Purification and characterization of the lipoprotein-associated coagulation inhibitor from human plasma.
The lipoprotein-associated coagulation inhibitor (LACI) has been isolated from human plasma using a combination of hydrophobic, ion-exchange, and affinity chromatography. The final purification required was greater than 500,000-fold with a yield of 13%. Plasma LACI, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contains major bands at 40 and 46 kDa and minor bands at 55, 65, 75, 90, and approximately 130 kDa. All of the molecular weight forms are recognized by antibodies to LACI's amino and carboxyl termini and are able to inhibit the factor VII(a)-tissue factor complex and factor Xa. Plasma LACI, reduced with beta-mercaptoethanol, migrates on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis as a doublet at 42 kDa and has an amino-terminal sequence essentially identical to that of HepG2 LACI. The difference in size between reduced plasma LACI (42 kDa) and HepG2 LACI (47 kDa) may be related to differing degrees of N-linked glycosylation. The 46-kDa and larger forms of unreduced plasma LACI are associated with apolipoprotein A-II (apoA-II) in mixed disulfide linkages. Studies using isolated lipoproteins show that low density lipoprotein (LDL) contains primarily the 40-kDa form of LACI, whereas high density lipoprotein (HDL) contains primarily the 46-kDa form of LACI (LACI/apoA-II complexes). Gel filtration of a fresh plasma sample showed approximately 50% of plasma LACI to be associated with LDL/very low density lipoprotein, 44% with HDL, and the remaining 6% to not be associated with lipoproteins.
Novotny
W F
WF
Division of Hematology/Oncology, Washington University School of Medicine, Jewish Hospital, St. Louis, Missouri 63110.
Girard
T J
TJ
Miletich
J P
JP
Broze
G J
GJ
D000595:Amino Acid Sequence; D016633:Apolipoprotein A-II; D001054:Apolipoproteins A / Q000097:blood*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000032:analysis; D002845:Chromatography; D004220:Disulfides / Q000097:blood; D004591:Electrophoresis, Polyacrylamide Gel; D005167:Factor VII / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D015942:Factor VIIa / Q000037:antagonists & inhibitors; D065427:Factor Xa Inhibitors; D006801:Humans; D008074:Lipoproteins / Q000302:isolation & purification* / Q000494:pharmacology; D008075:Lipoproteins, HDL / Q000097:blood*; D008077:Lipoproteins, LDL / Q000097:blood*; D008113:Liver Neoplasms / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013925:Thromboplastin / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D016633:Apolipoprotein A-II; D001054:Apolipoproteins A; D004220:Disulfides; D065427:Factor Xa Inhibitors; D008074:Lipoproteins; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; C051928:lipoprotein-associated coagulation inhibitor; D005167:Factor VII; D013925:Thromboplastin; D015942:Factor VIIa