Tissue Factor: molecular recognition and cofactor

function
DAVID M. A. MARTIN,* C. WILUAM G. BOYS,t AND WOLFRAM RUFt’
*Haemostasis Research Group, Medical Research Counsil Clinical Sciences Centre, Royal Postgraduate Medical
School, London, England; tDepai.tment of Biochemistry, The University of Edinburgh, Edinburgh, Scotland;
and tDepartments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California
92037, USA

ABSTRACT One aspect of the inflammatory re- proteolytically activates coagulation factors IX and X, trig-
sponse is the activation of the coagulation protease gering the downstream coagulation pathways. Ligand-in-
cascade resulting from the expression of tissue factor duced changes in intracellular calcium (1) and
(TF) on vascular cells. TF is the cell-surface receptor phosphorylation of specific serine residues in the TF cyto-
for the coagulation serine protease factor Vila, pro- plasmic domain (2) indicate a potential TF-dependent sig-
viding cofactor function by “switching on” the cata- nal-transduction pathway that may have a role in vascular
lytic site of the bound enzyme and by contributing to cell-cell interaction. In addition to situations of acute in-
the assembly with macromolecular substrate. The flammatory responses at the vascular interface, TF is ex-
recently determined crystal structure of the TF ex- pressed by cells in arteriosclerotic lesions consistent with
tracellular domain shows two 3-strand modules of C2 its postulated role in initiating the coagulation pathways
immunoglobulin-like topology that align at a 125#{176} that lead to thrombotic complication associated with plaque
angle with an extensive intermodule interface. Mu- rupture. In animal models of balloon injury to arteries, ex-
tagenesis studies have identified residues in both mod- posure of TF in deeper layers of the vessel wall has been
ules that are important for the binding of ligand. The implicated in initiation and prolonged triggering of a proco-
deduced ligand interface extends from the convex agulant response in the vasculature (3). TF makes diverse
side of the molecule into the concave side of the elbow contributions to the pathophysiology of thromboembolic
angle. Specific binding residues control the catalytic disease, so specific inhibition of the IF pathway may po-
activity of the bound protease. At the lower end of the tentially be exploited as a novel anticoagulant strategy to
carboxyl-terminal module, basic residues form part prevent and intervene in intravascular thrombotic events.
of a region that is important for both recognition and The biochemical characterization of function of the
activation of macromolecular substrate and, poten- TFFVIIa complex has progressed rapidly in recent years
tially, for modulation of proteolytic function. After and we review here the current understanding of the mo-
combining the biochemical data with the crystal struc- lecular basis for TF function within the context of the re-
hire, a model of TF function can be proposed in which cently solved 3-dimensional structure of TF. The structural
the catalytic activity of the active site of the protease biology of TF may provide a paradigm for the structural
and the extended recognition of macromolecular basis of function of other receptor and cofactor molecules
substrates are separately controlled by distinct struc- that are used by vascular cells to initiate downstream pro-
tural sites of the cofactor.-Martin, D. M. A., Boys, tease cascades involved in host defense and repair, regula-
C. W. G., Ruf, W. Tissue factor: molecular recogni- tion of migration, and cell-cell communication.
tion and cofactor function. FASEB J. 9, 852-859
(1995)
THE 3-DIMENSIONAL STRUCTURE OF TF
Key Words: receptor structure . ligand binding . macromolecular
cofactor . coagulation pathways . thrombosis TF has been classified on the basis of distant sequence
similarities as a member of the cytokine/hematopoietic
CELLULAR lNl’FIATION OF THE COAGULATION PATHWAYS is fre-
quently a component of the inflammatory response in the
vascular system. Mter cytokine- or gram-negative bacterial
endotoxin stimulation, endothelial cells and monocytes
1’fO whom conespondence and reprint requests should be addressed, at:
change to a procoagulant phenotype by the cell-surface
The Scripps Research Institute, Department of Immunology, IMM-17,
expression of the transmembrane receptor tissue factor
10666 N. Torrey Pines Road, La Jolla, CA 92037, USA.
(TF)2 (see this issue for review of transcriptional regulation 2Abbreviations: IF, tissue factor; FYIIa, factor V11a CRF, cytokine
of the TF gene). TF binds the serine protease coagulation receptor family; Kd, dissociation constant; CIa, -carboxyglutamic acid;
factor YlIa (FYIIa) and the resulting TF FVIIa complex EGF, epidermal growth factor-like.

852 0892-6638/95/0009.0852/SO 1.50. © FASEB

rom www.fasebj.org by Tulane Univ Howard-Tilton Lib (129.81.226.78) on December 10, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}
 REVIEW
growth factor receptor family (CRF) (4). Molecules of this strands-A, B, and E-or four strands: C, C’, F, and G. The
class are structurally related cell-surface proteins that are two modules are oriented with an angle of 125#{176}, as shown
usually anchored through a single transmembrane domain in the ribbon representation of IF in Fig. 1. The solvent-
followed by a structurally diverse cytoplasmic domain of exposed surface of each module has a similar number of
varying length. The extracellular domains typically contain charged residues with no obvious extended clustering of
two tandem 7 p-strand sandwich-type modules that show positive or negative charge. The disuffide bond between
structural similarity to the fibronectin III subclass of the strands F and G in the C-module is solvent-accessible, an
immunoglobulin superfamily. The CRF members show two unusual feature for modules with immunoglobulin-like to-
distinct patterns of disulfide bonding, allowing their subdi- pology. Trp is also partially solvent-exposed and packs
vision into either class 1 (human growth hormone receptor, toward Cys’, potentially contributing to the conforma-
most of the interleukin receptors, and the hematopoietic tional stability of this module. The aromatic side chains of
growth factor receptors including the erythropoietin recep- Trp, Phe50, TyrM, Phe76, Tyr78, and Tyr, together with
tor and MPL, the receptor for thromhopoietin) or class 2 the aliphatic side chains of Val33 and Pro92, form a sol-
(interferon receptors, IF, IL-b receptor). vent-exposed hydrophobic cluster predominantly on the
The extracellular domain of IF has been crystallized (5, four-stranded .-sheet in the N-module. Some of these
6) and the structure solved simultaneously by two groups to residues contribute to FVIIa binding. The N-linked glyco-
a resolution of 2.2A and 2.4A, respectively (7, 8). TF con- sylation sites in IF are at residue Asn124, which is largely
tains two C2-type immunoglobulin-like modules that are buried on strand B in the C-module, and at residues Asn
characterized by two 3-.sheets formed either by three and Asn137, which are well exposed to solvent and point

N-module N-module

C-module C-module

213 213

Figure 1. Stereo view of the architecture of the TF extracellular domain. N-linked glycosylation sites are shown in yellow. Major strands are labeled
A through C and the short antiparallel ribbon p (residues 133-136) and q (residues 138-141). Major heices are labeled al (residues 60-64) and a2
(residues 143-150); the helical turn of residues 102-105 is not labeled. Disuffide bridges are between residues 49 and 57 (N-module) or 186 and 209
(C-module). Residues involved in FYITa binding (>1 kcal/mol contribution to the free energy of binding), Lye20, lle, C1u24,Asp, Irp, Lys, A8re,
Phe76, Arg’, and Phe’40, are shown at the intermodule boundary; residues involved in the activation of macromolecular substrate, Lys’ and Lysise,
protrude to the right in the C-module. Figs. 1 and 2 were prepared using the program MOLSCRIPT.

MOLECULAR RECOGNF11ON BY TISSUE FACTOR 853

rom www.fasebj.org by Tulane Univ Howard-Tilton Lib (129.81.226.78) on December 10, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}
 REVIEW

away from functionally important regions of IF (Fig. 1).

Glycosylation is not required for function, though it may
influence cell-surface expression of TF.
An interesting feature of the IF extracellular domain is
the presence of short stretches of a-helical secondary
structure. An extended finger-like region not only contains
the a2-helix (Fig. 1) but also includes a short antiparallel
sheet (8), which protrudes from the side of the molecule
at the intermoclule interface. This loop is highly conserved
between mammalian sequences for IF, and is particularly
long in comparison to class b members of the CRF. Inter-
strand loops from both modules interdigitate in a manner
analogous to a dove-tailed joint, possibly conferring a high
degree of rigidity on the receptor with respect to the link
between the modules. The module interface is tightly
packed and forms a hydrophobic core involving residues
Phe19, Val, Va167, Pro102, lyrlOS, Thr106, Leu, Va1134,
Leu, Val, and Phe147 with almost 1000 A of buried
surface. This core region is depicted in Fig. 2. Phe19 packs
in a pocket formed by several of the residues that form the
Figure 2. A stereo diagram of the IF module interface. C-u backbone
hydrophobic core of the module interface (8). Mutation of this
in black, with the hydrophobic core residues highlighted in blue, showing
residue affects correct processing and expression from the cell central position of Phe’#{176}
(in red).
(9), potentially due to incorrect folding, suggesting that the
interface structure is critical for folding of the molecule. Other
specific mutations, particularly in the linker region from
Iyr’#{176}3-Thr’#{176}6,
affect function without apparent major struc- binding of FYIIa was found for IF on endotoxin-stimulated
tural alterations, indicating that the interface is also important monocytes (Kd = -80 pM) (19) or for purified TF in high-
for function of the molecule (9). The membrane linker region pressure dissociation experiments (10) and in functional
(Met#{176}-Glu219) has not been implicated in function and assay (11) (Kd = 3-7 pM). There appears to be only little
this region is disordered in both X-ray structures (7, 8). change in affinity for FVIIa binding when the phospholipid
insertion of IF is omitted by solubilization with detergent
(11). This indicates that protein-protein interactions are
THE MACROMOLECULAR LIGANDS FOR TF predominantly responsible for the tight interaction of FYIIa
with IF. However, phospholipid may influence the interac-
The macromolecular ligands for TF are the serine protease tion with the protease ligand, as several studies report co-
FYlla and its zymogen precursor FY11. The zymogen is operative binding characteristics particularly with anionic
composed of an amino-terminal, y-carboxyglutamic phospholipid (16, 20). The high Kd values reported in the
acid-rich (Gla) domain, two epidermal growth factor-like literature for binding of FYIIa by IF may reflect limitations
(EGF) modules, a short region in which the proteolytic of the methodology or potentially modulation of the affinity
activation cleavage occurs, followed by a typical serine pro- of ligand binding by the cell surface environment.
tease domain. FYIIa is a two-chain enzyme with the light Fragments consisting of the Gla domain and the first
chain (Gla- and EGF-modules) covalently linked to the EGF-module of FYIIa (21, 22) inhibit TF’F’VIIa complex
heavy chain protease domain via a disulfide bridge. Both formation. The light chain of FVIIa is further implicated in
the zymogen and the protease bind the extracellular domain IF interaction by 1) IF binding function of chimeric pro-
in a 1:1 stoichiometry (10) and the Kj of binding measured teins containing modules of both FVIIa and factor IX (23),
with soluble IF, which is truncated in the membrane linker 2) the localization of inhibitory antibody epitopes to the first
region, is approximately 2-5 nM. This value is consistently EGF-module (24), 3) characterization of FVII-Arg79--*Gln,
reported using different methodology. The methodology in- a hereditary dysfunctional mutation (14, 25), and 4) de-
cludes functional titration (11-13) or direct biophysical creased affinity for IF after proteolytic cleavage of the heli-
evaluation using surface plasmon resonance (14). Soluble cal aromatic stack region that connects Gla domain and the
TF has a 20-fold reduced affinity for FVIIa as compared to first EGF-module (15, 18, 21, 22, 26). Arg3#{176}4 in the pro-
full-length IF when analyzed with similar methodology un- tease domain of FVIIa has been shown to contribute to the
der identical conditions (15). binding interaction with IF after analysis of certain heredi-
The Kd estimates reported for FVIIa binding to full- tary FVII deficiencies (27, 28). Certain interactions of the
length IF vary considerably, the highest estimates being protease domain with IF require the proteolytic conversion
1-10 nM, derived from radioligand binding analysis with of the zymogen. There are distinct differences between the
purified and phospholipid reconstituted IF (16) or with TF binding of the protease and the zymogen after chemical and
expressed on certain tumor cells (17, 18). Much tighter proteolytic modifications (21). Although there is competi-

854 Vol.9 July 1995 The FASEB Journal MARTIN FTAL

rom www.fasebj.org by Tulane Univ Howard-Tilton Lib (129.81.226.78) on December 10, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}
 REVIEW

tion for TF binding between the protease FYIJa and the

zymogen FY11, it has not been established whether the in-
teraction of the two ligands with TF is on identical sites.
Subtle changes in the alignment of the light and heavy
chains or conformational changes in the protease domain as
a consequence of zymogen activation may result in altera-
tions in the docking of the multidomain ligand with TF.

HOW TF BINDS FYIIa

Alanine scanning mutagenesis has been used to assess the

role of specific amino acid side chains in the IF extracel-
lular domain for interaction with the macromolecular ligand
FYlla (9, 11, 13). The region implicated in ligand interac-
tion is consistent with independent approaches involving
antibody mapping (29), chemical cross-linking (30), and
proteolytic fragmentation of TF (31). Alanine substitution
identified a limited number of residue positions at which
alanine replacements caused 5- to 100-fold reduced affin-
ity for FYIIa binding. Most of these residue side chains
were found to be well exposed to solvent in the crystal
structure of TF, concordant with macromolecular ligand
interaction. Figure 3 shows the topographical organization
of the residues that make energetic contributions to the
interaction with FYIIa. The FYIIa ligand binding site is
located over an extensive region at the boundary between
the two modules. In the C-module, residues Arg’35 and
Phe0 located on the protruding B-C loop provide an inter-
dependent contact with ligand (11). Leu133 is located at the
base of the fingerlike structure and packed in the cleft
between the two modules. This provides continuity to a
major cluster of important binding residues consisting of
Lys20, Thr60, Asp, and 11e22. Thr60 is only partially solvent
exposed and may play a local structural role rather than
Figure 3. Space- filling (A) and ribbon (B) representations of the ligand
making a significant contact with ligand.
binding site of IF. Surfaces and ribbons are colored according to change
The binding site extends onto the concave side of the in binding energy (grey, not examined; pale blue, <0.4 kcallmol; light red,
intermodule angle involving G1u24 (9) and G1n1 10, and po- >0.4 and <1.0 kcal/mol; dark red, >1.0 kcal/mol). The disulfide bridge is
tentially the more distant residue Val207 (13). The binding shown in yellow. Key binding residues are labeled. Phe76 is not labeled
region extends from Asp onto a convex surface area because it is largely buried in this projection. Figs. 3, 4, and 5 were
prepared using Insight II (Biosym Inc., San Diego Calif).
formed by Lys, Lys, G1n37, Asp, and Trp45. Trp45 and
Asp”’ do not interact independently with FYIIa, indicating
that the mutational effect at the Trp45 position may reflect
structural importance of this side chain for the local pack-
ing of the adjacent Asp and G1n37 side chains (32). The face that contains a bound sodium ion. There are significant
interactive area further includes two surface-exposed aro- differences in the relative orientation of the two modules
matic residues, Phe76 and Tyr78, which form part of the between the neuroglian repeat and TF, probably reflecting
hydrophobic cluster in the N-module (32). the differences in function of the molecules. The module
alignment in neuroglian is compatible with an extended
fiberlike alignment of several fibronectin type III modules,
COMPARISON WITH RELATED RECEPTORS thus providing an example for functional diversity gener-
ated by variation in the spatial alignment of a fairly rigid
The structure of neuroglian, a cell adhesion molecule con- tandem repeat of similar structural modules.
taining tandem fibronectin type III modules, has recently Crystal structures have now been determined for growth
been solved (33). The neuroglian molecule shows some hormone in complex with two members of the class I CRF,
similarity to TF, i.e., it has a-helical segments within the the growth hormone receptor (34) and the prolactin receptor
n-strand framework, including one in the B-C loop of the (35). These two receptor structures show the highest degree
second module. Neuroglian has an extensive module inter- of structural similarity with TF. Although there is signifi-

MOLECULAR RECOGNITION BY TISSUE FACTOR 855

rom www.fasebj.org by Tulane Univ Howard-Tilton Lib (129.81.226.78) on December 10, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}
 cant variation between the three structures in the relative
orientation of the two 13-sandwich modules, all three recep-
tors have a similar buried surface area at the module inter-
face with a typical short helical stretch in the linker region.
This feature is quite distinct from other tight alignments of
immunoglobulin-like modules (e.g., CD4) in which the
G-strand of the amino-terminal module in each pair ex-
tends quite directly into the A-strands of the carboxyl-ter-
minal module.
Ligand binding residues of TF are located predominantly
on 13-strands, in contrast to the growth hormone and pro-
lactin receptors where the ligand binding interfaces are
composed predominantly from interstrand loops. The cen-
tral docking sites for the hormone on the prolactin and
growth hormone receptors are small and located on the
convex side of the elbow angle formed by the two modules.
Although the periphery of the IF ligand binding region
marked by Asp is located on the convex side of the cleft,
the IF ligand binding site extends substantially onto the
concave side of the molecule. The distribution of ligand
binding residues for IF and the growth hormone receptor
are detailed in Fig. 4. Residues making an energetic con-
tribution to ligand binding are highlighted on the backbone
of the molecule. The two proteins are aligned such that the
C-modules are superimposed and the hinge axis is normal
to the page. An orthogonal view is also shown with the
N-modules bending into the page.
The solvent-exposed area of the identified binding resi-
dues in TF is approximately 1200 A2, which is comparable
to ligand interfaces in the cytokine receptor family (34, 35).
Functionally determined epitopes are generally less ex-
tended than the epitope determination from structure solu-
tion of ligand-receptor complexes (36), so the ligand
interface in IF may be significantly larger than the minimal
functional site identified. The variations in shape and extent
of the ligand binding site architecture between IF and other
members of the CRF reflect adaption of the cytokine receptor
topology to interaction with different ligands. Human growth
hormone is a small, single module protein that interacts with
the receptor binding site via a pair of a-helices. FYIIa is a
larger multimodular protein with multiple interaction sites.
This is reflected in the close distribution of binding residues Figure 4. Orthogonal views (top and bottom) of the ligand binding site
in the growth hormone receptor and the more dispersed architecture in IF (left, blue) and human growth hormone receptor (right,
distribution in IF. Specific interaction with at least two green). Both proteins are oriented along the elbow angle with the C-mod-
ules superimposed. Disulfide bridges are shown in yellow. IF: Strong
distinct structural modules in the ligand FYIIa probably
binding residues (Lys20, Hen, G1u24, Asp, Trpu, Lys, Asp, ThrAO,
requires spatial separation and specific shape complemen-
Phe76, GIn’ Phe’#{176})
are colored purple. Weaker binding residues
tarity of the interface in IF, resulting in the location of (Lys, G1n37, 1yr78, and Leu) are colored mauve. Residues involved in
ligand binding residues on opposite sides of the modules. macromolecular substrate recognition (Lys’, Lys’) are colored blue.
Growth hormone receptor: coordinates are from file 3HHR deposited in
the Brookhaven Protein Data Bank (34). Only binding protein 1 is shown.
Binding residues with highest energetic contributions (G>4 kcal/mol)
HOW DOES IF FUNCTIONAS THE COFACIOR FOR
are shown in purple (Irp’#{176}4, Trp’); other significant binding contacts
FYIIa? (&G>1 kcal/mol) are shown in mauve (Arg”, Glu’’, He’#{176}3, lle’#{176}5,
Pro’#{176}6,
Asp, Glu’27, AspiM, I1e’), according to the recent study by Clackson
FYIIa undergoes a dramatic enhancement in proteolytic and Wells (54).
activity upon binding to IF. TF serves as a cofactor in at
least two ways: 1) by “switching on” the active site of macromolecular substrate. The enhancement in catalytic
FYIIa, which lacks significant catalytic function in the ab- function of the catalytic site is readily monitored with small
sence of cofactor and 2) by coordinating the assembly of peptidyl pseudosubstrates that are hydrolyzed with 20- to

856 Vol. 9 July 1995 The FASEBJournal MARTIN Er AL

rom www.fasebj.org by Tulane Univ Howard-Tilton Lib (129.81.226.78) on December 10, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}
 REVIEW

100-fold increased catalytic rates (kt) in the presence of chains in the protease domain (27, 28, 45). There is loss of
cofactor as compared to free enzyme F’VIIa (37). The acti- flexibility of FVIIa after complex fonnation with IF (46);
vation of serine protease domains typically results in the regions for extended substrate recognition in the light chain
formation of a critical salt bridge between the newly ex- might become optimally oriented relative to the active site
posed amino terminus and the aspartate side chain adjacent of the protease. A specific role of the cofactor in the activa-
to the catalytic serine. The protease domain amino-termi- tion of protein substrates was initially suggested from the
nal Ile’ in FYIIa can be carbamylated at the a-amino functional characterization of an inhibitory monoclonal an-
group (21), reflecting the labile nature of the Ile’-Asp’3 tibody to the C-module of IF. This antibody demonstrated
salt bridge in FYIIa. This modification reaction is inhibited immediate neutralization of the preformed IF’ FYIIa com-
by IF, indicating that the lability of the salt bridge may be plex by competitive inhibition of factor X activation (47).
the cause of the low catalytic activity of free FYIIa (38, 39). Site-specific mutagenesis has identified specific resi-
These data support a model where FYIIa is maintained dues in TF that coordinate the assembly of macromolecular
in an active conformation by cofactor interaction. Several substrate but are not involved in the binding of enzyme
key contacts in the central FYIIa binding region involving FYIIa or enhancement of F\TIIa activity against small sub-
the cofactor residues Lys20, 11e22, Arg’35, Phe’0, and Asp strates (48, 49). The functional defects in factor X activa-
(Fig. 3) can be replaced by alanine without compromising tion were not phospholipid surface-dependent, indicating
catalytic function of the mutant IF’ FYIIa complex despite that the mutations did not perturb phospholipid interactions
the 5000-fold reduction in affinity for ligand (11). In con- (48). Lys’65 and Lys’ are the most significant contributors
trast, the Asp’/Trp45 contact located toward the periphery to the activation of factor X (48, 50). The side chains of
of the FYIIa binding site is partly responsible for catalytic these residues point in opposite directions and are well
enhancement of substrate hydrolysis by FYIIa (13) (C. R. exposed to solvent at the lower end of the C-module
Kelly, J. R. Schullek, W. Ruf, I. S. Edgington, unpublished (Fig. 5). Tyr’57 and Trp’ are buried under the Lys resi-
results). The catalytic enhancement in the FYIIa protease dues. The reduction in IF activity after substitutions of
domain thus is not simply a result of the overall docking of these aromatic side chains is probably due to a conforma-
FYIIa with IF, but rather a consequence of interactions of tional perturbation of the functional site. The disulfide bond
specific side chains in IF that stabilize the conformation of may also be a structural component in the interaction with
corresponding structures located presumably in the pro- macromolecular substrate (51). Iyr is not required for
tease domain of F’VIIa. function, but the functional site extends toward the end of
the module encompassing residues G1y1M, Ser, and
Lys159. The Ala replacement should not significantly per-
IF AND MACROMOLECULAR SUBSTRATE turb the backbone conformation at the Gly’ position, in-
RECOGNITION dicating that the Gly residue may play a functional rather
than a structural role. Lys’59 appears to be more specifi-
The macromolecular substrates for the TF’ FYIIa complex cally involved in the activation of FY11 rather than factor X
are factors IX and X, as well as the zymogen FY11 in com- (49), indicating fine specificity in the interaction with
plex with IF. Factor X activation requires an intact Gla macromolecular substrates.
domain (40) that is essential for crucial Ca2+_dependent The majority of IF molecules are found on the cell
interactions of the macromolecular substrate with a phos- surface where they are capable of binding FYIIa and en-
pholipid surface (39,41). Macromolecular substrate assem- hancing the catalytic activity toward small peptidyl sub-
bly with the IF’FYIIa complex may, however, occur strates (17, 52). Cell-surface proteolytic activity of the
without direct membrane-substrate interaction, as demon- TF FYIIa complex, however, is modulated, and pools of
strated by the activation mechanism of the zymogen FY11. TF’VIIa that lack proteolytic activity have been proposed.
In this reaction, the FY11 forms a complex with IF, allowing One may speculate that the C-module could be involved in
activation after membrane-dependent assembly with the specific interactions on the cell surface that prevent its
enzymatic unit IF’FVIIa (42). Soluble IF, which lacks function to support the activation of macromolecular sub-
stable membrane anchoring, is a poor cofactor, supporting strate. The C-module may play a further role in the assem-
the conversion of FY11 only at extremely high phospholipid bly of TF pathway inhibitor bound to factor Xa with
concentrations (43). The assembly of macromolecular sub- IF #{149}
Ylla, a physiological feedback inhibition of the IF
strate is therefore a membrane-dependent reaction in pathway (53). TF function in the initiation of the coagula-
which substrate is presented bound either to phospholipid tion pathways may thus be regulated not only at the tran-
or bound to another IF molecule. scriptional level, but also after translocation to the surface
TF makes essential contributions to the recognition of by specific cellular modulation and functional inhibition.
macromolecular substrates by the IF’ FVIIa complex. Under
certain experimental conditions, activation of macromolecular
substrates can be severely reduced despite normal or only SUMMARY
slightly affected hydrolysis of small peptidyl pseudosubstrates.
Efficient recognition of macromolecular substrate requires the Interpreting the effects of specific receptor contacts on the
Gla domain of the enzyme (15, 44) and certain residue side function of the ligand and on the coordination of macro-

MOLECULAR RECOGNITION BY TISSUE FACTOR 857

rom www.fasebj.org by Tulane Univ Howard-Tilton Lib (129.81.226.78) on December 10, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}
 in proteolytic function. Perturbation of these interactions
has the potential to provide a useful mechanism for prevent-
ing thrombotic disease in vivo.

We would like to acknowledge Karl Harlos for providing the data for
the 3-dimensional structure of TF. C. W. G. B. is supported by the
Wellcome Trust, D. M. A. M. by a Medical Research Council student-
ship, and W. R. by National Institutes of Health grants HL-48752 and
HL- 16411.

REFERENCES

1. Rettingen, J. A., Enden, T., Camerer, E., Iversen, J., and Prydz. H. (1995)
Binding of human factor Vila to’ tissue factor induces cytosolic Ca2+ signals
in J82 cells, transfected COS-1 cells, Madin-Darby canine kidney cells and
in human endothelial cells induced to synthesize tissue factor. J. Biol. C/tern.
270,4650-4660
2. Zioncheck, T. F., Roy, S., and Vehar, C. A. (1992) The cytoplasmic domain
of tissue factor is phosphorylated by a protein kinase C-dependent mechanism.
J. Biol. Chem. 267,3561-3564
3. Speidel, C. M., Edgington, T. S., Eisenberg, P. R., and Ahendschein, D. R.
(1994) Tissue factor induces prolonged procoagulant activity on the luminal
surface of rabbit aortas after balloon-induced injury. Circulation 90, 1-
344(abstr.)
4. Bazan, J. F. (1990) Structural design and molecular evolution of a cytokine
receptor superfamily. Proc. Nat!. Acad. Sci. USA 87, 6934-6938
5. Boys, C. W. C., Miller, A., Harlos, K., Martin, D. M. A., Tuddenham, E. C.
D., and O’Brien, D. P. (1993) Crystallization and preliminary X-ray analysis
of human tissue factor extracellular domain. I. Mo!. Biol. 234, 1263-1265
6. Ruf, W., Stura, E. A., LaPolla, R. J., Syed, R., Edgington, T. S., and Wilson,
I. A. (1992) Purification, sequence and crystallization of an anti-tissue factor
Fab and its use for the crystallization of tissue factor. J. Cryst. Growth 122,
253-264
7. Harlos, K., Martin, D. M. A., O’Brien, D. P., Jones, E. Y., Stuart, D. I.,
Polikarpov, I., Miller, A., Tuddenham, E. C. D., and Boys, C. W. C. (1994)
Crystal structure of the extracellular region of human tissue factor. Nature
370,662-666
8. Muller, Y. A., Ultsch, M. H., Kelley, R. F., and Dc Vos, A. M. (1994) Structure
of the extracellular domain of human tissue factor location of the factor VIla
binding site. Biochemistry 33, 10864-10870
9. Ruf, W., Schullek, J. R., Stone, M. J., and Edgington, T. S. (1994) Mutational
mapping of functional residues in tissue factor: identification of factor VII
recognition determinants in both structural modules of the predicted cytokine
receptor homology domain. Biochemistry 33, 1565-1572
10. Waxman, E., Ross, J. B. A., Laue, T. M., Guha, A., Thiruvikraman, S. V., Lin,
T. C., Konigsberg, W. H., and Nemerson, Y. (1992) Tissue factor and its
extracellular solubte domain: the relationship between intermolecular asso-
ciation with factor Vhs and enzymatic activity of the complex. Biochemistry
31,3998-4003
Figure 5. Space-filling (A) and ribbon (B) representations of the sub- 11. Schullek, J. R., Ruf, W., and Edgington, T. S. (1994) Key ligand interface
strate coordination site at the membrane-proximal end of the extracellular residues in tissue factor contribute independently to factor Vila binding. I.
domain. Residues are colored purple (Lys’59, Lys, Lys1, Lyst); light Biol. Chem. 269, 19399-19403
yellow (disulfide bridge sulphur atoms); pale blue (Tyr’, Tyr’7, Trp); 12. Neuenschwander, P. F., and Morrissey, J. H. (1994) Roles of the membrane-
interactive regions of factor YhIa and tissue factor. J. Biol. Chem. 269,
dark yellow (Ser’, GlyiM).
8007-8013
13. Gibbs, C., McCurdy, S., Leung, L., and Paborsky, L. (1994) Identification of
the factor Vhha binding site on tissue factor by homologous loop swap and
alanine scanning mutagenesis. Biochemistry 33, 14003-14010
14. O’Brien, D. P., Kemball-Cook, G., Hutchinson, A. M., Martin, D. M. A.,
Johnson, D. J. D., Byfield, P. C. H., Takamiya, 0., Tuddenham, E.G. D., and
McVey, J. H. (1994) Surface plasmon resonance studies of the interaction
molecular assemblies in the context of the crystal structure
between factor VII and tissue factor. Demonstration of defective tissue factor
of IF has allowed novel insights into function of cellular binding in a variant FYI! molecule (FVII-R79Q). Biochemistry 33,
cofactor-enzyme complexes. The architecture of the ligand 14162-14169
15. Ruf, W., Kalnik, M. W., Lund-Hansen, T., and Edgington, T. S. (1991)
binding site of IF shows topological similarities to other Characterization of factor VII association with tissue factor in solution. High
members of the CRF, reflecting the evolution from a puta- and low affinity calcium binding sites in factor VII contribute to functionally
distinct interactions.). Biol. Chem. 266, 157 19-15725
tive common ancestor. The topographical organization has 16, Bach, R., Gentry, R., and Nemerson, Y. (1986) Factor VII binding to tissue
diverged considerably in the family to accommodate spe- factor in reconstituted phospholipid vesicles: Induction of cooperativity by
cific ligands. Complex formation of FYIIa with IF involves phosphatidylserine. Biochemistry 25,4007-4020
17. Le, D. T., Rapaport, S. I. and Rao, L. V. M. (1992) Relations between factor
a number of relatively subtle interactions that act to specifi- VHa binding and expression of factor Vila/tissue factor catalytic activity on
cally “switch on” the enzyme and present it against a small cell surfaces.). BiOL. Chem. 267, 15447-15454
18. Sakai, T., Lund-Hansen, T., Thim, L., and Kisiel, W. (1990) The gamma-car-
number of macromolecular substrates. Small modifications boxyglutamic acid domain of human factor VIla is essential for its interaction
in cofactor or enzyme structure can result in large changes with cell surface tissue factor.). Biol. Chem. 265, 1890-1894

858 Vol. 9 July 1995 The FASEB Journal MARTIN EFAL

rom www.fasebj.org by Tulane Univ Howard-Tilton Lib (129.81.226.78) on December 10, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}
 REVIEW

19. Broze, C. J. (1982) Binding of human factor VII and VIIa to monocytes.). 37. Lawson, J. H., Butenas, S., and Mann, K. C. (1992) The evaluation of
Clin. invest. 70, 526-535 complex-dependent alterations in human factor VIla.). Bid. Chem. 267,
20. Fair, D. S., and MacDonald, M. J. (1987) Cooperative interaction between 4834-4843
factor VII and cell surface-expressed tissue factor. J. Biol. Chem. 262, 38. Born, V. J. J., and Bertina, R. M. (1990) The contributions of Ca2, phos-
11692-1 1698 pholipids and tissue-factor apoprotein to the activation of human blood-co-
21. Higashi, S., Nishimura, H., Aita, K., and Iwanaga, 5. (1994) Identification of agulation factor X by activated factor VII. Biochem. J. 265,327-336
regions of bovine factor VII essential for binding to tissue factor.). Biol. Chem. 39. Ruf, W., Rehemtulla, A.. Morrissey, J. H., and Edgington, T. S. (1991)
269, 18891-18898 Phospholipid independent and dependent interactions required for tissue
22. Kazama, Y., Pastuszyn, A.. Wildgoose, P., Hamamoto, T., and Kisiel, W. factor receptor and cofactor function.). Bid. Chem. 266,2158-2166
(1993) Isolation and characterization of proteolytic fragments of human factor 40. Watzke, H. H., Lechner, K., Roberts, H. R., Reddy, S. V., Welsch, D. J.,
VIIa which inhibit the tissue factor-enhanced amidolytic activity of factor Friedman, P., Mahr, C., Jagadeeswaran, P., Monroe, D. M., and High, K. A.
VIla. J. Bid. Chem. 268, 1623 1-16240 (1990) Molecular defect (G1a1’t4_+Lys) and its functional consequences in a
23. Toomey, J. R., Smith, K. J., and Stafford, D. W. (1991) Localization of the hereditary factor X deficiency (factor X “Vorarlberg”). J. Bid. Chem. 265,
human tissue factor recognition determinant of human factor VIla. J. Bid. 11982-11989
Chern. 266, 19198-19202 41. Krishnaswamy, S., Field, K. A., Edgington, T. S., Morrissey, J. H., and Mann,
24. Clarke, B. J., Ofosu, F. A., Sridhara, S., Bona, R. D., Rickles, F. R., and K. C. (1992) Role of the membrane surface in the activation of human
Blajchman, M. A. (1992) The first epidermal growth factor domain of human coagulation factor X.). Biol. Chern. 267,26110-26120
coagulation factor VII is essential for binding with tissue factor. FEBS Lett. 42. Neuenschwander, P. F., Fiore, M. M., and Morrissey, J. H. (1993) Factor VII
298,206-210 autoactivation proceeds via interaction of distinct protease-cofactor and
25. Sridhara, S., Clarke, B. J., and Blajchman, M. A. (1993) Arginine-79 in the zymogen-cofactor complexes. J. Biol. Chern. 268,21489-21492
first epidermal growth factor domain of factor VII is essential for the interaction 43. Neuenschwander, P. F.,and Morrissey,J. H. (1992) Deletion of the membrane
with tissue factor. Blood Coagul. Fibrinolysis 4,505-506 anchoring region of tissue factor abolishes autoactivation of factor VII but not
26. Petersen, L. C., Schiodt. J.. and Christensen, U. (1994) Involvement of the cofactor function. Analysis of a mutant with a selective deficiency in activity.
hydrophobic stack residues 39-44 of factor Vhla in tissue factor interactions. ). Biol. Chem. 267, 14477-14482
FEBS Lett. 347, 73-79 44. Martin, D. M. A., O’Brien, D. P., Tuddenham, E.G. D., and Byfield, P. C. H.
27. O’Brien,D.P.,Gale,K.M.,Anderson,J.S.,McVey,J.H.,Miller,C.J.,Meade, (1993) Synthesis and characterization of wild-type and variant ‘y-carboxyglu-
T. W., and Tuddenham, E. C. D. (1991) Purification and characterization of tamic acid-containing domains of factor VII. Biochemistry 32, 13949-13955
factor VII 304-GIn: a variant molecule with reduced activity isolated from a 45. Ruf, W. (1994) Factor VIIa residue Arg29#{176} is required for efficient activation
clinically unaffected male. Blood 78, 132-140 of the macrornolecular substrate factor X’ Biochemistry 33, 11631-11636
28. Matsushita, T., Kojima, T., Emi, N., Takahashi, I., and Saito, H. (1994) Im- 46. Waxman, E., Laws, W. R., Lane, T. M., Nemerson, Y., and Ross, J. B. A.
paired human tissue factor-mediated activity in blood clotting factor (1993) Human factor VIIa and its complex with soluble tissue factor: evalu-
(Arg304-sTrp). Evidence that a region in the catalytic domain of factor VII is ation of asymmetry and conformational dynamics by ultracentrifugation and
important for the association with tissue factor. J. Biol. Chem. 269, fluorescence anisotropy decay methods. Biochemistry 32, 3005-3012
7355-7363 47. Ruf, W., and Edgington, T. S. (1991) An anti-tissue factor monoclonal
29. Ruf, W., Rehemtulla, A., and Edgington, T. 5. (1991) Antibody mapping of antibody which inhibits TF:VIIa complex is a potent anticoagulant in plasma.
tissue factor implicates two different exon-encoded regions in function. Bio- Thromb. Haemost. 66,529-533
chem.). 278,729-733 48. Ruf, W., Miles, D. J., Rehemtulla, A., and Edgington, T. S. (1992) Cofactor
30. Ruf, W., and Edgington, T. S. (1991) Two sites in the tissue factor extracellular residues lysine 165 and 166 arc critical for protein substrate recognition by the
domain mediate the recognition of the ligand factor VIla. Proc. Nat!. Acad. tissue factor-factor Viha protease complex.). Biol. Chem. 267,6375-6381
Sci. USA 88,8430-8434 49. Ruf, W., Miles, D. J., Rehemtulla, A., and Edgington, T. S. (1992) Tissue
31. O’Brien, D. P., Anderson, J. S., Martin, D. M. A., Byfield, P. C. H., and factor residues 157-167 are required for efficient proteolytic activation of
Tuddenham, E. C. D. (1993) Structural requirements for the interaction factor X and factor VII.). Bid. Chem. 267,22206-22210
between tissue factorand factor VII: characterization of chymotrypsin-derived 50. Roy, S., Hass, P. E., Bourell, J. H., Henzel, W. J., and Vehar, C. A. (1991)
tissue factor polypeptides. Biochem. J. 292, 7-12 Lysine residues 165 and 166 are essential for the cofactor function of tissue
32. Ruf, W., Kelly, C. R., Schullek, J. R., Martin, D. M. A., Polikai-pov, I., Boys, factor.). Biol. Chem. 266,22063-22066
C. W. C., Tuddenham, E. C. D., and Edgington, T. S. (1995) Energetic 51. Rehemtulla. A., Ruf, W., and Edgington, T. S. (1991) The integrity of the
contributions and topographical organization of ligand binding residues of Cys186-Cys209 bond of the second disulfide loop of tissue factor is required
tissue factor. Biochemistry In press for binding of factor VII.). Biol. Chem. 266, 10294-10299
33. Huber, A.. Wang, Y., Bieber, A., and Bjorkman, P. (1994) Crystal structure 52. Drake,T. A., Ruf, W., Momssey,J. H., and Edgington,T. S. (1989) Functional
of tandem type III fibronectin domains from drosophila neuroglian at 2.0 A. tissue factor is entirely cell surface expressed on lipopolysaccharide-stimu-
Neuron 12,717-731 lated human blood monocytes and a constitutively tissue factor-producing
34. Dc Vos, A. M., Ultsch, M., and Kossiakoff, A. A. (1992) Human growth neoplastic cell line.). Cell Biol. 109,389-395
hormone and extracellular domain of its receptor: Crystal structure of the 53. Rao, L V. M., Ruf, W., and Nordfang, 0. (1994) Importance of tissue factor
complex. Science 255, 306-312 residues 165, 166 for the accelerated inhibition of factor Vllatissue factor
35. Somers, W., Ultsch, M., Dc Vos, A. M., and Kossiakoff, A. A. (1994) The X-ray by tissue factor pathway inhibition in the presence of factor Xa. Circulation
structure of a growth hormone-prolactin receptorcomplex. Nature 372, 478-481 90, 1-617 (abstr.)
36. Cunningham, B. C., and Wells, J. A. (1993) Comparison of a structural and a 54. Clackson, T., and Wells, J. A. (1991) A hot spot of binding energy in a
functional epitope.). Mo!. Biol. 234, 554-563 hormone- receptor interface. Science 267, 383-386

MOLECULAR RECOGNITION BY TISSUEFACTOR 859

rom www.fasebj.org by Tulane Univ Howard-Tilton Lib (129.81.226.78) on December 10, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}