Document Detail

Tissue factor pathway inhibitor and protease nexin-1 are major factor Xa binding proteins on the HepG2 cell surface.
MedLine Citation:
PMID:  8381308     Owner:  NLM     Status:  MEDLINE    
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.
Y Kazama; Y Komiyama; W Kisiel
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Blood     Volume:  81     ISSN:  0006-4971     ISO Abbreviation:  Blood     Publication Date:  1993 Feb 
Date Detail:
Created Date:  1993-03-10     Completed Date:  1993-03-10     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  7603509     Medline TA:  Blood     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  676-82     Citation Subset:  AIM; IM    
Department of Pathology, University of New Mexico, School of Medicine, Albuquerque 87131.
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MeSH Terms
Amyloid beta-Protein Precursor
Binding Sites
Carcinoma, Hepatocellular
Carrier Proteins / isolation & purification,  metabolism*
Cell Membrane / metabolism
Factor Xa / isolation & purification,  metabolism*
Heparin / pharmacology
Immunoglobulin G / pharmacology
Lipoproteins / isolation & purification,  metabolism*
Liver Neoplasms
Plasminogen Inactivators / metabolism*
Protease Inhibitors / metabolism*
Receptors, Cell Surface
Tumor Cells, Cultured
Grant Support
35246//PHS HHS
Reg. No./Substance:
0/Amyloid beta-Protein Precursor; 0/Carrier Proteins; 0/Immunoglobulin G; 0/Lipoproteins; 0/Plasminogen Inactivators; 0/Protease Inhibitors; 0/Receptors, Cell Surface; 0/lipoprotein-associated coagulation inhibitor; 0/protease nexins; 9005-49-6/Heparin; EC Xa

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