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(Circulation. 2004;110:3567-3572.)
© 2004 American Heart Association, Inc.

Molecular Cardiology

Role for the Kunitz-3 Domain of Tissue Factor Pathway Inhibitor- in Cell Surface Binding

From the Division of Hematology, Washington University School of Medicine, St Louis, Mo.

Correspondence to George J. Broze, Jr, MD, BJH: 90-20-66, 216 S Kingshighway Blvd, St Louis, MO 63110. E-mail gbroze{at}im.wustl.edu

Received June 9, 2004; revision received August 17, 2004; accepted August 23, 2004.

Background— Tissue factor pathway inhibitor (TFPI)-, 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-, however, has remained an enigma.

Methods and Results— To determine the structures within TFPI- involved in its binding to cell surface, altered forms of TFPI- were expressed in C127 (mouse mammary) cells: C-terminal truncated forms TFPI- (252), TFPI- (242), and TFPI- (181), which also lacks the third Kunitz domain (K3); TFPI- (desK3), which lacks only the K3 domain; and TFPI- (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- (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- bound poorly and desK3 did not bind at all to the surface of ECV304 cells in which TFPI- expression had been "knocked down" by RNA interference.

Conclusions— Optimal cell binding of endogenously expressed TFPI- 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-.

Key Words: coagulation • inhibitors • endothelium-derived factors

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