Tissue factor pathway inhibitor and protease nexin-1 are major factor Xa binding proteins on the HepG2 cell surface.
Previous studies indicated that human factor Xa bound to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule. Factor Xa binding to this cell line was not measurably affected by pretreatment of the cells with anti-factor V IgG and to a large extent (approximately 70%) was calcium-independent, suggesting the presence of cell-surface binding proteins specific for factor Xa other than factor V/Va. In the present study, we have further characterized the interaction of factor Xa with the HepG2 cell and performed chemical cross-linking and immunoprecipitation studies to determine the identity of the HepG2 surface protein(s) interacting with factor Xa. Initial studies demonstrated that HepG2-bound 125I-factor Xa was not significantly displaced by unlabeled factor Xa blocked at the active site with dansyl-L-glutamyl-glycyl-L-arginine (DEGR)-chloromethyl ketone (DEGR-Xa), whereas DEGR-Xa effectively inhibited prothrombinase activity of cell-bound factor Xa (Ki = 5 nmol/L). Essentially no 125I-DEGR-Xa binding to the HepG2 cells was observed, suggesting that an intact factor Xa active site was a prerequisite for binding. 125I-factor Xa binding to HepG2 cells was inhibited approximately 70% by pretreatment of the cells with anti-tissue factor pathway inhibitor (TFPI) IgG in the presence or absence of calcium ions, but was without effect on the expression of prothrombinase activity. Immunoprecipitation of 125I-factor Xa chemically cross-linked to its cell-surface binding protein with anti-factor X IgG followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a complex with an apparent molecular weight of 96,000. An identical molecular weight complex was observed following immunoprecipitation of this radiolabeled complex with anti-TFPI IgG. In addition to TFPI, approximately 30% of cell-bound factor Xa appears to form a covalent complex with HepG2 cell-surface protease nexin-1 (PN-1) as shown by pretreatment of the HepG2 cell with murine anti-PN-1 IgG. These results suggest that approximately 1% to 2% of the factor Xa interacts with HepG2 cell-surface factor V/Va to form a productive prothrombinase complex, while the remaining factor Xa forms a non-productive complex with either TFPI or PN-1.
Department of Pathology, University of New Mexico, School of Medicine, Albuquerque 87131.
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Kazama
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Komiyama
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Kisiel
D016564:Amyloid beta-Protein Precursor; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D015951:Factor Xa / Q000302:isolation & purification / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D007700:Kinetics; D008074:Lipoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008113:Liver Neoplasms; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism*; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D058258:Serpin E2; D014407:Tumor Cells, Cultured
D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D007074:Immunoglobulin G; D008074:Lipoproteins; D015849:Plasminogen Inactivators; D011480:Protease Inhibitors; D058255:Protease Nexins; D011956:Receptors, Cell Surface; C545486:SERPINE2 protein, human; D058258:Serpin E2; C051928:lipoprotein-associated coagulation inhibitor; D006493:Heparin; D015951:Factor Xa