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REVIEW ARTICLE
The Fibrinogen Sequences That Interact With Thrombin
By Cameron G. Binnie and Susan T. Lord
T
HE SERINE PROTEASE thrombin interacts with a
variety of substrates and as a consequence is involved
in a number of physiologic processes. Fibrinogen is a particularly interesting substrate of thrombin for a number of
reasons. First, thrombin cleaves not just one but four peptide bonds in fibrinogen, apparently in four successivesteps.
As a result ofthese proteolytic cleavages, the soluble fibrinogen molecules are converted into an insoluble fibrous polymer. In addition, the affinity of the enzyme for the final
product, fibrin, is similar to that for the substrate, fibrinogen. Active thrombin is thus bound by the fibrin clot in a
form largely resistant to inactivation by thrombin inhibitors, a situation that has important clinical consequences
and complicates clinical anticoagulant therapy.
This review focuses on the specific sequences of fibrinogen and fibrin that interact with thrombin. As illustrated in
recent x-ray crystal structures of thrombin, the thrombin
active site can be considered as two separate domains: a
catalytic site that includes the Asp-His-Ser catalytic residues, the specificity pocket and an apolar pocket; and an
extended fibrinogen recognition site (FRS). The fibrin(ogen) regions responsible for binding to the catalytic
site and the FRS will be examined and the properties of
fibrin-bound thrombin discussed.
'3'
BACKGROUND
Fibrinogen is a dimeric glycoprotein, with each monomer
composed of three polypeptides: the Aa (6 I O residues), the
BO (46 1 residues), and the y (4 I 1 residues) chain^.^ The six
chains are connected by 29 disulfide bonds. Although the
complete primary structure of the protein is known, high
resolution structural analysis has not been achieved as the
available crystals of fibrinogen diffract poorly. In electron
micrographs, fibrinogen has an elongated, trinodular structure with three globular domains connected by two spacer
arms (Fig l).4,5The central globular domain contains the
dimer interface along with the amino termini of all six
chains, whereas the peripheral globular domains consist of
the carboxy termini of the BO and y chains.697
The Aa chain
carboxy termini extend freely in solution, although in some
electron micrographs they appear to form a separate,
smaller nodule near to the central domain.6
Thrombin cleaves fibrinogen at An Arg- 16 and at BP Arg-
From the Department of Pathology and Curriculum in Genetic.7.
University of North Carolina at Chapel Hill, Chapel Hill, NC.
Submitted December 10, 1992; accepted February 17, 1993.
Supported by Grant No. HL31048from the National Institutes of
Health, Department ofHealth and Human Services, Bethesda, MD.
Address reprint requests to Susan T. Lord, PhD, Department of
Pathology, CB# 7525,603 Brinkhaus-Bullitt Building, University oj"
North Carolina at Chapel Hill, Chapel Hill, N C 27599.
0 1993 by The American Society ef Hematology.
0006-4971/93/8112-0040$3.00/0
3186
14, releasing fibrinopeptides A (fpA) and B (fpB), respectively. FpA is initially released more rapidly than fpB, but
after a lag period in which fpB is released slowly, the rate of
fpB release accelerates. This accelerated rate of fpB cleavage
is thought to result from fibrin formation, which initiates
upon fpA release, although the exact mechanism is not yet
understood.*-" Cleavage of the fibrinopeptides leads to formation of fibrin monomers that spontaneously polymerize
to form a fibrin clot.
THE AMINO TERMINAL DISULFIDE KNOT
It is the central domain of fibrinogen (Fig I ) that binds to
thrombin. Chemical cleavage of fibrinogen with cyanogen
bromide generates a number of fragments, including one
called the amino terminal disulfide knot, or NDSK. NDSK,
which is composed of AN 1-51, BP 1-1 18, and y 1-78,"
represents the central globular domain along with some residues from the spacer arms and contains the fibrinopeptides
cleaved by thrombin. NDSK is a simple competitive inhibitor of thrombin,I2 which illustrates its involvement in
thrombin binding. Furthermore, active site inhibited
thrombin binds to NDSK-agarose but not to C-terminal
fragmentsi3 Thrombin cleaves fpA from NDSK almost as
efficiently as from fibrinogen, as the Michaelis constant
(K,) and the rate constant (kcat)for fpA cleavage in fibrinogen and NDSK are ~ i m i l a r . ' ~FpB
, ' ~ is also cleaved from
NDSK, but more slowly than from fibrin~gen.'~
FIBRINOPEPTIDE A
The NDSK-derived fragment Aa 1-5 1 is a good substrate
for thrombin, which suggests that much of the information
necessary for fpA cleavage is present in this ~ e q u e n c e . ' ~ , ' ~
Residues that identify Aa Arg-16-Gly-17 as a target for
thrombin cleavage are found, not surprisingly, within fpA
itselfand the sequence offpA is shown in Fig 2 . High resolution structures of fpA in thrombin-peptide complexes have
been determined in solution by NMRl7-I9and in crystals by
x-ray analysis.2032'
NMR spectra of synthetic peptides in the
presence of thrombin show that the first five residues of the
ACYchain do not interact with thrombin, whereas residues
7- 16 form a compact structure, with some residues interacting directly with thrombin.22 The crystal structure of the
synthetic peptide Aa 7-16 bound to thrombin has illustrated the precise requirements for fibrinopeptide recognition, ie, a short a-helical segment, a chain reversal at Ala- 10,
and multiple main chain hydrogen bonds within residues
7-12, such that the sidechains of Phe-8, Leu-9, and Val-1 5
form a hydrophobic group that binds to the thrombin apolar pocket. The side chain of Arg-16 is inserted into the
specificity (PI) pocket of thrombin.20,2'
Within the sequence of fibrinopeptide A there are both
critical (nonvariable) residues and residues that can be varied without cost. As previously stated, the first five residues
do not interact with thrombin and this is reflected in the
Blood, Vol81, No 12 (June 15). 1993: pp3186-3192
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3187
FIBRINOGEN SEQUENCES INTERACT WITH THROMBIN
Thrombin
C50A-I
I---
4
5
0
A
.
I
Fig 1. The trinodular structure of fibrinogen as modeled from
electron micrographs of negatively stained specimens.’.’ The dimensions of each domain have been drawn to scale. The central
domain, otherwise referred to as the amino terminal disulfide knot,
contains the fibrinopeptides that are cleaved by thrombin and is
also the region of fibrinogen that binds thrombin. The outer nodules
are composed of two globular domains, the Bg chain carboxy terminus (inner) and the y chain carboxy terminus (outer). The Aa
chain carboxy termini have not been drawn. For comparison,
thrombin, a sphere of diameter 45 A,* has been drawn alongside.
considerable variation in sequence among mammalian species for the amino terminal part ofthe fibrinopeptide, Ala-I
to G~u-S.’~
Also, the rate of cleavage of fpA phosphorylated
at Ser-3, a modification found in 20% to 30% of circulating
fibrin~gen?~
is quite similar to that of the nonphosphorylated form.25
By contrast, when fpA sequences from many mammalian
species are aligned, any interspecies differences within Gly6 to Arg- I6 are quite c o n ~ e r v a t i v e , ” * ~suggesting
’ ~ ~ ~ * ~ ~that
this region is critical to thrombin binding. When Asp-7 is
replaced by Asn, as in the genetic variant fibrinogen Lille I,
fibrinopeptide cleavage is redu~ed.’~*’’However, substitution of Asp7 with Ala had no detectable effect on the rate or
specificity of fpA cleavage from an ACY1-50 &galactosidase
fusion protein.” Also, analysis of fpA release from Asp-7 +
Asn synthetic peptides by kinetic methods or analysis of the
structure of the substituted peptide by NMR failed to show
differences within the native sequence.” Thus, the importance of Asp7 in thrombin interaction remains uncertain.
By contrast, several lines of evidence show that Phe-8 is
extremely important. Phe-8 is highly conserved in the fpA
sequence of many mammalian species.23Peptide mapping
studies showed that peptides containing Phe-8 were consid-
erably better substrates than those in which it was abIn fact, when Phe-8 is replaced by tyrosine in an Aa
1-50 fusion protein, thrombin does not cleave fpA at all.”
The role of this residue is illustrated by the crystal structure,
in which the Phe-8 sidechain projects into the apolar pocket
next to the catalytic site and provides binding affinity
through hydrophobic
The phenyl ring participates as a hydrophobic cluster consisting of residues
from both fpA and thrombin. This interaction apparently
provides binding energy and properly orients the scissile
bond for hydrolysis.
Gly- I2 is another nonvariable residue. The Gly- I2 + Val
substitution present in fibrinogen Rouen I” probably
disrupts the compact structure necessary for fpA binding,
and presumably alters the positioning of the Arg-Gly bond
in the active site, resulting in markedly slower cleavage.”
This reduced cleavage rate was also exhibited in a fusion
protein with a Gly-12 + Val substitution.28 Similarly, a
Gly-I3 + Val substitution in the fusion protein reduced the
cleavage rate, indicating that this substitution also alters the
structure and affects positioning of the scissile bond.” In
contrast, the substitution Gly-14 + Val had little effect on
the rate of fpA cleavage, as might be expected from the x-ray
data.28
Arg-16, which is the site of cleavage, is critically important. lfreplaced by His, as in fibrinogen Petosky I, thrombin
will cleave at this residue, although considerably more
~ l o w l y .The
~ ~ delayed
.~~
release from this variant can be explained by the poor fit of histidine to the PI specificity
pocket of thrombin. With the substitution Arg-16 + Leu, a
variant fusion protein was cleaved by thrombin at Arg-19
instead of Arg- I 6.28Upon extended digestion with high concentrations of thrombin, cleavage at Arg-19 occurs in normal fibrinogen after release of fpA, releasing the tripeptide
Gly- 17-Pro-Arg- I 9.34 Replacement of Arg- 16 with Cys, as
in Fibrinogen Metz I, completely abolishes fpA ~leavage.~’
When. as in homozygous patients, only the abnormal protein is present the two cysteine sidechains form a disulfide
bond that is not present in normal f i b r i n ~ g e nLocked
. ~ ~ into
a covalent disulfide bond, the cysteine sidechain cannot fit
into the specificity pocket of thrombin, and the scissile peptide bond is not hydrolyzed. The residues downstream of
fpA, ACT 17-20, may bind to thrombin, as presented in a
hypothetical model.” Variant fibrinogens have been identified with substitutions of residues 18 and 19. However, analyses of these variants show that it is generally the subsequent
step of fibrin polymerization that is defective, rather than
fpA release rate. For instance, Pro-I8 + Leu (Kyoto 11)
does not alter the rate of fpA relea~e.~’
and Arg-19 + Ser
- + ?+
ADSGEGDFLAEGGGVR-GPRV
? +
FpA:
Fig 2. Sequences of human fibrinopeptide A
and B. The site of cleavage by thrombin is marked
by the dash. X = pyroglutamate. Residues that
when substituted result in delayed cleavage are indicated
residues that can be substituted
without effect ” ,” and conflicting or uncertain
residues ”?.“
”+,“
-
-+ +
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 13141516
171819 20
+ +
FpB:
xGVNDNEEGFFSAR-GHRP
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 15161718
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3188
BlNNlE AND LORD
(Detroit)38 has fpA release that is normal.39 Nevertheless,
Arg- 19 + Gly (Aarhus I,40Mannheim 14’) did have reduced
fpA release. In Fig 2, the critical and variable residues within
the fpA sequence are illustrated.
FIBRINOPEPTIDE B
Fibrinopeptide B cleavage has not been as well studied as
fpA. The NDSK-derived peptide BP 1-1 18, in contrast to
the case for the Aa chain fragment Aa 1-5 1, is a poor substrate for t h r ~ m b i n , ~suggesting
’
that these residues are not
sufficient for efficient cleavage or have an altered, suboptimal conformation in this fragment. A comparison of the
sequence of fpB with fpA shows little similarity (Fig 2), an
indication that the structure of the thrombin-bound fpB is
different from fpA. Furthermore, mammalian fpB sequences are much less alike than PA.'^ No structural data is
available for the fpB-thrombin complex; however, it has
been suggested that the side chains of Phe-10 and Phe-1 1
bind to the apolar site in the opposite direction to fpA.*’
Support for the importance of Phe-1 1 comes from the observation that in mammalian species where Phe-l I is replaced, a hydrophobic residue, either Leu or Val, is almost
invariably present. Variant fibrinogens that alter fpB cleavage have only been detected at the point of cleavage: Arg- 14
( I j m ~ i d e nand
) ~ ~Gly- I5
However, variants that impair fpB cleavage may go undetected because removal of
fpA (by the enzyme Reptilase) is sufficient to form a clot
that is indistinguishable from that formed by t h r ~ m b i n . ~ ’
BINDING TO THE FRS
Although the fibrinogen sequences that bind to thrombin’s catalytic site are well defined, there is less agreement
about the fibrinogen sequence or sequences that bind to the
FRS of thrombin. It is clear that fibrinogen and fibrin bind
to a site distinct from the catalytic site because thrombin
that is chemically blocked at the catalytic site with D-PhePro-Arg-chloromethylketone (PPACK) binds to both fibrinogen-agarose and fibrin-agar~se.~~
Also, fibrin-bound
thrombin remains catalytically active against small peptide
substrate^.^',^^ It is a commonly held assumption that
thrombin binds to fibrinogen in a manner similar to fibrin.
In other words, the interactions that promote thrombin
binding to fibrinogen as a substrate are those that anchor
thrombin to fibrin as a product, with the obvious caveat that
in fibrin the fibrinopeptides are no longer present. Support
for this hypothesis comes from the similar affinity of thrombin for fibrin (I(d = 2 to 10 p m ~ l / L ) ~ ’ ,to
~ ~that
- ~ for
’ fibrinogen (K, = 8 to 10 p m ~ l / L ) . ~ ~ , ~ ~
Clues to the location of the FRS binding residues have
come from peptide studies. Although the fibrinogen fragment Aa 1-51 is a good substrate for thrombin, the rate of
cleavage at Arg-16 is markedly reduced in a smaller fragment, Aa l-23.53,54The removed residues, 24-51, do not
occupy the catalytic site, so the increased rate of cleavage of
the larger peptide may result from binding to another site on
thrombin, such as the FRS. (Alternatively, residues 24-5 1
may stabilize an optimal substrate conformation within
7- 16, thus enhancing catalysis.’’) Affinity chromatography
of proteolytic fragments of fibrinogen and fibrin on active
site inhibited thrombin-agarose identified the segment Aa
17-78 as a region that binds to thrombin,13 whereas a peptide analogous to Aa 27-50, but with the four cysteines replaced by alanine, inhibits clotting of fibrinogen by thrombin and also binds to P P A C K - t h r ~ m b i n .From
~ ~ these
studies it is clear that an FRS binding site is present within
the common sequence, Aa 27-50.
Insight into the FRS domain and the sequences that bind
to it has also come from the crystal structure of thrombin
complexed to the inhibitor hirudin.s7258
Hirudin, a 65 residue polypeptide of known sequence,59binds with its hydrophobic amino terminus at the catalytic site and its negatively charged carboxy terminus at the FRS. Examination of
the hirudin carboxy terminal sequence reveals a number of
negatively charged residues interspersed with hydrophobic
residues. In the thrombin-hirudin crystal structure, hirudin
C-terminal residues Asp-55, Glu-57, Glu-58, and Gln-65
participate in polar interactions with thrombin residues,
whereas multiple nonpolar interactions also contribute to
inhibitor-FRS binding. Although the structural data show
several apparently critical interactions, analyses of individual hirudin residues by site-directed mutagenesis indicate
that substitution of individual residues results in unexpectedly small changes in the affinity for thrombin.60*6’Thus,
although individual residues make small contributions, the
sum of these numerous interactions results in the significant
binding affinity of the hirudin-FRS interaction.61
In similar site-directed mutagenesis experiments, we also
found only small changes in the affinity of Aa 1-50-pgalactosidase fusion proteins with single amino acid substitutions (Fig 3). Substitutions of anionic residues with glycine caused a small decrease in binding affinity for
thrombin, less than would be expected if these charged
groups were critical for interaction with the thrombin FRS.
Single substitutions of hydrophobic residues also caused
small changes. The largest decrease in binding was seen with
doubly substituted proteins. Thus, our results mirror those
observed with hirudin. One final point: the Aa 1-50-pgalactosidase fusion protein has an affinity for thrombin,
Residue number
Inhibition constant
41
(PM)
Trp-Pro-Phe-Cys-Ser-Asp-Glu-Asp-Trp
9 f 2
33
34
35
36
37
38
39
40
Ser
Leu
Gly
GlY
Gly
Ser
Gly Gly
Leu
Ser
Ala S e r
13
19
12
15
19
13
21
28
35
f
f
f
f
f
f
f
f
f
2
1
2
3
2
3
3
6
4
Fig 3. Inhibition constants of Aa 1-50-8 galactosidase fusion
proteins for human a-thrombin (mean 2 SD, n = 4) were determined as previously described.28~’2The residue numbers correspond to the Aa chain of fibrinogen with the native sequence listed
in the first row.
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3189
FIBRINOGEN SEQUENCES INTERACT WITH THROMBIN
measured as K, (6 to 9 Gmol/L),28,62
that is similar to the
affinity of thrombin for fibrinogen. Substitutions in the region ACY
7- 16 decreased the rate of cleavage of the fibrinopeptide, but did not affect the binding affinity for thrombin.28This is clear evidence that while substitutions within
the fibrinopeptide region alter the rate of cleavage, a region
outside the fibrinopeptide region also provides a site of interaction and this site can accommodate ACY
residues 17-50
in the fusion protein.
Some evidence points to the BP chain as an additional site
of thrombin FRS binding. Des-BP 15-42 fibrin, which lacks
both fibrinopeptides and the additional segment BP 15-42,
has severely diminished thrombin binding.63 The loss of
thrombin binding suggests that the segment BP 15-42 binds
to the thrombin FRS. The sequence BP 15-42 has also been
shown to be essential for endothelial cell spreading on fibrin, although these investigators reported thrombin binding to des-BP 15-42 fibrin that was equal to normal fibrin.64
Fibrinogen New York I has a deletion of residues BP 9-72,
and the fibrin clot has dramatically reduced thrombin binding.65,66
The deletion of such a large segment with concomitant loss of two interchain disulfide bonds within the NDSK
may alter the conformation of the other chains, so a more
convincing example is provided by fibrinogen Naples I,
which has the single substitution BP Ala-68 + Thr.67When
fibrinogen Naples from a homozygous patient is clotted by
thrombin, both fpA and fpB release is reduced and both
after considerable lag periods. Binding of thrombin to the
Naples I clot is also greatly reduced, being less than 10% of
normal. The observation that not only is clot-bound thrombin reduced but also the cleavage rate of both fibrinopeptides is reduced emphasizes how important FRS binding is
for normal fibrinopeptide cleavage, further supporting the
assumption that thrombin-fibrinogen and thrombin-fibrin
interactions are the same.
One study suggests that all three chains bind to thrombin,
as thrombin bound to all three chains when the chains were
immobilized to agarose. However, the affinity for the isolated chains was weak and abolished at high ionic strength.68
The most attractive conclusion to be drawn from this
information is that the nonsubstrate binding site is composed from both the Aa and BP chains or perhaps all three
chains. It is well known that the three chains of fibrinogen
are evolutionarily
A number of charged residues
and hydrophobic residues that are conserved among chains
may all participate in the FRS binding site. The NDSK
sequences that have been discussed here as possible residues
important for thrombin binding are highlighted in Fig 4.
If the binding site exists on a combination of more than
one chain, then the approach of investigating isolated chain
segments will not be fruitful. More promising is an approach investigating substitutions within the whole molecule. Congenital dysfibrinogenemias are one source of
ready-made variant fibrinogens, and as the molecular basis
of these dysfibrinogenemias is expanded, it is expected that
these variant fibrinogens will be a source of great information. Another avenue that holds great promise is the generation of recombinant
with designed substitutions, an approach recently initiated in our 1aborato1-y.~~
COAGULANT PROPERTIES OF CLOT-BOUND THROMBIN
Active thrombin is bound by the clot during clot formation,49,74
apparently in two orientations. In one orientation,
both catalytic site and FRS are occupied by the fibrin; in the
other, only the FRS is occupied, so that the catalytic site is
accessible to other
The fibrin clot thus acts as a
hemostatic mechanism to ensure that clot formation is localized and thrombin is prevented from spreading to the general circulation. The importance of this mechanism is
shown by the dysfibrinogenemias Naples I and New York I,
whose patients have recurrent thromboses correlated with
the inability of the clot to bind t h r ~ m b i n . ~ ' , ~ ~
Bound thrombin also is well positioned to activate factor
XIII. In the last step ofclot formation, the transglutaminase
factor XIIIa covalently links fibrin molecules by introducing covalent bonds between y chains and between the CY
chains. In fibrin, the monomers overlap in a half staggered
manner so that the outer globular domains (to which factor
XI11 binds in circulating f i b r i n ~ g e n ~of~ two
, ~ ~ monomers
)
interact with the NDSK (which binds thrombin) of a third
monomer. This formation of a thrombin-fibrin-factor XI11
ternary complex greatly enhances factor XI11 a ~ t i v a t i o n . ~ ~
A fibrin clot acts as a reservoir for enzymatically active
thrombin and represents a localized thrombogenic surface.
This clot-bound thrombin is resistant to inactivation by circulating thrombin inhibitor^.^','^ During fibrinolysis, plasmin digests the fibrin clot into soluble fibrin degradation
products but does not inactivate thrombin.79 Thrombin is
thus released into the circulation complexed to macromolecular fibrin degradation products." As anticoagulant therapy
using heparin is not effective in preventing rethrombosis in
patients undergoing thrombolytic therapy," and as the
thrombin complexed to fibrin degradation products is not
inhibited by heparin-antithrombin 111, it appears that the
clot-bound thrombin exposed during thrombolytic therapy
may mediate r e t h r o m b ~ s i s . ~Hirudin,
' ~ ~ ~ is a more effective
ADSGEGDFLAEGGGVRGPRWERHQ.S&CZE
_
.
_
_
A
XGVNDNEEGFFSAR
ARPAKAAATQKKVERKAPDAGGCL
YVATRDNCC I
Fig 4. Primary structures of the six chains of fibrinogen up to the first disulfide knots. Shaded sequences are those proposed to be
important in thrombin binding: Aa 27-50, E@ 15-42, and E@ Ala-68. Charged and hydrophobic residues conserved between the chains are
boxed. Fibrinopeptides A and E are in bold characters.
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3190
BlNNlE AND LORD
inhibitor against clot-bound thrombin?'.*' and may be effective in preventing rethrombosis during thrombolytic therapy.8'
MOLECULAR MECHANISM
The clotting of fibrinogen by thrombin is unusually complex because of the multistep reaction sequence. It is not
known if thrombin dissociates between steps or if a single
thrombin molecule binds to one molecule of fibrinogen,
cleaves the first Aa Arg- 16-Gly- I7 bond, then the second,
then successively the two BP Arg- 14-Gly- 1 5 bonds, and remains bound after polymerization. As currently depicted,
the symmetric molecular models predict that each fibrinogen molecule contains two equivalent sites for thrombin
binding, one on each half of the molecule. This model favors thrombin dissociation between steps as it infers that
thrombin binds and cleaves each half of the molecule separately. Perhaps the symmetry is misleading. In an alternative model, residues at the dimer interface may form a single
domain that binds to the thrombin FRS site with the catalytic site accessible for hydrolysis of each of the four scissile
bonds. After cleavage of the fibrinopeptides, thrombin remains bound by this single domain. These speculations
show that although the recent high resolution structural
analyses of thrombin have provided important information
relevant to the molecular basis for thrombin activity and
specificity, there are many questions that remain unanswered when considering the details for thrombin cleavage
of fibrinogen.
ACKNOWLEDGMENT
We are grateful to Dr Oleg Gorkun for helpful discussions and to
Dr Gilbert White for critical review of this manuscript.
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1993 81: 3186-3192
The fibrinogen sequences that interact with thrombin
CG Binnie and ST Lord
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