Williams Hematology, 8e > Chapter
115. Molecular Biology and Biochemistry of the Coagulation Factors and
Pathways of Hemostasis >
Blood coagulation is a very delicately balanced
When it functions as it should, the blood is maintained in a fluid
state in the vasculature, yet rapidly clots to seal an injury. When
hemostatic functions fail, hemorrhage or thromboembolic phenomena
This chapter addresses molecular and biochemical features of the
proteins of the coagulation system, and how they interact with cells
and with one another to provide hemostasis in the living organism.
We have grouped the coagulation factors as (1) the vitamin-K-dependent
zymogens (prothrombin, and factors VII, IX, X, and protein C); (2)
the soluble cofactors (protein S, factor V, factor VIII, and von
Willebrand factor); (3) factor XI and the other "contact" factors;
(4) cell-associated cofactors (tissue factor and thrombomodulin);
(5) fibrinogen; (6) factor XIII and TAFI; and (7) the plasma coagulation
protease inhibitors. Table 115–1 shows
the major features of the coagulation factors addressed in this
chapter. A model of the coagulation pathway is presented that is
based on current understanding of cell–cell and cell–protein
interactions that regulate hemostasis. This scheme emphasizes the
importance of cellular localization and plasma protease inhibitors
in confining the coagulation reactions to a specific site of vascular
Table 115–1 Characteristics
of Coagulation Proteins
(factor II)||100–150 mcg/mL||60–70||11p11-q12|
|Factor VII||0.5 mcg/mL||3–6||13q34|
|Factor IX||4–5 mcg/mL||18–24||Xq27.1-q27.2|
|Factor X||8–10 mcg/mL||30–40||13q34|
|Protein C||4–5 mcg/mL||6||2q13-q14|
| non-GLA||Factor XI||5 mcg/mL||52||4q32-q35|
|Factor XII||30 mcg/mL||60 hours||5q33|
|Factor XIII-A chainb,c|
|Factor XIII-B chainb|
|22 mcg/mL|| ||1q31-q32.1|
|Thrombin activatable fibrinolysis inhibitor||6 mcg/mL|| ||13q14|
| Soluble||Factor Vc|
|Factor VIII||0.1–0.2 mcg/mL||8–12||Xq28|
|Protein S||25 mcg/mLd|
|Protein Z||2–3 mcg/mL||60||13q34|
|High-molecular-weight kininogen||70 mcg/mL||150||3q26|
| Cellular||Tissue factor||—||—||1p21-p22|
PROTEIN||Fibrinogen||2000–4000 mcg/mL||72–120|| |
| A chain|| || ||4q23-q32|
| B chain|| || ||4q23-q32|
| Chain|| || ||4q23-q32|
|Tissue factor pathway
Z-dependent protease inhibitor||1–1.6 mcg/mL||e|
of the factor XIII-A chain is in complex
with factor XIII-B chain; only half of factor XIII-B chain is in
complex with factor XIII-A chain, the rest is free in plasma.
significant amounts of factor
XIIIa (roughly half of the total factor XIII activity) and factor
V (20% of circulating factor V).
dApproximately 60% of the protein S
is in complex with C4b binding protein.
eProtein Z-dependent protease inhibitor
in complex with protein Z.
Acronyms and Abbreviations
Acronyms and abbreviations
appear in this chapter include: ADAMTS, a disintegrin and
with thrombospondin motifs; APC, activated protein C; aPTT, activated
partial thromboplastin time; AT, antithrombin; BiP,
protein; CYP2C9, cytochrome P450 complex that metabolizes warfarin;
C/EB, CCAAT/enhancer binding protein; EGF, epidermal
growth factor; EPCR, endothelial cell protein C receptor; ERGIC,
endoplasmic reticulum-Golgi intermediate compartment; GLA, -carboxyglutamic
acid; HC II, heparin cofactor II; HK, high-molecular-weight kininogen;
HNF, hepatic nuclear factor; IL, interleukin; LMAN1, mannose-binding
lectin-1 gene product; MCFD2, multiple coagulation deficiency protein
2; MZF, myeloid-enriched transcription factor; NF-B,
nuclear factor kappa-light-chain-enhancer of activated B cells; PAR,
proteolytically activated receptor; PK, prekallikrein; PS,
RFLP, restriction fragment length polymorphism; SCR, short consensus
repeat; Serpin, serine protease inhibitor; TAFI, thrombin-activatable
fibrinolytic inhibitor; TF, tissue factor; TFPI, tissue factor pathway
inhibitor; TM, thrombomodulin; VKORC1, vitamin K epoxide reductase
complex 1; VWF, von Willebrand factor; ZPI, protein Z-dependent
Biochemistry, and Life Span of the Coagulation Factors
Zymogens (Prothrombin and Factors VII, IX, X, and Protein C)
and Functional Features
vitamin K-dependent coagulation zymogens are precursors of
serine proteases that must be proteolytically activated to express
their enzymatic activity. They all share a similar protein domain
structure (Fig. 115–1). Each of
the mature vitamin K-dependent coagulation zymogen proteins has
an amino-terminal -carboxy glutamic acid (GLA)
domain with 9 to 12 GLA residues. This is followed by a hydrophobic
region. All except prothrombin have two epidermal growth factor
(EGF)-like domains, and all have a serine protease domain in their
carboxy-terminal region. Prothrombin has two kringle domains instead
of EGF-like domains. Specific functions are associated, at least
in part, with specific domains.
addition to the functional modules found in the mature protein,
each vitamin K-dependent factor is synthesized with an amino-terminal
sequence directing it to the endoplasmic reticulum, followed by a
19- to 25-amino-acid propeptide that is recognized by the -glutamyl
carboxylase that catalyzes carboxylation of glutamic acid residues
in the amino-terminal portion of the molecule. Following translocation
into the endoplasmic reticulum, the signal sequence is removed by
a microsomal signal peptidase. The propeptide is cleaved following
carboxylation before the mature protein is secreted.
Not only are the proteins
homologous, but their gene structures
also are highly similar. The coding regions of the vitamin K-dependent
factors are quite similar in size. However, the intron lengths vary
substantially and account for the differences in the overall size
of the genes (20 kb for prothrombin, 13 kb for factor VII, 33 kb
for factor IX, 25 kb for factor X, and 10 kb for protein C). Although
the complementary DNA (cDNA) of all of the vitamin K-dependent factors
has been sequenced, the noncoding regions have only been characterized
to varying degrees of detail. The vitamin K-dependent coagulation
zymogens are synthesized primarily by the liver and thus they all
have regulatory elements that direct liver-specific expression.
The regulatory elements vary however among the proteins.
In factors VII, IX, and X and
proteins C and Z, the introns are
located in identical positions in the genes,1–3 suggesting
that these enzymes evolved by duplication of a common ancestral
precursor gene. The regions of the molecules that constitute
tend to be encoded in their entirety by a single exon, that is,
the signal peptide by one exon, the propeptide and GLA region by
the next exon, and so forth. This "modular" design
suggests how "exon shuffling" could splice together
intact functional units of different proteins to give rise to new
proteins with novel properties.
The GLA domain that is characteristic of the vitamin
factors mediates interaction of the protein with lipid membranes.
The GLA domain is named for the modified amino acids found in the
first 42 residues of the mature protein. GLA residues are produced
by the posttranslational modification of glutamic acid residues
carried out by a specific -glutamyl carboxylase4 in
the endoplasmic reticulum (Fig. 115–2).
This carboxylase requires oxygen, carbon dioxide, and the reduced
form of vitamin K for its action. For each glutamyl residue that
is carboxylated, one molecule of reduced vitamin K is converted
to the epoxide form. The propeptide sequence is required for -carboxylation
to take place, and is highly conserved among the vitamin K-dependent
factors. Amino acids at positions –18, –17, –16, –15,
and –10 are critical to recognition by the carboxylase.5,6
of the carboxylase can lead to low levels of all of the GLA-containing
the -glutamyl carboxylase is directly
responsible for modification of the vitamin K-dependent factors,
a separate enzyme complex, the vitamin K epoxide reductase, is required
to convert the epoxide form of vitamin K back to the reduced form.
Warfarin inhibits the activity of the vitamin K epoxide reductase
and prevents recycling of vitamin K back to the reduced form. The
effect of warfarin is, therefore, to inhibit -glutamyl
carboxylation, resulting in the presence of a heterogeneous population
of undercarboxylated forms of the GLA-containing factors in the
circulation. These undercarboxylated forms have reduced activity.
Because warfarin blocks the reductase (rather than blocking the
carboxylase) and prevents recycling of vitamin K, the effects of
warfarin poisoning can be (temporarily) reversed by administration
of vitamin K. The gene for the epoxide reductase has been sequenced.8
in this gene have been linked both to warfarin resistance and to
a combined deficiency of the vitamin K-dependent clotting factors
variants in the vitamin K epoxide reductase complex 1
(VKORC1), as well as in the cytochrome P450 complex that metabolizes
warfarin (CYP2C9), are associated with the dose of warfarin required
to achieve therapeutic anticoagulation.11 Interestingly,
polymorphisms in the -glutamyl carboxylase do
not seem to contribute to individual differences in warfarin
are large variations in the effective warfarin dose among patients,
and significant clinical consequences when the degree of anticoagulation
is either insufficient or excessive. Thus, individualization of
warfarin dosing appears to be one of the most promising clinical
applications of pharmacogenetics.
The calcium-bound form of the GLA domain is
responsible for mediating
association with phospholipid membranes. Lipids with negatively
charged head groups, primarily phosphatidylserine (PS), are required
for this binding. Even in the absence of the appropriate protein
cofactor, binding to phospholipids increases the proteolytic activity
of GLA-containing proteases. PS is required for activity on synthetic
membranes. The role of PS in mediating coagulation reactions on
cellular membranes, such as platelets, is more complex. PS is not
normally exposed on the outer membrane leaflet of cells in contact
with flowing blood. Further, activation of cells (particularly
is often accompanied by exposure of PS on the outer leaflet of cell
membranes. Because this activation enhances the ability to support
reactions, it has often been assumed that exposure of PS on the
outer surface of cells is sufficient to account for the ability
of a cell to support coagulation reactions. However, other studies
show that the level of coagulant activity on cells does not directly
correlate with the amount of PS exposure. This result is in direct
contrast to studies with phospholipid membranes in which the level
of coagulant activity is directly related to the amount of PS expressed.
From these studies, it can be concluded that PS exposure is necessary
for cells to support coagulation reactions, but that other features,
such as cell receptors and/or binding proteins, are also
is very high homology in the amino acid sequence in the
first 42 residues of GLA-containing proteins. This implies that
the three-dimensional structure of the GLA-domain is highly conserved
and that few specific interactions are determined by this region.
It was once thought that the binding of GLA-containing proteins
to phospholipids was mediated by calcium ion "bridging" between
the negatively charged GLA residues and negatively charged phospholipid.
This mechanism provided a good explanation for why both calcium
and negatively charged phospholipid were required for binding. It
is currently believed, however, that binding of GLA-containing factors
to lipid surfaces is mediated by membrane insertion of hydrophobic
residues in the first 10 amino acids of the GLA domain. Calcium
is essential for this to occur because calcium binding to some of
the GLA residues induces a dramatic conformational change that exposes
the hydrophobic amino acid residues in a contiguous patch. This
hydrophobic patch is located at the tip of a structural feature
that sticks out from the surface of the GLA-containing protease
like the keel of a ship. This conformation allows insertion of the
a phospholipid membrane.14,15 A computer modeling
study suggests a refinement of this hypothesis.16 This model
suggests that the "keel" is inserted more deeply
into the membrane than previously thought. The hydrophobic tip is
buried in the membrane, but (as shown in Fig.
115–3) the keel is inserted in the membrane up to
a level that allows surface-exposed GLA-residues to interact with
PS molecules in the outer leaflet of the membrane by calcium bridging.
In addition, lysine and arginine residues in the GLA-domain can
interact directly with PS headgroups in the membrane.
striking degree of homology among the GLA domains of the
vitamin K-dependent clotting factors suggest that the affinity of
the calcium–GLA complexes for phospholipids would also
be very similar. However, this turns out not to be the case. Factor
IX and factor X bind much more strongly to
vesicles than does factor VII.17 The reasons for
these marked differences are not clear, but may be related to
in presence of positively charged amino acids in the GLA-domain that
might interact directly with PS.
The first EGF domain of the vitamin K-dependent
a calcium ion binding site that does not involve GLA residues, but
does involve a -hydroxy aspartic acid. This conserved
aspartic acid residue is modified posttranslationally by a -hydroxylase about
which little is known. Binding of calcium to this EGF-1 site appears
to be important for activity and probably serves to orient the GLA
domain relative to the rest of the molecule. The EGF-1 and EGF-2
domains serve, at least in part, to space the serine protease domain
above the lipid membrane surface. Factor VIIa interaction with its
cofactor, tissue factor, is mediated at least in part, by direct
interaction between tissue factor and both EGF domains of factor VIIa,
as shown in Figure 115–4.
All of the GLA-containing
zymogens undergo activation by cleavage
of at least one peptide bond (see Fig. 115–1). Activation is indicated
the letter "a" to the name of the factor, except
for protein C which is often abbreviated APC. The cleavage that
leads to activation generates a new amino terminal that folds back
and interacts with specific residues in the serine protease domain.
This interaction changes the conformation of the protein such that
the active site residues (His, Ser, Asp) are aligned and the protease
activity of the factor is expressed.
The serine protease domains of all the
show a high homology to each other and to chymotrypsin and trypsin;
all have trypsin-like activity, with an almost absolute specificity
for cleaving at the carboxy terminal of arginyl residues. However,
trypsin which shows little specificity beyond cleaving after an
arginyl or lysyl residue, the activated coagulation factors have
extended substrate specificity pockets, such that only a small number
of amino acid sequences are recognized by each activated factor.
Despite the high degree of homology between the protease domains
of protein C, prothrombin, and factors VII, IX and X, each of these
factors has a highly specific function in coagulation that is mediated
by surface loops that are not highly homologous.
The activated forms of factors VII, IX, and X each
with a specific cofactor. Tissue factor (TF) is the cofactor for
factor VIIa; factor VIIIa is the cofactor for factor IXa; and factor
Va is the cofactor for factor Xa. The factors and cofactors associate
on cell membranes to form proteolytically active complexes. Thrombin
does not require a cofactor for its coagulant activity. However,
upon association with the cofactor thrombomodulin (TM), its specificity
is changed from coagulant (clotting fibrinogen) to anticoagulant
and activating protein C). Although each of the proteases has some
activity in the absence of its cofactor, association with cofactor
dramatically enhances its activity. This is illustrated in Table 115–2,
which shows the enhancement
of factor IXa activity by calcium, activated platelets, and the
factor VIII cofactor.18,19 Thus, the physiologic
coagulant activity of factors VIIa, IXa, and Xa is only expressed
as a part of a complete procoagulant complex (Table
115–3). The complexes are sometimes named for their physiologic
substrate: the factor IXa/VIIIa complex is termed the "tenase" or
tenase" complex; the factor VIIa/tissue factor
complex the "extrinsic tenase" complex; and the
factor Xa/Va complex the "prothrombinase" complex. The
cofactors enhance proteolytic activity by two basic mechanisms:
(1) they have binding sites for both substrate and enzyme and bring
the two into close proximity, and (2) they associate with the protease
and induce a conformational change that enhances enzymatic activity.
The structure of the factor VIIa/TF complex has been determined
by x-ray crystallography.20 Figure
115–421–23 illustrates the
projected change in conformation of the factor VIIa molecule when
it binds to its cofactor, tissue factor. The factor IXa/VIIIa
and Xa/Va complexes have not been crystallized, but it
is likely that generally similar conformational changes occur during
formation of these complexes.
Table 115–2. Cofactor
of Factor IXa Activity
were activated with thrombin.
are given as kcat/Km.
Table 115–3. Protease/Cofactor
factor||Factor X||Many cellsa|
|Factor IXa||Factor VIIIa||Factor X||Platelets|
|Factor Xa||Factor Va||Prothrombin||Plateletsb|
|Activated protein C||Protein
is constitutively expressed on many extravascular cells (e.g., stromal
cells, epithelial cells, astrocytes) and is induced by inflammatory
mediators in many other cells (e.g., monocytes, endothelial cells).
other cells have low levels of factor
Xa/factor Va activity.
Like the other
vitamin K-dependent zymogens, plasma prothrombin
is primarily synthesized in the liver. It circulates as a single-chain
zymogen of Mr ~72,000 and has a plasma half-life of about 60 hours.
Figure 115–5 is a schematic representation.
It has 10 GLA residues. Instead of the EGF region present in most
vitamin K-dependent zymogens, prothrombin has two kringle domains.
Kringle domains are structures held together by three disulfide
bonds that schematically resemble a Danish pastry called a "kringle."
primary function of kringle structures appears to be to bind other
proteins such as activators, substrates, cofactors, or receptors.24
The human prothrombin gene has
been localized to chromosome 11,
near the centromere.25 It has been completely sequenced
and is composed of 14 exons separated by 13 introns (Fig.
115–6). The 5' flanking
region of the prothrombin gene contains the promoter region and
two or more cis-acting enhancer sequences. Cis-acting
sequences are portions of the DNA that act as promoters, enhancers,
or silencers. Unlike many other promoters, the promoter region of
the prothrombin gene does not contain a TATA box. It has multiple
potential sites of transcription initiation extending from 3 to
38 base pairs (bp) upstream from the initial methionine. The site
at –31 is the most likely start site. The region between –887 and –875
is likely to be a binding site for hepatic nuclear factor 1 (HNF-1),
a DNA-binding protein that plays a role in the liver-specific expression
of a number of genes.26 HNF-1 is an example of
a trans-acting factor, which is a molecule that
binds to a DNA sequence and affects expression of the associated
gene. An additional site in the prothrombin promoter region with
non–tissue-specific enhancer activity lies just upstream
to the HNF-1 site.
One unusual feature of the
prothrombin gene is the presence of
many repetitive sequences in its 5' flanking
region.27 Approximately 41 percent of the gene
and upstream sequence consists of Alu repeats. The function of these
repetitive sequences, if any, is not known.
Several polymorphisms of the prothrombin gene have
and one of these is now recognized to have important functional
consequences. This G-to-A transition in the 3' untranslated
region (20210 GA)
of the prothrombin
gene is associated with higher-than-normal levels of plasma prothrombin.28
prothrombin levels are associated with an increased risk of venous
thromboembolism (see Chap. 131).
Knockout of the prothrombin gene in a mouse model
intrauterine and neonatal lethality.29
cleaved by the factor Xa/Va prothrombinase
complex in two places (Arg 271 and Arg 320), as shown in Figure 115–7.14,30–32
domain (thrombin), Mr 36,600, is released from the remainder of
the molecule (prothrombin fragment 1.2). Because one molecule of
fragment 1.2 is released for each molecule of thrombin, assays for
fragment 1.2 reflect the level of prothrombin activation.
Thrombin cleaves a number of
biologically important substrates.
It removes fibrinopeptides A and B from fibrinogen to form fibrin
monomers, which then spontaneously polymerize to form a fibrin clot
(see Chap. 126). The anion-binding exosite
spans residues 387 to 398 and is involved in binding to fibrinogen,
thrombomodulin, hirudin, heparin cofactor II (HC II), and the
activated thrombin receptors. Interestingly, this region of thrombin
is identical in human, bovine, rat, and mouse.33 In
addition to directly clotting fibrinogen, thrombin has a procoagulant
effect by participating in positive feedback loops by activating
platelets and coagulation factors V, VIII, XI, and XIII.
Thrombin is a potent platelet
activator through at least two
types of receptors. These include the G-protein-linked proteolytically
activated receptors, PAR-1 and PAR-4, as well as platelet glycoprotein
Ib (see Chap. 114).
Another function of thrombin is to activate a
B-like enzyme to its active state, a reaction enhanced by
active carboxypeptidase inhibits plasmin-mediated fibrinolysis by
C-terminal lysine residues from fibrin, which facilitate plasminogen
binding, from partially degraded fibrin. Thus, the carboxypeptidase
has been termed thrombin-activatable fibrinolysis inhibitor
addition to its procoagulant activity, thrombin has an anticoagulant
Thus, thrombin binds to the cofactor thrombomodulin on endothelial
cells, allowing it to activate protein C which inactivates factors
Va and VIIIa (see Chap. 116).36,37 Thrombin
also has growth factor and cytokine-like activities that may play
a role in atherosclerosis, wound healing, and inflammation.38
The primary plasma inhibitor
of thrombin in coagulation is antithrombin
(AT). HC II also inhibits thrombin, and may serve as an extravascular
thrombin inhibitor that regulates the growth factor and cytokine-like
activities of thrombin.38
Factor VII circulates as a single-chain zymogen of
It has the shortest half-life of the procoagulant factors, approximately
3 to 6 hours (see Table 115–1),
and has 10 GLA residues.
The human factor VII gene is located on chromosome
13, very close
to the gene for factor X. The gene consists of eight exons and seven
introns, with an overall size of about 13 kb, and an organization
similar to the other vitamin K-dependent factors (Fig.
The major transcription start
site in the factor VII gene is
at –51. Three other minor start sites have been described.39 A
hormone-responsive element and binding sites for the trans-acting
factors HNF-4 and Sp-1 are located between –233 and –58
in the promoter region of the factor VII gene.
In contrast to the intrauterine lethality observed
mice, embryos deficient in factor VII developed normally, without
evidence of hemorrhage. However, factor VII-deficient newborns sometimes
to intraabdominal or intracranial
binds to tissue factor with a kDa in the subnanomolar range.
Once bound to its cofactor, factor VII can be activated by a number
of different proteases that cleave between Arg152 and Ile153. The
physiologic activator of factor VII is thought to be factor Xa, although
significant autoactivation by factor VIIa can occur.41 Unlike
prothrombin, the catalytic domain of factor VII is linked to the rest
of the molecule by a disulfide bond, so no portion is cleaved from the
protein (see Fig. 115–1). The factor
VIIa/TF complex activates both factors IX and X. It is
inhibited by tissue factor pathway inhibitor (TFPI) in complex with
factor Xa. It is also inhibited by AT, but only in the presence
Factor IX is synthesized in
hepatocytes and circulates as a single-chain
zymogen of Mr ~57,000 with a plasma half-life of 18 to 24 hours.
It has 12 GLA residues. Only approximately 40 percent of factor
IX molecules are hydroxylated at aspartic acid 64 in the EGF-1 domain.
All the other GLA-containing zymogens have complete hydroxylation
of the homologous residues (Fig. 115–9).
Factor IX contains N- and
O-linked carbohydrate moieties found mostly in the activation peptide.
In the mature molecule, the tyrosine residue at position 155 is
sulfated, whereas the serine residue at position 158 is phosphorylated.
Factor IX, unlike other vitamin K-dependent factors, has been shown
to bind effectively to collagen IV in vitro.42 The
molecule appears to bind to collagen IV in vivo,
which may account for the observation that when factor IX is infused
into hemophilia B patients, recovery is only 50 percent of that
expected (see Chap. 124).43 The
physiologic relevance of this observation remains to be precisely
determined, but studies suggest that factor IX mutants lacking collagen
IV binding exhibit a greater recovery but may be associated with
a mild bleeding tendency.44,44a
The gene for factor IX is
located on the tip of the long arm
of the X chromosome at position Xq27.1-q27.2.45 Therefore,
deficiency of factor IX (hemophilia B) is sex linked. The gene contains
eight exons, seven introns, and a long 1.4 kb 3'-untranslated
region, for an overall size of 33 kb (Fig. 115–10).
Eight polymorphisms have been
described within or flanking the
factor IX gene. These polymorphisms can be useful for antenatal
diagnosis and carrier detection of hemophilia B by restriction fragment
length polymorphism (RFLP) analysis.46
The promoter activity of the 5' untranslated
region of the factor IX gene resides 274 bp upstream of the major
transcription start site.47 Binding sites for several trans-acting
factors have been identified, including sites for CCAAT/enhancer
binding protein (C/EBP),48 D-site binding
protein,49 HNF-4,50 and HNF-1.51
Factor IX can
be activated either by factor XIa or by the factor
VIIa/TF complex. Full activation requires cleavage of two
bonds (Arg 145 and Arg 180) releasing an activation peptide of Mr
~10,000 (see Fig. 115–9). This results
in an Mr 17,000 light chain connected to an Mr 30,000 heavy chain
that contains the active sites Asp, His, and Ser.
In complex with its cofactor, factor VIIIa, on a
membrane surface, factor IXa activates factor X. Physiologically
this activity is primarily expressed on the surface of activated
platelets, and there is preliminary evidence suggesting that platelets
a receptor/binding protein for factor IXa that promotes
assembly of the factor IXa/VIIIa complex.52
The primary plasma inhibitor
of factor IXa appears to be AT.
Inhibition of factor IXa by AT is slow compared to AT inhibition
of thrombin. However, it is enhanced in the presence of heparin.
Factor X circulates as a 2-chain, disulfide-linked
Mr 59,000 (see Fig. 115–1) with
a plasma half-life of approximately 34 to 40 hours. A 3-amino-acid
sequence between the light and heavy chains (Arg140-Lys141-Arg142)
is cleaved from the protein during intracellular processing. The
resulting light chain Mr is ~17,000 and the heavy chain Mr is ~40,000.
The light chain contains the GLA domain, with its 11 GLA residues,
and the two EGF domains. The heavy chain contains the 52-amino-acid
activation peptide and the catalytic domain. Like all other vitamin
K-dependent factors, it is synthesized in the liver.
The gene for human factor X is
on chromosome 13q34-qter53 in
close proximity to the factor VII gene. It is composed of eight
exons and seven introns,1 with a size of ~25 kb
(Fig. 115–11). The 3' untranslated
region is unusually short, with only 10 base pairs. A number of
potentially useful polymorphisms have been identified.54
The factor X promoter region
has been sequenced and characterized.
It lacks a typical TATA box, but contains a CCAAT sequence at –120
to –116. Factor X appears to have multiple start sites
of transcription.55 This finding is consistent
with multiple start sites reported for other promoters lacking a
TATA box. Like the factor IX gene, a binding site for HNF-4 has
been identified.56 However, unlike the factor IX
gene, there does not appear to be a binding site for C/EBP.
Complete deficiency of FX
induced by targeted gene disruption
in a mouse model often resulted in embryonic lethality. Most of
the deficient mice that survived to term succumbed to hemorrhage
within 5 days after birth.57
Factor X can be
activated to fully active factor Xa by
factor VIIa/TF or factor IXa/VIIIa by cleavage
at the Arg 194–IIe 195 bond in the heavy chain. Further autocatalytic
cleavage near the carboxy terminus of the heavy chain releases a
19-amino-acid peptide to yield "-factor
Xa" which is also enzymatically active.
Factor Xa in complex with factor Va on a
surface activates prothrombin to thrombin by cleaving two peptide
bonds. Factor Xa may also play a physiologic role in activation
of factors VII,58 VIII,59 and
V.60 Although any membrane surface that expresses
anionic phospholipid can support prothrombinase complex assembly,
the activated platelet surface is especially well suited for this
purpose. Prothrombinase assembly on platelets is not strictly a
function of phospholipid composition, but is likely coordinated
by one or more specific binding proteins.61
Like thrombin, factor X has
biologic activities not directly
related to coagulation. It is reported to have mitogenic activity
for smooth muscle cells.62 Factor Xa also possesses
receptor-mediated proinflammatory activities.63 The
primary plasma inhibitor of factor Xa is the serine protease inhibitor
(Serpin) AT. The inhibition of factor Xa by AT is accelerated by
heparin. Tissue factor pathway inhibitor (TFPI) is also a potent
inhibitor of factor Xa, as shown in Table 115–4.
TFPI and AT Inhibition of Coagulation Factors
| || ||Time
to 50% Inhibitiona (min)|
|Inhibitor||Protease||– Heparin||+ Heparin|
| ||Factor Xa ||4||<0.1|
| ||Factor IXa||60||0.6|
|Tissue factor pathway inhibitor||Factor Xa||0.3||<0.1|
aTime to 50%
inhibition in plasma. In
vivo, natural glycosaminoglycan molecules on endothelium
and other cells accelerate the rate of inhibition.
Protein C, unlike the other
vitamin K-dependent zymogens, is
not a procoagulant, but controls coagulation, when activated, by
inactivating factors Va and VIIIa (see Chap. 116).
It circulates as a two-chain disulfide-linked zymogen with nine
GLA residues (see Fig. 115–1). It
has an Mr of 59,000 and a short plasma half-life of approximately
The gene for
human protein C is on chromosome 2q13–14.64 It
was originally described as being composed of eight exons with a
size of ~10 kb.65 Other workers have described
it as having nine exons and eight introns,66 with
the first exon corresponding to the 5'-noncoding
region (Fig. 115–12). Thus, the
first exon is transcribed from the gene into messenger ribonucleic
acid (mRNA), but is not translated into protein. The gene structure
is very similar to the other vitamin K-dependent factors, with
close homology to factor IX.
Protein C is
activated by thrombin in complex with the cell-surface
cofactor thrombomodulin. A single cleavage at Arg169-Leu170 releases
a 12-amino-acid activation peptide leading to APC with a molecular
of 56,000. Activation of protein C is modulated in part by the
cell protein C receptor (EPCR).67 EPCR concentrates
protein C on the endothelial cell membrane, thereby enhancing the
activation of protein C by the thrombin–thrombomodulin complex.68
EPCR is preferentially expressed by the endothelium of larger vessels.
APC, in complex with its
cofactor protein S, proteolytically
inactivates factors Va and VIIIa. Data suggest that inactivation
of these factors is much more likely to occur on endothelial cells
than on platelet surfaces.69 Thus, APC primarily
acts to prevent thrombin generation on intact endothelial cells that
might lead to thrombosis. It has also been reported that factor
V can act as a cofactor for the inactivation of factors Va and VIIIa
by APC.70 The primary inhibitor of APC is the serpin
protein C inhibitor, also known as plasminogen activator inhibitor-3
(PAI-3) and SERPINA5.71
In addition to its antithrombotic effects, APC has a
of antiinflammatory activities (see Chap. 116).
It suppresses inflammatory cytokine production in animal models
of sepsis, inhibits leukocyte adhesion and chemotaxis, reduces
cell apoptosis, helps maintain endothelial cell barrier function,
and minimizes hypotension associated with severe sepsis.72
of these functions require binding to EPCR and cleavage of
activated receptor (PAR)-1. Although the antiinflammatory functions
of APC require proteolytic activity, they are not mediated by its
inhibitory effects on thrombin generation.73 Overall
the protein C system has important roles in controlling inappropriate
or excessive activity of both the coagulation and inflammatory
(Protein S, Factor V, Factor VIII, and von Willebrand Factor)
Protein S is a single-chain
plasma glycoprotein of Mr ~75,000
with a plasma half-life of approximately 42 hours. It is dependent
on vitamin K for its synthesis and contains 11 GLA residues in the
amino-terminal region. Its structure is quite different from the
GLA-containing zymogens (Fig. 115–13).
Protein S is organized into a GLA domain, a thrombin-sensitive finger
region, four EGF domains, and a region with homology to
proteins. Unlike the other vitamin K-dependent factors, it does
not contain a serine protease domain and so does not have the potential
to catalyze reactions. Each EGF domain contains a modified amino
acid, either -hydroxyaspartic acid or -hydroxyasparagine.
Protein S circulates both in the free form (~40% of the
total amount) and in a form bound to the complement regulatory protein
C4b-binding protein. The glucocorticoid hormone-binding globulin-like
region of protein S is involved in binding to the subunit
of C4b-binding protein. Like the GLA-containing zymogens, protein
S is synthesized with a signal peptide that directs it to the
reticulum, and a propeptide that binds to the -glutamyl
carboxylase. The signal sequence and propeptide are removed before
the mature protein is secreted.
Protein S is synthesized
primarily by hepatocytes,74 as
well as by endothelial cells,75 megakaryocytes,76
cells,77 and osteoblasts.78
The human protein S gene is on chromosome 3,
spanning the centromere from
p11.1 to q11.2. It is more than 80 kb in length and contains 15
exons and 14 introns (see Fig. 115–13).79 Exons
1 to 8 encode protein domains that are homologous to the GLA-containing
zymogens. The intron–exon structure is typical of the members
of this family. Exons 9 to 15 encode protein segments homologous
to glucocorticoid hormone-binding globulin. There is also a pseudogene
of protein S located on the same chromosome. It is approximately
55 kb in size and contains coding sequences for regions corresponding
to amino acids 46 to 635 of protein S.
Protein S serves as a cofactor for the cleavage and
of factors Va and VIIIa by APC. In contrast to
factors V and VIII, it does not require proteolytic activation for
its cofactor activity. Until recently, it had been thought that
protein S can only serve as a cofactor for APC when in the free
form, rather than when bound to C4b-binding protein. However,
of the data suggests that C4b-binding protein-bound protein S does,
indeed, express APC-cofactor activity.80
Protein S alone also has a low level of
by virtue of its ability to compete with factor Xa for binding to
factor Va81 and this activity is not reduced by
binding to C4b-binding protein.82 In addition,
data show that protein S can act to enhance the effectiveness of
TFPI in inhibiting the factor VIIa/TF complex.83
Factors V and VIII are homologous in their gene
acid sequences, and protein domain structures. They have similar
mechanisms of intracellular processing in the endoplasmic reticulum
(ER) and Golgi apparatus and defects in these mechanisms can result
in combined deficiency of factors V and VIII. The mannose-binding
lectin-1 gene product LMAN1 (also called endoplasmic reticulum-Golgi
compartment [ERGIC]-53) is a protein found in
the intermediate compartment of the Golgi apparatus that facilitates
secretion of both factor V and factor VIII.84 Mutations
in the LMAN1 gene lead to a hereditary deficiency of both factors
V and VIII85 which accounts for about two-thirds
of the cases of combined deficiency (see Chap. 125). Analysis of
patients without defects in LMAN1 suggests
that mutations of a second protein, MCFD2 (multiple coagulation
deficiency protein 2), also account for a number of the cases of
combined factors V and VIII deficiency.86 The gene
product of MCFD2 is an Mr 16,000 protein localized to the ERGIC
through a direct, calcium-dependent interaction with LMAN1. The
MCFD2–LMAN1 complex appears to form a specific cargo receptor
for the ER-to-Golgi transport of selected proteins, including both
factors V and VIII.
V is a large glycoprotein of Mr ~330,000 and has a plasma
half-life of approximately 12 hours, with some reports of a half-life
of up to 36 hours.87 It has the following domain
organization: A1-A2-B-A3-C1-C2 (Fig. 115–14).
The three A domains have significant homology to the copper-binding
plasma protein ceruloplasmin. The C domains have some homology to
fat globule proteins. The C2 domain of factor V mediates binding to
lipid membranes.88 The A and C domains of factor V
are approximately 40 percent identical to
the homologous regions in factor VIII. In contrast, the B domains
show little homology between the two proteins and are not known
to be homologous to any other proteins. In factor V, unlike factor
VIII, sequences in the B domain appear to be important in promoting
its activation by thrombin. The acidic regions of factor V have
a high proportion of Asp and Glu residues. These regions are thought
to be important in promoting activation, possibly by providing a site
of interaction with the anion binding exosite of thrombin.
Factor V shows five potential
sites for tyrosine sulfation at
residues 696, 698, 1494, 1510, and 1565. Sulfation of factor V also
plays a role in factor V activity by enhancing activation by thrombin
and by promoting maximal factor Xa activation of prothrombin.89
V contains both N- and O-linked carbohydrate moieties, most of which
are clustered in the B domain.
The gene for factor V is located on chromosome 1q21
to q25. It
is located very close to the genes for the selectin family of leukocyte
adhesion molecules. The factor V gene spans approximately 70 kb
and consists of 25 exons (see Fig. 115–14).
The gene structure is very similar to that of the factor VIII gene,
with exon–intron boundaries occurring at exactly the same
location in 21 of 24 cases.90 The mechanisms governing
factor V gene transcription and translation are not clear.
circulates in plasma as a single-chain molecule. As
much as 20 percent of the circulating factor V pool is found in
platelet granules, where it is localized by uptake
from the plasma.91 Platelet factor V appears to
be sufficient for hemostatic function, at least in mice.92
the origin of factor V in mouse platelets is different from human.
Mouse platelet factor V is synthesized in megakaryocytes and packaged
into the granules before platelet release from
the marrow.92,93 This very clear difference between
mouse and human platelets should serve to remind us that one should
not assume, without validation, that any animal model reproduces
human biology and pathophysiology.
Platelet factor V is heterogeneous because of
cleavages in the
B domain by calpain and other platelet proteases. These cleavages
produce a partially activated form of platelet factor V. Activated
platelet factor V is also more resistant to inactivation by activated
protein C.69,94 In platelets, but not in plasma,
factor V is complexed to a large multimeric protein called multimerin.95
has a massive repeating structure, with some of the multimers having
molecular weights of several million. Multimerin has structural
features that suggest it may mediate adhesive interactions. Although
multimerin interactions with factor V are functionally similar to
von Willebrand factor (VWF) interactions with factor VIII, multimerin
and VWF share no structural homology.
Full factor V cofactor activity is achieved only
at several bonds (Fig. 115–15).
Factor V is believed to be primarily activated by thrombin in
vivo, although it can be activated by factor Xa as well,60
factor Xa appears to be the preferred activator of factor V released
from platelet granules.94 Thrombin
cleaves factor V at Arg 709 and Arg 1545 to produce a two-chain
heterodimeric molecule consisting of an A1-A2 heavy chain (Mr 110,000)
that is associated with an A3-C1-C2 light chain (Mr 73,000). The
two chains are noncovalently linked through metal ions (probably
calcium). APC catalyzes inactivation of factor Va by proteolysis
of Arg 306 and Arg 506, resulting in dissociation of the cleaved
A2 fragments87 (Fig. 115–15).
A common Arg 506 Gln mutation in factor V leads to resistance
to inactivation by APC (factor V Leiden) and is associated with
an increased risk of venous thromboembolism (see Chap. 131).96
Disruption of the factor V gene in
mice leads to the death of approximately half of the homozygous factor
V-deficient embryos dying at embryonic days 9 to 10. The remaining
factor V-deficient embryos survive to term, but die from massive
bleeding within 2 hours of birth.97
The domain organization of the
factor VIII protein is A1-A2-B-A3-C1-C2,
like that of factor V (Fig. 115–16).
In factor VIII, the B domain does not appear to play a significant
stability or activation. B-domainless factor VIII has been used
successfully as a therapeutic agent in patients with classic hemophilia.
Factor VIII is synthesized in
the liver,98 although
not in hepatocytes. Hepatic endothelial cells appear to be a major
site of synthesis.99 This conclusion is supported
by the effects of transplantation in hemophilia A (factor VIII
Hemophilia A has been cured by liver transplantation in human and
canine subjects.100 However, spleen transplantation
was not curative in a dog model.101 Transplantation
of isolated hepatocytes did not correct hemophilia A in a mouse
model, but transplantation of a cellular fraction enriched in liver
endothelial cells did.102
Factor VIII is secreted into the plasma as a
of partially cleaved forms resulting from different cleavages in
the B domain. Factor VIII circulates in a noncovalent complex with
VWF. The normal half-life of factor VIII is 8 to 12 hours when
with VWF. The half-life is markedly reduced in the absence of VWF,
accounting for the reduced factor VIII levels observed in many patients
a deficiency of VWF.
VIII is not secreted very efficiently from the cell. In
addition to LMAN1 and MCDF2, several molecular chaperone proteins
have been identified that appear to play a role in regulating transit
of the large factor VIII protein through secretory and/or
degradative pathways. Calnexin and calreticulin are chaperone proteins
that preferentially interact with glycoproteins containing
N-linked oligosaccharides. These proteins bind to the heavily
of factor VIII and enhance both its intracellular degradation and
secretion.103 Factor V associates with calreticulin,
but not calnexin. Factor VIII, but not factor V, also interacts
through its A1-domain with another chaperone protein,
protein (BiP). Association with BiP appears to enhance the stability
of factor VIII, but also retards its secretion.104
Factor VIII has six tyrosine
residues that are modified by sulfation (residues
346, 718, 719, 723, 1664, and 1680). Sulfation of these residues is
required for optimal activation by thrombin, maximal activity in
complex with factor IXa, and maximal affinity of factor VIIIa for
The acidic regions
of factor VIII appear to promote activation
by interacting with the anion-binding exosite of thrombin. In addition,
the site for factor VIII binding to VWF is in the acidic domain
in the light chain of factor VIII.105
The factor VIII gene is on the X chromosome at q28.
of factor VIII results in classic sex-linked hemophilia A. The factor
VIII gene contains 26 exons (see Fig. 115–16),
one more than factor V. Exon 5 of factor V corresponds to exons
5 and 6 of the factor VIII gene.106 The gene for
factor VIII is much larger than that for factor V, spanning
190 kb. This is largely because six of the introns in the factor
VIII gene are much larger than the corresponding introns in the
factor V gene. The mRNA for factor VIII is also much larger than
that for factor V because of a 1.8-kb 3'-untranslated
region in the factor VIII message.
Factor VIII is
activated by thrombin or factor Xa by cleavages
at arginyl residues 372, 740, and 1689 (Fig. 115–17).
This produces a heterotrimeric molecule consisting of A1 and A2
domains noncovalently linked with an A3-C1-C2 light chain through
calcium ions. Activation also results in the release of factor VIIIa
from VWF. The factor VIIIa molecule is thermodynamically unstable
and dissociation of the A2 domain results in the spontaneous loss
of activity. Factor VIIIa is also inactivated by thrombin or APC
through additional cleavages at arginyl residues 336 and 562 (Fig.
discusses the structure, molecular
biology, and activities of VWF in greater detail.
VWF is a
large multimeric glycoprotein that serves as a carrier
for factor VIII and is required for normal platelet adhesion to
components of the vessel wall. It is synthesized as a prepropolypeptide
with a 22-amino-acid signal sequence, a 741-amino-acid precursor
called VWF antigen II, and the mature VWF polypeptide chain.107
mature VWF protein contains three A domains, three B domains, two C
domains, and four D domains. The A domains are structurally homologous
to a family of proteins involved in extracellular matrix or
functions.108 Factor VIII binds to the amino-terminal region
of VWF, within the first 272 amino acids of the mature protein subunit.109
In the ER, the pro-VWF
monomers form disulfide-stabilized dimers.
The dimers move to the Golgi apparatus where they assemble into
high-molecular-weight multimers, which are also held together by
bonds. The propeptide is essential for multimerization to occur.
It is usually removed before secretion of the mature VWF multimers.
After secretion from its cells of origin, endothelial cells, VWF
is further cleaved by a metalloprotease named ADAMTS13 (a disintegrin
and metalloproteinase with thrombospondin domain 13).110 The
circulating VWF multimers range in size from Mr ~500,000 to larger
than 20,000,000.111 The higher-molecular-weight
multimers are most effective in promoting platelet adhesion. However,
all multimers can bind factor VIII and enhance its stability. The
plasma half-life of VWF is approximately 12 hours.
The VWF gene is located on chromosome 12 and spans
180 kb. It contains 52 exons.112 VWF is synthesized
only in endothelial cells and megakaryocytes.
Factor XI and
the Contact Factors
Factor XI, along with factor
XII, high-molecular-weight kininogen
(HK) and prekallikrein (PK) are sometimes referred to as the contact
factors. Factor XI is a zymogen precursor of a serine protease.
Factor XI circulates in complex with the nonenzymatic cofactor,
Factor XI is
synthesized in the liver and has a plasma mean half-life
of approximately 52 hours. Although synthesized as a single chain,
it circulates as a homodimer held together by a disulfide bond through
residues.113 Each monomer has a Mr ~80,000, including
approximately 5 percent carbohydrate and contains four repeats of
a structural motif called an apple domain, as shown
in Figure 115–18. Each apple domain
contains 90 or 91 amino acids held together by 3 disulfide bonds.
Specific functions have been assigned to the different apple domains
factor XI,114–117 including sites for
binding to HK, prothrombin, platelets, factor IX, thrombin, and
The human factor XI gene is 23
kb in length and is localized
to chromosome 4q32–35.118 It consists
of 15 exons and 14 introns (Fig. 115–19).119 The
gene lacks canonical CAAT and TATA boxes, which may account for
the multiple transcription initiation sites that have been identified.
Exon 1 encodes a 5' untranslated region
that is transcribed into mRNA, but not translated into protein.
The signal peptide is encoded in exon 2. Each of the four apple domains
is encoded in two exons. The light chain is encoded in five exons,
with an organization similar to the homologous proteins PK, tissue
plasminogen activator, urokinase, and factor XII. An HNF-4 binding
site required for liver-specific expression is located between –375
and –363 bp.120
Factor XI can
be activated by more than one mechanism in
vitro. In vitro, factor XI can be activated
by factor XIIa. In the fluid phase and on charged surfaces thrombin
can activate factor XI even in the absence of the other contact
factors.121,122 Factor XI can also be activated by
thrombin on the surface of activated platelets, and this pathway
is the most likely mechanism of activation during hemostasis in
vivo.123 At least in a mouse model, factor
XI may play a more significant role in thrombosis than in hemostasis.124
As discussed below in the
section "A Cell-Based Model of Coagulation," factor XI serves as a
enhancing platelet surface thrombin generation. Knockout of the
factor XI gene in mice does not result in intrauterine death.125
deficiencies of factor XI in humans can lead to a bleeding tendency,126
it is not as severe as in hemophilia A or B. This reflects the
role of factor XI in hemostasis, in contrast to the other contact
factors (see below).
of factor XI by either factor XIIa or thrombin is
caused by cleavage of the Arg369–Ile370 bond in the factor
XI subunits. This yields two active sites in each factor XIa dimer.
Each subunit has a heavy chain containing the apple domains and
a light chain containing the catalytic domain (see Fig.
115–18). Both the heavy and light chains interact
with the substrate, factor IX.127 Factor XIa activation
of factor IX is calcium dependent, but does not require any other
cofactor. Factor XIa binds with high affinity to activated platelets
and can activate factor IX with the same efficiency as unbound factor
XIa.128 Binding to activated platelets could serve
to localize factor XIa to the site of clot formation, as well as
protect it from plasma protease inhibitors.
Factor XIa is susceptible to inhibition by several
inhibitors that circulate in high concentrations. The Serpin protease
nexin 1 has the highest affinity for factor XIa, followed by C1-esterase
inhibitor, antithrombin, 1-protease inhibitor,
and 2-plasmin inhibitor.129 Platelets
also contain a tight-binding Kunitz-type inhibitor of factor XIa,
protease nexin 2.130
Prekallikein, and High-Molecular-Weight Kininogen
Factor XII and PK are zymogen precursors of
proteases. PK has
four apple domains, and is highly homologous to factor XI. Factor
XII is homologous to plasminogen activators. HK is a nonenzymatic
cofactor that circulates in complex with Factor XI and with PK.
In addition to its nonenzymatic role in contact activation, HK acts
as a thiol protease inhibitor and as an antiadhesive protein. HK
is cleaved at two sites by kallikrein to release the bioactive
bradykinin, a potent vasodilator. Table 115–1 shows
the plasma levels, plasma half-lives, and chromosomal locations
of factor XII, PK, and HK.131 All three proteins
are synthesized in the liver.
The gene for factor XII is located on chromosome
spans approximately 12 kb. It contains 14 exons. The intron–exon
structure of the gene is similar to the plasminogen activator family
of serine proteases. Portions of the gene are homologous to domains
found in fibronectin and tissue-type plasminogen activator. The
gene for PK is located on chromosome 4q35, close to the factor XI
gene. It spans 30 kb and has 15 exons with 14 introns and is homologous
to the factor XI gene.132 The gene for HK is located
on chromosome 3 and contains 11 exons and spans 27 kb. HK and
kininogen are produced from the same gene by alternative splicing.
Both proteins serve as precursors to bradykinin, but
has no interaction with the coagulation proteins.
Factor XII, HK,
and PK are responsible for the contact activation
of blood coagulation as seen in the activated partial thromboplastin
time test (aPTT). In this clinical laboratory test, plasma is mixed
with a reagent such as glass, kaolin, celite, or ellagic acid that
a negatively charged surface. Contact activation involves both
protein–surface interactions that lead to the activation
of factor XII. Factor XIIa activates factor XI, which then activates
IX. In spite of the fact that factor XII, HK, and PK are required
for a normal aPTT, they do not appear to be required for normal
hemostasis. Individuals who are deficient in any of these factors
do not have a bleeding tendency, even after significant trauma or
However, factor XII, HK, and PK, along with complement factor C1q,
do participate in inflammatory responses that involve the blood
clotting system, fibrinolysis, and generation of kinins.133,134
deletion of factor XII in a mouse model does not impair hemostasis,135
does result in reduced generation of inflammatory mediators. Factor
XII deficiency is associated with an increased tendency toward
in animal models,136,137 and perhaps in patients.138,139
activation of the contact factors likely plays a much greater role
in thrombosis than hemostasis. The available data suggest that the
contact system may normally function on the surface of endothelial
cells. PK and factor XI, through its binding to HK, assemble on
a multiprotein receptor complex on endothelial cells.140 PK
is activated to kallikrein by a membrane-expressed carboxypeptidase,
releasing bradykinin in the process. Kallikrein activates factor
XII, which, in turn, initiates fibrinolysis by activating urokinase.
Thus, activation of the contact system in vivo on
cell surfaces is mechanistically quite different from activation
on a charged surface in the aPTT. The kallikrein/kinin
system has been hypothesized to serve in vivo as
a physiologic counterbalance to the renin–angiotensin system
by lowering blood pressure and preventing thrombosis.141
TF is the cellular receptor
and cofactor for factor VII and VIIa
(see Fig. 115–4). TF is composed
of 263 amino acids, and consists of a 219-amino-acid extracellular
domain, a 23-residue transmembrane portion, and a 21-residue
domain (Fig. 115–20).142 A
cysteine in the intracytoplasmic domain is linked to a palmityl
fatty acid, which downregulates phosphorylation of the cytoplasmic
domain.143 Although many of the coagulation factors share
a high degree of homology, the structure of TF is unique. It is
the only one of the procoagulant proteins that is an integral membrane
protein, and it is homologous to the type 2 cytokine receptors.144
includes the receptors for interleukin-10 and interferons , ,
and . The extracellular domain of TF complexed
with factor VIIa has been crystallized, and the extracellular domain
has been found to fold in a manner typical of the cytokine receptor
homology unit (see Fig. 115–4).22,145 These
structural features suggest that TF may be a multifunctional protein
with both signal transducing and procoagulant functions.
The human TF gene is located
on chromosome 1p21-p22.146 The
DNA sequence of the TF gene has been determined and consists of
6 exons and 5 introns that span approximately 12 kb.147 The
first exon codes for the signal peptide, whereas the second through
encode the extracellular domain. The sixth exon codes for the
and cytoplasmic domains, as well as a relatively long 3' untranslated
region. An alternatively spliced form of TF that lacks the
domain has been described in humans and mice.148,149 It
is reported to be present in the blood and to possess cofactor activity,
but its role in hemostasis or thrombosis remains to be clarified.
The initiation site for
transcription of the TF gene is well
defined, and the region with promoter activity is from –383
to –121 bp relative to the start site.150 The promoter
contains a serum response element with a putative binding site for
Sp-1, and a lipopolysaccharide responsive element with AP-1 and
nuclear factor B (NF-B)-like
TF is expressed
constitutively on many extravascular tissues.
Although it is not normally expressed by cells in contact with flowing
blood, TF expression can be induced on blood monocytes by bacterial
products, inflammatory cytokines, and engagement of P-selectin
on monocytes.151–154 Small amounts of
TF protein have been reported to be present in platelets, but the
physiologic and/or pathophysiologic roles of this phenomenon
are not yet clear.155
VIIa/TF complex is thought to be the major
physiologic initiator of blood coagulation. TF is normally expressed
on pericytes surrounding blood vessels and by epidermal, stromal,
and glial cells.156,157 It has also been shown
that monocytes, which normally have no TF activity, can express
TF when exposed to vessel media or collagen.158 The process
of coagulation is initiated when an injury ruptures a vessel and
allows blood to come in to contact with extravascular TF. It is
often said that release of blood from the vessel allows factor VII
to bind to extravascular TF and initiate coagulation. However, it
is very likely that TF around vessels in most sites already has
bound factor VIIa in the absence of injury.159 An
injury allows the extravascular factor VIIa/TF complexes
to come into contact with platelets, and initiate large-scale thrombin
generation on platelet surfaces.
The binding of factor VIIa to TF enhances its
almost three orders of magnitude.160,161 The factor
VIIa/TF complex can activate both factor IX and factor
X.162 However, unlike binding of factor IXa or
Xa to their cofactors, binding of factor VIIa to TF does not strictly
require calcium,163 and the affinity of the interaction
is only slightly enhanced by the presence of anionic phospholipid.164,165
the cleavage of factor IX or X by factor VIIa/TF is enhanced
by anionic phospholipid.165 This effect is a result
of the enhanced binding of the substrate, rather than of any effect
of the phospholipid on the catalytic efficiency of the VIIa/TF
The affinity of
factor VIIa binding for TF on cells is very high
(20–80 pM). Binding of factor VIIa to TF that is reconstituted
into synthetic phospholipid vesicles always results in enhanced
factor VIIa proteolytic activity. However, binding of factor VIIa
to cellular sources of TF does not always correlate with enhanced
enzymatic activity. This suggests that cells can regulate the cofactor
activity of TF in a manner that is not reproduced by synthetic
TF does not
require proteolytic activation to express its activity.
However, it appears that TF can occur in a latent or "encrypted" form166,167;
that is, TF detected as antigen on the cell surface may not express
clotting activity. It has been hypothesized that the TF could form
dimers that block access to the substrate binding site on TF. Dimerized
("encrypted") TF could still bind factor VII,
but would be inactive because it could not bind either factor IX
or X. The physiologic regulators that control TF encryption are not
clear and it remains to be determined whether this is an important
regulatory mechanism in vivo.
In addition to its role as a cofactor in
factor is thought to play important roles in cell signaling. This
signaling can occur both through proteolytic activity of factor
VIIa bound to tissue factor168 and through the
formation of a VIIa/TF/Xa complex.169 This
cell signaling has been suggested to play important roles in the
cell migration needed for wound healing, inflammation, and
The importance of this signaling in vasculogenesis is suggested
by the observation that mice in which the tissue factor gene has
been knocked out die during embryonic development partly as a result
of disorganization of the yolk sac vasculature.170
Thrombomodulin is a
transmembrane protein of Mr of 78,000.171 It
is the cellular cofactor for thrombin.172 TM has
a leader sequence followed by lectin-like domains homologous to
the asialoglycoprotein receptor (Fig. 115–21).173 However,
TM has no known lectin-like activity. Following the lectin-like
domain are six EGF-like domains, the fourth, fifth, and sixth of
which are responsible for both thrombin-binding and protein-C activating
activities (Fig. 115–21).174 A
serine-and threonine-rich region follows the EGF domains and is
the site of O-linked glycosylation. A chondroitin sulfate moiety,
which enhances TM anticoagulant activity, is attached to Ser492
in this region.175 The 23-amino-acid transmembrane
domain follows the serine and threonine-rich region, followed by
a short cytoplasmic tail.
The human TM gene is located
on chromosome 20p12-cen176 and spans
approximately 3.5 kb. It consists of a single exon (see Fig. 115–21).
Intronless genes are
uncommon and include rhodopsin, angiogenin, mitochondrial genes,
interferons - and -adrenergic
receptors. The functional significance of the lack of introns is
cleave a number of substrates without a cofactor,
such as fibrinogen, factors V and VIII, and the protease-activated
thrombin receptors. However, binding to the cofactor TM localizes
thrombin to endothelial cell surfaces and induces a conformational
such that its ability to activate protein C is enhanced 1000- to
2000-fold. Thrombin bound to TM no longer activates platelets, nor
does it cleave fibrinogen or activate factor V or factor VIII.177
TM changes the activity of thrombin from procoagulant to anticoagulant.
TM also enhances the ability of thrombin to activate TAFI.34
TM is expressed on the
surface of vascular endothelial cells.
In conjunction with EPCR, TM appears to play a major role in preventing
from occurring on intact endothelium in the microcirculation.178
mice, knocking out either TM or EPCR creates an embryonically lethal
phenotype correlated with increased placental fibrin deposition.179,180
has also been detected in mesothelial cells,181 mononuclear
phagocytes,182 squamous epithelium,183
and malignant cells,36,184 where its function is unknown.
The level of TM expression differs among endothelial cells from
different sites.185 Endothelial TM and TF expression
are regulated by inflammatory cytokines in a reciprocal fashion.
Thus, thrombosis may be favored at sites of inflammation by a concurrent
elevation of endothelial TF and depression of endothelial TM.
Protein C inhibitor is an
effective inhibitor of the thrombin/TM
Fibrinogen, when converted to
fibrin, forms the structural meshwork
that consolidates an initial platelet plug into a solid hemostatic
clot. The physiologic importance of fibrinogen is underscored by
the bleeding diathesis associated with afibrinogenemia187,188
some dysfibrinogenemias189 (see Chap. 126). Other
dysfibrinogenemias are associated with thromboembolic
disease.188,190 Although afibrinogenemia is associated
with a bleeding tendency, it is usually not as severe as classical
hemophilia. This is possibly explained by findings in mice demonstrating
fibronectin can accumulate in platelets and assume the adhesive
functions of fibrinogen to a limited extent.191
Fibrinogen is a dimeric
glycoprotein whose dominant form has
an Mr of 340,000. It is found in plasma and in platelet granules.
Each of the two subunits contains three disulfide-linked polypeptide
chains192 that are referred to as the A (Mr
66,500), B (Mr 52,000), and (Mr
46,500) chains. Fibrinopeptides A and B are released from the amino
termini of the A and B chains
by thrombin cleavage of the Argl6-Glyl7 and Argl4-Glyl5 bonds,
respectively.193 The central
globular domain of fibrinogen is called the E-domain. It includes
the disulfide-linked amino-termini of all six polypeptide chains
referred to as the N-terminal disulfide knot.194 The
E domain is linked by helical, coiled-coil domains to the
domains of the three chains, designated the D domains. A trinodular
model of fibrinogen structure has been established from the crystal
structure of fibrinogen (Fig. 115–22).195 N-linked
glycosylation occurs at Asn364 of the B chain
and Asn52 of the chain.
Because human fibrinogen is
subject to modification at a number
of different sites both during and after biosynthesis, the fibrinogen
present in the circulation is a heterogeneous mixture of molecules.
These normal variants are caused by alternative splicing, modification
of certain amino acids by sulfation, phosphorylation, and hydroxylation,
different degrees of glycosylation, and proteolysis. It has been
estimated that the number of nonidentical fibrinogen molecules that
can be produced by these mechanisms is in excess of 1 million.196
of these variations may have significant functional consequences.
For example, the level of one variant of fibrinogen with an
spliced chain (fibrinogen-)
is associated with a decreased risk of venous thrombosis,197,198
an increased risk of myocardial infarction.199
In normal individuals, the
plasma half-life of fibrinogen is
3 to 5 days,200 with only a small proportion of
the catabolism caused by consumption. Plasma fibrinogen is synthesized
in the liver. Fibrinogen is an acute-phase reactant and its synthesis
can be increased up to 20-fold with a strong inflammatory stimulus.201,202
Interleukin-6 (IL-6) is an important mediator of increased fibrinogen
synthesis during an acute-phase response,203 and
IL-6 secretion can be upregulated by fibrin(ogen)-degradation products.
The genes for the three chains
of fibrinogen are found within
a 50-kb length of DNA on chromosome 4 at q23-q32204 (Fig.
115–23). The genes for all
three chains have been sequenced. The genomic sequences show a high
degree of homology, suggesting they
were derived through duplication of a common ancestral gene.205,206
homology extends to sites upstream of the gene, suggesting that
common regulatory elements may reside in these areas, thus helping
to coordinate synthesis of the three chains.207
Studies of tissue-specific
expression and acute-phase regulation
of the mRNA of the fibrinogen chains have revealed some surprises.
The expression of the chain is regulated by ubiquitous
factors such as SP1, whereas transcription of the A and
B genes requires the liver-specific factor HNF-1.208
B-chain promoter contains an IL-6–responsive element209
appears to be present in the upstream sequences of the other chains
as well. Because of the differences in the promoter regions of the
genes for the three chains, the tissue distribution differs. The highest
levels of mRNA for all three chains are found in the liver. However, -chain
transcripts have been found in a number of organs that lack transcripts
of the other chains. mRNA for A and B has
been found in the kidney, consistent with the presence of HNF-1
of the presence of fibrinogen in the granules
of platelets, it was initially assumed that megakaryocytes synthesized
fibrinogen. However, although some -chain transcripts
are present in marrow precursors, it appears that most of the fibrinogen
within platelets is taken up from the plasma by endocytosis (see Chap.
to the central domain of fibrinogen213 and
proteolytically releases two fibrinopeptides A (A 1–16)
and two fibrinopeptides B (B 1–14) from
each fibrinogen molecule. Release of the fibrinopeptides exposes
binding sites in the E domain that have complementary sites in the D
domains of other fibrin monomers.214,215 These
complementary binding sites lead to the initial formation of
protofibrils with a half-staggered overlap configuration (Fig. 115–24).
then aggregate into thick fibers consisting of 14 to 22 protofibrils
that branch into a meshwork of interconnected thick fibers.216
half-staggered overlap of the fibrin monomers gives a characteristic
cross-banded pattern on electron micrographs.217 Calcium
appears to enhance lateral fiber growth by binding to sites on human
During fibrin monomer
polymerization, other plasma proteins also
bind to the surface of the developing meshwork. These include elements
of the fibrinolytic system and a variety of adhesive proteins, such
as fibronectin, thrombospondin, and VWF. These surface proteins
influence the generation, cross-linking, and lysis of fibrin.
also has specific integrin-binding sites that are essential for
platelet binding (see Chaps. 114 and 126). The thrombin that initiates
fibrin polymerization also
activates factor XIII, which stabilizes the fibrin polymer by
Factor XIIIa also crosslinks other bound proteins, for example,
plasminogen activator-1, vitronectin, fibronectin, and 2-antiplasmin,
to the fibrin network.
formed, the fibrin mesh can be degraded by the fibrinolytic
system. Plasmin cleaves fibrin and fibrinogen in an ordered sequence
at arginyl and lysyl bonds, giving rise to a series of soluble
degradation products.220 The
plasmin digestion of fibrinogen initially cleaves the A polar
appendage and the B 1–42 fragment, generating
fragment X (Mr 250,000), which can still form a clot, albeit slowly.
Further action of plasmin releases a D fragment (Mr 100,000) from
fragment X to form fragment Y (Mr 150,000). Fragment Y is further
cleaved to form another fragment D and a fragment E (Mr 50,000).
Similar fragments are generated during plasmin digestion of cross-linked
fibrin, with two exceptions: (1) the B 15–42
is released from the des 1–14 B chain
of fibrin, and (2) D-dimer and other covalently cross-linked degradation
products are cleaved from the cross-linked fibrin polymer. Monoclonal
antibodies recognizing the fibrin D-dimer fragments can help to
fibrin-degradation products from fibrinogen-degradation products.221
the large X fragment can still polymerize into a weak clot,222
Y and D fragments inhibit normal fibrin monomer polymerization.223
inhibition of polymerization can prolong the thrombin time and lead
to spuriously low values of fibrinogen when measured by
In addition to
its obvious procoagulant role in stabilizing the
initial platelet hemostatic plug, fibrin can also act as an important
inhibitor of thrombin generation. Fibrin functions as "antithrombin
I" by sequestering thrombin in the developing fibrin clot,
and also by reducing the catalytic activity of fibrin-bound thrombin.224
Factor XIII is a 320,000 Mr
glycoprotein composed of A and B
subunits with a plasma half-life of approximately 10 days. It is
a protransglutaminase that is activated by thrombin in the presence
of calcium.225 The A chain contains the cysteine
active site, whereas the B chain is not enzymatically active and
functions as a carrier protein. The cDNA and protein sequences of
both subunits have been determined.226–228
The factor XIII A chain is a
unique member of the transglutaminase
family, which is composed of calcium- and thiol-dependent enzymes
found in all human tissues and fluids. Factor XIIIa crosslinks proteins
between the -carbon of glutamine in one protein
and the -amino group of lysine in the other. The
A chain contains 731 amino acids with a Mr of ~83,000 (Fig.
XIII B chain is homologous to complement regulatory
proteins. It is synthesized as a chain of 661 amino acids starting
with a signal peptide. The mature B chain comprises 641 amino acids,228
Mr of ~76,500, including 8.5 percent carbohydrate. The B chain contains
10 short consensus repeat (SCR) units (also called GP-1 or Sushi
domains; Fig. 115–26). Each SCR
contains 60 to 70 amino acids, containing 4 conserved cysteine residues
with a characteristic pattern of disulfide bonds.229 In
plasma, the B subunit is found in molar excess over the A component.
So all circulating A subunit is bound to B subunit. The B subunit
can also be found free in circulation.
In addition to plasma, factor
XIII also is present in platelets,
monocytes, and monocyte-derived macrophages. The plasma factor is
a heterotetramer consisting of paired A and B subunits (A2B2). In
platelet and other cells, factor XIII exists as an A2 dimer and
lacks the B domain. Monocytes/macrophages can synthesize
factor XIII,230 and the factor XIII found in platelets
is probably synthesized by megakaryocytes.231 Cells
of marrow origin seem to be the primary site for the synthesis of
A in plasma factor XIII, but hepatocytes might also contribute.225
B subunit of plasma factor XIII is synthesized in the liver.
The factor XIII A chain gene
has been localized to chromosome
6 p24-p25.232 It contains 15 exons and 14 introns
and is larger than 160 kb in size (see Fig. 115–25).227 The
fibrin-binding domain is encoded by exons 2 to 12. The active site,
with its reactive thiol at Cys314, is present in exon 7. Although
the structure of the factor XIII A-chain gene is quite similar to
that of other transglutaminases, it has unique regulatory
mechanisms. Transcription is regulated by a myeloid-enriched
factor (MZF-1–like protein) and two ubiquitous transcription
factors (NF-1 and SP-1).233 The myeloid-enriched
factor GATA-1 acts as an enhancer, as does Ets-1. The transcription
initiation site for the A-chain is 76 bp upstream from the first
The factor XIII B chain has been localized to
It has 12 exons separated by 11 introns and is about 28 kb in size
(see Fig. 115–26).226 Each
SCR is encoded by a single exon. The regulation of the factor XIII
B-chain expression is poorly understood. A total of 30 potential
start sites are located upstream of the initial methionine.
XIII circulates in association with its substrate,
fibrinogen. The key step in the activation of plasma factor XIII
is thrombin cleavage of the Arg37-Gly38 bond in the A chain to release
a Mr 4500 activation peptide. This leads to dissociation of the
A and B subunits and exposure of the active site on the free A subunits.
Cellular factor XIII in platelets becomes activated through a
process. When intracytoplasmic Ca2+ is
elevated during platelet activation, the zymogen, in the absence
of the B-chain, assumes an active configuration.225 The
main physiologic function of plasma factor XIIIa is to crosslink
the and chains of fibrin to
stabilize the fibrin plug.234 In the absence of
factor XIII a clot forms, but is inadequate for hemostasis. Additional
protein substrates of factor XIIIa include components of the clotting
and fibrinolytic system, as well as multiple adhesive and contractile
proteins. Factor XIIIa also protects fibrin from fibrinolysis by
crosslinking it to 2-antiplasmin.235 Plasma
factor XIII is also involved in wound healing and tissue repair,
and is essential to maintaining pregnancy.
TAFI is the
zymogen precursor to a zinc-bound metalloprotease,
and has been called carboxypeptidase B, R, and U in the literature.
It has an Mr of 60,000 with 20 percent of that mass a result of
carbohydrate attached to 4 sites within the first 92 amino acids.
In sequence alignments with other members of the carboxypeptidase
A family, active site residues (Glu 271 and Arg 125), and zinc-binding
residues (His 67, Glu 70, and His 196) in TAFI are conserved.
The gene for TAFI has been
localized to 13q14. The gene contains
11 exons with 10 introns and spans 48 kb.236 TAFI
is synthesized in the liver. The TAFI promoter has a C/EBP
binding site that regulates liver synthesis.237 The
promoter lacks a consensus TATA box and transcription is initiated
from multiple sites. Plasma concentration of TAFI in individuals
can vary from 4 to 15 mcg/mL with a strong correlation between
plasma levels and polymorphisms in the promoter and 3' region.238
activated by cleavage by plasmin or thrombin, a reaction
that is accelerated 1000-fold when thrombin is bound to thrombomodulin.
Both enzymes cleave after Arg-92 to give a Mr 37,000 activated form
(TAFIa) with release of the large activation peptide. TAFIa catalyzes
removal of carboxy-terminal lysine and arginine residues from fibrin
and fibrin cleavage products. These residues are important for binding
and activation of plasminogen. Removal of these residues by TAFIa
reduces clot catalyzed formation of plasmin resulting in decreased
clot lysis. TAFIa may also have an antiinflammatory role as it can
efficiently cleave bradykinin. A polymorphism in TAFI is associated
with lower blood pressure in individuals homozygous for this
Inhibitors of TAFIa have not been identified,
however the molecule
is thermodynamically unstable with a half-life at 37°C (98.6°F)
of less than 15 minutes.240
There are many protease inhibitors in plasma, but
the two most
specifically involved in inhibition of coagulation factors are TFPI
and AT (see Table 115–4). The protein
Z/protein Z-dependent protease inhibitor (ZPI) system is
also emerging as a potentially important regulator of the coagulation
116 discusses coagulation inhibitors
TFPI is a
single-chain polypeptide with a Mr of 34,000 to 40,000,
depending upon the degree of proteolysis of the carboxy-terminal
region. TFPI contains three Kunitz-type protease inhibitor domains. The
second Kunitz domain binds and inhibits factor Xa; this is required
for the first Kunitz domain to bind and inhibit the factor VIIa/TF
complex. The function of the third Kunitz domain is not clear, but
it may be involved in binding to glycosaminoglycans. Thus, TFPI
is unique among the coagulation protease inhibitors in two respects.
First, it has inhibitory sites for both factor Xa and for the factor
VIIa/TF complex. Second, TFPI cannot inhibit the factor
VIIa/TF complex unless it has also bound factor Xa.242,243
The primary site of plasma
TFPI synthesis is endothelial cells.244 Most
circulating TFPI is bound to lipoproteins. A second pool of TFPI
is bound to heparan sulfates on the surface of endothelial cells.
Administration of heparin releases the endothelial cell bound TFPI
and raises the plasma level severalfold.245 A third
pool is an alternatively spliced form of TFPI (TFPI-)
that is anchored to endothelial cells via a glycosyl
is only present in the plasma at about 2.5 nM, compared
to AT at about 2 M. However, its rate of reaction
with factor Xa in plasma is similar to that of AT. Therefore, TFPI
contributes significantly to the inhibition of factor Xa in
The gene for
human TFPI is located on chromosome 2q31-q32.1 and
has 9 exons that span 70 kb. The first two exons code for a 5'
region, and coding begins at exon 3. No TATA box is present in the
region of the TFPI gene. DNA sequences that are consistent with
binding sites for the transcription factors GATA-2, AP-1, and NF-1
are present in the 5' untranslated region
of the TFPI gene. It is thought that GATA-2 binding is necessary
for constitutive expression of TFPI by endothelial cells.244
TFPI is synthesized in two
alternatively spliced forms, and . TFPI- lacks
the third Kunitz domain and instead has a unique carboxy-terminal.
TFPI- is the predominant form in circulation. Although
a significant fraction of TFPI- is linked to the
endothelial cells via a glycosyl phosphatidylinositol anchor,246
is also found in plasma and has inhibitory activity similar to that
AT is a
member of the large family of serine protease inhibitors
(serpins) and in the systematic nomenclature is SERPINC1. These
inhibitors act as "suicide" substrates for their
target proteases through a surface-exposed structure termed a reactive
siteloop. An amino acid sequence in the
reactive site loop of AT is cleaved by the target protease to form
a 1:1 covalent complex that blocks the active site of the protease.
AT is an important physiologic inhibitor of the blood coagulation
proteases, as its deficiency leads to a significantly increased
risk of thrombosis. The primary proteases targeted by AT are thrombin,
factor Xa, and factor IXa.248–250 Inhibition
of these proteases is accelerated by heparin. Factor VIIa is resistant
to inhibition by AT unless it is complexed to TF in the presence
of heparin or cell surface glycosaminoglycans.251,252
Heparin and related molecules
accelerate inhibition of proteases
by AT by two distinct mechanisms, as illustrated in Figure
115–27. As shown in the middle
panel, binding of a specific pentasaccharide sequence in a heparin
molecule results in a conformational change in the AT molecule that
increases the accessibility of the reactive site loop to a target
protease. This results in an increased rate of inhibition of proteases
by AT. In addition, as shown in the right panel of the figure, a
larger heparin molecule can bind to both AT and a target protease.
This serves to align the two molecules and facilitates their
again increasing the rate of inhibition of the protease by AT.
The protease–serpin complex
is cleared from the circulation
by receptor-mediated endocytosis in the liver.253
The gene for AT is on the long
arm of chromosome 1. The gene
has seven exons and spans about 13.5 kb. Little is known about
regulation of AT. The region from –89 to –68 has
been implicated in the binding of transcription factors from rat
liver,254 but the specific transcription factors
involved are not clear.
Z-Dependent Protease Inhibitor
The ZPI is a
Mr 72,000 serine protease inhibitor (SERPINA10 in
the systematic nomenclature) that inhibits coagulation factors XIa
and Xa. Its inhibition of factor Xa is enhanced over 1000-fold in
the presence of a vitamin K-dependent plasma protein, protein Z.255
Z is a plasma glycoprotein of Mr 62,000. Like the other GLA-containing
proteins, it consists of a GLA domain, hydrophobic region, and two
EGF domains. However, instead of a catalytic domain, the
region of protein Z contains a domain that, while homologous to
the catalytic domain of the other GLA containing proteins, lacks
the His and Ser residues characteristic of the catalytic triad of
trypsin-like serine proteases. Thus, protein Z has no protease activity.
In normal plasma, which has a molar excess of ZPI over protein Z,
all protein Z circulates in complex with ZPI.256
The physiologic role of
protein Z/ZPI in the coagulation
system is not yet clear. Deficiency of protein Z in a mouse model
does not lead to thrombosis, but dramatically worsens the thrombotic
tendency of mice who simultaneously express the factor V Leiden
genotype, a known risk factor for thrombosis.257
The chromosomal location of
the gene for ZPI is not known. The
gene for protein Z is on the long arm of chromosome 13 (q34) in
close proximity to the genes for factor X and factor VII.3
gene spans 14 kb and consists of 9 exons and 8 introns. The intron/exon
boundaries are identical to the other GLA-containing coagulation
proteins. There is an alternative exon that codes for a unique peptide
of 22 amino acids in the preproleader sequence. The gene is transcribed
into a 1.6 kb mRNA.
1960s, two groups proposed a model of coagulation that
envisaged a sequential series of steps in which activation of one
clotting factor led to the activation of another, finally leading
to a burst of thrombin generation.258,259 Each
clotting factor was thought to exist as a proenzyme that could be
converted to an active enzyme.
The original cascade models were subsequently
modified to include
the observation that some procoagulants were cofactors and did not
possess enzymatic activity. In addition the clotting sequences were
divided into so-called extrinsic and intrinsic systems, as shown
in Figure 115–28. As can be seen,
the extrinsic system consisted of factor VIIa and tissue factor,
the latter being viewed as extrinsic to the circulating blood. The
factors in the intrinsic system were all viewed as being intravascular.
Both pathways could activate factor X, which, in complex with its
cofactor Va, could convert prothrombin to thrombin. Although these
earlier concepts of coagulation were extremely valuable, investigators
recognized that the intrinsic and extrinsic systems could not operate
independently of one another and that all the clotting factors were
somehow interrelated. Only in this way could hemostasis in
vivo be explained.
the Coagulation Models
observations made by several groups have led to a revision
of earlier models of coagulation. A major observation was that a
complex of factor VIIa/TF activated not only factor X but
also factor IX.162 Furthermore, it was observed
that thrombin could directly activate factor XI.121,122
with other important observations, this led to the conclusion that
the major initiating event in hemostasis in vivo was
the formation of a factor VIIa/TF complex at the site of
injury.260–262 This led to the belief
that factor VIII and IX deficiency, which resulted
in hemophilia A and B, respectively, were in fact, abnormalities
of the VIIa/TF pathway, even though factors IX and VIII
were considered components of the intrinsic system. It was also
recognized that in vivo coagulation was regulated
by control mechanisms, one of which was the localization of the
reactions to cell surfaces. In addition, earlier and more recent
observations emphasized the importance of plasma inhibitors of each
step of the coagulation process. These include TFPI, which inhibits
the factor VIIa/TF/Xa complex,243,263 proteins
C and S, which inactivate factors Va and VIIIa,178,264,265
AT, which inhibits thrombin and other coagulation proteases.266
Model of Coagulation
the TF-Bearing Cell
goal of hemostasis it to produce a fibrin clot to seal a
site of injury or rupture in the blood vessel wall. This process
is initiated when TF-bearing cells are exposed to blood at a site
of injury. TF is anchored to cells via a transmembrane domain and
acts as a receptor for plasma factor VII. Once bound to TF, zymogen
VII is rapidly converted to factor VIIa through mechanisms incompletely
understood, but which may involve factor Xa and autoactivation.
TF is expressed around vessels and in the epithelium, where it has been
described as forming a "hemostatic envelope." The
TF around vessels has likely already bound factor VII(a), even in
the absence of an injury.159 The factor VIIa/TF
complex catalyzes two very important reactions: (1) activation of
factor X to factor Xa and (2) activation of factor IX to IXa. The
factor Xa and IXa formed on the TF-bearing cells have very distinct
and separate functions in initiating the process of blood coagulation.13
a vessel is injured, the blood delivers platelets to the site of
injury. They bind to extravascular matrix components to produce
the primary hemostatic plug and become partially activated in the
process. The platelets are thereby localized in close proximity
to active factor VIIa/TF complexes.
The factor Xa formed on the TF-bearing cell
interacts with factor
Va released from the activated platelets to form prothrombinase
complexes sufficient to generate a small amount of thrombin in the
vicinity of the TF cells (Fig. 115–29).
Although this amount of thrombin may not be sufficient to clot
it is sufficient to initiate events that "prime" the
clotting system for a subsequent burst of thrombin generation.
using a cell-based model have shown that minute amounts of thrombin
are formed in the milieu of TF-bearing cells exposed to plasma
of procoagulants, even in the absence of platelets. The small amounts
of factor Va required for prothrombinase assembly on the TF-bearing
cells are likely provided by release from platelets, or activated
by factor Xa60 or noncoagulation proteases elaborated
by the cells.267 The small amounts of thrombin
generated on the TF-bearing cells are capable of accomplishing the
following: (1) activating platelets; (2) activating factor V; (3)
activating factor VIII and dissociating factor VIII from VWF; and
(4) activating factor XI (Fig. 115–30).123,268 The
activity of the factor Xa formed by the factor VIIa/TF
complex is restricted to the TF-bearing cell. Factor Xa that diffuses
off the cell surface is rapidly inhibited by TFPI or AT.
Unlike factor Xa, the primary
site of activity of the factor
IXa formed by factor VIIa/TF is on activated platelets
in close proximity to the TF-bearing cell. Factor IXa can diffuse
to adjacent cell surfaces because it is not inhibited by TFPI and
is inhibited much more slowly by AT than is factor Xa (see Table 115–4).
In addition to the pool of
extravascular, cell-anchored TF, many
reports now document the presence of TF antigen and active TF protein
in the circulating blood. This tissue factor can be found either
associated with so-called microparticles269 or
as an alternatively spliced form that has no membrane association.
The microparticles are membrane vesicles that can be shed from many
cell types, particularly in the setting of inflammation or during
apoptosis. Their presence in the blood has been reported in association
with a wide range of inflammatory and prothrombotic states, including
atherosclerotic vascular disease, severe infections and malignancy. Less
available about the alternatively spliced form. However, it is not
clear that the alternatively spliced form of tissue factor can
factor Xa and thrombin generation.270
Tissue factor mRNA has also been reported to be
present and transcribed
in platelets after they are strongly activated.271 The
time course of these events appears to be too slow to play a role
in normal hemostasis, but could play a role in thrombosis or
and wound healing following injury.
There are data showing that thrombi formed in
flowing blood accumulate
significant amounts of tissue factor as they develop.158 This
factor accumulation is very different than what is seen in wounds where
the only detectable tissue factor is present at the periphery of
the hemostatic clot where it contacts the injured tissue.272
animal models of thrombosis induced by stasis, microparticles containing
tissue factor enhanced thrombus formation in a dose-dependent fashion.273
in a mouse model suggested that tissue factor in microparticles
to thrombus formation in vivo.274 However,
another study in mice, using a different model of thrombosis, suggested
that circulating tissue factor did not contribute significantly
to thrombus formation.275
Thus, based on the available data, it can be
that circulating tissue factor is present at a low level in normal
individuals and higher levels in some disease states. The circulating
tissue factor likely contributes to thrombosis in some settings,
but is less likely to play a significant role in normal hemostasis.
The Role of
also play a major role in localizing clotting reactions
to the site of injury, as they adhere and aggregate at the same
sites where TF is exposed. Platelet localization and activation
are mediated by VWF, thrombin, platelet receptors, and vessel wall
components such as collagen (see Chap. 114).276
Once platelets are activated,
the cofactors Va and VIIIa are
rapidly localized to the platelet membrane surface (see Fig. 115–30).
Cofactor binding is
mediated in part by the exposure of phosphatidyl serine on the platelet
membrane, a process resulting from a flip-flop mechanism whereby
phosphatidyl serine on the inner leaflet of the membrane bilayer
flips to the outside.277 In addition, it appears
that the cofactors bind to platelet surface before the binding of
the respective enzymes.278
The factor IXa formed by the factor VIIa/TF complex
to the surface of activated platelets (Fig. 115–31).
Specific receptors on the activated platelets bind factor IXa and
promote formation of active factor IXa/VIIIa complexes.279,280
the platelet "tenase" complex is assembled, factor
X is recruited from the plasma and is activated to factor Xa on
the platelet surface. Factor Xa then associates with factor Va on the
surface to generate a burst of thrombin sufficient to clot fibrinogen
and form a hemostatic plug (Fig. 115–31).
Factor XIII, activated by thrombin, crosslinks fibrin and stabilizes
the hemostatic plug, rendering it impermeable. Thrombin also activates
TAFI which helps to stabilize the fibrin clot.
can directly activate factor XI.121,122,280a This
reaction is enhanced when factor XI and thrombin bind to platelet
surfaces,123,128,281 thus bypassing the need for
factor XIIa in hemostasis. The platelet-bound factor XIa can then
activate more factor IX to IXa. Thus, it appears that factor XI
activation enhances the platelet tenase activity and serves as a
to enhance thrombin generation. The enhanced thrombin generation
resulting from the effect of factor XIa probably ensures activation
role of factor XI in hemostasis has been a point of major
interest, as even severe factor XI deficiency does not result in
a hemorrhagic tendency comparable to that seen in severe factor
VIII or IX deficiency. This observation can be explained if factor
XI is viewed as an "enhancer" or "booster" of
thrombin generation. In factor VIII and IX deficiency, the individual
has a markedly decreased ability to generate factor Xa on the platelet
surface. Thus, one would expect that patients with a severe deficiency
of either factor VIII or factor IX would generate insufficient thrombin
for hemostasis as the tenase and hence prothrombinase activity would
be markedly reduced. In contrast, patients with factor XI deficiency
would always possess some baseline tenase activity. Such patients
only lack the ability to "boost" platelet surface factor
X activation by producing extra factor IXa.
Our knowledge of the platelet contribution to
has been expanded. There is evidence that there is more than one
population of activated platelets, one of which has been referred
to as COAT (collagen and thrombin stimulated) platelets.283
platelets have enhanced thrombin-generating ability as a result
of enhanced binding of both tenase and prothrombinase complexes.284,285
vivo relevance of these findings is not clear.
Even though each step of the model has been
depicted as an isolated
set of reactions including initiation, amplification, and propagation,
they should be viewed as an overlapping continuum of events, as
illustrated in Figure 115–32.
The Role of
fibrin/platelet clot is formed over an area of
injury, the clotting process must be terminated to avoid thrombotic
occlusion in adjacent normal areas of the vasculature. If the
mechanism were not controlled, clotting could occur throughout the
entire vascular tree after even a modest procoagulant stimulus.
Endothelial cells play a major
role in confining the coagulation
reactions to a site of injury and preventing clot extension to areas
where an intact endothelium is present (see Chap. 116). Endothelial
cells have two major types of anticoagulant/antithrombotic
activities, as illustrated in Figure 115–33. The
system is activated in response to thrombin generation.69
of the thrombin formed during the coagulation process can diffuse
away or be swept downstream from a site of injury. When thrombin reaches
an intact endothelial cell, it binds to TM on the endothelial surface.
The thrombin/TM complex, in conjunction with the EPCR,
then activates protein C, which binds to its cofactor protein S
and inactivates any factor Va or VIIIa that finds its way to the
endothelial cell membrane. This prevents the generation of additional
thrombin in the vasculature. The endothelial cell also possesses
other anticoagulant features. The protease inhibitors AT and TFPI
are always present bound to heparan sulfates expressed on the
surface where they can inactivate proteases near an intact endothelium.286
(GPI)-anchored TFPI- may also play a role in controlling
intravascular thrombin generation. Endothelial cells also inhibit
platelet activation by releasing the inhibitors prostacyclin (PGI2)
and nitric oxide (NO), as well as degrading ADP by their membrane
Role of Plasma
cell-based coagulation, circulating protease inhibitors
are also critical in localizing the coagulation reactions to specific
cell surfaces by directly inhibiting proteases that escape into
the fluid phase. Not only are the plasma protease inhibitors key players
a clot to the proper location, they also impose a threshold effect
on the coagulation process.288 Thus, in the presence
of inhibitors, coagulation does not proceed unless procoagulant
factors are generated in sufficient amounts to overcome the effects
of inhibitors. If the triggering event is insufficiently strong,
the system returns to baseline rather than continuing through the
coagulation process. Under pathologic conditions the trigger for
clotting may be so strong as to overwhelm the control mechanisms,
to disseminated intravascular coagulation or thrombosis (see Chaps. 116
hemostatic clot has been formed, some provision must be
made for its eventual removal as wound healing takes place. Dissolution
of clots is accomplished by the fibrinolytic system, as discussed
in detail in Chap. 136. In addition, small
clots formed inappropriately within the vasculature can sometimes
be removed by fibrinolysis.
of Basal Coagulation and Anticoagulation
The coagulation process only proceeds when enough
generated on or near the TF-bearing cell to trigger activation of
platelets and cofactors. One wonders, however, if minute hemostatic
plugs are not constantly formed throughout the body to maintain the
integrity of the vascular tree. A low level of coagulation factor
activation probably occurs at all times.289 It
was shown more than 30 years ago that fibrinopeptides are continuously
cleaved from fibrinogen at low levels in normal individuals.290
has also been shown that there are low levels of circulating factor
VIIa, as well as low levels of the activation peptides from factors
IX and X in the blood of normal individuals.291–293 This
has been called basal coagulation or idling.
Some have speculated that circulating TF drives this idling process.294
authors of this chapter favor the explanation that basal activation of
coagulation factors results from minor injuries that occur during
normal daily activities, and as the coagulation factors percolate
through the extravascular spaces.159
This basal coagulation must be balanced by basal
the anticoagulation and fibrinolytic systems. This is evidenced
by the presence of low levels of the protein C activation peptide
and tissue plasminogen activator activity in normal individuals.295
as a Part of the Host Defense Mechanism
The process of hemostasis is only one part of the
response to injury. Although different parts of the host response
are presented as if they were truly separate processes, in fact,
coagulation, fibrinolysis, inflammation, the immune response, and
wound healing are interrelated parts of the overall response to
injury. This close interaction is reflected in the close structural
relationships between many proteins of the coagulation and
systems. For example, tissue factor is structurally analogous to type
2 cytokine receptors.296 Furthermore, a number of
coagulation proteins have multiple diverse activities in the host
response to injury. Thrombin not only acts as a procoagulant to
clot fibrinogen, but also as a growth factor and cytokine that promotes
monocyte, fibroblast, and endothelial cell influx and proliferation
in an area of recent injury. By generating thrombin, the coagulation
process not only stops bleeding in the short-term, but also sets
the stage for removal of damaged tissue and wound healing in the
long-term.38 Platelets also have multiple roles
in the response to injury. They release growth factors and cytokines
upon activation, some of which play key roles in wound healing and
atherosclerosis. Factor Xa, TF, and fibrinogen fragments similarly
seem to have roles as inflammatory mediators and cell growth regulators.
The contact factors (factor XII, PK, and HK) also may play a role
as a bridge between the coagulation reactions and other host defense
mechanisms. The roles of the coagulation system in the host response
to injury are emphasized by the finding that wound healing is impaired
in hemophilia.297 No doubt the list of multifunctional
molecules will grow as understanding of the blood clotting mechanism
|1. Leytus S, Foster D, Kurachi K,
Davie E: Gene
for human factor X, a blood coagulation factor whose gene organization
is essentially identical to that of factor IX and protein C. Biochemistry
|2. Yoshitake S, Schach BG, Forter DC,
Nucleotide sequence of the gene for human factor IX (antihemophilic
factor B). Biochemistry 24:3736, 1985.
|3. Fujimaki K, Yamazaki T, Taniwaki
M, Ichinose A: The gene
for human protein Z is localized to chromosome 13 at band q34 and
is coded by eight regular exons and one alternative exon. Biochemistry
|4. Wu SM, Cheung WF, Frazier D,
Stafford DW: Cloning and expression
of the cDNA for human gamma-glutamyl carboxylase. Science
|5. Jorgensen M, Cantor A, Furie B,
et al: Recognition
site directing vitamin K-dependent gamma-carboxylation residues
on the propeptide of factor IX. Cell 48:185, 1987.
|6. Huber P, Schmitz T, Griffin J, et
of amino acids in the gamma-carboxylation recognition site on the
propeptide of prothrombin. J Biol Chem 265:12467,
|7. Brenner B, Sánchez-Vega B, Wu SM,
al: A missense mutation in gamma-glutamyl carboxylase gene causes
combined deficiency of all vitamin K-dependent blood coagulation
factors. Blood 92:4554, 1998.
|8. Li T, Chang CY, Jin DY, et
of the gene for vitamin K epoxide reductase. Nature 427:541,
|9. Oldenburg J, von Brederlow B,
Fregin A, et
al: Congenital deficiency of vitamin K dependent coagulation factors
in two families presents as a genetic defect of the vitamin
Haemost 84:937, 2000.
|10. Rost S, Fregin A, Ivaskevicius V,
Mutations in VKORC1 cause warfarin resistance and multiple coagulation
factor deficiency type 2. Nature 427:537, 2004.
|11. Kamali F: Genetic influences on
the response to warfarin. Curr
Opin Hematol 13:357, 2006.
|12. Cooper GM, Johnson JA, Langaee TY,
KA genome-wide scan for common genetic variants with a large influence
on warfarin maintenance dose. Blood 112:1022, 2008.
|13. Monroe DM, Hoffman M, Roberts HR:
Platelets and thrombin
generation. Arterioscler Thromb Vasc Biol 22:1381,
|14. Soriano-Garcia M, Padmanabhan K,
de Vos AM, Tulinsky A:
The Ca2+ ion and membrane binding structure
of the Gla domain of Ca-prothrombin fragment 1. Biochemistry
|15. Sunnerhagen M, Forsen S, Hoffren
al: Structure of the Ca(2+)-free Gla domain sheds light on
membrane binding of blood coagulation proteins. Nat Struct
Biol 2:504, 1995.
|16. Ohkubo YZ, Tajkhorshid E:
Distinct structural and adhesive
roles of Ca2+ in membrane binding of blood coagulation
factors. Structure 16:72, 2008.
|17. Nelsestuen G, Kisiel W, RG DS:
Interaction of vitamin K-dependent
proteins with membranes. Biochemistry 12:2134,
|18. Rawal-Sheikh R, Ahmad SS, Ashby
B, Walsh PN: Kinetics of
coagulation factor X activation by platelet-bound factor IXa. Biochemistry
|19. Gilbert GE, Arena AA: Partial
activation of the factor VIIIa-factor
IXa enzyme complex by dihexanoic phosphatidylserine at submicellar
concentrations. Biochemistry 36:10768, 1997.
|20. Kirchhofer D, Guha A, Nemerson Y,
Activation of blood coagulation factor VIIa with cleaved tissue
factor extracellular domain and crystallization of the active complex. Proteins
|21. Muller Y, Ultsch M, de Vos A: The
crystal structure of the
extracellular domain of tissue factor refined to 1.7 Å resolution. J
Mol Biol 256:144, 1996.
|22. Banner DW, D'Arcy A, Chene C, et
al: The crystal structure of the complex of blood coagulation factor
VIIa with human soluble tissue factor. Nature 380:41,
|23. Brandstetter H, Bauer M, Huber R,
X-ray structure of clotting factor IXa: Active site and module structure
related to Xase activity and hemophilia B. Proc Natl Acad
Sci U S A 92:9796, 1995.
|24. Patthy L, Trexler M, Vali Z, et
Modules specialized for protein binding. Homology of the gelatin-binding
region of fibronectin with the kringle structure of proteases. FEBS
Lett 171:131, 1984.
|25. Royle N, Irwin D, Koschinsky ML,
Human genes encoding prothrombin and ceruloplasmin map to 11p11-q12
and 3q21-q24, respectively. Somat Cell Mol Genet.
|26. Chow B-C, Ting V, Tufaro F,
MacGillivray R: Characterization
of a novel liver-specific enhancer in the human prothrombin gene. J
Biol Chem 266:18927, 1991.
|27. Degen S: The prothrombin gene and
its liver-specific expression. Semin
Thromb Hemost 18:230, 1992.
|28. Poort S, Rosendaal F, Bertina R: A
common genetic variant
in the 3'-untranslated region of the prothrombin
gene is associated with elevated plasma prothrombin levels and an
increase in venous thrombosis. Blood 88:3698, 1996.
|29. Sun WY, Witte DP, Degen JL, et
al: Prothrombin deficiency
results in embryonic and neonatal lethality in mice. Proc
Natl Acad Sci U S A 95:7597, 1998.
|30. Bode W, Mayr I, Baumann U, et
al: The refined 1.9 Å crystal
structure of human alpha-thrombin: Interaction with D-Phe-Pro-Arg
chloromethylketone and significance of the Try-Pro-Pro-Trp insertion
J 8:3467, 1989.
|31. Martin PD, Malkowski MG, Box J,
New insights into the regulation of the blood clotting cascade derived
from the X-ray crystal structure of bovine meizothrombin des F1
in complex with PPACK. Structure 5:1681, 1997.
|32. Vijayalakshmi J, Padmanabhan KP,
Mann KG, Tulinsky A: The
isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin:
accompanying activation and exosite binding to thrombin. Protein
Sci 3:2254, 1994.
|33. Banefield D, MacGillivray R:
Partial characterization of
vertebrate prothrombin cDNAs: Amplification and sequence analysis
of the B chain of thrombin from nine different species. Proc
Natl Acad Sci U S A 89:2779, 1992. |
|34. Nesheim M:
Fibrinolysis and the plasma carboxypeptidase. Curr
Opin Hematol 5:309, 1998.
|35. Boffa MB, Nesheim ME, Koschinsky
ML: Thrombin activatable
fibrinolysis inhibitor (TAFI): Molecular genetics of an emerging
potential risk factor for thrombotic disorders. Curr Drug
Targets Cardiovasc Haematol Disord 1:59, 2001.
|36. Dittman W, Nelson S:
Thrombomodulin, in Molecular
Basis of Thrombosis and Hemostasis, edited by KA High,
HR Roberts, p 425. Marcel Dekker, New York, 1995. |
|37. Esmon CT: The protein C pathway. Chest
|38. Church FC, Hoffman MR: Heparin
cofactor II and thrombin:
Heparin-binding proteins linking hemostasis and inflammation. Trends
Cardiovasc Med 4:140, 1994. |
|39. Pollak E,
Hung H, Godin W, et al: Functional characterization
of the human factor VII 5'-flanking region. J
Biol Chem 271:1738, 1996.
|40. Rosen ED, Chan JC, Idusogie E,
et al: Mice
lacking factor VII develop normally but suffer fatal perinatal bleeding.
|41. Hedner U, Kisiel W: Use of human
factor VIIa in the treatment
of two hemophilia A patients with high-titer inhibitors. J
Clin Invest 71:1836, 1983.
|42. Wolberg AS, Stafford DW, Erie DA:
Human factor IX binds
to specific sites on the collagenous domain of collagen IV. J
Biol Chem 272:16717, 1997.
|43. Cheung WF, van den Born J, Kuhn K,
Identification of the endothelial cell binding site for factor IX. Proc
Natl Acad Sci U S A 93:11068, 1996.
|44. Gui T, Lin HF, Jin DY, et
and binding characteristics of wild-type factor IX and certain Gla
domain mutants in vivo. Blood 100:153,
|44a. Gui T, Reheman A, Ni H, et al:
Abnormal hemostasis in a
knock-in mouse carrying a variant of Factor IX with impaired binding
to collagen type IV. J Thromb Haemost 2009:epub
ahead of press. |
|45. Camerino G,
Grzeschik K, Jaye M, et al:
Regional localization on the human X chromosome and polymorphism
of the coagulation factor IX gene (hemophilia B locus). Proc
Natl Acad Sci U S A 81:498, 1984.
|46. Winship P, Rees D, Alkan M:
Detection of polymorphisms at
cytosine phosphoguanidine dinucleotides and diagnosis of haemophilia
B carriers. Lancet 1:631, 1989.
|47. High K, Roberts H: Factor IX, in Molecular
of Thrombosis and Hemostasis, edited by KA High, HR Roberts,
p 215. Marcel Dekker, New York, 1995. |
W, Johnson P, McKnight S: Homologous recognition
of a promoter domain common to the MSV LTR and the HSV tk gene. Cell
|49. Mueller C, Maire P, Schibler U:
DBP, a liver-enriched transcriptional
activator, is expressed late in ontogeny and its tissue specificity
is determined posttranslationally. Cell 61:279,
|50. Sladek F, Zhong W, Lai E, Darnell
JJ: Liver-enriched transcription
factor NHF-4 is a novel member of the steroid hormone receptor
Dev 4:2353, 1990.
|51. Paonessa G, Gounari F, Frank R,
Cortese R: Purification
of a NF1-like DNA-binding protein from rat liver and cloning of
the corresponding cDNA. EMBO J 7:3115, 1988.
|52. London FS, Walsh PN: Activation
dependent appearance of
a platelet protein that recognizes coagulation factor IXa. Circulation
|53. Scambler P, Williamson R: The
structural gene for human
coagulation factor X is located on chromosome 13q34. Cytogenet
Cell Genet 39:231, 1985.
|54. Watzke H, High K: Factor X, in Molecular
Thrombosis and Hemostasis, edited by KA High, HR Roberts,
p 239. Marcel Dekker, New York, 1995. |
|55. Huang M,
Hung H, Stanfield-Oakley S, High K: Characterization
of the human coagulation factor X promoter. J Biol Chem
|56. Hung H, High K: Liver-enriched
transcription factor HNF-4
and ubiquitous factor NF-Y are critical for expression of blood
coagulation factor X. J Biol Chem 271:2323, 1996.
|57. Dewerchin M, Liang Z, Moons L,
et al: Blood
coagulation factor X deficiency causes partial embryonic lethality
and fatal neonatal bleeding in mice. Thromb Haemost 83:185,
|58. Rao L, Rapaport SI: Activation of
factor VII bound to tissue
factor: A key early step in the tissue factor pathway of blood
Natl Acad Sci U S A 85:6687, 1988.
|59. Neuenschwander PF, Jesty J:
Thrombin-activated and factor
Xa-activated human factor VIII: Differences in cofactor activity
and decay rate. Arch Biochem Biophys 296:426, 1992.
|60. Monkovic DD, Tracy PB: Activation
of human factor V by factor
Xa and thrombin. Biochemistry 29:1118, 1990.
|61. Bouchard BA, Catcher CS, Thrash
al: Effector cell protease receptor-1, a platelet activation-dependent
membrane protein, regulates prothrombinase-catalyzed thrombin
Biol Chem 272:9244, 1997.
|62. Gasic GP, Arenas CP, Gasic TB,
Gasic GJ: Coagulation factors
X, Xa, and protein S as potent mitogens of cultured aortic smooth
muscle cells. Proc Natl Acad Sci U S A 89:2317,
|63. Altieri DC, Edgington TS:
Identification of effector cell
protease receptor-1. A leukocyte-distributed receptor for the serine
protease factor Xa. J Immunol 145:246, 1990.
|64. Patrucchini P, Aiello V, Palazzi P,
al: Sublocalization of the human protein C gene on chromosome 2q13-q14. Hum
Genet 81:191, 1989. |
|65. Foster D,
Yoshitake S, Davie E: The nucleotide sequence
for the gene for human protein C. Proc Natl Acad Sci U S
A 82:4673, 1985.
|66. Plutzky J, Hoskins J, Long G,
Crabtree G: Evolution and
organization of the human protein C gene. Proc Natl Acad
Sci U S A 83:546, 1986.
|67. Esmon CT: Inflammation and
thrombosis. J Thromb
Haemost 1:1343, 2003.
|68. Stearns-Kurosawa DJ, Kurosawa S,
Mollica JS, et al:
The endothelial cell protein C receptor augments protein C activation
by the thrombin-thrombomodulin complex. Proc Natl Acad Sci
U S A 93:10212, 1996.
|69. Oliver JA, Monroe DM, Church FC,
Activated protein C cleaves factor Va more efficiently on endothelium
than on platelet surfaces. Blood 100:539, 2002.
|70. Shen L, Dahlbäck B: Factor V and
protein S as synergistic
cofactors to activated protein C in degradation of factor VIIIa. J
Biol Chem 269:18735, 1994.
|71. Cooper S, Church F: PCI: Protein C
Exp Med Biol 425:45, 1997.
|72. Esmon CT: Inflammation and the
activated protein C anticoagulant
pathway. Semin Thromb Hemost 32 Suppl 1:49, 2006. |
|73. Kerschen EJ, Fernandez JA, Cooley BC, et
al: Endotoxemia and sepsis mortality reduction by non-anticoagulant
activated protein C. J Exp Med 204:2439, 2007.
|74. Fair D, Marlar R: Biosynthesis
and secretion of factor VII,
protein C, protein S and the protein inhibitor from a human hepatoma
cell line. Blood 6:64, 1986. |
|75. Fair D,
Marlar R, Levin E: Human endothelial cells synthesize
protein S. Blood 67:1168, 1986.
|76. Ogura M, Tanabe N, Nishioka J,
et al: Biosynthesis
and secretion of functional protein S by a human megakaryoblastic
cell line. Blood 70:301, 1987.
|77. Dahlback B: Protein S and
C4b-binding protein: Components
involved in the regulation of the protein C anticoagulant system. Thromb
Haemost 66:49, 1991.
|78. Maillard C, Berruyer M, Serre C,
Protein S, a vitamin K-dependent protein, is a bone matrix component
synthesized and secreted by osteoblasts. Endocrinology 130:1599,
|79. Edenbrandt C-M, Lundvall A, Wydro
R, Stenflo J: Molecular
analysis of the gene for vitamin K-dependent protein S and its
Cloning and partial characterization. Biochemistry 29:7861,
|80. Castoldi E, Hackeng TM:
Regulation of coagulation by protein
S. Curr Opin Hematol 15:529, 2008.
|81. Hackeng T, van't Veer C, Meijers
J, Bouma B: Human
protein S inhibits prothrombinase complex activity on endothelial
cells and platelets via direct interactions with factors Va and
Xa. J Biol Chem 269:21051, 1994.
|82. Rezende SM, Simmonds RE, Lane DA:
and apoptosis: Different roles for protein S and the protein S-C4b
binding protein complex. Blood 103:1192, 2004.
|83. Hackeng TM, Sere KM, Tans G,
Rosing J: Protein S stimulates
inhibition of the tissue factor pathway by tissue factor pathway
inhibitor. Proc Natl Acad Sci U S A 103:3106, 2006.
|84. Moussalli M, Pipe SW, Hauri HP,
Mannose-dependent endoplasmic reticulum (ER)-Golgi intermediate
compartment-53-mediated ER to Golgi trafficking of coagulation factors
V and VIII. J Biol Chem 274:32539, 1999.
|85. Nichols W, Seligsohn U, Zivelin A,
Mutations in the ER-Golgi intermediate compartment protein ERGIC-53
cause combined deficiency of coagulation factors V and VIII. Cell
|86. Zhang B, Ginsburg D: Familial
multiple coagulation factor
deficiencies: New biological insights from rare genetic bleeding
disorders. J Thromb Haemost 2:1564, 2004.
|87. Ortel T, Keller F, Kane W: Factor
V, in Molecular
Basis of Thrombosis and Hemostasis, edited by KA High,
HR Roberts, p 19. Marcel Dekker, New York, 1995. |
|88. Ortel TL, Quinn-Allen MA, Keller FG, et
al: Localization of functionally important epitopes within the second
C-type domain of coagulation factor V using recombinant chimeras. J
Biol Chem 269:15898, 1994.
|89. Pittman DD, Tomkinson KN,
Michnick D, et
al: Posttranslational sulfation of factor V is required for efficient
thrombin cleavage and activation and for full procoagulant activity. Biochemistry
|90. Cripe L, Moore K, Kane W:
Structure of the gene for human
factor V. Biochemistry 31:3777, 1992.
|91. Gould WR, Simioni P, Silveira JR,
Megakaryocytes endocytose and subsequently modify human factor V
in vivo to form the entire pool of a unique platelet-derived cofactor. J
Thromb Haemost 3:450, 2005.
|92. Sun H, Yang TL, Yang A, et
al: The murine
platelet and plasma factor V pools are biosynthetically distinct
and sufficient for minimal hemostasis. Blood 102:2856,
|93. Yang TL, Pipe SW, Yang A,
Ginsburg D: Biosynthetic origin
and functional significance of murine platelet factor V. Blood
|94. Gould WR, Silveira JR, Tracy PB:
Unique in vivo modifications
of coagulation factor V produce a physically and functionally distinct
platelet-derived cofactor: Characterization of purified platelet-derived
V/Va. J Biol Chem 279:2383, 2004.
|95. Hayward C: Multimerin: A
bench-to-bedside chronology of
a unique platelet and endothelial cell protein—From discovery
to function to abnormalities in disease. Clin Invest Med 20:176,
|96. Bertina RM, Koeleman BP, Koster T,
Mutation in blood coagulation factor V associated with resistance
to activated protein C. Nature 369:64, 1994.
|97. Cui J, O'Shea KS, Purkayastha A,
al: Fatal haemorrhage and incomplete block to embryogenesis in mice
lacking coagulation factor V. Nature 384:66, 1996.
|98. Shaw E, Giddings JC, Peake IR,
Bloom AL: Synthesis of procoagulant
factor VIII, factor VIII related antigen and other coagulation factors
by the isolated perfused rat liver. Br J Haematol 41:585,
|99. Hellman L, Smedsrod B, Sandberg
H, Pettersson U: Secretion
of coagulant factor VIII activity and antigen by in vitro cultivated
rat liver sinusoidal endothelial cells. Br J Haematol 73:348,
|100. Bontempo FA, Lewis JH, Gorenc TJ,
Liver transplantation in hemophilia A. Blood 69:1721,
|101. Marchioro TL, Hougie C, Ragde H,
Hemophilia: Role of organ homografts. Science 163:188,
|102. Kumaran V, Benten D, Follenzi A,
Transplantation of endothelial cells corrects the phenotype in
A mice. J Thromb Haemost 3:2022, 2005.
|103. Pipe S, Morris J, Shah J,
Kaufman R: Differential interaction
of coagulation factor VIII and factor V with protein chaperones
calnexin and calreticulin. J Biol Chem 273:8537,
|104. Swaroop M, Moussalli M, Pipe S,
Kaufman R: Mutagenesis
of a potential immunoglobulin-binding protein-binding site enhances
secretion of coagulation factor VIII. J Biol Chem 272:24121,
|105. Lollar P, Hill-Eubanks E, Parker
C: Association of the
FVIII light chain with von Willebrand factor. J Biol Chem
|106. Gitschier J, Wood W, Goralka T,
Characterization of the human factor VIII gene. Nature 312:326,
|107. Bonthron D, Handin R, Kaufman R,
Structure of pre-pro-von Willebrand factor and its expression in
heterologous cells. Nature 324:270, 1986.
|108. Colombatti A, Bonaldo P: The
superfamily of proteins with
von Willebrand factor type A-domains: One theme common to components
of extracellular matrix, hemostasis, cellular adhesion, and defense
mechanisms. Blood 77:2305,
|109. Foster P, Fulcher C, Marti T,
et al: A
major factor VIII binding domain resides within the amino-terminal
272 amino acid residues of von Willebrand factor. J Biol
Chem 262:8443, 1987.
|110. Dong JF, Moake JL, Nolasco L,
et al: ADAMTS-13
rapidly cleaves newly secreted ultralarge von Willebrand factor
multimers on the endothelial surface under flowing conditions. Blood
|111. Zimmerman T, Roberts J,
Edgington T: Factor VIII-related
antigen: Multiple molecular forms in human plasma. Proc
Natl Acad Sci U S A 72:5121, 1975.
|112. Mancuso D, Tuley E, Westfield L,
Structure of the gene for human von Willebrand factor. J
Biol Chem 264:19514, 1989.
|113. Fujikawa K, Chung DW: Factor XI,
in Molecular Basis
of Thrombosis and Hemostasis, edited by KA High, HR Roberts,
p 257. Marcel Dekker, New York, 1995. |
|114. Baglia FA,
Seaman FS, Walsh PN: The Apple 1 and Apple 4
domains of factor XI act synergistically to. Blood 85:2078,
|115. Baglia FA, Jameson BA, Walsh PN:
Identification and characterization
of a binding site for platelets in the Apple 3 domain of coagulation
factor XI. J Biol Chem 270:6734, 1995.
|116. Baglia FA, Jameson BA, Walsh PN:
Identification and characterization
of a binding site for factor XIIa in the Apple 4 domain of coagulation
factor XI. J Biol Chem 268:3838, 1993.
|117. Baglia FA, Walsh PN: A binding
site for thrombin in the
apple 1 domain of factor XI. J Biol Chem 271:3652,
|118. Kato A, Asaki R, Davie E, Aoki
N: Factor XI gene (F11)
is located on the distal end of the long arm of chromosome 4. Cytogenet
Cell Genet 52:77, 1989.
|119. Asakai R, Davie E, Chung D:
Organization of the gene for
human factor XI. Biochemistry 26:7221, 1987.
|120. Tarumi T, Kravtsov DV, Zhao M,
Cloning and characterization of the human factor XI gene promoter:
Transcription factor hepatocyte nuclear factor 4alpha (HNF-4alpha)
is required for hepatocyte-specific expression of factor XI. J
Biol Chem 277:18510, 2002.
|121. Gailani D, Broze Jr. GJ: Factor
XI activation in a revised
model of blood coagulation. Science 253:909, 1991.
|122. Naito K, Fujikawa K: Activation
of human blood coagulation
factor XI independent of factor XII. Factor XI is activated by thrombin
and factor XIa in the presence of negatively charged surfaces. J
Biol Chem 266:7353, 1991.
|123. Oliver J, Monroe D, Roberts H,
Hoffman M: Thrombin activates
factor XI on activated platelets in the absence of factor XII. Arterioscler
Thromb Vasc Biol 19:170, 1999.
|124. Rosen ED, Gailani D, Castellino
FJ: FXI is essential for
thrombus formation following FeCl3-induced injury of the carotid
artery in the mouse. Thromb Haemost 87:774, 2002.
|125. Gailani D, Lasky NM, Broze GJ
Jr: A murine model of factor
XI deficiency. Blood Coagul Fibrinolysis 8:134,
|126. Ragni MV, Sinha D, Seaman F, et
of bleeding tendency, factor XI coagulant activity, and factor XI
antigen in 25 factor XI-deficient kindreds. Blood 65:719,
|127. Sinha D, Seaman FS, Walsh PN:
Role of calcium ions and
the heavy chain of factor XIa in the activation of human coagulation
factor IX. Biochemistry 26:3768, 1987.
|128. Sinha D, Seaman FS, Koshy A, et
coagulation factor XIa binds specifically to a site on activated
human platelets distinct from that for factor XI. J Clin
Invest 73:1550, 1984.
|129. Knauer DJ, Majumdar D, Fong PC,
Knauer MF: SERPIN regulation
of factor XIa. The novel observation that protease nexin 1 in the
presence of heparin is a more potent inhibitor of factor XIa than
C1 inhibitor. J Biol Chem 275:37340, 2000.
|130. Cronlund AL, Walsh PN: A low
molecular weight platelet
inhibitor of factor XIa: Purification, characterization, and possible
role in blood coagulation. Biochemistry 31:1685,
|131. Saito H, Kojima T: Factor XII,
prekallikrein and high-molecular-weight
kininogen, in Molecular Basis of Thrombosis and Hemostasis,
edited by KA High, HR Roberts, p 269. Marcel Dekker, New York, 1995. |
|132. Yu H, Anderson PJ, Freedman BI, et al:
Genomic structure of the human plasma prekallikrein gene, identification
of allelic variants, and analysis in end-stage renal disease. Genomics
|133. Colman RW: Biologic activities
of the contact factors in
vivo—potentiation of hypotension, inflammation, and fibrinolysis,
and inhibition of cell adhesion, angiogenesis and thrombosis. Thromb
Haemost 82:1568, 1999.
|134. Schmaier AH: The plasma
kallikrein-kinin system counterbalances
the renin-angiotensin system. J Clin Invest 109:1007,
|135. Pauer HU, Renne T, Hemmerlein B,
Targeted deletion of murine coagulation factor XII gene—A
model for contact phase activation in vivo. Thromb
Haemost 92:503, 2004.
|136. Renne T, Gailani D: Role of
Factor XII in hemostasis and
thrombosis: Clinical implications. Expert Rev Cardiovasc
Ther 5:733, 2007.
|137. Kleinschnitz C, Stoll G,
Bendszus M, et
al: Targeting coagulation factor XII provides protection from
thrombosis in cerebral ischemia without interfering with hemostasis. J
Exp Med 203:513, 2006.
|138. Castaman G, Ruggeri M, Tosetto A,
et al: Thrombosis
in patients with heterozygous and homozygous factor XII deficiency
is not explained by the associated presence of factor V Leiden. Thromb
Haemost 76:275, 1996.
|139. Dyerberg J, Stoffersen E:
Recurrent thrombosis in a patient
with factor XII deficiency. Acta Haematol 63:278,
|140. Mahdi F, Madar ZS, Figueroa CD,
Factor XII interacts with the multiprotein assembly of urokinase
plasminogen activator receptor, gC1qR, and cytokeratin 1 on endothelial
cell membranes. Blood 99:3585, 2002.
|141. Shariat-Madar Z, Mahdi F,
Schmaier AH: Assembly and activation
of the plasma kallikrein/kinin system: A new interpretation. Int
Immunopharmacol 2:1841, 2002.
|142. Morrissey JH, Fakhrai H,
Edgington TS: Molecular cloning
of the cDNA for tissue factor, the cellular receptor for the initiation
of the coagulation protease cascade. Cell 50:129,
|143. Dorfleutner A, Ruf W: Regulation
of tissue factor cytoplasmic
domain phosphorylation by palmitoylation. Blood 102:3998,
|144. Martin D, Boys C, Ruf W: Tissue
factor: Molecular recognition
and cofactor function. FASEB J 9:852, 1995.
|145. Minazzo AS, Darlington RC, Ross
JB: Loop dynamics of the
extracellular domain of human tissue factor and activation of factor
VIIa. Biophys J 96:681, 2009.
|146. Kao F-T, Hartz J, Horton R, et
assignment of human tissue factor gene (F3) to chromosome 1p21–22. Somat
Cell Mol Genet 14:407, 1988.
|147. Mackman N, Morrissey JH, Fowler
B, Edgington TS: Complete
sequence of the human tissue factor gene, a highly regulated cellular
receptor that initiates the coagulation protease cascade. Biochemistry
|148. Bogdanov VY, Balasubramanian V,
Hathcock J, et
al: Alternatively spliced human tissue factor: A circulating, soluble,
thrombogenic protein. Nat Med 9:458, 2003.
|149. Bogdanov VY, Kirk RI, Miller C,
Identification and characterization of murine alternatively spliced
tissue factor. J Thromb Haemost 4:158, 2006.
|150. Mackman N, Fowler B, Edgington
TS, Morrissey JH: Functional
analysis of the human tissue factor promoter and induction by serum. Proc
Natl Acad Sci U S A 87:2254, 1990.
|151. Gregory SA, Morrissey JH,
Edgington TS: Regulation of tissue
factor gene expression in the monocyte procoagulant response to
endotoxin. Mol Cell Biol 9:2752, 1989.
|152. Schecter AD, Rollins BJ, Zhang
al: Tissue factor is induced by monocyte chemoattractant protein-1
in human aortic smooth muscle and THP-1 cells. J Biol Chem
|153. Conway EM, Bach R, Rosenberg RD,
Konigsberg WH: Tumor necrosis
factor enhances expression of tissue factor mRNA in endothelial
cells. Thromb Res 53:231, 1989.
|154. Hoffman M, Cooper S: Thrombin
enhances monocyte secretion
of tumor necrosis factor and Interleukin-1 beta by two distinct
mechanisms. Blood Cells Mol Dis 21:156, 1995.
|155. Key NS: Platelet tissue factor:
How did it get there and
is it important? Semin Hematol 45: S16, 2008. |
|156. Drake TA, Morrissey JH, Edgington TS: Selective
expression of tissue factor in human tissues. Implications for disorders
of hemostasis and thrombosis. Am J Pathol 134:1087,
|157. Eddleston M, de la Torre J,
Oldstone M, et
al: Astrocytes are the primary source of tissue factor in the murine
central nervous system. A role for astrocytes in cerebral hemostasis. J
Clin Invest 92:349, 1993.
|158. Giesen PLA, Rauch U, Bohrmann B,
et al: Blood-borne
tissue factor: Another view of thrombosis. Proc Natl Acad
Sci U S A 96:2311, 1999.
|159. Hoffman M, Colina CM, McDonald
al: Tissue factor around dermal vessels has bound factor VII in
the absence of injury. J Thromb Haemost 5:1403,
|160. Shigematsu Y, Miyata T, Higashi
S: Expression of human
soluble tissue factor in yeast and enzymatic properties of its complex
with factor VIIa. J Biol Chem 267:21329, 1992.
|161. Lawson JH, Butenas S, Mann KG:
The evaluation of complex-dependent
alterations in human factor VIIa. J Biol Chem 267:4834,
|162. Østerud B, Rapaport SI:
Activation of factor IX by
the reaction product of tissue factor and factor VII: Additional
pathway for initiating blood coagulation. Proc Natl Acad
Sci U S A 74:5260, 1977. |
Neuenschwander PF, Morrissey JH: Roles of the membrane-interactive
regions of factor VIIa-tissue factor. J Biol Chem 269:8007,
|164. Neuenschwander PF, Morrissey JH:
Deletion of the membrane
anchoring region of tissue factor abolishes autoactivation of factor
VII but not cofactor function. Analysis of a mutant with a selective
deficiency in activity. J Biol Chem 267:14477,
|165. Krishnaswamy S, Field KA,
Edgington TS, et
al: Role of the membrane surface in the activation of human coagulation
factor X. J Biol Chem 267:26110, 1992.
|166. Bach R, Moldow C: Mechanism of
tissue factor activation
on HL-60 cells. Blood 89:3270, 1997.
|167. Bach R, Rifkin DB: Expression of
tissue factor procoagulant
activity: Regulation by cytosolic calcium. Proc Natl Acad
Sci U S A 87:6995, 1990.
|168. Versteeg HH, Sorensen BB,
Slofstra SH, et
al: VIIa/tissue factor interaction results in a tissue
factor cytoplasmic domain-independent activation of protein synthesis,
p70, and p90 S6 kinase phosphorylation. J Biol Chem 277:27065,
|169. Riewald M, Ruf W: Mechanistic
coupling of protease signaling
and initiation of coagulation by tissue factor. Proc Natl
Acad Sci U S A 98:7742, 2001.
|170. Mackman N: Role of tissue factor
in hemostasis, thrombosis,
and vascular development. Arterioscler Thromb Vasc Biol 24:1015,
|171. Wen D, Dittman W, Ye R, et
al: Human thrombomodulin:
Complete cDNA sequence and chromosome localization of the gene. Biochemistry
|172. Esmon N, Owen W, Esmon C:
Isolation of a membrane-bound
cofactor for thrombin-catalyzed activation of protein C. J
Biol Chem 257:859, 1982.
|173. Patthy L: Detecting distant
homologies of mosaic proteins:
Analysis of thrombomodulin, thrombospondin, complement components
C9, C8 alpha, C8 beta, vitronectin and plasma cell membrane glycoprotein
PC-1. J Mol Biol 202:689, 1988.
|174. Stearns D, Kurosawa S, Esmon C:
310–486 from the epidermal growth factor homology domain
of rabbit thrombomodulin will accelerate protein C activation. J
Biol Chem 264:3352, 1989.
|175. Parkinson J, Vlahos C, Yan S,
Bang N: Recombinant human
thrombomodulin: Regulation of cofactor activity and anticoagulant
function by a glycosaminoglycan side chain. Biochem J 283:151,
|176. Espinosa R, Sadler J, LeBeau M:
Regional localization of
the human thrombomodulin gene to 20p12-cen. Genomics 5:649,
|177. Esmon C, Esmon N, Hams K:
Complex formation between thrombin
and thrombomodulin inhibits both thrombin-catalyzed fibrin formation
and factor V activation. J Biol Chem 257:7944,
|178. Cadroy Y, Diquelou A, Dupouy D,
The thrombomodulin/protein C/protein S anticoagulant pathway
modulates the thrombogenic properties of the normal resting and
stimulated endothelium. Arterioscler Thromb Vasc Biol 17:520,
|179. Healy AM, Rayburn HB, Rosenberg
RD, Weiler H: Absence of
the blood-clotting regulator thrombomodulin causes embryonic lethality
in mice before development of a functional cardiovascular system. Proc
Natl Acad Sci U S A 92:850, 1995.
|180. Gu JM, Crawley JT, Ferrell G,
et al: Disruption of
the endothelial cell protein C receptor gene in mice causes placental
thrombosis and early embryonic lethality. J Biol Chem 277:43335,
|181. Verhagen HJ, Heijnen-Snyder GJ,
Pronk A, et al:
Thrombomodulin activity on mesothelial cells: Perspectives for
cells as an alternative for endothelial cells for cell seeding on
vascular grafts. Br J Haematol 95:542, 1996.
|182. McCachren SS, Diggs J, Weinberg
JB, Dittman WA: Thrombomodulin
expression by human blood monocytes and by human synovial tissue
lining macrophages. Blood 78:3128, 1991.
|183. Raife TJ, Demetroulis EM, Lentz
SR: Regulation of thrombomodulin
expression by all-trans retinoic acid and tumor necrosis factor-alpha:
Differential responses in keratinocytes and endothelial cells. Blood
|184. Ishii H, Nakana M, Tsubouchi J,
Distribution of thrombomodulin in human tissues and characterization
of thrombomodulin in plasma. Nippon Ketsueki Gakkai Zasshi
|185. Dichek D, Quertermous T:
Variability in mRNA levels in
HUVECs of different lineage and time in culture. In Vitro
Cell Dev Biol 25:289, 1989.
|186. Rezaie A, Cooper S, Church F,
Esmon C: Protein C inhibitor
is a potent inhibitor of the thrombin-thrombomodulin complex. J
Biol Chem 270:25336, 1995.
|187. Neerman-Arbez M: The molecular
basis of inherited afibrinogenaemia. Thromb
Haemost 86:154, 2001.
|188. Hanss M, Biot F: A database for
human fibrinogen variants. Ann
N Y Acad Sci 936:89, 2001.
|189. Carrell N, McDonagh J.
Functional defects in abnormal fibrinogens,
in Fibrinogen: Structural Variants and Interaction,
edited by A Henschen, B Hesse, J McDonagh, T Saldeen, p 155. Walter
DeGruyter, Berlin, 1985. |
|190. Egeberg O:
Inherited fibrinogen abnormality causing thrombophilia. Thromb
Diath Haemorrh 17:176, 1967.
|191. Ni H, Papalia JM, Degen JL,
Wagner DD: Control of thrombus
embolization and fibronectin internalization by integrin alpha IIb
beta 3 engagement of the fibrinogen gamma chain. Blood 102:3609,
|192. Gardlund B, Hessel B, Marguerie G,
al: Primary structure of human fibrinogen. Characterization of
cyanogen-bromide fragments. Eur J Biochem 77:595,
|193. Blomback B: Studies on the
action of thrombotic enzymes
on bovine fibrinogen as measured by N-terminal analysis. Ark
Kemi 12:321, 1958. |
|194. Blomback B,
Blomback M, Henschen A, et
al: N-terminal disulfide knot of human fibrinogen. Nature
|195. Doolittle RF: Determining the
crystal structure of fibrinogen. J
Thromb Haemost 2:683, 2004.
|195a. Côté HC, Lord ST, Pratt KP:
Molecular structure-function relationships of naturally occurring
mutations in the gamma chain of human fibrinogen. Blood 92:2195,
|196. Henschen AH: Human
and functional sites. Thromb Haemost 70:42, 1993.
|197. Mosesson MW, Cooley BC,
Hernandez I, et
al: Thrombosis risk modification in transgenic mice containing the
human fibrinogen thrombin-binding gamma' chain
sequence. J Thromb Haemost 7:102, 2009.
|198. Uitte de Willige S, de Visser
MC, Houwing-Duistermaat JJ, et
al: Genetic variation in the fibrinogen gamma gene increases the
risk for deep venous thrombosis by reducing plasma fibrinogen gamma'
levels. Blood 106:4176,
|199. Mannila MN, Lovely RS,
Kazmierczak SC, et
al: Elevated plasma fibrinogen gamma' concentration
is associated with myocardial infarction: Effects of variation in
fibrinogen genes and environmental factors. J Thromb Haemost
|200. Collen D, Tytgat C, Claeys H:
Metabolism and distribution
of fibrinogen I. Fibrinogen turnover in physiological conditions
in humans. Br J Haematol 22:681, 1972.
|201. Reeve K, Franks J: Fibrinogen
synthesis, distribution and
degradation. Semin Thromb Hemost 1:129, 1974. |
|202. Fuller G, Otto J, Woloski B: The effects of
factor on fibrinogen biosynthesis in hepatocyte monolayers. J
Cell Biol 101:1481, 1985.
|203. Huber P, Laurent M, Dalmon J:
Human beta-fibrinogen gene
expression. Upstream sequences involved in its tissue specific
and its dexamethasone and interleukin-6 stimulation. J Biol
Chem 265:5695, 1990.
|204. Chung D, Harris I, Davie E:
Nucleotide sequences of the
three genes coding for human fibrinogen, in Advances in
Experimental Medicine and Biology, edited by C Liu, S Chien
S, p 39. Plenum, New York, 1990. |
R, Watt KW, Cottrell BA, et al: The amino acid
sequence of the alpha-chain of human fibrinogen. Nature 280:464,
|206. Kant I, Fornace A, Saxe D:
Evolution and organization of
the fibrinogen locus on chromosome 4: Gene duplication accompanied
by transposition and inversion. Proc Natl Acad Sci U S A 82:2344,
|207. Morgan J, Courtois G, Fourel G:
Spl, a CAAT binding factor
and the adenovirus major late promoter transcription factor interact
with functional regions of the gamma-fibrinogen promoter. Mol
Cell Biol 8:2628, 1988.
|208. Courtois G, Morgan J, Campbell L,
Interaction of a liver-specific nuclear factor with the fibrinogen
and a1 antitrypsin promoters. Science 238:688,
|209. Dalmon J, Laurent M, Courtois G:
The human b fibrinogen
promoter contains a HAF-1 dependent IL-6 responsive element. Mol
Cell Biol 13:1183, 1993.
|210. Haidaris P, Courtney M:
Molecular biology and regulation
of the fibrinogen gene: Tissue-specific and ubiquitous expression
of fibrinogen gamma-chain mRNA. Blood Coagul Fibrinolysis 1:433,
|211. Handagama PJ, Shuman MA, Bainton
DF: In vivo defibrination
results in markedly decreased amounts of fibrinogen in rat
and platelets. Am J Pathol 137:1393, 1990.
|212. Louache F, Debili N, Cramer E,
Fibrinogen is not synthesized by human megakaryocytes. Blood
|213. Vali Z, Scheraga H: Localization
of the binding site on
fibrin for the secondary binding site of thrombin. Biochemistry
|214. Olexa S, Budzynaski A: Evidence
for four different polymerization
sites involved in human fibrin formation. Proc Natl Acad
Sci U S A 77:1374, 1980.
|215. Kaczmarek E, McDonagh J:
Thrombin binding to the A alpha-,
B beta-, and gamma-chains of fibrinogen and to their remnants contained
in fragment E. J Biol Chem 263:13896, 1988.
|216. Weisel I, Phillips G, Cohen C:
The structure of fibrinogen
and fibrin: II. Architecture of the fibrin clot. Ann N Y
Acad Sci 408:367, 1983.
|217. Hantgan R, Fowler R, Erickson H,
Hermans J: Fibrin assembly:
A comparison of electron microscopic and light scattering results. Thromb
Haemost 44:119, 1980.
|218. Dang C, Shin C, Bell W:
Fibrinogen sialic acid residues
are low affinity calcium-binding sites that influence fibrin assembly. J
Biol Chem 264:1989, 1989. |
Nieuwenhuizen W, van Ruijven-Vermneer J, Nooijen W: Recalculation
of calcium-binding properties of human and rat fibrin(ogen) and
their degradation products. Thromb Res 22:653,
|220. Marder V, Budzynski A:
Degradation products of fibrinogen
and crosslinked fibrin: Projected clinical applications. Thromb
Diath Haemorrh 32:49, 1974.
|221. Elms M, Bunce I, Bundesen P, et
of cross-linked fibrin degradation products: An immunoassay using
monoclonal antibodies. Thromb Haemost 50:591, 1983.
|222. Hermans J, McDonagh J: Fibrin:
Structure and interactions. Semin
Thromb Hemost 8:11, 1982.
|223. Williams J, Hantgan R, Hermanns
J, McDonagh J: Characterization
of the inhibition of fibrin assembly by fibrinogen fragment D. Biochemistry
|224. Mosesson MW: Update on
antithrombin I (fibrin). Thromb
Haemost 98:105, 2007.
|225. Lai T-S, Greenberg C: Factor
XIII, in Molecular
Basis of Thrombosis and Hemostasis, edited by KA High,
HR Roberts, p 287. Marcel Dekker, New York, 1995. |
|226. Bottenus R, Ichinose A, Davie E: Nucleotide
the gene for the b subunit of human factor XIII. Biochemistry
|227. Ichinose A, Davie E:
Characterization of the gene for the
a subunit of human factor XIII (plasma transglutaminase) a blood
coagulation factor. Proc Natl Acad Sci U S A 85:5829,
|228. Ichinose A: Amino acid sequence
of the b subunit of human
factor XIII, a protein composed of 109 repetitive segments. Biochemistry
|229. Ichinose A, Bottenus R, Davie E:
Structure of transglutaminase. J
Biol Chem 265:13411, 1990.
|230. Henricksson P, Becker S,
McDonagh J: Identification of
intracellular factor XIII in human monocytes and macrophages. J
Clin Invest 76:528, 1985. |
|231. McDonagh J,
McDonagh R, Deleage J, Wagner R: Factor XIII
in human plasma and platelets. J Clin Invest 48:940,
|232. Weisberg L, Shiu D, Greenberg C,
Localization of the gene for coagulation factor XIII a-chain to
chromosome 6 and identification of sites of synthesis. J
Clin Invest 79:649, 1987.
|233. Kida M, Souri M, Yamamoto M, et
regulation of cell type-specific expression of the TATA-less A subunit
gene for human coagulation factor XIII. J Biol Chem 274:6138,
|234. Lewis KB, Teller DC, Fry J, et
al: Crosslinking kinetics
of the human transglutaminase, factor XIII[A2],
acting on fibrin gels and gamma-chain peptides. Biochemistry
|235. Sakata Y, Aoki N: Cross-linking
of alpha 2-plasmin inhibitor
to fibrin by fibrin-stabilizing factor. J Clin Invest 65:290,
|236. Boffa MB, Reid TS, Joo E, et
of the gene encoding human TAFI (thrombin-activatable fibrinolysis
inhibitor; plasma procarboxypeptidase B). Biochemistry 38:6547,
|237. Boffa MB, Hamill JD, Bastajian N,
A role for CCAAT/enhancer-binding protein in hepatic expression
of thrombin-activatable fibrinolysis inhibitor. J Biol Chem
|238. Henry M, Aubert H, Morange PE,
Identification of polymorphisms in the promoter and the 3' region
of the TAFI gene: Evidence that plasma TAFI antigen levels are strongly
genetically controlled. Blood 97:2053, 2001.
|239. Koschinsky ML, Boffa MB, Nesheim
al: Association of a single nucleotide polymorphism in CPB2 encoding
the thrombin-activatable fibrinolysis inhibitor (TAF1) with blood
pressure. Clin Genet 60:345, 2001.
|240. Schneider M, Boffa M, Stewart R,
Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ
substantially with respect to thermal stability and antifibrinolytic
activity of the enzyme. J Biol Chem 277:1021, 2002.
|241. Broze GJ Jr: Protein Z-dependent
regulation of coagulation. Thromb
Haemost 86:8, 2001.
|242. Broze GJ Jr, Warren LA, Novotny
al: The lipoprotein-associated coagulation inhibitor that inhibits
the factor VII-tissue factor complex also inhibits factor Xa: Insight
into its possible mechanism of action. Blood 71:335,
|243. Warn-Cramer B, Rao L, Maki S,
Rapaport SI: Modifications
of extrinsic pathway inhibitor (EPI) and factor Xa that affect their
ability to interact and to inhibit factor VIIa/tissue factor:
Evidence for a two-step model of inhibition. Thromb Haemost
|244. Ameri A, Kuppuswamy M, Basu S,
Bajaj S: Expression of tissue
factor pathway inhibitor by cultured endothelial cells in response
to inflammatory mediators. Blood 79:3219, 1992.
|245. Sandset P, Abildgaard U, Larsen
M: Heparin induces release
of extrinsic coagulation pathway inhibitor (EPI). Thromb
Res 50:803, 1988.
|246. Zhang J, Piro O, Lu L, Broze GJ
Jr: Glycosyl phosphatidylinositol
anchorage of tissue factor pathway inhibitor. Circulation
|247. Chang J-Y, Monroe DM, Oliver JA,
Roberts HR: TFPIbeta,
a second product from the mouse tissue factor pathway inhibitor
(TFPI) gene. Thromb Haemost 81:45, 1999.
|248. Griffith MJ: Measurement of the
III/thrombin reaction rate in the presence of synthetic
substrates. Thromb Res 25:245, 1982.
|249. Fuchs HE, Trapp HG, Griffith MJ,
Regulation of Factor IXa in vitro in human and
mouse plasma and in vivo in the mouse. J
Clin Invest 73:1696, 1984.
|250. Sheffield W, Wu Y, Blajchman M:
and function, in Molecular Basis of Thrombosis and Hemostasis,
edited by KA High, HR Roberts, p 355. Marcel Dekker, New York, 1995. |
|251. Hamamoto T, Kisiel W: The effect of cell surface
(GAGs) on the inactivation of factor VIIa—Tissue factor
activity by antithrombin III. Int J Hematol 68:67,
|252. Rao LV, Rapaport SI, Hoang AD:
Binding of factor VIIa to
tissue factor permits rapid antithrombin III/heparin inhibition
of factor VIIa. Blood 81:2600, 1993.
|253. Pizzo S: Serpin receptor 1: A
hepatic receptor that mediates
the clearance of antithrombin II protease complexes. Am
J Med 87:10S, 1989. |
|254. Ochoa A,
Brunel F, Mendelson D, et al:
Different liver nuclear proteins bind to similar DNA sequences in
the 5' flanking regions of three hepatic genes. Nucleic
Acids Res 17:116, 1989. |
|255. Han X,
Fiehler R, Broze GJ Jr: Isolation of a protein Z-dependent
plasma protease inhibitor. Proc Natl Acad Sci U S A 95:9250,
|256. Tabatabai A, Fiehler R, Broze GJ
Jr: Protein Z circulates
in plasma in a complex with protein Z-dependent protease inhibitor. Thromb
Haemost 85:655, 2001.
|257. Yin ZF, Huang ZF, Cui J, et
al: Prothrombotic phenotype
of protein Z deficiency. Proc Natl Acad Sci U S A 97:6734,
|258. MacFarlane RG: An enzyme cascade
in the blood clotting
mechanism, and its function as a biological amplifier. Nature
|259. Davie EW, Ratnoff OD: Waterfall
sequence for intrinsic
blood clotting. Science 145:1310, 1964.
|260. Nemerson Y, Esnouf MP:
Activation of a proteolytic system
by a membrane lipoprotein: Mechanism of action of tissue factor. Proc
Natl Acad Sci U S A 70:310, 1973.
|261. Nemerson Y: The tissue factor
pathway of blood coagulation. Semin
Hematol 29:170, 1992.
|262. Repke D, Gemmell CH, Guha A, et
as a defect of the tissue factor pathway of blood coagulation: Effect
of factors VIII and IX on factor X activation in a continuous-flow
reactor. Proc Natl Acad Sci U S A 87:7623, 1990.
|263. Broze GJ Jr, Girard TJ, Novotny
WF: Regulation of coagulation
by a multivalent Kunitz-type inhibitor. Biochemistry 29:7539,
|264. Hockin MF, Kalafatis M, Shatos
M, Mann KG: Protein C activation
and factor Va inactivation on human umbilical vein endothelial cells. Arterioscler
Thromb Vasc Biol 17:2765, 1997.
|265. Fay PJ, Smudzin TM, Walker FJ:
Activated protein C-catalyzed
inactivation of human factor VIII and VIIIa. J Biol Chem
|266. Pieters J, Willems G, Hemker HC,
Lindhout T: Inhibition
of factor IXa and factor Xa by antithrombin III/heparin
during factor X activation. J Biol Chem 263:15313,
|267. Allen DH, Tracy PB: Human
coagulation factor V is activated
to the functional cofactor by elastase and cathepsin G expressed
at the monocyte surface. J Biol Chem 270:1408,
|268. Monroe DM, Hoffman M, Roberts
HR: Transmission of a procoagulant
signal from tissue factor-bearing cells to platelets. Blood
Coagul Fibrinolysis 7:459, 1996.
|269. Eilertsen KE, Osterud B: Tissue
and cellular biology. Blood Coagul Fibrinolysis 15:521,
|270. Censarek P, Bobbe A, Grandoch M,
Alternatively spliced human tissue factor (asHTF) is not pro-coagulant. Thromb
Haemost 97:11, 2007.
|271. Schwertz H, Tolley ND, Foulks JM,
Signal-dependent splicing of tissue factor pre-mRNA modulates the
thrombogenicity of human platelets. J Exp Med 203:2433,
|272. Hoffman M, Whinna HC, Monroe DM:
Circulating tissue factor
accumulates in thrombi, but not in hemostatic plugs. J Thromb
Haemost 4:2092, 2006.
|273. Biro E, Sturk-Maquelin KN, Vogel
al: Human cell-derived microparticles promote thrombus formation in
vivo in a tissue factor-dependent manner. J Thromb
Haemost 1:2561, 2003.
|274. Chou J, Mackman N,
Merrill-Skoloff G, et
al: Hematopoietic cell-derived microparticle tissue factor contributes
to fibrin formation during thrombus propagation. Blood 104:3190,
|275. Day SM, Reeve JL, Pedersen B,
et al: Macrovascular
thrombosis is driven by tissue factor derived primarily from the
blood vessel wall. Blood 105:192, 2005.
|276. Falati S, Gross P,
Merrill-Skoloff G, et
al: Real-time in vivo imaging of platelets, tissue factor
and fibrin during arterial thrombus formation in the mouse. Nat
Med 8:1175, 2002.
|277. Williamson P, Bevers EM, Smeets
al: Continuous analysis of the mechanism of activated transbilayer
lipid movement in platelets. Biochemistry 34:10448,
|278. Monroe DM, Roberts HR, Hoffman
M: Platelet procoagulant
complex assembly in a tissue factor-initiated system. Br
J Haematol 88:364, 1994.
|279. Ahmad SS, Rawala-Sheikh R, Walsh
PN: Platelet receptor
occupancy with factor IXa promotes factor X activation. J
Biol Chem 264:20012, 1989.
|280. Ahmad SS, Rawala-Sheikh R, Ashby
B, Walsh PN: Platelet
receptor-mediated factor X activation by factor IX. High-affinity
factor IXa receptors induced by factor VIII are deficient on platelets
in Scott syndrome. J Clin Invest 84:824, 1998. |
|280a. Kravtsov DV, Matafonov A, Tucker EI: Factor XI
to thrombin generation in the absence of factor XII. Blood
|281. Baglia FA, Walsh PN: Prothrombin
is a cofactor for the
binding of factor XI to the platelet surface and for platelet-mediated
factor XI activation by thrombin. Biochemistry 37:2271,
|282. von dem Borne PA, Bajzar L,
Meijers JC, et
al: Thrombin-mediated activation of factor XI results in a
fibrinolysis inhibitor-dependent inhibition of fibrinolysis. J
Clin Invest 99:2323, 1997. |
|283. Alberio L,
Safa O, Clemetson KJ, et al:
Surface expression and functional characterization of alpha-granule
factor V in human platelets: Effects of ionophore A23187, thrombin,
collagen, and convulxin. Blood 95:1694, 2000.
|284. Dale GL, Friese P, Batar P, et
al: Stimulated platelets
use serotonin to enhance their retention of procoagulant proteins
on the cell surface. Nature 415:175, 2002.
|285. Kempton CL, Hoffman M, Roberts
HR, Monroe DM: Platelet
heterogeneity: Variation in coagulation complexes on platelet
Thromb Vasc Biol 25:861, 2005.
|286. de Agostini A, Watkins S,
Slayter H, et
al: Localization of the anticoagulantly active heparan sulphate
proteoglycans in vascular endothelium: Antithrombin binding on cultured
endothelial cells and perfused rat aorta. J Cell Biol 111:1293,
|287. Marcus AJ, Broekman MJ,
Drosopoulos JHF, et al:
The endothelial cell ecto-ADPase responsible for inhibition of platelet
function is CD39. J Clin Invest 99:1351, 1997.
|288. Jesty J, Beltrami E, Willems G:
Mathematical analysis of
a proteolytic positive-feedback loop: Dependence of lag time and
enzyme yields on the initial conditions and kinetic parameters. Biochemistry
|289. Brakman P, Albrechtsen OK,
Astrup T: A comparative study
of coagulation and fibrinolysis in blood from normal men and women. Br
J Haematol 12:74, 1966.
|290. Nossel H, Yudelman I, Canfield
Rea: Measurement of fibrinopeptide
A in human blood. J Clin Invest 54:43, 1974.
|291. Bauer KA, Kass BL, ten Cate H,
Factor IX is activated in vivo by the tissue factor
mechanism. Blood 76:731, 1990.
|292. Bauer KA, Kass BL, ten Cate H,
Detection of factor X activation in humans. Blood 74:2007,
|293. Morrissey JH: Tissue factor
modulation of factor VIIa activity:
Use in measuring trace levels of factor VIIa in plasma. Thromb
Haemost 74:185, 1995.
|294. Jesty J, Beltrami E: Positive
feedbacks of coagulation:
Their role in threshold regulation. Arterioscler Thromb
Vasc Biol 25:2463, 2005.
|295. Conard J, Bauer KA, Gruber A,
et al: Normalization
of markers of coagulation activation with a purified protein C
in adults with homozygous protein C deficiency. Blood 82:1159,
|296. Harlos K, Martin DM, O'Brien DP,
al: Crystal structure of the extracellular region of human tissue
factor. Nature 370:662,
|297. Hoffman M, Harger A, Lenkowski A,
Cutaneous wound healing is impaired in hemophilia B. Blood