Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2195-2212
REVIEW ARTICLE
-Chain Dysfibrinogenemias: Molecular Structure-Function
Relationships of Naturally Occurring Mutations in the Chain of
Human Fibrinogen
By
Hélène C.F. Côté,
Susan T. Lord, and
Kathleen P. Pratt
From the Department of Biochemistry, University of Washington,
Seattle; and the Department of Pathology and Laboratory Medicine,
Curriculum in Genetics and Molecular Biology, University of North
Carolina at Chapel Hill, Chapel Hill, NC.
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FIBRINOGEN IN HEMOSTASIS |
FIBRINOGEN IS A 340-kD glycoprotein that
circulates in the blood at approximately 3 mg/mL, making it one of the
most abundant blood proteins. It is composed of six polypeptide chains,
(
, 
, 
)2, which are held together by disulfide
bonds and organized in a symmetrical dimeric fashion
(Fig 1A).1-6 Limited plasmin proteolysis of fibrinogen generates fragments E and D.7
Fragment E represents the central domain of the molecule and contains
the amino termini of all six chains. The chains extend in coiled coils toward the distal globular regions (fragments D), which contain the
carboxyl-terminal regions of the and chains. The
carboxyl-terminal region of the chain has been shown to be
noncovalently associated with the E domain.8,9
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| Fig 1.
Schematic representation of human
fibrinogen. The chain is shown in blue, the chain in green, and
the chain in red. The black arrowheads indicate thrombin cleavage
sites on the chain. Glycosylation sites are shown as purple
hexagons, while the calcium ions within the chains are represented
as purple spheres (A). Schematic diagram showing the initial process of
fibrin polymerization. The central nodules contain the amino-termini of
all six chains(,,)2 and are referred to as the
"E" regions, named after a fragment obtained by limited plasmin
digestion of fibrin. They are flanked by two symmetric coiled coils
that terminate in the distal "D" nodules. After the cleavage of
fibrinopeptide A by thrombin, the newly exposed polymerization site
"A" binds to the polymerization pocket "a" that is part of
the chain of fibrin(ogen). The fibrin monomers thus align in a
half-staggered, two-stranded arrangement to form long fibrils. Branch
points and junctions occur sporadically (only one type is depicted
here), contributing to the formation of a three-dimensional mesh
(B).
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In the last stage of blood coagulation, thrombin cleaves the
amino-termini of the and chains of fibrinogen, releasing fibrinopeptides A and B, respectively, and converting fibrinogen to
fibrin monomers. The spontaneous polymerization of fibrin monomers initiates fibrin clotting with the formation of protofibrils. The newly
exposed -chain amino-terminus (GPR) of one fibrin molecule, which is
referred to as the "A" polymerization site, binds noncovalently to a complementary polymerization pocket, termed the "a" site, in
the carboxyl-terminal region of the chain of a second fibrin(ogen) molecule. This "A-a" interaction aligns the fibrin monomers into half-staggered, two-stranded protofibrils (Fig 1B). Subsequently, the
growing fibrils aggregate in a lateral fashion to form fibers, and this
presumably involves interactions between the amino terminus of the chain, the "B" polymerization site, and a complementary site,
"b," whose location is uncertain.10-14 Under
physiological conditions, the release of fibrinopeptide B follows the
release of fibrinopeptide A and correlates with lateral
aggregation.15-17 This sequential release of
fibrinopeptides, such that the "A" site appears before the
"B" site, may control the final clot structure.18
After polymerization, the transglutaminase factor
XIIIa19-21 forms covalent bonds between specific lysine and
glutamine residues located within the carboxyl-terminal regions of
adjacent chains22,23 and between chains24-27 to form - dimers and
-polymers.28 These intermolecular bonds crosslink the
fibrin gel network and, together with the factor XIIIa-mediated
crosslinking of 2-antiplasmin to fibrin, solidify the clot, and
render it more resistant to fibrinolysis.29-32
Fibrinolysis, or the lysis of the clot, is initiated by tissue-type
plasminogen activator (t-PA)-mediated activation of plasminogen to
plasmin. Both t-PA and plasminogen bind to fibrin,33 and the activation of plasminogen by t-PA is stimulated by its substrate, fibrin, which acts as an "effector" for plasmin
formation.34,35 The rate of t-PA-catalyzed activation of
plasminogen has been shown to depend on the formation of
double-stranded protofibrils during fibrin
polymerization.36,37 Plasmin then proteolyzes the fibrin
mesh, thereby dissolving the clot. Hemostasis is achieved through
maintenance of the delicate balance between the procoagulant, anticoagulant, and fibrinolytic reaction pathways. In whole blood, fibrin(ogen) also binds to the receptor GPIIbIIIa
(IIb3)38 on the surface of
activated platelets and thereby mediates the formation of the platelet
plug.
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| Fig 2.
Bead model of the globular carboxyl-terminal region of
the human fibrinogen chain, from Val143 to Val411. Mutations
responsible for -dysfibrinogenemias are presented in red. Colored
beads indicate stretches of helix or strand structure.
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Apart from its role in hemostasis, fibrinogen is also involved in
inflammation, wound healing, cell migration, and cell proliferation via
a number of interactions. These include the binding of fibrinogen to
endothelial cells,39-43 to leukocytes,44-47 and
to components of the extracellular matrix.48
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-CHAIN-RELATED FUNCTIONS |
Many of the important intermolecular interactions of fibrin(ogen)
are localized partly or entirely within the globular carboxyl-terminal region of the chain. The primary polymerization pocket "a," the -chain calcium-binding site, and the - factor XIIIa
crosslinking site all reside in this region.22,23,49-56 The
platelet-surface integrin GPIIbIIIa57-60 and the leukocyte
integrin MAC-1 (M2, CD11b/CD18)61-63 have been shown to interact with specific
sites within this region of the fibrin(ogen) molecule. The chain of fibrin has also been implicated in plasminogen binding64
and in t-PA binding and stimulation.65-68
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FIBRINOGEN AND DISEASE |
An elevated plasma fibrinogen level is associated with coronary
atherosclerosis and is recognized as a significant independent risk
factor for cardiovascular diseases.69-73 In fact, the
predictive value of high fibrinogen levels for ischemic heart
disease,69 myocardial infarction, and stroke74
appears to be as important as high cholesterol levels. In vitro, high
levels of fibrinogen have been shown, among other things, to correlate
with lower fibrin gel porosity,75 to affect the rheological
properties of blood,76 and to influence the thickness of
the fibrin fibers.77
Dysfibrinogenemia refers to the presence in plasma of functionally
abnormal fibrinogen due to a structural defect in the molecule. Inherited dysfibrinogenemia is recognized as a cause of hemorrhagic diathesis and bruising and is now also emerging as a significant risk
factor for familial thrombophilia, heart disease, and
stroke.78-86 The assessment of the risks that
dysfibrinogenemias represent is complicated by the fact that most of
the affected individuals are heterozygous for the mutation, with a
circulating mixture of normal and abnormalfibrinogen. The molecular
basis for the defect and the resulting symptoms vary greatly and those
symptoms, when recognized, are usually episodic in nature. Further, to
correlate the fibrinogen abnormality with the disease requires the
thorough examination of several family members, often over long periods of time. Finally, the correlation with disease depends inherently on
the diagnostic tests and their proper interpretation.
The analysis of dysfibrinogens at the molecular level has been
challenging because of the size and complexity of the fibrinogen molecule. Over the last 15 years, the molecular defects of numerous inherited dysfibrinogenemias have been elucidated. These can be separated into two groups: those that affect the release of the fibrinopeptides A and B, and those that do not. The first group includes dysfibrinogenemias that are generally caused by the
substitutions of amino acids situated at the amino-terminal regions of
the and chains, specifically at or near the thrombin-cleavage
sites, and tend to associate with bleeding complications.87
These mutations interfere with the initial conversion of soluble
fibrinogen to fibrin monomer, and they account for the majority of
abnormal fibrinogens identified to date.88
The second group of dysfibrinogenemias is heterogeneous. It comprises
mutations within the globular carboxyl-terminal regions of the three
chains as well as mutations at the "A" and "B"
polymerization sites that affect the "A-a" or "B-b"
interactions. The clinical manifestations associated with this class of
mutations vary from severe bleeding, to asymptomatic, to severe
thrombophilia. Generally speaking, the mechanisms by which mutations in
these regions of fibrin may cause these clinical manifestations have
not been characterized as thoroughly as those belonging to the first
group.
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FIBRIN(OGEN) MOLECULAR STRUCTURE |
Much valuable information on the dimensions and arrangement of domains
within the molecule has been gained through electron microscopic and
low-resolution crystallographic studies.89-94 These have
provided a wealth of structural information detailing the overall size
of the molecule, the arrangement of the six chains into a symmetric
trinodular structure, and the structures of intermediates formed during
polymerization and fibrinolysis. The final fibrin mesh can be
visualized by electron microscopy, providing an important tool for
analyzing abnormal fibrinogens. Seemingly minor structural abnormalities in fibrinogen can lead to drastic changes in the clot
structure, causing alterations in fiber thickness, length, branching,
and porosity.95,96
Although the high-resolution crystal structure of the entire 340-kD
fibrinogen molecule remains an elusive goal, several high-resolution crystallographic structures of fibrinogen fragments have been published
recently: the 2.1-Å structure of a recombinant 30-kD protein
corresponding to the carboxyl-terminal region of the chain,55 the same 30-kD protein complexed with the peptide
GPRP,56 the 2.9-Å structure of the 86-kD proteolytic
fragment D, and the 172-kD factor XIIIa-crosslinked D-dimer complexed
with the peptide GPRP.97 The peptide GPRP mimics the amino
terminus of the thrombin-cleaved fibrin chain (the "A"
polymerization site). The protein-peptide complex structures showed the
exact position of the polymerization pocket "a" within the chain and identified the amino acids that interact with the fibrin chain GPR residues.56,97
A recombinant protein (rFbgC30) encoding the 30-kD carboxyl-terminal
fragment of the chain of human fibrinogen (Val143-Val411) was
expressed in a soluble secreted form98 in the yeast
Pichia pastoris, purified, and crystallized.55 A
similar fragment of the chain, (the -module Ile148-Val411) was
also expressed in Escherichia coli as an insoluble protein and
refolded in vitro.63 Both of the recombinant proteins were
shown to be functional with respect to calcium binding, crosslinking by
factor XIIIa, and platelet binding.63,98 These studies
represent a "modular" approach to fibrinogen research where small
regions within this complex molecule can be expressed and characterized
without the complicating factors such as partial degradation and
heterogeneity that are associated with fibrinogen isolated from plasma.
The three-dimensional x-ray structures of human fibrinogen fragment D
and of the factor XIIIa-crosslinked D dimer complexed with the peptide
GPRP amide were determined to 2.9-Å resolution.97 This
86-kD fragment D comprises the carboxyl-terminal regions of the and
chains, as well as a short section of the chain (111-197,
134-461, 88-406). In fragment D, the carboxyl-terminal regions of
the and chains are globular, with the chain comprising the
distal end of the outer nodule.97 The overall fold of the and carboxyl terminal regions is similar, as was expected from
their strong sequence similarity.99
The fragment D dimer was seen to have an extensive : end-to-end
"self-association" interface that is formed as the distal nodules
of fibrin become aligned during polymerization, before crosslinking by
factor XIIIa. The characteristics of this : interface are of
great interest, as it has been hypothesized that some fibrinogen
mutations delay polymerization by interfering with the alignment of the
distal nodules at this interface.100,101 The final 8 to 20 residues at the carboxyl-terminal end of the chains were not
visible in the electron density for the -chain fragment and fragment
D crystal structures. Several studies have indicated that this region
can adopt multiple conformations.55,102-106
The recent crystallographic studies have led to new insights into the
structure-function relationships of the complex molecule that is
fibrin(ogen). In particular, we can now correlate the clinical
manifestations and the biochemical information available for the
dysfibrinogens with what is now known about the three-dimensional structure of the molecule.
| |
-DYSFIBRINOGEMIAS |
Dysfibrinogenemias and their pathologies have been reviewed
previously.78,84,87,107-110 The goal of this review is to
examine the dysfibrinogemias, 37 to date, with identified molecular
defects situated within the carboxyl-terminal region of the chain
(Fig 2). Table 1 summarizes the available
biochemical and clinical information. For all of the
-dysfibrinogens, the thrombin time was prolonged, fibrin
polymerization was impaired, and the release of fibrinopeptides A and B
was normal. The mutations were identified either by proteolytic peptide
sequencing or by sequencing of PCR-amplified genomic DNA. The
structural information presented below is derived from the rFbgC30
structures,55,56 unless specified otherwise.
G268E.
Fibrinogen Kurashiki I was identified in a 58-year-old man who was
homozygous for this molecular defect and experienced no major bleeding
or thrombosis.101 Crosslinking of the fibrin chains
appeared to proceed normally. In contrast, the crosslinking of
fibrinogen Kurashiki I by factor XIIIa was delayed, compared with normal fibrinogen. The rate of plasminogen activation by t-PA was
not enhanced by fibrin Kurashiki I to the same extent as is typically
seen with normal fibrin.101 The investigators concluded
that the G268E substitution would disturb the D:D association, possibly
by introducing repulsive forces at a : interaction site between
two dysfibrinogen molecules. This would in turn lead to alterations in
the protofibril alignment and to the observed delay in fibrin
polymerization.101 This hypothesis was based in part on the
close proximity of G268 to R275, which has been proposed to be an
important participant in the D:D interaction.100
In the crystal structure of rFbgC30, the backbone nitrogen of G268
is hydrogen bonded to the carbonyl oxygen of R275, and these two
residues flank a surface-exposed chain reversal comprising residues 269 through 274, with a sharp turn at A271 and D272. Mutations at R275
have also been identified in dysfibrinogens (see next section). G268
exhibits no backbone strain, so the effects of mutations at this site
are not likely to alter the backbone conformation. Because G268 is
exposed to the solvent, a glutamic acid side chain can be modeled in
rFbgC30, without encountering atomic overlaps with neighboring
residues. The fact that bovine111 and
lamprey112 fibrinogens both encode a threonine at position 268 further supports the hypothesis that the observed defect in
polymerization is likely caused by the introduction of a negative charge here, rather than by strain caused by the bulkiness of the
glutamic acid residue.101
Because G268 is approximately 24 Å from the "a" site and 20 Å from the calcium atom, respectively, it is unlikely that the G268E
substitution would affect either the polymerization pocket or the
calcium site directly. The normal crosslinking of fibrin Kurashiki I
would imply that the D:E interaction provides enough stabilization
energy to overcome the proposed D:D repulsion caused by the mutated
residue. Thus, the half-staggered arrangement of mutant protofibrils
would presumably be close to the normal fibrin structure.
R275C, H.
The amino acid R275 is the site of mutation for at least 18 dysfibrinogenemias (Table 1), making it the most commonly mutated site
within the carboxyl-terminal region of the chain. Fibrinogens Osaka
II,113 Tochigi I,114 Morioka I,115
Baltimore IV,116,117 Tokyo II,100,118 Milano
V,119 and Villajoyosa I120 were also
characterized as R275C substitutions. In the case of fibrinogen Osaka
II,113 the new cysteine residue was shown to be linked to a
free cysteine. R275C-fragment D1 that was prepared from
fibrinogen Osaka II failed to prolong significantly the fibrinogen clotting time,113 in contrast to normal fragment
D1.121 The binding of t-PA and plasminogen to
fibrin Villajoyosa I was found to be normal.120 The normal
factor XIIIa-catalyzed crosslinking of fibrin Tokyo II was interpreted
as indicating a normal D:E interaction.100 Electron
microscopic images of crosslinked fibrinogen Tokyo II showed that it
failed to form elongated, double-stranded fibrils. Furthermore, in
contrast to normal fibrin, fibrin Tokyo II formed tapered terminating
fibers with extensive branching of the clot network. The investigators
concluded that the mutation R275C causes a functionally abnormal D:D
association site100 that results in impaired fibrin
assembly.
In contrast to the asymptomatic R275C propositi listed above,
fibrinogen Bologna I was identified in a 20-year-old woman who suffered
multiple episodes of venous thrombosis. Protein C, protein S, and
antithrombin III deficiencies were ruled out as risk
factors.84 Recently, fibrinogen Cedar Rapids (R275C) was
identified in three second-generation family members with severe
pregnancy-associated thrombophilia.122 These patients were
also heterozygous for the Factor V Leiden defect.123-125
Interestingly, first-generation family members who carried only the
Factor V Leiden defect or only the dysfibrinogen had no history of
thrombophilia. These findings suggested that thrombophilia is
associated via the presence of both defects.122
The molecular substitution R275H is also commonly encountered. The
R275H dysfibrinogens Essen I,126 Perugia I,126
Osaka III,127 Barcelona IV,120 and Claro
I128 were isolated from asymptomatic patients. Fibrinogen
Bergamo II was isolated from a patient who, along with several members of her family, suffered from recurrent thromboembolism,126
while Fibrinogen Saga I was found in a patient with
hematuria.129 Fibrinogen Haifa I was described in a
30-year-old woman who experienced severe intermittent claudication
after short walks, due to bilateral occlusion of the superficial
femoral arteries.130 The Fibrinogen Barcelona III
propositus suffered a single postsurgical thrombotic event at age
32,120 and the Claro I propositus had two spontaneous abortions.128 These findings are suggestive, but not
conclusive, of a possible association between mutations at R275 and a
tendency to thrombosis.
Calcium binding, platelet aggregation, and factor XIIIa-mediated
crosslinking appeared to be normal in Fibrinogen Haifa.131 However, the presence of calcium ions did not protect its fragment D
against plasmin cleavage at position K302-F303,131 as would be expected for normal fragment D.132 An electron
microscopic study of polymerized fibrin Haifa I showed an abnormal,
highly branched matrix, with the fibers appearing generally thinner
than those seen in normal polymerized fibrin.95 In contrast
to fibrinogen Haifa I, fragment D1 derived from fibrinogen
Saga I was fully protected by calcium ions against plasmin degradation,
but it failed to inhibit fibrinogen clotting.129
In the rFbgC30 structure, residue R275 is surface-exposed, flanking
a chain reversal comprising residues 269-274. As mentioned previously,
a turn between residues 270 and 271 brings the backbone of R275 in
close proximity to G268, allowing a hydrogen bond between the carbonyl
oxygen of R275 and the nitrogen of G268. A salt link between R275 and
D272 further stabilizes this chain reversal. Thus, it is reasonable to
assume that amino acid substitutions that alter this turn may cause
similar disruptions in the protein structure. This could in turn affect
the interactions of this region with other proteins. The participation
of R275 in a D:D interface was confirmed by the three-dimensional
structure of the factor XIII-crosslinked D dimer.97
The side chain of R275 is involved in another very important
interaction, because it forms two hydrogen bonds with the backbone of
G309, thus clearly providing extra stabilization for this region of the
protein. The backbone nitrogen atom of G309 is hydrogen bonded to the
backbone carbonyl atom of L276 as well as to the side chain oxygen of
N308. The N308 side chain in turn shares two hydrogen bonds with the
backbone of Y278. The importance of the proper alignment of residues in
this region is highlighted further by the localization here of several
other molecular defects associated with dysfibrinogenemias; ie, N308I,
N308K, and M310T (see below).
G292V.
Dysfibrinogen Baltimore I was diagnosed more than 30 years ago in a
29-year-old woman suffering from femoral vein thrombosis after minor
trauma.133-135 The patient had a history of mild
hemorrhagic diathesis characterized by frequent bruising, epitaxis, and
hemorrhage as well as previous severe recurrent thrombosis and
pulmonary embolism. Fibrinogen Baltimore I showed delayed fibrin
polymerization that could be partially corrected by addition of
calcium.133 The defective formation of factor
XIIIa-crosslinked -polymers was corrected by increased calcium
concentration or lowered pH conditions.136 Plasmin
degradation of fibrinogen Baltimore I was normal and its fragment D was
protected effectively by calcium. The amino acid substitution G292V was
identified as the molecular defect.137
G292 lies in the middle of a highly conserved stretch of amino
acids,99 approximately 10 Å from the polymerization pocket and 18 Å from the calcium atom.55 Upon first inspection of
the crystal structures,55,56 G292 appears to be isolated
from the clusters of residues linked to other dysfibrinogenemias. G292 is located at the surface of the protein, and the backbone dihedral angles of (
,
) = (101o,164o) indicate that
substitution of a nonglycine residue at this site would lead to
backbone strain and destabilize the structure. Further, the side chain
of N337, which points away from the polymerization pocket, is
only 7 Å from G292. It is plausible that insertion of a bulky valine
side chain at position 292 would alter the region surrounding N337.
N337 is part of an unusual, highly strained region of the the chain. Its dihedral angles indicate backbone strain, and it lies
immediately adjacent to a cis peptide bond between residues
K338 and C339. This strained conformation enables the backbone of C339
to form an additional hydrogen bond with the fibrin chain
amino-terminal residues GPR.56 This strained conformation
is stabilized in part by an extensive network of hydrogen bonds,
including the hydrogen bonds between the N337 side chain and its
neighbors. Therefore, any disruption of the environment of the N337
side chain could destabilize its backbone, altering the polymerization
pocket and thereby fibrin polymerization.
Residues F303 and F304 are positioned in between G292 and N337. Both
the phenyl rings are directed to the surface of the molecule. It has
been well established from solvent transfer studies138,139 that the phenylalanine side chain is reasonably soluble in both polar
and nonpolar solvents; hence, its occurrence both in the hydrophobic
cores of proteins and at their solvent-exposed surfaces. The valine
side chain, by contrast, occurs almost exclusively in hydrophobic
environments, and is typically buried in solvent-inaccessible protein
cores. The substitution of a valine for G292 would promote hydrophobic
interactions between the valine and phenylalanine side chains, thus
altering the molecular surface significantly. Further, the surface
exposure of the hydrophobic valine side chain would decrease the
entropy, and thus the stability of the protein. Therefore, we propose
that the substitution G292V leads to the structural destabilization of
a fairly large region of the protein, and that this affects the
polymerization pocket directly.
N308I, K.
The dysfibrinogenemia Baltimore III was diagnosed in an asymptomatic
30-year-old woman who had a prolonged thrombin time that was corrected
by excess calcium.140 The defect was identified as an N308I
mutation.141 The fibrinogen showed normal crosslinking by
FXIIIa, normal calcium binding, and delayed fibrin monomer polymerization.140,141
The N308K mutation was identified in fibrinogens Kyoto
I,142,143 Bicêtre II,144 and Matsumoto
II.145 The clinical manifestations associated with the
N308K substitution varied greatly. Fibrinogen Kyoto I was isolated from
an asymptomatic, hypofibrinogenic male who had a family history of both
thrombotic and bleeding problems142,143. Fibrinogen
Bicêtre II was isolated from a man who suffered a spontaneous
deep-vein thrombophlebitis and a pulmonary embolism.144 Finally, fibrinogen Matsumoto II was isolated from a woman with Graves' disease who had a tendency to bruise easily and who had experienced moderate to severe bleeding after each of her three deliveries.145 For fibrinogen Bicêtre II, it was
shown that the mutation did not affect binding to t-PA or to
plasminogen, thus eliminating these possible explanations for the
thrombotic incidents.144
The residue N308 is situated on the surface of rFbgC30. For that
reason, the introduction of a hydrophobic amino acid such as isoleucine
at this position would appear to be very destabilizing. In
structure-based sequence alignments, N308 is conserved in all of the
-extended, -, and -chain sequences except in the lamprey fibrinogen chain, where it is replaced by a
leucine.55,97,99 Although a lysine residue should be
accommodated easily on the surface of rFbgC30, a positive charge
here could disrupt interactions with other fibrinogen domains. The new
lysine residue could also change the pattern of cleavage by plasmin,
thereby affecting fibrinolysis. This possibility has not been addressed
experimentally.
The side chain of N308 forms hydrogen bonds with the backbone nitrogen
and carbonyl oxygen of Y278.56 An additional hydrogen bond
connects the side-chain oxygen of N308 with the backbone nitrogen of
G309, as discussed in the context of the mutations at G268 and R275. A
cluster of residues including N308, G309, G268, R275, and M310 is
exposed to the solvent in both the rFbgC30 and fragment D
structures, and is in the vicinity of the D:D interface of the factor
XIIIa-crosslinked D dimer structure.97 This interaction site is presumably buried during the initial contact between the D
domains, in the early stages of fibrin polymerization. Thus, the
introduction of bulky or charged side chains at N308 would be expected
to disrupt the initial alignment of fibrin monomers into protofibrils.
We would expect the clot structure in these patients to be altered in a
manner similar to that seen with fibrinogen Tokyo II.100
M310T.
Dysfibrinogenemia Asahi I was diagnosed in a 33-year-old man
sufferering from posttraumatic bleeding after a traffic accident. He
had experienced moderate hemorrhage related to injuries and delayed
wound healing since adolescence.146 The mutation M310T creates a consensus sequence, N308-G309-T310, that directs N-linked glycosylation, and, indeed, the attachment of a carbohydrate moiety to
N308 was confirmed.146 The factor XIIIa-mediated -
crosslinking of fibrinogen and of fibrin Asahi I were both markedly
delayed, even though the chain amine acceptor Q398 functioned
normally when assayed by monodansylcadaverine incorporation. The
delayed fibrin(ogen) crosslinking rate indicated therefore that the
abnormal molecules were unable to align properly.146,147
The extent of fibrinogen glycosylation affects fibrin polymerization
and lateral aggregation, as well as the structural and mechanical
properties of the clot.148 For example, desialylated fibrin
polymerizes faster than normal fibrin149 and clots
containing deglycosylated fibrin(ogen) form faster and display thicker,
underbranched fibers, resulting in a more porous mesh.150
Conversely, clots formed from hyperglycosylated fibrinogen (as would be
the case for fibrinogen Asahi I) assemble more slowly.151
Alignment of the human -chain sequence with a series of homologous
sequences55,97,99 shows that the methionine at position 310 is strictly conserved among the fibrinogen sequences. Nevertheless, the
substitution M310T would appear to be structurally benign. Because the
residue is surface-exposed and is not involved in any hydrogen bonds,
it is difficult to argue compellingly for the unique contribution of
M310 to the structural integrity of this region. Therefore, we propose
that the primary consequence of mutations at this site is in the
disruption of D:D interactions by the new glycosylation, and that the
effects of this mutation are analogous to those resulting from
substitutions at the chain positions R275, G268, G292, and N308.
D318G.
Fibrinogen Giessen IV (sometimes referred to as Kassel), was identified
in an 18-year-old woman who suffered from recurrent venous thrombosis
as well as mild bleeding.84 She was later found to be
heterozygous for the factor V Leiden defect (personal communication
from Drs F. Haverkate and R.M. Bertina, Leiden, The Netherlands,
December 1997). The only relative of the patient who was
found to have this dysfibrinogenemia did not experience thrombosis; it
is not known whether this individual was also heterozygous for the
Factor V Leiden defect. The side chain of aspartate 318 is directly
involved in binding to the calcium ion in the chain.55 The removal of this carboxyl group would obviously have a detrimental effect on calcium binding, presumably impairing the regulation of
fibrin polymerization and the protection of the chain by calcium
ions.
N319,D320.
Fibrinogen Vlissingen I was identified in a 23-year-old woman who was
hospitalized with a massive pulmonary embolism.152 The
father and daughter of the patient also showed prolonged fibrinogen clotting times, although they were asymptomatic. The same 2-amino acid
deletion was found in fibrinogen Frankfurt IV, in a patient with
thrombosis. A subsequent family history showed that these two patients
were related.84 Fibrin polymerization was delayed both in
the presence and absence of calcium. Calcium was only partially
effective in protecting this fibrinogen against plasmin degradation.
Calcium binding was measured by equilibrium dialysis, and showed the
loss of one high-affinity Ca2+-binding site within
fibrinogen Vlissingen I fragment D.152 DNA sequence
analysis showed that the patient was heterozygous for a deletion of the
six nucleotides encoding N319 and D320. It was concluded that these
residues were essential for calcium ion binding. Clotting of the
mutant fibrinogen in the presence of EDTA was also delayed relative to
normal fibrinogen, suggesting that the deletion affected not only the
calcium-binding site, but also the polymerization site "a" within
the chain. In rFbgC30, the polymerization site appears to
function relatively independently from the calcium-binding site. This
is illustrated by the fact that the fragment can bind GPRP even after
treatment with EDTA.98 Nevertheless, the Vlissingen I data
suggest that a disruption of the structure at one site could well
affect the folding of the other, leading to a polymerization defect.
Q329R.
Fibrinogen Nagoya I was found in three generations of a clinically
asymptomatic Japanese family.153 Fibrin monomer
polymerization was defective, while factor XIIIa-catalyzed
crosslinking of fibrin was normal. The chain of fibrinogen Nagoya I
showed abnormal behavior on CM-cellulose chromatography and sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE).154 A single amino acid substitution of
glutamine by arginine at position 329 was shown by peptide amino acid
sequence analysis.155
A comparison of the 30-kD -chain structures, both
uncomplexed55 and complexed with the fibrin chain-mimicking peptide GPRP,56 shows that upon binding of
GPRP, Q329 shifts its position to accommodate arginine 3 of the peptide
and forms a hydrogen bond with it.56 The recombinant chain fragment with the substitution Q329R (rFbgC30-Q329R) was not
protected against plasmin degradation in the presence of the peptide
GPRP and EDTA, presumably because the mutation abolished GPRP
binding.98 This suggests that the mutant arginine side
chain blocks access to the "a" site of fibrin(ogen), quite
possibly by occupying the preexisting arginine-binding site within the
polymerization pocket.
D330V, Y.
Fibrinogen Milano I (D330V) was identified in a young girl and her
father, both of whom were clinically asymptomatic.156 A
second dysfibrinogenemia involving this amino acid, fibrinogen Kyoto
III (D330Y), has been described in an asymptomatic 50-year-old woman.157 Her two sons showed the same abnormality, and
fragment D1 isolated from the second son was not effective
in inhibiting fibrinogen clotting. Because aspartic acid and tyrosine
have very different pKa's, it was hypothesized that the
point mutation perturbed the local folding of the chain, thus
altering the structure required for fibrin
polymerization.157
The crystal structure of the rFbgC30-GPRP complex56
showed that D330 forms a salt link with the arginine side chain of the
peptide, providing an interaction essential for D:E binding. Furthermore, the carboxyl group of D330 is hydrogen-bonded to the side
chain of H340. The backbone carbonyl oxygen of H340 forms an additional
hydrogen bond with the peptide amino terminus, and the backbone
nitrogen is hydrogen-bonded to the peptide glycine carbonyl oxygen.
Therefore, the replacement of D330 by an uncharged amino acid residue
such as tyrosine or valine would significantly alter the electrostatic
environment of the polymerization pocket. The addition of these bulky
side chains would necessarily alter the extensive and precise network
of hydrogen bonds within the polymerization pocket. These disruptions
are consistent with the markedly prolonged clotting times of patients
carrying this mutation.
N337K.
Fibrinogen Bern I was discovered in a 24-year-old asymptomatic
woman.158 Two-dimensional gel electrophoresis showed an
abnormal mobility for the chain of fragment D derived from
fibrinogen Bern I.159 N337 lies in a region of the chain that is highly conserved among mammals. It is part of a very
constrained chain reversal within the chain,56 as
described earlier in the G292V section. The side chain of N337
participates in three hydrogen bonds to S306 and F303 and points away
from the polymerization pocket, toward the solvent. Although a lysine
residue could be accommodated in this position, several N337 hydrogen
bonds that stabilize the strained backbone conformation at the
polymerization pocket would be lost.
Paris I (insertion at 350).
Fibrinogen Paris I is characterized by impaired fibrin polymerization
and clot retraction.160 Fibrin Paris I monomers inhibited the polymerization of normal fibrin monomers, did not form - dimers, and did not incorporate dansylcadaverine in the presence of
factor XIIIa.161,162 Approximately 2/3 of the Paris I
fibrinogen chain exhibited an apparent molecular weight higher
(
2.5 kD) than normal.161 Electron microscopic studies on
fibrin Paris I showed abnormal clumps connected by thin irregular
fibers, with frequent terminations.163 Fibrinogen Paris I
also failed to support adenosine diphosphate (ADP)-induced
platelet aggregation.164 The molecular defect was
determined by polymerase chain reaction (PCR) analysis to be an A
G point mutation within intron 8 of the chain gene. It
is hypothesized that alternative splicing, due to this nucleotide
change in the Paris I mRNA, leads to the insertion of 15 amino acids
after Q350, including two cysteine residues, and to the substitution of
G351 for a serine.165 No association with bleeding or
thrombosis has been reported for this dysfibrinogenemia.
The insertion of 15 amino acids at residue Q350 cannot be
modeled with reliability. Nevertheless, we can speculate
that this large insertion within the polymerization domain would cause
a major structural reorganization, quite possibly precluding the formation of functional polymerization and crosslinking sites.
S358C.
Fibrinogen Milano VII was identified in an asymptomatic 21-year-old
woman.166 No family member had a hemorrhagic or thrombotic tendency, but several had a prolonged thrombin time. The fibrin Milano
VII clots had an abnormal "transparent" appearance when compared
with clots obtained from normal fibrinogen.166 The S358C mutation creates an unpaired cysteine residue, and immunoblotting analysis determined that the abnormal fibrinogen circulated as a
disulfide-linked complex with the abundant blood protein albumin. Interestingly, removal of albumin failed to normalize the fibrin polymerization profile. This suggested that the defect was not due to
steric hindrance created by albumin, but rather was attributable directly to the substitution.166
The three-dimensional structure of rFbgC30 shows that S358 is indeed
on the protein surface, making the new cysteine residue available to
react with other free cysteines.55,56 S358 does not
interact directly with either the polymerization pocket or the calcium
site, and it does not appear to be part of the D:D interaction site. It
is possible that the substitution leads to a general structural
destabilization of the polymerization domain, but there is no direct
evidence for this. The observed transparency of fibrin Milano VII clot
suggests to us a possible effect on the lateral aggregation of fibrils.
D364H,V.
Dysfibrinogenemia Matsumoto I was identified recently in a 1-year-old
boy who had Down Syndrome and congenital heart disease. The young
patient showed no signs of bleeding or thrombotic tendencies, nor did
his two relatives who also showed prolonged thrombin times. The D364H
mutation was identified in the propositus and his affected relatives.167
Another dysfibrinogenemia involving amino acid D364, Melun I, was
identified in a 40-year-old woman following a routine blood coagulation
assay.168 The patient had no sign of hemorrhage or thrombosis at the time of the assay, but at 67 years of age the patient
developed superficial venous thrombosis on her right foot. This was
followed 2 years later by another episode of superficial venous
thrombosis in her right leg. The propositus later had a stroke that did
not cause long-term effects.168 Further tests eliminated
other possible inherited causes of thrombophilia such as protein C,
protein S, or antithrombin III deficiencies, or activated protein C
(APC) resistance. The patient's mother, sisters, and
brother all suffered from repeated deep vein thrombosis or pulmonary
thromboembolic episodes. An exhaustive study of four generations showed
that most, but not all, of the family members carrying the
dysfibrinogenemia suffered from various forms of thrombosis, with
episodes beginning as young as age 11. DNA sequencing of the three
chains of fibrinogen Melun I led to the discovery of a D364V
mutation.168 Again, two dysfibrinogenemias involving the
same amino acid were associated with different phenotypes, one (D364H)
devoid of symptoms and the other (D364V) associated with thrombophilia.
Two recent independent investigations showed the crucial role of D364
in the early stages of fibrin polymerization, ie, during the "A-a
site" interaction.98,169 In the first study, the
substitution D364A was introduced into the 30-kD fragment rFbgC30
and, unlike the wild-type rFbgC30, this mutant molecule
(rFbgC30-D364A) did not inhibit fibrinogen clotting. rFbgC30 was
protected against plasmin digestion by addition of the peptide GPRP or
by calcium, as is the case for fibrinogen.170,171 The
rFbgC30-D364A mutant molecule was not protected by the peptide,
suggesting that the "a" site is substantially altered in this
mutant.98 In the second study, fibrin polymerization of
recombinant, fully assembled fibrinogen containing the D364A and D364H
mutations was significantly impaired, as expected if the "a" site
is not functional.169 Fibrin polymerization of recombinant
fibrinogen with the D364H mutation was almost undetectable. Clottability of the D364A mutant was essentially normal but that of the
D364H mutant was substantially reduced. In fact, a fibrin gel did not
form when the D364H-mutant fibrinogen was clotted.169 These
data suggest that both protofibril formation and lateral aggregation
were disrupted in the D364H mutant, indicating that the
carboxyl-terminal region of the chain plays a role in both polymerization steps.169
The crystal structures of rFbgC30-GPRP56 and fragment D
dimer-GPRPamide97 showed that the side chain of D364 forms
a critical salt link with the charged amino terminus of the -chain sequence GPR. The loss of the carboxylate group in the D364H and D364V
mutations would eliminate an electrostatic bond that is crucial to the
"A-a" interaction.
R375G.
Fibrinogen Osaka V was identified fortuitously in an asymptomatic
44-year-old woman.172 The polymerization of fibrin monomers was prolonged in the absence of calcium but normal polymerization occurred at 5 mmol/L CaCl2. Digestion of fibrinogen Osaka V
fragment D by plasmin resulted in a more complete degradation of the
molecule than was observed for normal fragment D. Further, equilibrium dialysis and Scatchard analysis showed that the mutant fibrinogen contained only one calcium-binding site, whereas three calcium sites
have been found in normal fibrinogen.173-176
In the GPRP-complexed structures,56,97 the side chain of
R375 forms hydrogen bonds that are critical for the stability of the
polymerization pocket. The loss of this large side chain would
drastically alter the shape and charge of this region. It would also
remove an important salt link between R375 and D297. R375 is absolutely
conserved among all the known chain sequences,55,97,99 indicating the importance of this residue. The polymerization pocket
appears to be biochemically98 and
structurally56,97 distinct from the calcium-binding site.
However, we suggest that the loss of the R375 side chain, which lines
one side of the polymerization pocket, would exert a destabilizing
effect on the entire region. This would explain the observed
calcium-binding defect.
K380N.
Fibrinogen Kaiserslautern was identified in a 34-year-old woman who
suffered from thrombosis in the cerebral sinus after a cesarean
delivery, prior to which she had no history of bleeding or
thrombosis.177 Her family included individuals who were
homozygous as well as heterozygous for this defect, all of whom were
asymptomatic. A K380N substitution was shown and glycosylation at N380,
directed by the new consensus sequence (N-K-T), was
confirmed.177
K380 is a surface-exposed residue, occurring at a site that is well
removed from the polymerization pocket, the calcium-binding site, and
the D:D interaction surfaces. Therefore, the polymerization defect is
likely to be an altered packing of fibrils caused by the addition of an
extra carbohydrate moiety.
| |
FUNCTIONAL SITES WITHIN THE FIBRINOGEN CHAIN |
In view of the biochemical, clinical, and structural information
available to date on fibrinogen, at least five major functional sites
can be distinguished within the globular carboxyl-terminal region of
the chain. These are: (1) the calcium-binding site; (2) the
polymerization site "a"; (3) the D:D interaction surface; (4) the
: crosslinking site; and (5) the platelet-binding site. Other
functions have been reported to involve the chain of fibrinogen, such as t-PA binding, plasminogen binding, and interactions with cell-surface receptors; these await further characterization at the
molecular level. Each of the five sites appears to function fairly
independently from the others. However, a mutation at one site may
destabilize the overall structure and thus affect function at a second
site.
Calcium-binding site.
A single high-affinity calcium binding site has been found within
fragment D,49,174-176,178 and localized to the globular
carboxyl-terminal region of the chain.51,52,55
Figure
3 presents a schematic of the interactions between the metal ion and
the protein. The calcium ion is liganded by the side chains of D318 and
D320, as well as by the backbone carbonyl oxygens of F322 and
G324.55 Two water molecules provide the fifth and sixth
coordinating ligands. Although fibrinogen can clot in the presence of
EDTA, it does so less efficiently than in the presence of
calcium.179 Thus, the calcium-bound conformation of the chain favors fibrin polymerization and protects this region from
plasmin degradation.132,173 Two dysfibrinogens, Giessen IV
(D318G) and Vlissingen I (N319,D320), alter the calcium-binding
site. These mutations disrupt the metal binding, and both of these
dysfibrinogenemias correlate with a thrombotic tendency. Fibrinogen
Osaka V (R375G) is also associated with altered calcium binding,
presumably resulting from a regional disruption of the protein
structure, but not with thrombosis.
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| Fig 3.
Specific molecular interactions between the fibrinogen
chain and the calcium ion. The calcium ion is liganded by two
aspartate side chains, two carbonyl oxygen atoms, and two water
molecules.55
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| Fig 4.
Close-up stereo views of the chain polymerization site "a" showing the interactions within the
polymerization pocket for both the uncomplexed protein (A) and for the
complex with GPRP (B), based on the rFbgC30 structures. The mutation
sites that affect the polymerization pocket are indicated in
ball-and-stick representation. In (A), the water molecules that form
hydrogen bonds with the displayed side chains are shown as pink
spheres. In (B), the peptide GPRP is represented in pink. The calcium
ion is shown in green, and is enlarged for emphasis.
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| Fig 5.
Space-filling model showing a close-up view of the
: (or D:D) interface between adjacent molecules in the
crosslinked D dimer complexed with the peptide GPRP (A). (Adapted and
reprinted with permission from Nature [Spraggon G, Everse
SJ, Doolittle RF: Crystal structures of fragment D from human
fibrinogen and its crosslinked counterpart from fibrin. Volume 389, page 455, 1997].97 Copyright 1997 Macmillan Magazines
Limited.) Space-filling (B) and ribbon (C) models of the globular
carboxyl-terminal chain region, based on the rFbgC30 structures
(143-392). Mutation sites at the : interface are colored
orange (B and C), the calcium ion is green, and the peptide GPRP is
shown in magenta. The side chains of residues F303 and F304 are shown
in white. We hypothesize that these residues may form part of an
extended interaction surface between the D and E regions of fibrin.
Residues G292, S358, and K380 are also shown; G268 and G292 are
indicated by asterisks (C).
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|
Polymerization site "a."
The localization of the primary polymerization site "a" to the
carboxyl-terminal region of the chain was established several years
ago.49,50,52,54 The "a" site binds the GPR sequence that is exposed upon the release of fibrinopeptide A from the chain, and several short peptides having sequences related to GPR
also bind to the "a" site.12,180-182 The
peptides GPRP and GPRP-amide bind to the chain of fragment D with
an affinity that actually exceeds that of the peptide having the native
sequence, GPRV.182 In addition, the peptide GPRP protects
the chain of fragment D against plasmic degradation, even in the
presence of EDTA.63,98,170 Figure 4 shows in detail some of
the important molecular interactions at the polymerization site before
and after binding to the GPRP sequence.
Several dysfibrinogens involve mutations of -chain residues that
either bind to the GPR sequence directly, or that constitute the
architecture of the polymerization site. These include Fibrinogens Nagoya I (Q329R), Milano I (D330V), Kyoto III (D330Y), Bern I (N337K),
Matsumoto I (D364H), Melun I (D364V), and Osaka V (R375G).
D:D interaction surface.
During the alignment of the fibrin monomers into fibrils (Fig 1),
surface-exposed regions on the chains of two adjacent molecules
abut each other and form the D:D interface (Fig 5A). The - (or
D:D) interface is extensive,97 and encompasses a number of
amino acids that are substituted in dysfunctional fibrinogens (Fig 5B
andC). These dysfibrinogens include Fibrinogens Kurashiki I (G268E),
Baltimore IV, Bellingham I, Bologna I, Milano V, Morioka I, Osaka II,
Tochigi I, Tokyo II, and Villajoyosa I (R275C), Barcelona III and IV,
Bergamo II, Claro I, Essen I, Haifa I, Osaka III, Perugia I, and Saga I
(R275H), Japanese I (R275S), Baltimore III (N308I), Bicêtre I,
Kyoto I, and Matsumoto II (N308K), and Asahi I (M310T). Disruption of
this : interaction has been hypothesized to disrupt the fibrin
alignment.100,101 Scanning electron microscopic images of
fibrin Tokyo II (Fig 6) show unequivocally
that the structure of the fibrin clot was altered drastically by a
mutation at the : interface, R275C.100
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| Fig 6.
Scanning electron micrograph images of Tokyo II fibrin
(R275C) (A and B) and normal fibrin (C and D). Fibrin formed in
HEPES pH 7 buffer (A and C) and formed in the same buffer containing 10 mmol/L CaCl2. (Reproduced from The Journal of
Clinical Investigation, 1995, vol. 96, p. 1053, by copyright
permission of The American Society for Clinical
Investigation.100)
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|
We also note that F303 and F304 form a surface patch on the chain
that could interact favorably with a hydrophobic patch on the surface
of another molecule, and that these residues lie between the :
surface and the polymerization pocket (Fig 5B). Therefore,
F303 and F304 could potentially be involved in D:E and/or D:D
interactions.
- crosslinking site.
Factor XIIIa catalyzes reciprocal covalent crosslinks between the
glutamine donors Q398 and/or Q399 of one molecule and the lysine acceptor K406 of an adjacent fibrin(ogen)
molecule.22,183,184 There are no reported
dysfibrinogenemias associated with mutations in this region of the chain (also referred to as XL:XL). Such
mutants would likely be "silent" in standard thrombin time
assays, and this may explain why they have not been reported in the
literature.
Platelet-binding site.
Fibrinogen interacts with the platelet receptor GPIIbIIIa through the
last several amino acids at the carboxyl end of the chain.57-59,185,186 Although other regions may interact
with this receptor as well, the sequence 408-411 (AGDV) is
absolutely required for fibrinogen binding.60 The
interaction with platelets has also been visualized through electron
microscopic studies.187 Crystallographic studies have not
shown a unique conformation for the carboxyl-terminal 19 residues of
the chain. In the rFbgC30 protein preparations, this region was
very susceptible to proteolysis.55 The gaps in
interpretable electron density in some of the crystal structures of
this molecule were likely caused by heterogeneity at the carboxyl
terminus, and it is probable that this region also has some inherent
flexibility.
| |
-CHAIN DYSFIBRINOGENEMIAS ASSOCIATED WITH BLEEDING |
In general, mutations within the chain of fibrinogen are not
associated with serious bleeding disorders. Two patients, Baltimore I
(G292V) and Giessen IV (D318G), who experienced mild bleeding symptoms,
also suffered from thrombotic tendencies. The only
-dysfibrinogenemia associated with a serious bleeding diathesis is
Asahi I (M310T). In this instance, the bleeding symptoms were probably
related to the extra glycosylation resulting from the substitution. As discussed earlier, hypoglycosylation increases the rate and extent of
clotting.150 Therefore, one can speculate that
hyperglycosylation could decrease the clotting rate and thereby cause a
bleeding disorder. The molecular explanation would be that the charged carbohydrates lead to a repulsive force between adjacent molecules, thus hindering the assembly and polymerization of fibrin. Also, the
bulkiness of the extra carbohydrate could preclude the proper alignment
of the fibrin chains.
| |
-DYSFIBRINOGENEMIAS ASSOCIATED WITH THROMBOSIS |
The proposed mechanisms by which abnormal fibrinogens may contribute to
a thrombotic tendency are numerous.80,84 For example, an
increased resistance of the mutant fibrin to plasmin proteolysis may
arise from alterations in the clot structure and permeability. These
mutations may restrict the accessibility of fibrin to
plasmin.96,188,189 Other mutations may reduce
fibrin-mediated enhancement of fibrinolysis by altering the binding of
plasminogen or t-PA to fibrin.190,191 For the
-dysfibrinogenemias, the most probable reason for the association of
mutations with thrombophilia is an altered clot structure. In
heterozygous individuals, fibrin polymerization is perturbed but
functional, leading to clots with abnormal fiber thickness and
porosity. As fibrin structure has been shown to influence the
fibrinolytic rate,192 these clots may not be dissolved effectively by the fibrinolytic system, resulting in an increased tendency to thrombosis. We speculate that many of these mutations, if
present in the homozygous state, would cause bleeding disorders as
well.
| |
DYSFIBRINOGENEMIA AS A HYPERCOAGULABLE STATE |
Thrombosis is a major cause of mortality and morbidity. The risk
factors for thrombosis can be transient or permanent, acquired, congenital, or inherited. Among the identified inherited risks factors
associated with thrombophilia are protein C, protein S, antithrombin
III, and heparin cofactor II deficiencies; factor Va resistance to APC;
thrombomodulin defects; factor II 20210 allele; and
hypoplasminogenemia.79,80,83,85,86,193,194 However, taken
together, these conditions account for only 40% to 60% of all
familial thrombophilias.
Dysfibrinogenemias are also recognized as possible risk factors for
thrombosis. The majority of diagnosed individuals are asymptomatic,
whereas approximately 30% of dysfibrinogenemias are associated with
bleeding and 10% are associated with thrombotic tendencies.
However, if one considers only dysfibrinogenemias with mutations in the
carboxyl-terminal region of the chain, then the distribution
changes considerably. An examination of the clinical symptoms
associated with -dysfibrinogenemias shows that 5% (2 of 37) of
the individuals experienced significant bleeding, and 30% (11 of
37) showed thrombotic tendencies. Approximately 60% (23 of 37) of
patients with -dysfibrinogenemias were asymptomatic at the time of
diagnosis. Although an association is found between certain
dysfibrinogenemias and specific symptoms, a direct causal relationship
is difficult to demonstrate. Often, the investigation of other risk
factors was omitted, incomplete, or not reported. Furthermore, the
family history for these mutations, which can be difficult to gather,
was often not documented. Unfortunately, data on long-term follow-up of
dysfibrinogemic patients and their relatives are rarely available.
Typically, dysfibrinogenemias are discovered fortuitously during
routine coagulation tests. At present, there is no practical, simple,
and cost-effective clinical test that could allow the diagnosis of
thrombophilic dysfibrinogenemias that do not affect fibrin
polymerization. One must consider the possibility that mutant
dysfibrinogens exist that affect fibrinolysis but not fibrin polymerization. If these dysfibrinogenemias exist, then
thrombosis-associated dysfibrinogenemias are surely underdiagnosed, and
these may represent a more important determinant of hereditary
hypercoagulable states than is generally recognized.
A relatively small number of the diagnosed dysfibrinogenemias have been
characterized at the molecular level. Numerous others that are
associated with mild to severe familial thrombosis still await
characterization at the molecular level. These include fibrinogens Date
I,191 Richfield,195 Tampere I,196
and London I.197,198
It has been suggested that an inherited predisposition to thrombosis is
often the result of mutations in two or more genes encoding proteins
involved in hemostasis.79 Similarly, a
-dysfibrinogenemia may well act in concert with other risk factors
such as stasis, surgery, pregnancy, trauma, lupus, malignancy, oral
contraceptives, hyperhomocysteinemia, or elevated factor VIII or
fibrinogen levels. Such combinations could trigger the occurrence of a
thrombotic episode in an otherwise healthy individual. Examples
illustrating this phenomenon would be the heterozygous dysfibrinogemias
Cedar Rapids (R275C)122 and Giessen IV
(D318G),84 both of which were found in conjunction with a
heterozygous factor V Leiden trait. In the Cedar Rapids family, neither
defect alone was associated with symptoms, but the double phenotype was
strongly associated with pregnancy-related thrombophilia. The complex
interactions between potential risk factors would explain to some
degree the variability observed in the clinical symptoms associated
with -dysfibrinogenemias. In the case of the R275, N308, and D364 mutants, the clinical manifestations associated with a given molecular defect varied greatly from one patient to another. A mutation associated with mild hemorrhage in one patient may appear to be silent
in another, and yet may be associated with severe thrombophilia in
another individual.
Finally, the analysis of the various fibrinogen chain mutants is
complicated by many factors. First, most dysfibrinogens were identified
in heterozygous individuals; therefore, the circulating fibrinogen is a
heterozygous mixture of normal and mutant molecules. Second, apart from
commonly used assays such as thrombin time and fibrinopeptide release,
the biochemical characterization of these defects has not been
standardized. Experiments are performed using different protocols, on
plasma in some cases, and on purified fibrinogen in others, under
varying conditions. This situation complicates the direct comparison of
results from different laboratories, and may explain some of the
apparent discrepancies between the observed effects of a given
molecular defect.
| |
CONCLUSIONS |
As proposed in the fibrinogen subcommittee study,84 it
would be valuable, in approaching a case of thrombophilic
dysfibrinogenemia, to evaluate all other known contributing risk
factors and to rigorously document the family history. This would help
determine if the diagnosed dysfibrinogenemia is the sole thrombotic
risk factor present. The same analysis should be performed for patients
with bleeding symptoms and for asymptomatic individuals, if we are to
determine with any degree of certainty the influence of the dysfibrinogenemia on the long-term manifestations of these defects. Finally, every effort should be made to accurately establish the diagnosis of a thrombosis. By combining well-documented family histories with correct diagnoses of symptoms, the possible associations between the various dysfibrinogenemias and thrombosis can then be
assessed more accurately.
The recent determination of several crystal structures of fibrinogen
fragments has shed light on the arrangement of domains at the distal
nodules of fibrinogen, the architecture of the "a" polymerization
pocket, and the interactions between amino acid residues that are
critical for the various functions of this fascinating and complex
molecule. As an early outgrowth of this work, the effects of mutations
that cause dysfibrinogenemias can now be explored through further
structure-function studies. Hypotheses originating from a consideration
of the patients' symptoms and from an examination of the fibrinogen
structure can now be explored and refined by experiments using
recombinant protein expression systems. We expect that future studies
will lead to a broader overall understanding of fibrinogen and its
pathologies, and hopefully to improvements in the diagnosis and
management of dysfibrinogenemic patients.
| |
FOOTNOTES |
Submitted December 29, 1997;
accepted May 27, 1998.
Supported in part by National Institute of Health Grants No. HL31048
(to S.T.L.) and HL16919. H.C.F.C. was supported in part by a Research
Fellowship from the Heart and Stroke Foundation of Canada.
Address reprint requests to Kathleen P. Pratt, PhD,
Department of Biochemistry, Box 357350, University of Washington,
Seattle, WA; 98195; e-mail: kpratt{at}u.washington.edu.
| |
ACKNOWLEDGMENT |
The authors thank Drs E.W. Davie, D.W. Chung, and F. Haverkate for
critical reading of the manuscript. We are also grateful to Dr B. Stoddard for access to computing resources and to Jeff Harris for
technical assistance. We thank Dr M. Mosesson for providing us with the
EM photographs and for helpful comments, and Dr R.F. Doolittle and
coworkers for permission to reproduce Fig 5A.
The coordinates of the various fibrinogen fragment crystal structures
are available from the Brookhaven Data Bank, http://www.pdb.bnl.gov, with accession nos. 1FIB, 2FIB, 3FIB, 1FZA, and 1FZB.
| |
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