Immunoaffinity purification and characterization of lipoprotein-associated coagulation inhibitors from Hep G2 hepatoma, Chang liver, and SK hepatoma cells. A comparative study.
A polyclonal antibody against a synthetic peptide corresponding to amino acids 3-25 of mature lipoprotein-associated coagulation inhibitor (LACI) was raised in rabbits. The antibody was used to study the production of LACI by Hep G2 hepatoma, Chang liver, and SK hepatoma cells, and to purify LACI from the culture media. By using an amidolytic assay for factor Xa, it was found that the culture media from these liver-derived cell lines contain inhibitors of factor Xa. In Hep G2 hepatoma culture medium, approximately 50% of Xa inhibitory activity was due to LACI. In the Chang liver and SK hepatoma culture media over 95% of the Xa inhibitory activity was due to LACI. The LACIs were purified from these media by immunoaffinity chromatography on an anti-LACI-lg-Sepharose 4B column and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified LA-CIs varied in molecular weight depending on whether the media were concentrated before chromatography. An Mr approximately 38,000 LACI was obtained by chromatography of unconcentrated media. Chromatography of concentrated media yielded a LACI of Mr approximately 35,000 with the same amino-terminal sequence, suggesting partial proteolysis in the carboxyl-terminal region. In addition, an Mr approximately 25,000 form of LACI was also present. The purified Mr approximately 38,000 and approximately 35,000 LACI species from the above cells possess similar specific activities when measured by an anti-Xa/amidolysis assay. To study the role of LACI in the control of coagulation, pooled human plasma was depleted of LACI antigen by immunoaffinity absorption and reconstituted with varying amounts of purified LACI to examine the effect on tissue factor (TF)-induced coagulation. LACI depletion shortens the time of TF-induced clotting of plasma and the clotting time is linearly related to the LACI concentration after reconstitution. These results suggest that LACI plays an important role in limiting TF-induced coagulation in human plasma. Comparison of the potencies of various purified LACIs in the prolongation of TF-induced coagulation revealed that LA-CIs from different sources are not equivalent. The plasma LACI, SK hepatoma LACI, and Chang liver LACI are approximately 7-, 6-7, and 1.3-fold higher in specific activity than Hep G2 hepatoma LACI in the TF-induced clotting assay when compared on an anti-Xa/amidolysis unit basis, suggesting possible differences in post-translational modification of these LA-CIs.
Wun
T C
TC
Monsanto Company, St. Louis, Missouri 63198.
Huang
M D
MD
Kretzmer
K K
KK
Palmier
M O
MO
Day
K C
KC
Bulock
J W
JW
Fok
K F
KF
Broze
G J
GJ
D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005167:Factor VII / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D065427:Factor Xa Inhibitors; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000302:isolation & purification* / Q000494:pharmacology; D008099:Liver; D008113:Liver Neoplasms; D008970:Molecular Weight; D011517:Prothrombin Time; D013925:Thromboplastin / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology
D065427:Factor Xa Inhibitors; D008074:Lipoproteins; C051928:lipoprotein-associated coagulation inhibitor; D005167:Factor VII; D013925:Thromboplastin