|
|
Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 3983-3996
REVIEW ARTICLE
The Life Cycle of Coagulation Factor VIII in View of Its Structure
and Function
By
Peter J. Lenting,
Jan A. van Mourik, and
Koen Mertens
From the Departments of Plasma Protein Technology and Blood
Coagulation, CLB, Sanquin Blood Supply Foundation, Amsterdam, The
Netherlands.
 |
INTRODUCTION |
THE PAST TWO DECADES have brought
remarkable progress in our understanding of the molecular basis of
hemophilia A. This disease, which has already been documented as a
familial bleeding tendency in the fifth century,1 still
persists as the most common hemorrhagic disorder, affecting 1 in
approximately 5,000 males.2 Hemophilia has been associated
with deficiency of a plasma component since 1937, when Patek and
Taylor3 showed that the clotting defect of hemophilic
plasma could be corrected by plasma of a normal individual. This
component was called "antihemophilic factor," or "factor
VIII" according to the more recent nomenclature. Subsequent studies
using preparations enriched in factor VIII activity have established
factor VIII as being the cofactor of activated factor IX in the factor
X-activating complex of the intrinsic coagulation
pathway.4 However, the molecular entity of factor VIII has
remained unidentified until the early 1980s, when the protein was
purified to complete homogeneity, and its cDNA was
cloned.5-8 This breakthrough has triggered numerous studies
on the genetic and molecular basis of hemophilia A, and consequently
our knowledge on the structure and function of the factor VIII protein
has been rapidly expanding since then.
The present review focuses on the "life cycle" of factor VIII,
which comprises the sequence of events between biosynthesis and
clearance of the protein (see Fig 1). These
processes are discussed in view of our current knowledge on factor VIII
structure and function, with particular reference to the proteolytic
modulation of factor VIII, and its assembly into the factor X
activating complex.
View larger version (20K):
[in this window]
[in a new window]
| Fig 1.
The lifespan of factor VIII. Factor VIII is synthesized
by various tissues, including liver, kidney, and spleen, as an inactive
single-chain protein. After extensive posttranslational processing,
factor VIII is released into the circulation as a set of heterodimeric
proteins. This heterogenous population of factor VIII molecules readily
interacts with vWF, which is produced and secreted by vascular
endothelial cells. Upon triggering of the coagulation cascade and
subsequent generation of serine proteases, factor VIII is subject to
multiple proteolytic cleavages. These cleavages are associated with
dramatic changes of the molecular properties of factor VIII, including
dissociation of vWF and development of biological activity. After
conversion into its active conformation, and participation in the
factor X activating complex, activated factor VIII rapidly looses its
activity. This process is governed by both enzymatic degradation and
subunit dissociation.
|
|
| |
BIOSYNTHESIS AND SECRETION OF FACTOR VIII |
Factor VIII gene.
The gene of factor VIII is located at the tip of the long arm of the X
chromosome.5 It spans over 180 kb, and as such is one of
the largest genes known. Its transcription may require several hours
assuming a transcription rate of 10 nucleotides per second, and yields
a 9-kb mRNA product.5 The factor VIII gene comprises 26 exons, which encode a polypeptide chain of 2351 amino
acids.6-8 This includes a signal peptide of 19 and a mature protein of 2332 amino acids. Analysis of the deduced primary structure determined from the cloned factor VIII cDNA showed the presence of a
discrete domain structure:
A1-a1-A2-a2-B-a3-A3-C1-C26-8 (Fig
2). The A domains display approximately 30% homology to each other.
These domains further display a similar extent of homology to the
copper-binding protein ceruloplasmin and to factor V, the cofactor in
the prothrombinase complex.9 The A domains are bordered by
short spacers (a1, a2, and a3) that contain
clusters of Asp and Glu residues, the so-called acidic regions. The C
domains are structurally related to the C domains of factor V. In
addition, the lipid-binding lectin discoidin I, human and murine milk
fat globule proteins, and a putative neuronal cell adhesion molecule from Xenopus laevis share amino acid sequence similarity to the factor VIII C domains.10-12 The B domain is unique in that
it exhibits no significant homology with any other known protein.
Biosynthesis of factor VIII.
Several tissues have the potential of expressing the factor VIII gene.
Factor VIII mRNA has been demonstrated in a variety of tissues,
including spleen, lymph nodes, liver, and kidney.13-15 Transplantation studies in hemophilic animals showed that organs such
as lung and spleen indeed contribute to the presence of circulating factor VIII.16,17 However, the liver most likely provides
the primary source of factor VIII. This view is supported by liver perfusion18,19 and by liver transplantation studies in both animals and humans.20-22 A number of cases concerning
hemophilic patients have been reported in which transplantation
resulted in sustained, normalized levels of factor
VIII.21,22
Several lines of evidence indicate that, within the liver, hepatocytes
are the major factor VIII-producing cells. First, factor VIII mRNA is
present in hepatocytes but not in sinusoidal cells.13 Second, the promotor region of the factor VIII gene comprises responsive elements that are characteristic for hepatocyte-specific expression.23 Finally, in immuno-ultrastructural studies
factor VIII protein was detected in the rough endoplasmatic reticulum and the Golgi apparatus of hepatocytes.24 It should be
mentioned that other reports showed the presence of factor VIII in
hepatic endothelium using histochemical techniques.25-27
This is unexpected because these cells appear to lack factor VIII mRNA.
It is possible that the latter observation reflects surface binding of
factor VIII or internalization rather than factor VIII biosynthesis.
Secretion of factor VIII.
Studies on factor VIII biosynthesis and secretion have been limited by
the lack of human cell lines that properly express significant amounts
of factor VIII. Analysis of the factor VIII secretion process has
therefore been restricted to autologous gene expression.28
These studies showed that, in general, factor VIII is poorly expressed.
Low expression is associated with a low level of steady-state
mRNA29 and inefficient secretion.30 The initial
stage of secretion involves the translocation of the mature 2332 amino
acid polypeptide into the lumen of the endoplasmatic reticulum (ER),
where N-linked glycosylation occurs. Within the ER, factor VIII
appears to interact with a number of chaperone proteins, including
calreticulin, calnexin, and the Ig-binding protein
(BiP).31-34 Due to the interaction with these chaperone proteins, a significant proportion of the factor VIII molecules is
retained within the ER, thereby limiting the transport of factor VIII
to the Golgi apparatus. The mechanism reponsible for the transport from
the ER to the Golgi apparatus is not elucidated yet. However, recent
studies indicate that this step involves an intracellular membrane
lectin: endoplasmatic reticulum-Golgi intermediate compartment-53
(ERGIC-53).35
Within the Golgi apparatus, factor VIII is subject to further
processing, including modification of the N-linked
oligosaccharides to complex-type structures, O-linked
glycosylation, and sulfation of specific Tyr-residues (Fig 2). In
addition, factor VIII is among the many proteins that undergoes
intracelullar proteolysis.36-39 The middle part and the
carboxyterminal region of the B domain comprise a motif (Arg-X-X-Arg),
which is similar to the motif that is recognized by intracellular
proteases of the subtilisin-like family.38,39 The
responsible endoprotease mediating intracellular factor VIII
proteolysis, however, has not been identified. The Arg-X-X-Arg motif
allows proteolysis at Arg1313 and at Arg1648.
The latter event disrupts the covalent linkage of the factor VIII heavy
chain (A1-a1-A2-a2-B) and light chain
(a3-A3-C1-C2), giving rise to the heterodimeric molecule that
circulates in plasma.
The factor VIII heavy and light chain remain noncovalently associated
through the A1 and A3 domain in a metal-ion-dependent manner.7,40-42 Considering the structural homology of
factor VIII to the copper-binding protein ceruloplasmin, it is not
surprising that copper ions have been found in factor VIII as
well.43,44 One molecule of copper is present per molecule
of factor VIII. Most likely, the copper-ion binding site is composed of
residues His265, Cys310, His315,
and Met320 within the A1 domain.44 Binding of
copper ions to this site may allow the A1 domain to adopt an A3-domain
binding conformation. Alternatively, copper ions may directly bridge
the A1 and A3 domain by interacting with both domains simultaneously.
Whether only copper ions are involved in the association between heavy
and light chain is unclear. Whereas in the absence of other metal ions
copper ions are ineffective in promoting reassembly of dissociated heavy and light chain, calcium or manganese ions are considerably more
efficient in this respect.45-47a However, specific activity
of factor VIII dimers that were reassociated in the presence of calcium
ions, is markedly enhanced upon the addition of copper
ions.47a Apparently, copper ions serve an auxiliary role to
enhance cofactor function of factor VIII. These observations suggest
that multiple sites may be involved in the association between heavy
and light chain. Irrespective of the precise mechanism, it is clear
that metal ions serve an important role in maintaining the
heterodimeric structure of secreted factor VIII.
Factor VIII secretion and hemophilia A.
Aberrant biosynthesis or secretion may result from several defects.
Obviously, gross deletions or rearrangements may result in impaired
transcription, RNA processing, or translation. No data are reported on
the secretion of such gene products or on the stability within the
circulation provided that these gene products are actually secreted.
Defective secretion may further be caused by apparently minor gene
defects like single missense mutations. Frequently known missense
mutations associated with low levels of factor VIII are located in
codon 2307, resulting in replacement of Arg2307 by Gln or
Leu.48,49 Both mutations have been analyzed using recombinant factor VIII mutants expressed in mouse
fibroblasts50 and COS-1 monkey kidney cells.51
Both proteins appear to be functionally normal, but are poorly
secreted. The majority of the retained mutants are targeted into an
ER-associated degradation pathway. The mechanism responsible for
intracellular retention of these mutated factor VIII molecules is
unknown.
Reduced levels of factor VIII protein may also result from defects
located outside the factor VIII gene. One striking example concerns
patients having combined factor V and VIII deficiency. The gene
responsible for combined factors V and VIII deficiency has been mapped
to the long arm of chromosome 18, between markers D18S849 and
D18S1103,52,53 whereas the genes for factors V and VIII are
located at chromosomes 1 and X, respectively. Recently, the unknown
gene has been idenfied to encode the intracellular membrane lectin
ERGIC-53, a resident protein of the ER-Golgi intermediate compartment.35 Indeed, affected individuals displayed
mutations in this gene, in association with a complete lack of
expression of ERGIC-53.35 Apparently, ERGIC-53 contributes
to the secretion process of factors V and VIII, presumably by acting as
a chaperone protein. Identification of the underlying mechanism should
provide more insight in the intracellular routing and secretion of both cofactors.
| |
ASSEMBLY OF THE FACTOR VIII-VON WILLEBRAND FACTOR (vWF) COMPLEX |
Binding sites for vWF.
Immediately after its release into the circulation, the factor VIII
heterodimer interacts with its carrier protein vWF to form a tight,
noncovalent complex. Each monomer of the multimeric vWF protein is able
to bind one factor VIII molecule with high affinity (kd < 0.5 nmol/L).54-57 In vivo, however, the stoichiometry is limited by the number of factor VIII molecules present, resulting in
approximately a 1:50 ratio.
Two peptide regions of factor VIII are implicated to be involved in
binding vWF: one at the aminoterminal end of intact factor VIII light
chain,55-59 and one at the carboxyterminal end (residues 2303-2332).57,60-62 Using proteolytically derived fragments
of factor VIII light chain, it was shown that both these individual regions are capable of binding vWF.57 However, the affinity of these fragments for vWF is markedly lower compared with the intact
factor VIII heterodimer (two and three orders of magnitude for the
aminoterminal and carboxyterminal end, respectively).57 Apparently, both ends of factor VIII light chain act synergistically in
the binding of vWF.
With respect to vWF binding to the aminoterminal region of factor VIII
light chain, it appears that residues 1649 to 1671, thus including
sulfated residue Tyr1664, are dispensable for vWF
binding.57,63,64 In addition, the Arg1689-Ser1690 cleavage site has to be
intact,57 suggesting that residues carboxyterminal of this
thrombin cleavage site contribute to binding as well. Recombinant
factor VIII synthesized in the presence of an inhibitor of sulfation
displays reduced binding to vWF,63,65 suggesting a role for
sulfated Tyr1680 in the interaction with the carier
protein. Indeed, replacement of Tyr1680 by Phe results in
loss of high-affinity binding to vWF, allowing only a low-affinity
interaction.63,65,66 The presence of Tyr1680 in
its sulfated form thus contributes to optimal binding of factor VIII to
vWF.
Factor VIII interactions modulated by vWF.
One functional aspect of factor VIII-vWF complex formation may be to
prevent premature binding of factor VIII to components of the factor
X-activating complex. For instance, binding of factor VIII light chain
to factor IXa is inhibited by vWF.67 Because the affinity
of factor VIII for vWF exceeds that for factor IXa by approximately
100-fold,56,67 factor VIII highly favors vWF binding over
factor IXa binding. The mechanism by which vWF inhibits factor IXa
binding is not yet elucidated. Because binding of both proteins by
factor VIII light chain seems to involve distinct parts of the
molecule, it is unlikely that vWF competes with factor IXa for binding
to the same site. Mechanisms that could contribute to inhibition
include sterical hindrance and alteration of the factor VIII
conformation so that factor IXa cannot bind.
Whereas factor IXa and vWF bind at different sites, vWF and
phospholipids both bind to the C2-domain region
2303-2332.62,68 Using a human anti-factor VIII antibody,
it has been shown that these sites, although in very close proximity,
do not completely overlap.69 Nevertheless, the close
proximity of these sites may explain the observations that binding of
factor VIII to vWF is incompatible with factor VIII binding to membrane
surfaces.70-73 It should be noted that, in comparison with
noncomplexed factor VIII, factor VIII in complex with vWF is less
susceptible to proteolytic attack by various lipid-binding
proteases.74-77 These vitamin K-dependent serine
proteases, which include activated protein C (APC) and factor Xa,
require to assemble with factor VIII at a membrane surface for
efficient proteolysis of factor VIII.76,78 In contrast, thrombin displays proteolytic activity independent of a membrane surface. Indeed, vWF does not protect factor VIII against cleavage by
thrombin.79-81 Cleavage of factor VIII by thrombin results
in loss of vWF binding and conversion of factor VIII into its active conformation.
vWF and hemophilia A.
The association with vWF serves an important role in factor VIII
physiology, as vWF functions as a stabilizer of the heterodimeric structure of factor VIII.45,82-84 The physiological
relevance of complex formation is particularly apparent in patients
with von Willebrand disease (vWD) (type 3), who have no detectable vWF
protein. Not only do these patients have a secondary deficiency of
factor VIII, but they also have a considerably reduced half-life of
intravenously administred factor VIII.83,85-88 This
phenotype is also observed in patients with vWD with the so-called
Normandy variant, which is defined as type 2N. Despite normal levels of circulating vWF, factor VIII levels are severely
reduced.89,90 These patients harbor a mutation in the
factor VIII-binding domain of vWF, which results in defective binding
to vWF.
With regard to factor VIII, two distinct basepair substitutions have
been reported that are associated with impaired complex assembly.49,91,92 Both mutations result in the replacement of one single amino acid, Tyr1680, and are associated with
hemophilia A. So far, no mutations located within the C2 domain region
have been reported that are associated with reduced affinity for vWF.
| |
ACTIVATION OF FACTOR VIII |
Cleavage sites associated with factor VIII activation.
Within the factor X-activating complex, the proteolytic activity of
factor IXa is markedly enhanced by factor VIII. It has been well
established that proteolysis of factor VIII is required for the
generation of its cofactor activity.79,93-97 The uncleaved factor VIII procofactor lacks the ability to enhance factor IXa activity.47,98 Enzymes that are able to endow factor VIII
with its cofactor activity include thrombin and factor
Xa.79,93,98-102 Thrombin cleaves factor VIII at one
specific site within the light chain, Arg1689, and at two
sites in the heavy chain: Arg372 and Arg740
(Fig 3).79
Proteolysis of factor VIII heavy chain by factor Xa involves three
sites: Arg336, and the two thrombin-cleavage sites
Arg372 and Arg740
(Fig 3).79 With regard to
factor VIII light chain, factor Xa is able to cleave at
Arg1689, a site that is shared with thrombin, but also at
Arg1721, a site that is specific for factor
Xa.79 It should be noted that this site is cleaved in human
factor VIII, but not in porcine factor VIII.103 It has been
unclear whether cleavage at Arg1721 contributes to factor
VIII activation or inactivation, because prolonged incubation with
factor Xa results in loss of factor VIII activity in parallel with
cleavages at positions 336 and 1721.79 This has been
resolved by studying reassociated dimers of intact factor VIII heavy
chain with either thrombin- or factor Xa-cleaved factor VIII light
chain.47 The resulting factor VIII dimers were functionally
indistinguishable, demonstrating that factor Xa-cleavage of the light
chain is not associated with inactivation. It has been reported that
factor Xa-activated factor VIII displays less activity than
thrombin-activated factor VIII.79,98,102 Because cleavage
of the light chain cannot be responsible for this phenomenon, this is
most likely due to additional factor Xa-cleavage at Arg336
in the factor VIII heavy chain.
View larger version (12K):
[in this window]
[in a new window]
| Fig 2.
The factor VIII protein. Mature factor VIII consists of
2332 amino acids, which are arranged in a discrete domain structure: A1
(residues 1-336), A2 (373-710), B (741-1648), A3 (1690-2019), C1
(2020-2172), and C2 (2173-2332). The A domains are bordered by acidic
regions a1 (337-372), a2 (711-740), and a3
(1649-1689). Disulfide Bridges: Using B-domainless factor VIII, seven
disulfide bonds have been identified: residues 153 and 179, 248 and 329 (A1 domain), 528 and 554, 630 and 711 (A2 domain), 1832 and 1858, 1899 and 1903 (A3 domain), and 2021 and 2169 (C1 domain).196
Within the C2 domain, residues 2174 and 2326 most likely also form a
disulfide bridge. Free cysteine-residues have been identified at
positions 310, 692, and 2000.196 Cys528 and
Cys1858 may be present as free cysteines, because these
residues are reactive toward a sulfhydryl-specific
fluorphor.197 With regard to the Cys-residues in the
B-domain it is unknown whether they are free or linked.
N-Linked Glycosylation: Factor VIII contains 25 consensus
sequences (Asn-Xxx-Thr/Ser) that allow N-linked glycosylation. Using
either full-length or B-domainless factor VIII, the majority of these
sites have been shown to be glycosylated: residues 42 and 239 (A1
domain), residues 757, 784, 828, 900, 963, 1001, 1005, 1055, 1066, 1185, 1255, 1259, 1282, 1300, 1412, and 1442 (B domain), residue 1810 (A3 domain), and residue 2118 (C2 domain).198-200
Nonglycosylated residues are present at positions 943 and 1384 (B
domain) and at position 1685 (a3 acidic region). Residue 582 (A2 domain) has been reported to be nonglycosylated in two
studies,199,200 whereas one study reported this residue to
be partially glycosylated.198 Finally, it remains to be
investigated whether residue 1512 (B domain) is glycosylated. Tyrosine
Sulfation: The acidic regions contain consensus sequences that allow
sulfation of Tyr-residues at positions 346 (a1 region), 718, 719, 723 (a2 region), 1664, and 1680 (a3 region).
Analysis using recombinant proteins established that all sites indeed
can be sulfated.
|
|
View larger version (5K):
[in this window]
[in a new window]
| Fig 3.
Limited proteolysis of factor VIII. The major part of
factor VIII circulates as a set of heterogenous dimers, consisting of a
light (a3-A3-C1-C2) and heavy chain
(A1-a1-A2-a2-B). The heavy chain is variably sized due
to limited proteolysis within the B domain. Some of these cleavages may
occur intracellularly at positions 1313 and 1648 (open downward
arrows). Factor VIII can be converted into its active form by
proteolysis in both the heavy and light chain by various serine
proteases (closed downward arrows), including thrombin and factor Xa.
Because proteolysis by factor Xa but not thrombin is inhibited by vWF,
thrombin is probably the physiological activator of factor VIII.
Proteolytic degradation of factor VIIIa proceeds through cleavages
within the A1 and A2 domains by various serine proteases (upward
arrows), and results in release of the a1 acidic region and
bisecting of the A2 domain. In contrast to what has previously been
assumed, cleavages within the light chain by factor IXa or factor Xa do
not result in inactivation of factor VIII, but contribute to the
development of factor VIII cofactor activity.
|
|
Because activation of factor VIII involves proteolysis of both its
heavy and light chain, it is of interest to compare the relative
contribution of each cleavage to the development of factor VIII
cofactor function. The contribution of cleavage at Arg740
to factor VIII activation is limited, because mutations at this position do not interfere with factor VIII activation or
function.97 Selective cleavage of factor VIII at
Arg372 by a snake venom-derived protease generates a
factor VIII molecule that displays 60% of the activity of fully
activated factor VIII.80 Factor VIII dimers cleaved within
the light chain only have 25% to 30% of the activity displayed by
fully activated factor VIII.47,104 These findings allow the
conclusion that both cleavage at Arg372 and
Arg1689 are required to exert full cofactor activity. This
view is supported by the observation that recombinant factor VIII
mutants containing replacements at either Arg372 or
Arg1689 are unable to correct the clotting time of factor
VIII-deficient plasma.97
Acidic regions.
With regard to the cleavage sites at positions 372, 740, and 1689, it
is noteworthy that these residues are located at the carboxyterminal
end of an acidic sequence interconnecting adjacent domains. These
acidic regions (a1, a2, and a3, see Fig 2)
contain several Tyr residues in a sequence that meets the consensus
features for tyrosine sulfation.105,106 Sulfated Tyr
residues may serve a role in various processes, including
protein-protein interactions.106 Because thrombin is known
to interact with a variety of acidic, sulfated sequences, it has been
proposed that acidic regions within factor VIII serve a role in
thrombin activation of factor VIII.107 Replacement of
Tyr346 (a1 region) or Tyr1664
(a2 region) indeed results in factor VIII mutants that are
activated by thrombin less efficiently.66 It should be
mentioned that deletion of Tyr1664 leaves the activation
kinetics of factor VIII by thrombin unaffected.108,109
Replacement of Tyr-residues by Phe in the a2 acidic region
(residues 718, 719, and 723) did not affect thrombin activation, but
rather resulted in mutants that displayed reduced ability to stimulate
factor IXa enzymatic activity.66 In contrast, recombinant factor VIII variants (both full-length and B-domainless) containing nonsulfated Tyr at these positions are activated normally and display
full cofactor activity.110,111 Although both approaches reveal apparently contrasting findings with regard to the effect of
Tyr-sulfation in the a2 region on factor VIII cofactor
function, they allow the conclusion that sulfation of these
Tyr-residues does not contribute to activation of factor VIII. However,
the acidic nature of this a2 sequence appears to be of
importance for activation of factor VIII. First, a monoclonal antibody
directed against a2 inhibits thrombin activation of factor
VIII, but does not interfere with factor VIII cofactor
activity.112,113 Secondly, deletion of a2 or part
thereof in B-domainless factor VIII results in molecules that require
higher thrombin concentrations than normal factor VIII for efficient
activation, but display normal factor IXa cofactor
activity.110,112 Interestingly, in both cases it was
observed that reduced thrombin activation was caused by a reduced
cleavage efficiency at Arg372 and Arg1689,
suggesting that the a2 acidic region influences cleavage in remote regions in the factor VIII molecule. This hypothesis is supported by observations using a factor VIII chimera with a
replacement in the a2 region. In this chimeric molecule,
residues 716-736 of a2 have been replaced by a sequence that is
known to have a high affinity for thrombin, ie, the amino acid sequence
51-80 of heparin cofactor II.114 This chimeric protein
proved more potent than normal factor VIII in correcting the clotting
time of factor VIII-deficient plasma. The increased intrinsic activity is caused by an increased rate of thrombin cleavage at
Arg372 and Arg1689 compared with normal factor
VIII. Thus, these findings suggest that the a2 region promotes
proteolytic activation of factor VIII.
Apart from their role in thrombin activation, the acidic regions
a1 and a3 contribute to factor VIII function also in an
additional manner. The a1 region has been described to be
involved in maintaining the stability of the factor VIII
heterotrimer115 and in binding of factor
X116,117 (see Factor VIII Inactivation section). The a3 region is important for high affinity binding of vWF (see
Assembly of the Factor VIII-vWF Complex section). Further, factor VIII heterodimers consisting of uncleaved heavy and light chain do not
posses any cofactor activity.47,48 In contrast, factor VIII
exclusively cleaved at position 1689, thus lacking the a3 region, displays significant cofactor activity (approximately 25% of
fully activated factor VIII).47,104 Apparently, the acidic region a3 functions as an activation peptide that needs to be cleaved off for exposure of cofactor activity.
Defective activation and hemophilia A.
Mutations resulting in replacement of amino acids at the factor VIII
activation sites should predispose to hemorraghic diathesis. Indeed,
missense mutations at Arg372, Ser373, and
Arg1689 are associated with hemophilia
A.49,118-124 With regard to the Arg1689
mutation, biochemical data predict residual activity to be at least
half of that of normal factor VIII, as cleavage at Arg372
accounts for approximately 60% of total activity.80
However, residual activity appears to range between <1% and  12%,
thus lower than expected.49 Several possibilities may be
considered to explain this apparent discrepancy. First, due to
substitution of Arg1689, the vWF binding site including
sulfated Tyr1680 is not cleaved from the light chain.
Therefore, the factor VIII-vWF complex may fail to dissociate upon
thrombin treatment. As a consequence, factor VIII is unable to interact
with factor IXa and phospholipids, and thus cannot assemble into the
factor X-activating complex (see below). Secondly, in most of the
documented patients with a mutation at Arg1689, this
residue is replaced by a Cys.49 This Cys-residue has the
potential to form an extra disulfide bridge within the factor VIII
light chain.125,126 It seems conceivable that this results in misfolding within the factor VIII light chain, which precludes biological activity. Finally, residual activity may be affected by the
type of amino acid substitution. This view is supported by the notion
that a similar discrepancy exist for substitutions at
Arg372. For instance, Arg372 to Pro
substitutions exclusively result in severe hemophilia A, whereas
Arg372 to His substitutions result in a mild to moderate
phenotype.
| |
ASSEMBLY OF THE FACTOR X-ACTIVATING COMPLEX |
Regulation of complex assembly.
To activate factor X, factor IXa and factor VIIIa assemble into a
membrane-bound complex. To maintain the hemostatic balance, this
complex should only assemble after initiation of the coagulation cascade, implying that participation of factor VIII in this complex is
subject to a delicate regulatory mechanism. In this regard, vWF plays a
central role. Although factor IXa displays similar affinity for
nonactivated and activated factor VIII,127 the factor IXa
binding site within nonactivated factor VIII is unlikely to be
accesible when factor VIII is in complex with vWF.67
Furthermore, binding of factor VIII to the membrane surface is
inhibited by vWF,70-73 suggesting that only activated
factor VIII that is dissociated from vWF is able to bind to the
membrane surface. However, it should be mentioned that the affinity of
factor VIII for the membrane surface is dependent on the membrane
composition, as affinities have been reported that differ 10- to
100-fold (10 9 to 1011
mol/L).72,128-131 Thus, under particular conditions the
affinity of vWF and the membrane surface for factor VIII is similar.
Apparently, a delicate balance may exist between factor VIII being in
complex with vWF or the membrane surface. Because in direct binding
studies vWF prevents binding of factor VIII to the membrane
surface,70-73 a minor part of the factor VIII population
probably is in complex with the membrane surface. This situation
changes dramatically upon cleavage of factor VIII light chain, which
results in a 1,600-fold decrease in affinity for vWF.57
Because of this event, the balance will readily shift toward factor
VIIIa binding to the membrane surface. This subsequently favors binding
of factor IXa to factor VIIIa, which is no longer associated with vWF.
Ultimately, this leads to the assembly of the membrane-bound factor
VIIIa-factor IXa complex that activates factor X.
The role of the membrane surface in complex assembly.
The notion that in the absence of a membrane surface the generation of
factor Xa by the factor VIIIa-factor IXa complex is negligible132 underscores the essential role of the
membrane surface in the factor X-activating complex. The membrane
surface may act in two distinct ways: first by positioning the
enzyme-cofactor complex into an active conformation or, alternatively,
by locating the enzyme and cofactor at the same site. At present, data
have been reported that are in support of both mechanisms. On the one hand, it has been shown that the affinity of factor IXa for factor VIIIa is increased 2,000-fold in the presence of
phospholipids,133 which suggests that the second mechanism
is dominant. On the other hand, the affinity of (activated) factor VIII
for factor IXa is reported to be similar in the presence
(kd = 108 to 109
mol/L)104,127,132,134,135 and absence (kd = 108 mol/L)67,132 of a phospholipid
surface. Gilbert and Arena132 showed that in the presence
of phospholipids the catalytic activity of the enzyme-cofactor complex
is increased 1,500-fold. These data favor the view that the membrane
surface positions the enzyme and cofactor in a conformation that allows
efficient substrate cleavage.
Location of factor IXa interactive sites.
The interaction between factor VIIIa and factor IXa has been
investigated in several elegant studies using factor IXa molecules that
carry a fluorescent-label in the active site.127,134-139 It became evident that in the presence of phospholipids, factor VIIIa induces a conformational change in the factor IXa protease domain. In
addition, maximal changes in the factor IXa protease domain require the
presence of the factor VIIIa A2-domain,134 suggesting that
the A2 domain contains a factor IXa interactive site. Indeed, studies
using a series of synthetic peptides showed that factor IXa binding can
be attributed to the A2-domain sequence 558 to 565.137
Furthermore, a region within the carboxyterminal part of the A2 domain
(residues 698 to 710) also has been proposed to comprise a
factor IXa-binding site.140,141
Besides factor VIII heavy chain, the light chain also contributes to
factor IXa binding. In equilibrium binding studies, isolated factor
VIII light chain proved to bind factor IXa with high
affinity.67 Moreover, factor VIII light chain and the
intact factor VIII heterodimer are indistinguishable in terms of
affinity for factor IXa, indicating that high-affinity binding to
factor IXa is mediated by the factor VIII light chain. Binding of
factor VIII light chain to factor IXa was found to be inhibited by the
A3-domain directed monoclonal antibody CLB-CAg A, a strong inhibitor of
factor VIII activity.67,113,142 By using synthetic
peptides, the A3-domain sequence 1811 to 1818 has been identified as a
site that binds factor IXa.142 Thus, interaction with
factor IXa involves at least three sites on factor VIII: residues
558-565, 698-710, and 1811-1818.
Three-dimensional model of factor VIII and factor IXa.
Three-dimensional representations of the enzyme and cofactor have been
published, based on factor IXa crystallography143 and
factor VIII homology modeling144,145
(Fig 4). So far, it is unknown which
residues in the factor IXa molecule are involved in binding factor
VIII. However, the location of these sites should fit with the location
of their counterparts on the factor VIII molecule. Although the amino
acid numbering suggests that the factor IXa-binding regions are
located in completely different parts of the factor VIII protein, the
three-dimensional model indicates that the factor IXa-binding sites
are in close vicinity, and are exposed at the same side of the molecule
(Fig 4). Matching of the factor VIII and factor IXa models suggests
that factor IXa comprises distinct sites involved in factor VIIIa
binding, interacting with the A2 or A3 domain. Factor VIIIa binding
appears to be mediated by both the factor IXa light chain and protease domain. The involvement of the protease domain is in agreement with the
observation by Bajaj et al146,147 that the stimulation of
factor IXa proteolytic activity by factor VIIIa is inhibited by a
monoclonal antibody directed against the protease domain residues
231-265. This suggests that this protease domain region comprises a
factor VIII-binding site. However, recombinant factor IXa molecules in
which residues in the antibody-binding epitope have been mutated
combine a strongly reduced affinity for the antibody with normal
biological activity.148 Because normal activity is
associated with normal factor VIII binding, these findings leave the
exact location of the factor VIII binding site in the factor IXa heavy
chain unidentified. Irrespective of its precise location, this site on
the factor IXa heavy chain presumably interacts with the factor VIII A2
domain, because this domain induces the largest change in the
conformation of the factor IXa active site.134
View larger version (94K):
[in this window]
[in a new window]
| Fig 4.
Model of the factor VIII and factor IXa molecules. Shown
are representiations of porcine factor IXa (Protein Data Bank accession
code ) and the triplicated A-domains of human factor VIII
(Hemophilia A web site, http://europium.mrc.rpms.ac.uk), which are
derived from crystallography and homology modeling, respectively.
Factor IXa binding region in the factor VIII A3 domain (residues
1811-1818) is shown in white, whereas the binding regions in the A2
domain (residues 558-565 and 698-710) are shown in dark and light blue,
respectively (space-filling representations). These sites are in close
vicinity, and are exposed at the same side of the molecule. The factor
VIII A2 domain is required to induce significant changes within the
factor IXa protease domain, indicating that it binds to the factor IXa
protease domain. The A3 domain of factor VIII has been proposed to
interact with the factor IXa light chain. Within the factor IXa light
chain, residues 12, 64, 69, 78, 92, and 94 (see refs 150 to 155) are
indicated (red, space-filling representation). These residues have been
reported to be associated with an abnormal response to factor VIIIa in
factor X activation.
|
|
Assuming an interaction between the factor IXa protease domain and the
factor VIII A2 domain, it seems conceivable that the A3 domain
interacts with a region within the factor IXa light chain. This is in
line with recent observations that the light chain of factor VIII binds
to the light chain of factor IXa.138,149 Furthermore,
mutations within the factor IXa light chain have been described that
are associated with an abnormal response to factor VIIIa in factor X
activation.150-154 These mutations are dispersed over the
factor IXa light chain, indicating that multiple sites may contribute
to binding of the factor VIII A3 domain. Alternatively, some mutations
may destabilize the factor VIII binding site by affecting the
conformation of the factor IXa light chain. This latter possibility has
been reported for two distinct factor IX mutations.154,155
Collectively, by combining biochemical data with the three-dimensional
models of factor IXa and factor VIII, it appears that the factor VIII
A2 domain binds to the factor IXa heavy chain, and the factor VIII A3
domain to the factor IXa light chain (Fig 4). It is of importance to
realize that for factor VIII as well as for factor IXa the current
models not fully represent the biologically active molecules. The
factor IXa structure has been determined in the absence of calcium
ions,143 which are obligatory for optimal exposure of the
factor VIII light chain binding site and of the catalytic
centre.133,155 The factor VIII model provided by Pemberton et al145 comprises the A domains only and lacks the B and C
domains and the acidic domain spacers. It cannot be excluded that these domains affect the structure of the A domains. In addition, this factor
VIII model does not distinguish between the inactive procofactor or
activated factor VIII. It is obvious that both factor VIII species will
have different structural properties, because only factor VIII, which
is cleaved at specific positions, is able to stimulate factor IXa
activity. Despite these restrictions, both the factor VIII and factor
IXa model provide an important basis for proper selection of residues
that may be investigated for their contribution in the assembly of the
factor VIII-factor IX complex.
Factor IXa binding and hemophilia A.
Inspection of the hemophilia A database shows that several mutations
have been reported that are in or close to the factor IXa binding
sites: codons 558, 565, and 566, codons 698, 701, and 704 in the heavy
chain, and codons 1789, 1796, 1823, and 1825 in the factor VIII light
chain.49 It seems reasonable to assume that the bleeding
tendency which is associated with these mutations is caused by
suboptimal assembly of the factor IXa-factor VIIIa complex. With
regard to the 558 and 566 mutation, this view has been supported by
studies using recombinant factor VIII.156 Interestingly, a
mutation outside the factor IXa-binding regions, ie, at codon Arg527, also has been reported to be associated with
inefficient stimulation of factor IXa proteolytic
activity.157,158 Examination of the three-dimensional model
shows that Arg527 is located in the immediate vicinity of
the factor IXa binding sequence 558 to 565.145 The exposure
of this factor IXa binding site may be affected by substitution of
Arg527. Alternatively, Arg527 may be part of an
extensive factor IXa-binding interface, involving multiple sites of
the A2 domain.
| |
INACTIVATION OF FACTOR VIII |
Mechanisms of inactivation.
Downregulation of the factor X-activating complex may involve
inactivation or inhibition of either the enzyme factor IXa or the
cofactor, factor VIIIa. Inactivation of the cofactor comprises two
distinct pathways: proteolytic degradation and spontaneous dissociation. Once activated, factor VIII cofactor activity is rapidly
lost.135,159-162 Compared with activated factor VIII, the procofactor is markedly more stabile, which is illustrated by its
dissociation rate being 100-fold lower (kdiss 4 to 6 × 104 s1 and 4 × 106 s1 for factor VIIIa and
factor VIII, respectively).47,163 The intrinsic instability
of factor VIIIa can be attributed to the weak interaction between the
A2 domain and the metal ion-linked A1/A3-C1-C2
dimer.164-166 The kd for this interaction is approximately 0.2 µmol/L.42 Because this value exceeds the factor VIII
concentration in plasma 100- to 1,000-fold, equilibrium is in favor of
the inactive, dissociated state of factor VIIIa.
Proteolytic degradation of factor VIIIa involves cleavages in the heavy
chain at positions 336 and 562 by various enzymes, such as factor IXa,
factor Xa, and APC.74-79,167-170 Cleavage at position 336 in factor VIIIa releases a1, the acidic sequence that
interconnects the A1 and A2 domain. Because of this release, the A2
domain dissociates more rapidly from the factor VIIIa
heterotrimer.115 This acidic spacer has been proposed to
comprise a binding site for the substrate factor X,116,117
indicating that release of this site results in impaired substrate
binding. Thus, cleavage at Arg336 affects both
intramolecular (A2 domain dissociation) and intermolecular (factor
VIII-factor X) interactions. Arg562, which is part of the
A2 domain sequence that comprises a factor IXa interactive site, is
exclusively cleaved by APC.168 It seems conceivable that
loss of cofactor activity due to cleavage at this site reflects the
loss of the ability to interact with factor IXa.
One intriguing question is whether proteolytic degradation or
spontaneous dissociation dominates the inactivation of factor VIII in
vivo. At present the relative contribution of each mechanism to factor
VIII inactivation is not fully understood, although some reports
indicate that spontaneous dissociation is
dominant.162,171,172 This view is underscored by the
observation that dissociation of the factor VIIIa heterotrimer may be
accelerated by binding of the A2 domain to the low-density lipoprotein
receptor-related protein.173 To describe the process of
factor VIIIa inactivation, it should further be considered that factor
IXa plays a dual role. It stabilizes factor VIIIa by linking the A2
domain to the A3 domain,137,142,159 and protects factor
VIII against inactivation by APC.136,167,174 On the other
hand, under certain conditions factor IXa may inactivate factor VIIIa
by cleavage at position 336,169,170 a site that is shared
with factor Xa and APC. The fact that factor IXa is involved both in
stabilization and in inactivation of factor VIII complicates a final
assessment of the regulatory role of factor IXa in intrinsic factor Xa
formation.
Defects in factor VIII inactivation.
Impaired inactivation of factor VIIIa or its homologue factor Va may be
associated with a disturbed balance between procoagulant and
anticoagulant systems. With respect to factor Va this view is supported
by the notion that mutation at Arg506, a site that is
cleaved by APC, predisposes to venous
thromboembolism.175-178 It has been investigated whether
patients displaying venous thromboembolism carry analogous mutations at
the APC cleavage sites in factor VIII (ie, Arg336 and
Arg562).179,180 However, this association has
not been observed, which suggests that mutations at these positions are
rare. Alternatively, such mutations may not predispose to thrombotic
disorders, indicating that proteolytic inactivation of factor VIIIa is
less important than inactivation of factor Va with regard to the
hemostatic balance. This would be in agreement with the fact that
murine factor VIII lacks the inactivation site at position
336.15 In addition, in vitro data using genetically
engineered factor VIII with mutations in the APC cleavage sites showed
that these mutations were not associated with reduced clotting times in
APC-resistance assays.181
Although APC-resistant factor VIII molecules have not been identified
in patients, the possibility remains open that APC-resistance may
modulate factor VIII inactivation in an indirect manner. Inactivation of factor VIII by APC is enhanced in the presence of the APC-cofactor protein S.74,167 Several investigators have reported that
the factor V procofactor enhances the cofactor effect of protein S in
factor VIII inactivation.162,182,183 This link between
factor V and factor VIII inactivation becomes even more apparent by the finding of Váradi et al,183 who reported that factor
V, which carries the Arg506 to Gln mutation, has impaired
cofactor activity in APC- and protein S-dependent factor VIII
inactivation. The physiological significance of this observation
remains unclear. However, the possibility that APC-resistance also
affects factor VIII inactivation is challenging and deserves further
study.
Another intriguing finding is that APC-resistant factor V has the
potential to bypass the absence of factor VIII activity to some extent
in in vitro thrombin generation studies.184 Therefore, it
cannot be excluded that hemophilic patients which carry the factor V
Arg506 to Gln mutation display a less severe phenotype than
expected. This indeed has been shown for some hemophilic patients as
described by Nichols et al.185 However, Arbini et
al186 did not find an association between the presence of
APC-resistant factor V and the severity of the bleeding tendency in a
population of 295 hemophilic patients. The ability of APC-resistant
factor V to bypass factor VIII deficiency may be restricted to
specific, thus far unrecognized conditions.
| |
CLEARANCE OF FACTOR VIII |
At present, little is known about the mechanism by which factor VIII is
cleared from the circulation. Obviously, vWF serves an important role,
because in patients with severe vWD the factor VIII half-life is
considerably decreased.83,86-88 As vWF protects factor VIII
against proteolytic degradation in vitro (see Assembly of the Factor
VIII-vWF Complex section), it cannot be excluded that the decreased
half-life in the absence of vWF factor is associated with proteolytic
degradation of factor VIII. However, experimental data in support of
this possibility are lacking thus far. Another explanation for the
rapid clearance could be binding of noncomplexed factor VIII to the
surface of cells. In this respect it is of interest to mention that
hepatic endothelial cells have been reported to contain factor VIII
protein, but not its mRNA (see Biosynthesis and Secretion of Factor
VIII section). It may be noteworthy that the copper-binding protein
ceruloplasmin, which is structurally related to the factor VIII A
domains, is internalized in the hepatic endothelial cells through a
receptor-mediated process.187 It seems possible that factor
VIII is taken up by hepatic endothelium by a similar mechanism.
Because the effect of factor V proteolysis on its survival has been
investigated in a nonhuman primate model,188 it is of interest to compare factor VIII survival with that of factor V. Whereas
the half-life of the factor V procofactor is approximately 14 hours,
the half-life of its thrombin-activated derivative is dramatically
different. The heavy and light chain are cleared very rapidly
(t1/2 < 20 minutes). The half-life for factor V light chain is remarkably close to the half-life of 10 minutes reported for
the isolated light chain of factor VIII after infusion into hemophilic
dogs.189 Perhaps, clearance of both proteins involves a
similar mechanism. Clearance of the factor V activation peptide, ie,
the B domain, is considerably slower (t1/2 > 30 hours),188 suggesting a previously unrecognized role of
this domain in preventing premature clearance of factor V. Whether this
is also true for the factor VIII B domain is not clear. The half-lifes
of normal factor VIII and B-domain deleted factor VIII are similar in
hemophilic patients190 as well in hemophilic
dogs,87,191,192 indicating that the B domain does not
contribute to factor VIII survival. However, it is important to note
that these data reflect factor VIII survival in the presence of vWF.
Therefore, it would be of interest to investigate survival of
B-domainless factor VIII in patients with vWD type 3.
| |
FUTURE DIRECTIONS |
Structure-function studies have contributed significantly to our
current understanding of factor VIII biology and the molecular background of hemophilia A. As such this has provided the basis for the
development of second generation recombinant factor VIII molecules that
may find a therapeutical application. Pertinent to this point are the
B-domain-deleted factor VIII variants, which are subject to clinical
(factor VIII-SQ)190 or preclinical108,191,192 testing. Furthermore, various factor VIII variants have been designed which in the future may be particular useful in the treatment of
hemophilia A. Variants have been described which are less prone to
inhibitor neutralization.193,194 Other examples include
variants with enhanced hemostatic potency114 or
stability.195
Despite the rapid accumulation of information regarding the structure
and function of factor VIII, a number of questions remain to be
answered. For instance, it is still unclear why factor VIII becomes a
potent cofactor of factor IXa once it is cleaved within the heavy and
light chain. What is the structural basis for such a dramatic increase
in biological activity? How does factor VIIIa, together with the
membrane surface, push factor IXa into an extremely potent
configuration? It seems obvious that studies on three-dimensional modeling techniques based on crystal structures of factor VIII or
factor VIII fragments complexed with their ligands (eg, factor IX)
should provide a solid basis for a better understanding of the
molecular aspects of factor VIII function and dysfunction. Because both
factor VIII and factor IX are now potentially available in substantial
quantities, determination of the crystal structure of complexes
comprising wild-type or mutant proteins should be feasible and
undoubtedly will lead to significant advances in this field. This
approach will also facilitate the design of antagonistic inhibitors of
the factor VIII system, which could provide novel anticoagulants for
the treatment of thrombotic disorders.
The question of why exposure to factor VIII concentrates is associated
with allo-immune reponses in some of the hemophilia A patients remains
unanswered. What is the mechanism that causes these adverse reactions?
Why and how is the immune system challenged under these conditions? Why
do certain specific polypeptide regions of the factor VIII molecule
play a prominent role in these immune reactions? Finally, little is
known about the origin and expression of factor VIII at the cellular
level. Which mechanisms trigger the specific cellular responses that
govern elevations of the factor VIII plasma level under a variety of
clinical conditions? Similarly, the mechanism of factor VIII clearance
is an unexplored subject. Finding the answers to these questions is not
only of fundamental, merely scientific appeal, but also has the
potential of further improving our current strategies for the treatment of hemophilia A.
| |
ACKNOWLEDGMENT |
We express our gratitude to Drs W.G. van Aken, J. Voorberg, O.D.
Christophe, and K. Fijnvandraat for helpful discussions and critical
reading of the manuscript. We also thank Dr G. Kemball-Cook for
providing the coordinates of the factor VIII.
| |
FOOTNOTES |
Submitted June 5, 1997;
accepted July 17, 1998.
Address reprint requests to Peter J. Lenting, PhD,
Department of Plasma Protein Technology, CLB, Sanquin Blood Supply
Foundation, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands;
e-mail: P_Lenting{at}clb.nl.
| |
REFERENCES |
1.
Rosner F:
Hemophilia in the talmud and rabbinic writings.
Ann Intern Med
70:833, 1969
2.
Sadler JE, Davie EW:
Hemophilia A, Hemophilia B, and von Willebrand's disease, in
Stamatoyannopoulos G,
Nienhuis A,
Leder P,
Majerus P
(eds):
The Molecular Basis of Blood Diseases. Philadelphia, PA, Saunders, 1987, p 576.
3.
Patek AJ, Taylor FHL:
Hemophilia. II. Some properties of a substance obtained from normal plasma effective in accelerating the clotting of hemophilic blood.
J Clin Invest
16:113, 1937
4.
Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy S:
Surface dependent reactions of the vitamin K-dependent enzyme complexes.
Blood
76:1, 1990[Abstract/Free Full Text]
5.
Gitschier J, Wood WI, Goralka TM, Wion KL, Chen EY, Eaton DH, Vehar GA, Capon DJ, Lawn RM:
Characterization of the human factor VIII gene.
Nature
312:326, 1984[Medline]
[Order article via Infotrieve]
6.
Wood WI, Capon DJ, Simonson CC, Eaton DL, Gitschier J, Keyt B, Seeburg PH, Smith DH, Hollingshead P, Wion KL, Delwart E, Tuddenham EGD, Vehar GA, Lawn RM:
Expression of active human factor VIII from recombinant DNA clones.
Nature
312:330, 1984[Medline]
[Order article via Infotrieve]
< |
Top
Introduction
References