation of TFPI after TFPI binding to the hepatoma cell surface. To examine wheth er the carboxy terminus of TFPI was required for interacting with hepa toma cells, a mutant of TFPI lacking the third Kunitz-type domain and basic carboxy terminus was generated, We found that this mutant, TFPI- 160, did not compete with full-length I-125-TFPI-160 for binding to he patoma cells, We were also unable to demonstrate specific binding of I -125-TFPI-160 to hepatoma cells at 4 degrees C, At 37 degrees C, signi ficantly less I-125-TFPI-160 was internalized and degraded via low den sity lipoprotein receptor-related protein than full-length I-125-TFPI, Pull-length I-125-TFPI binding to hepatoma cells could be inhibited > 90% by heparin and other highly charged molecules, Since TFPI, but not TFPI-160, was capable of effectively binding to cultured hepatoma cel ls, the fates of TFPI and TFPI-160 in vivo were examined, Both I-125-T FPI and I-125-TFPI-160 disappeared rapidly from the circulation after their intravenous administration into rats, The initial plasma half-li fe of I-125-TFPI was similar to 30 s whereas the half-life of I-125-TF PI-160 was similar to 4 min, I-125-TFPI was cleared predominantly by t he liver, In contrast, I-125-TFPI-160 accumulated in the outer cortex of the kidney, Using microscopic autoradiography, we demonstrate that I-125-TFPI clearance is largely hepatocellular, whereas I-125-TFPI-160 accumulates mainly in the cells of the kidney proximal tubules, Toget her our findings demonstrate that the carboxy-terminal region(s) dista l to amino acid 160 of TFPI mediates TFPI binding to hepatoma cells bo th in vitro and in vivo.