The carboxy terminus of tissue factor pathway inhibitor is required for interacting with hepatoma cells in vitro and in vivo

I. Warshawsky, Guojun D Bu, A. Mast, J. E. Saffitz, G. J. Broze, A. L. Schwartz

Research output: Contribution to journalArticle

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Abstract

Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that directly inhibits coagulation Factor Xa and also inhibits tissue factor-initiated coagulation. Normal human plasma TFPI exists both as the full-length molecule and as variably carboxy-terminal truncated forms. We reported recently that the low density lipoprotein receptor- related protein mediates the cellular degradation of TFPI after TFPI binding to the hepatoma cell surface. To examine whether the carboxy terminus of TFPI was required for interacting with hepatoma 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 125I- TFPI-160 for binding to hepatoma cells. We were also unable to demonstrate specific binding of 125I-TFPI-160 to hepatoma cells at 4°C. At 37°C, significantly less 125I-TFPI-160 was internalized and degraded via low density lipoprotein receptor-related protein than full-length 125I-TFPI. Full-length 125I-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 cells, the fates of TFPI and TFPI-160 in vivo were examined. Both 125I-TFPI and 125I-TFPI- 160 disappeared rapidly from the circulation after their intravenous administration into rats. The initial plasma half-life of 125I-TFPI was ~ 30 s whereas the half-life of 125I-TFPI-160 was ~ 4 min. 125I-TFPI was cleared predominantly by the liver. In contrast, 125I-TFPI-160 accumulated in the outer cortex of the kidney. Using microscopic autoradiography, we demonstrate that 125I-TFPI clearance is largely hepatocellular, whereas 125-TFPI-160 accumulates mainly in the cells of the kidney proximal tubules. Together our findings demonstrate that the carboxy-terminal region(s) distal to amino acid 160 of TFPI mediates TFPI binding to hepatoma cells both in vitro and in vivo.

Original languageEnglish (US)
Pages (from-to)1773-1781
Number of pages9
JournalJournal of Clinical Investigation
Volume95
Issue number4
StatePublished - 1995
Externally publishedYes

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Hepatocellular Carcinoma
In Vitro Techniques
lipoprotein-associated coagulation inhibitor
LDL-Receptor Related Proteins
LDL Receptors
Half-Life
Kidney Cortex
Proximal Kidney Tubule
Factor Xa
Serine Proteinase Inhibitors

Keywords

  • binding
  • carboxy terminus
  • clearance
  • tissue factor pathway inhibitor

ASJC Scopus subject areas

  • Medicine(all)

Cite this

The carboxy terminus of tissue factor pathway inhibitor is required for interacting with hepatoma cells in vitro and in vivo. / Warshawsky, I.; Bu, Guojun D; Mast, A.; Saffitz, J. E.; Broze, G. J.; Schwartz, A. L.

In: Journal of Clinical Investigation, Vol. 95, No. 4, 1995, p. 1773-1781.

Research output: Contribution to journalArticle

Warshawsky, I. ; Bu, Guojun D ; Mast, A. ; Saffitz, J. E. ; Broze, G. J. ; Schwartz, A. L. / The carboxy terminus of tissue factor pathway inhibitor is required for interacting with hepatoma cells in vitro and in vivo. In: Journal of Clinical Investigation. 1995 ; Vol. 95, No. 4. pp. 1773-1781.
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T1 - The carboxy terminus of tissue factor pathway inhibitor is required for interacting with hepatoma cells in vitro and in vivo

AU - Warshawsky, I.

AU - Bu, Guojun D

AU - Mast, A.

AU - Saffitz, J. E.

AU - Broze, G. J.

AU - Schwartz, A. L.

PY - 1995

Y1 - 1995

N2 - Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that directly inhibits coagulation Factor Xa and also inhibits tissue factor-initiated coagulation. Normal human plasma TFPI exists both as the full-length molecule and as variably carboxy-terminal truncated forms. We reported recently that the low density lipoprotein receptor- related protein mediates the cellular degradation of TFPI after TFPI binding to the hepatoma cell surface. To examine whether the carboxy terminus of TFPI was required for interacting with hepatoma 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 125I- TFPI-160 for binding to hepatoma cells. We were also unable to demonstrate specific binding of 125I-TFPI-160 to hepatoma cells at 4°C. At 37°C, significantly less 125I-TFPI-160 was internalized and degraded via low density lipoprotein receptor-related protein than full-length 125I-TFPI. Full-length 125I-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 cells, the fates of TFPI and TFPI-160 in vivo were examined. Both 125I-TFPI and 125I-TFPI- 160 disappeared rapidly from the circulation after their intravenous administration into rats. The initial plasma half-life of 125I-TFPI was ~ 30 s whereas the half-life of 125I-TFPI-160 was ~ 4 min. 125I-TFPI was cleared predominantly by the liver. In contrast, 125I-TFPI-160 accumulated in the outer cortex of the kidney. Using microscopic autoradiography, we demonstrate that 125I-TFPI clearance is largely hepatocellular, whereas 125-TFPI-160 accumulates mainly in the cells of the kidney proximal tubules. Together our findings demonstrate that the carboxy-terminal region(s) distal to amino acid 160 of TFPI mediates TFPI binding to hepatoma cells both in vitro and in vivo.

AB - Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that directly inhibits coagulation Factor Xa and also inhibits tissue factor-initiated coagulation. Normal human plasma TFPI exists both as the full-length molecule and as variably carboxy-terminal truncated forms. We reported recently that the low density lipoprotein receptor- related protein mediates the cellular degradation of TFPI after TFPI binding to the hepatoma cell surface. To examine whether the carboxy terminus of TFPI was required for interacting with hepatoma 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 125I- TFPI-160 for binding to hepatoma cells. We were also unable to demonstrate specific binding of 125I-TFPI-160 to hepatoma cells at 4°C. At 37°C, significantly less 125I-TFPI-160 was internalized and degraded via low density lipoprotein receptor-related protein than full-length 125I-TFPI. Full-length 125I-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 cells, the fates of TFPI and TFPI-160 in vivo were examined. Both 125I-TFPI and 125I-TFPI- 160 disappeared rapidly from the circulation after their intravenous administration into rats. The initial plasma half-life of 125I-TFPI was ~ 30 s whereas the half-life of 125I-TFPI-160 was ~ 4 min. 125I-TFPI was cleared predominantly by the liver. In contrast, 125I-TFPI-160 accumulated in the outer cortex of the kidney. Using microscopic autoradiography, we demonstrate that 125I-TFPI clearance is largely hepatocellular, whereas 125-TFPI-160 accumulates mainly in the cells of the kidney proximal tubules. Together our findings demonstrate that the carboxy-terminal region(s) distal to amino acid 160 of TFPI mediates TFPI binding to hepatoma cells both in vitro and in vivo.

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