Document Detail

Role for the Kunitz-3 domain of tissue factor pathway inhibitor-alpha in cell surface binding.
MedLine Citation:
PMID:  15557366     Owner:  NLM     Status:  MEDLINE    
BACKGROUND: Tissue factor pathway inhibitor (TFPI)-alpha, a key regulator of tissue factor-induced coagulation, contains 3 tandem Kunitz-type inhibitory domains. Kunitz-1 binds and inhibits factor VIIa in the factor VIIa/tissue factor complex, and Kunitz-2 binds and inhibits factor Xa. The role of the Kunitz-3 domain of TFPI-alpha, however, has remained an enigma. METHODS AND RESULTS: To determine the structures within TFPI-alpha involved in its binding to cell surface, altered forms of TFPI-alpha were expressed in C127 (mouse mammary) cells: C-terminal truncated forms TFPI-alpha (252), TFPI-alpha (242), and TFPI-alpha (181), which also lacks the third Kunitz domain (K3); TFPI-alpha (desK3), which lacks only the K3 domain; and TFPI-alpha (R199L), in which the putative P1 site in K3 is changed from arginine to leucine. By flow cytometry (fluorescence-activated cell sorting), the altered forms 252, 242, and R199L showed significantly reduced binding, whereas the forms 181 and desK3 completely failed to bind to the cell surface. Transient expression of WT-, desK3-, and K3/K2-TFPI-alpha (in which K3 is replaced with K2) in another cell line (b-end3, mouse endothelial) produced comparable results. Exogenously added C-terminal truncated and R199L forms of TFPI-alpha bound poorly and desK3 did not bind at all to the surface of ECV304 cells in which TFPI-alpha expression had been "knocked down" by RNA interference. CONCLUSIONS: Optimal cell binding of endogenously expressed TFPI-alpha requires its K3 and C-terminal domains, and within the K3 domain, the P1 (R199) residue plays an important role. Thus, one role of the K3 domain involves the cell surface localization of TFPI-alpha.
Orlando Piro; George J Broze
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, P.H.S.     Date:  2004-11-22
Journal Detail:
Title:  Circulation     Volume:  110     ISSN:  1524-4539     ISO Abbreviation:  Circulation     Publication Date:  2004 Dec 
Date Detail:
Created Date:  2004-12-07     Completed Date:  2005-06-03     Revised Date:  2007-11-15    
Medline Journal Info:
Nlm Unique ID:  0147763     Medline TA:  Circulation     Country:  United States    
Other Details:
Languages:  eng     Pagination:  3567-72     Citation Subset:  AIM; IM    
Division of Hematology, Washington University School of Medicine, St Louis, Mo, USA.
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MeSH Terms
Cell Line
Cell Membrane / metabolism
Flow Cytometry
Heparin / pharmacology
Lipoproteins / genetics,  metabolism*,  pharmacology
Protein Binding
Protein Structure, Tertiary
Type C Phospholipases / pharmacology
Grant Support
Reg. No./Substance:
0/Lipoproteins; 0/lipoprotein-associated coagulation inhibitor; 9005-49-6/Heparin; EC 3.1.4.-/Type C Phospholipases

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