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Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4397-4418
REVIEW ARTICLE
Bernard-Soulier Syndrome
By
José A. López,
Robert K. Andrews,
Vahid Afshar-Kharghan, and
Michael C. Berndt
From the Departments of Medicine and Molecular and Human Genetics,
Baylor College of Medicine and VA Medical Center, Houston, TX; and the
Hazel and Pip Appel Vascular Biology Laboratory, Baker Medical Research
Institute, Prahran, Australia.
 |
INTRODUCTION |
IN 1948, BERNARD AND SOULIER described a
young male patient with a severe bleeding disorder that was
characterized by a prolonged bleeding time, thrombocytopenia, and
extremely large platelets.1 They termed the disorder "la
dystrophie thrombocytaire-hémorragipare congénitale."
Since then, an identical or similar disorder has been described in a
large number of individuals, virtually always transmitted in an
autosomal recessive manner and often occurring in persons whose parents
are close relatives.
The first clue to the molecular abnormality affecting the platelets of
patients with this disorder (now known as the Bernard-Soulier syndrome
[BSS]) came in 1969 from the work of Gröttum and
Solum,2 who noted reduced electrophoretic mobility of the
platelets due to a marked decrease in the concentration of sialic acid
on their membranes. Subsequently, Howard et al3 and Caen
and Levy-Toledano4 found that the platelets of BSS patients
failed to aggregate to ristocetin, a peptide antibiotic known to
aggregate normal platelets but not the platelets of patients suffering
from von Willebrand disease. Weiss et al5 in 1974 extended
this observation by demonstrating a defect in the ability of BSS
platelets to adhere to rabbit aortic subendothelium. They also
suggested that the defect resulted from absence of a receptor for von
Willebrand factor (vWF) on the platelet surface. Numerous other
phenotypic abnormalities have been described in BSS, including
defective platelet aggregation to bovine vWF,3,6
abnormalities of membrane phospholipid content7,8 and
coagulant activity,6,8 and morphological characteristics
that include large size and disordered cytoskeletal
structure.9,10
The nature of the missing vWF receptor was suggested in 1975 when
Nurden and Caen11 demonstrated that 1 of the 3 major
carbohydrate-containing proteins on the platelet surface, glycoprotein
I, was virtually absent in the platelets of BSS patients. The
biochemical defect was defined further in the laboratories of Clemetson
et al12 and Berndt et al,13 when they
demonstrated, in unrelated patients with BSS, deficiencies of 4 polypeptides: glycoproteins (GP) Ib , Ib , IX, and V. These
polypeptides all associate on the platelet surface to form a receptor
called the GP Ib-IX-V complex.
The importance of this receptor for normal hemostasis is perhaps best
illustrated by the clinical history of the original patient described
by Bernard and Soulier.14 As both a child and a young man,
this patient suffered numerous bleeding problems, including prolonged
bleeding after tooth extraction, life-threatening cerebrospinal
hemorrhage, and orbital and periorbital hematomas after an accident. He
died at 28 years of age of intracranial hemorrhage after a barroom
brawl.
| |
THE GP Ib-IX-V COMPLEX: STRUCTURE AND FUNCTION |
The GP Ib-IX-V complex has two important roles in platelet function
that explain the often severe bleeding observed in BSS: it mediates
adhesion to the blood vessel wall at sites of injury by binding vWF and
it facilitates the ability of thrombin at low concentrations to
activate platelets.15 The interaction with vWF underlies
another potentially important function that may be more relevant to
thrombosis than to hemostasis: shear-induced platelet
aggregation.16 Furthermore, the GP Ib-IX-V complex may have
important roles in the process by which platelets are generated and
possibly in platelet turnover, as evidenced by the decreased number and
abnormal size of platelets from BSS patients.
The key structural features of the GP Ib-IX-V complex are depicted
schematically in Fig 1. The complex
comprises 4 distinct transmembrane polypeptide subunits, GP Ib, GP
Ib, GP IX, and GP V, with a stoichiometry based on monoclonal
antibody binding of 2:2:2:1, respectively.17-20 Each of the
4 subunits is a member of the leucine-rich repeat motif superfamily,
members of which are involved in such diverse processes as cell
signaling, cell adhesion, and development.21,22 In the
polypeptides of the GP Ib-IX-V complex, the leucine-rich repeat
sequences are approximately 24 amino acids in length, occur singly or
in tandem repeats, and are flanked by conserved N- and C-terminal
disulfide loop structures.22 However, despite these
structural similarities, the polypeptides comprising the GP Ib-IX-V
complex all arise from distinct genes residing in different regions of
the genome.23-27
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| Fig 1.
Schematic view of the platelet GP Ib-IX-V complex. Key
structural features of the complex are shown. The leucine-rich repeats of the four polypeptides are drawn based on the structure determined for the porcine ribonuclease inhibitor, a protein made up entirely of
leucine-rich repeats.32 The depicted polypeptide
arrangement is based on the published stoichiometry determined by
monoclonal antibody binding17-19 and on the associations
determined for the polypeptides.47,112 A caveat about this
depiction: the quantity of GP V on the platelet surface has only been
determined using 2 GP V monoclonal antibodies,18,20 which
could lead to overestimates or underestimates of true polypeptide
number. In addition, no quantitation has ever been performed to
indicate that every GP V molecule on the platelet surface is associated
with the complex. Complexes of greater complexity having the same
stoichiometry are also possible.22,82 Diamonds on stalks
represent N-linked carbohydrates and circles on stalks
represent O-linked carbohydrate.
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GP Ib (135 kD, 610 amino acids) consists of an N-terminal globular
domain28 that contains 7 tandem leucine-rich repeats and
their flanking sequences, a 19-amino acid sequence rich in negatively
charged aspartate and glutamate residues, and 3 sulfated tyrosines,29,30 a highly glycosylated, macroglycopeptide
mucin core, a single transmembrane sequence, and a cytoplasmic tail of
96 amino acid residues.31 The structure of the leucine-rich repeats depicted in Fig 1 is based on the x-ray crystal structure of
porcine ribonuclease inhibitor, a protein made up entirely of
leucine-rich repeats.32 In this structure, each repeat
forms a - structural unit (a short -strand parallel to an
-helix), resulting in a horseshoe-shaped molecule in which the
helices form the outer circumference and the -strands form the inner surface. If the GP Ib-IX-V leucine-rich repeats adopt a similar structure, this produces a fan-shaped surface with most of the amino
acid side chains exposed to solvent, a property that may maximize
surface interactions with target proteins and that also has the effect
of bringing the flanking sequences into proximity. The
macroglycopeptide contains an O-linked, sialylated
hexasaccharide on average every 3 to 4 amino acids,33-35
creating a scaffold that extends the N-terminal globular domain and vWF
binding site approximately 45 nm from the surface of the platelet
plasma membrane.28 This region is highly polymorphic. In
any individual, its length depends on which combination of 4 possible
alleles is inherited. The products of these alleles differ in having 1, 2, 3, or 4 tandemly repeated copies of a 13-amino acid
sequence,36,37 each of which has been predicted to add
about 32 Å to the length of the macroglycopeptide.36
GP Ib (25 kD, 181 amino acids) has a single leucine-rich repeat and
is disulfide-linked to GP Ib immediately proximal to the platelet
plasma membrane.38 The cytoplasmic sequence of 34 amino
acids contains a protein kinase A phosphorylation site at
Ser16639 that appears to regulate platelet cytoskeletal
rearrangement in response to agonist stimulation.40
GP IX (22 kD, 160 amino acids), like GP Ib, has a single
leucine-rich repeat motif41 and remains associated with GP
Ib as a 1:1 complex when purified in Triton X-100.42 It has
a short cytoplasmic tail of 5 amino acids. The cytoplasmic sequences of GP Ib and GP IX both have a membrane-proximal cysteinyl residue that
can be palmitoylated in vitro, a modification that may provide additional anchorage for the complex in the platelet
membrane.43 Analysis of guinea pig megakaryocyte proteins
suggests that GP IX is primarily myristoylated rather than
palmitoylated.44
GP V (82 kD, 544 amino acids) has 15 leucine-rich repeats and a short
cytoplasmic tail of 16 amino acids.45,46 It is thought to
bridge adjacent GP Ib-IX complexes through an interaction with GP
Ib.47 The other feature of GP V is that it is one of a
limited set of thrombin substrates on the platelet plasma membrane,
with a major fragment, GP Vf1 (69.5 kD), released
from the surface of thrombin-treated platelets.48 The
functional significance of this cleavage in platelet physiology remains
unclear.
The principal function of the GP Ib-IX-V complex in hemostasis is to
initiate the arrest of platelets at sites of vascular injury. Like
other adhesion receptors, ligation of the GP Ib-IX-V complex by vWF can
transduce signals to the platelet cytoplasm, initiating the cascade of
events that leads to the formation of a hemostatic platelet plug.
However, unlike other adhesion receptors, the GP Ib-IX-V complex is a
unique adhesive system unrelated in structure to members of the
integrin, selectin, or Ig superfamilies, which mediate other aspects of
blood cell-vessel wall interaction.49 The binding site for
the GP Ib-IX-V complex resides within the A1 domain of
vWF,50,51 included within residues 480-718 of the mature
sequence.52 Mature vWF has a subunit molecular weight of
230,000 (2,050 amino acids)53 and circulates in a
nonadhesive form consisting of disulfide-linked multimers of up to 20 × 106 in molecular weight.54 vWF bound to
the subendothelial matrix is believed to undergo a conformational
change that reveals a normally cryptic binding site for the GP Ib-IX-V
complex within its A1 domain.55 vWF also binds to the GP
Ib-IX-V complex under the influence of high shear forces16
by induction of conformational changes in either the receptor or vWF or
in both.56,57 Consistent with this finding,
gain-of-function mutations occur in both the receptor and in vWF that
enhance the receptor-ligand interaction. In platelet-type (or pseudo)
von Willebrand disease, mutations of GP Ib
(Met239 Val58 or
Gly233Val59) result in a receptor complex with
higher affinity for circulating vWF.60,61 In type 2B von
Willebrand disease, point mutations in the vWF A1 domain clustered
around the Cys509-Cys695 disulfide bond and between Met540 and Arg578
yield a form of vWF with enhanced avidity for the native GP Ib-IX-V
receptor on platelets.62 A number of modulators have been
identified that also enhance the interaction between vWF and the GP
Ib-IX-V complex.63 These include the antibiotic ristocetin,
from the gram-negative bacterium Nocardia lurida, which appears
to function, at least in part, by binding to proline-rich sequences
flanking the disulfide bond between Cys509 and Cys695 in the vWF A1
domain.64-66 A second modulator, botrocetin (a
disulfide-linked heterodimer of 28 kD from the venom of the South
American pit viper, Botrops jararaca) activates vWF adhesive
function towards platelets by binding to noncontiguous sequences within
the A1 domain loop.66,67
The regions involved in the binding of vWF to GP Ib have only been
partially defined and appear to be dependent, in part, on
conformational structure in both the ligand and receptor. In vWF, both
the peptide sequence, Asp514 to Glu542,66 and the region
encompassing Glu596 and Lys59968 have been proposed as
receptor recognition sites. In GP Ib, the vWF binding site is
located within the N-terminal approximately 300 amino
acids.30,69,70 Three regions within this domain appear to
be important for vWF binding (Fig 2). One
corresponds to the anionic sulfated-tyrosine
sequence,29,30,71,72 which appears to be preferentially
involved in botrocetin-dependent binding of vWF.30,71
Sulfation of tyrosine residues in this sequence is more critical for
botrocetin-dependent than for ristocetin-dependent binding of
vWF,72 but both modulators require the modification for
optimum effect. An Escherichia coli-produced GP Ib fragment containing the sequence encompassing Gln221-Leu318 has been reported to
contain the ristocetin-dependent binding site for vWF, with a
disulfide-bond between Cys248 and Cys264 critical for
function.73 Because Cys248 and Cys264 are normally
disulfide-bonded to Cys209 and Cys211, respectively,74 the
significance of this finding is not clear. The leucine-rich repeats
also appear to have an important role in vWF binding, as suggested by
studies of BSS patients who express mutant GP Ib-IX-V complexes on
their platelets. Platelets expressing these mutant complexes, which
both result from mutations in the GP Ib leucine-rich repeats
(Leu47Phe75 and
Ala156Val76), bound vWF less efficiently than did
normal platelets. Finally, two gain-of-function mutations (Gly
233Val59 and Met 239Val58)
in platelet-type von Willebrand disease are located in the flanking
sequence C-terminal to the leucine-rich repeats.22 Both of
these mutants spontaneously bind vWF in the absence of ristocetin,
botrocetin, or shear, implying that this domain may be directly
involved in vWF binding or could regulate that function.
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| Fig 2.
The GP Ib N-terminus with the regions shown to be
important for vWF binding. Asterisks indicate that the tyrosines are
sulfated.
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Available evidence indicates that the GP Ib-IX-V-vWF interaction may
in many ways be similar to the interaction between selectins and their
ligands. Similar to the rolling of leukocytes mediated by selectins,
recent observations indicate that the GP Ib-IX-V complex can mediate
translocation of platelets along a surface coated with vWF. Such a
phenomenon requires that the bonds be able to form and break rapidly.
In the studies of Savage et al,77 the vWF-GP Ib-IX-V
interaction could slow the platelets in this way, but a further
interaction between vWF and the GP IIb-IIIa complex was required to
fully arrest the platelets. As yet, it is unclear how this in vitro
phenomenon relates to the situation in vivo, because vWF in the
environment of the subendothelium may adapt a different conformation
than when immobilized on glass. The influence of vWF conformation on
platelet translocation was nicely demonstrated in the studies of Moroi
et al78 (in a system similar to that of Savage et
al77), who demonstrated that addition of botrocetin to vWF
immobilized on glass markedly decreased platelet translocation,
presumably because it increased the affinity of the interaction.
Thrombin also binds within the N-terminal sequence, His1-Glu282, of GP
Ib, specifically to the anionic sulfated-tyrosine sequence.30,79 Thrombin recognition, in contrast to the
binding of vWF, has a greater stringency requirement for tyrosine
sulfation in that all 3 tyrosine residues must be sulfated for
effective binding of thrombin to GP Ib.72 High-affinity
binding to the GP Ib-IX-V complex may also involve recognition of
segments of the leucine-rich repeat C-terminal flanking
sequence80,81 and GP V.82 Although the agonist
action of thrombin towards platelets primarily involves signaling
through the 7-transmembrane PAR-1 and/or PAR-3 thrombin
receptors,83,84 binding of thrombin to the GP Ib-IX-V
complex facilitates the platelet response to low concentrations of
thrombin.85-87 A defective response to thrombin undoubtedly
contributes to the bleeding diathesis of patients with BSS. A detailed
discussion of the nuances of thrombin's association with the GP
Ib-IX-V complex is beyond the scope of this review. The interested
reader is referred to the recent review of Jamieson.88
Although it is unknown how thrombin signals through the GP Ib-IX-V
complex, there is increasing evidence that ligation of vWF initiates
signaling events that result ultimately in inside-out activation of the
integrin, GP IIb-IIIa, and platelet aggregation.16,89 Signaling by other adhesion receptors can be initiated by receptor cross-linking90; recent evidence suggests that a similar
mechanism may be operative in GP Ib-IX-V-dependent signaling in
platelets. First, a monomeric 39/34-kD proteolytic fragment of vWF is
able to bind to the GP Ib-IX-V complex and inhibit binding of
multimeric native vWF, but does not activate platelets.52
Second, GP Ib is arranged on the cell surface as part of a larger
receptor complex, with two or more GP Ib subunits forming a cluster
with the other glycoproteins of the complex.22 Third, GP
Ib is associated via its cytoplasmic region with actin-binding
protein and 14-3-3 protein (see below and Fig 1), both of which form
noncovalent dimers. Finally, the 50-kD (presumably bivalent) viper
venom protein, alboaggregin, binds to GP Ib and activates platelets,
whereas structurally related monomeric 25-kD venom proteins bind to the
same domain on GP Ib, but do not activate platelets.91
The signaling events induced by vWF binding to GP Ib-IX-V in the
presence of shear, ristocetin, or botrocetin include elevation of
cytosolic Ca2+ and activation of protein
kinases.92-95 Ser/Thr protein kinases become activated, as
2 of their substrates, pleckstrin and the myosin light chain, are
rapidly phosphorylated.92 Two major tyrosine kinase
substrates (~76 and ~36 kD) also become
phosphorylated,92 but the identity of neither is
known.95 Interestingly, both species are also
phosphorylated in response to 50-kD alboaggregin.91 Other
consequences of vWF binding to GP Ib-IX-V include association of
activated phosphatidylinositol 3-kinase (PI 3-kinase) and src with the
cytoskeleton,94 breakdown of phosphatidylinositol
4,5-bisphosphate, generation of phosphatidic acid, activation of
phospholipase A2, and synthesis of arachidonic acid and
thromboxane A2.92
One of the interesting features of the GP Ib-IX-V complex is that none
of the cytoplasmic sequences of its 4 constituent polypeptides contains
motifs known to interact with signaling proteins. Nevertheless, these
regions do interact with proteins of the platelet membrane cytoskeleton, providing a potential means for the complex to transduce activation signals. The cytoplasmic domain of GP Ib contains a
binding site for actin-binding protein within the sequence Thr536 to
Leu554.96 This association with actin-binding protein links the complex with a network of short submembranous actin
filaments.97,98 This membrane skeleton of quiescent
platelets contains other cytoskeletal proteins, including spectrin,
dystrophin, talin, vinculin, and protein 4.1, and several signaling
proteins, including the tyrosine kinases src, yes, and syk, the small G
protein, p21 ras, and the tyrosine phosphatase, SHP
1.99-101 In unstimulated platelets, much of the GP IIb-IIIa
complex is also attached to the membrane skeleton,99 suggesting that one of the functions of this structure may be to
preassemble key signaling elements, allowing transmission of signals
after GP Ib-IX-V ligation, eventually leading to GP IIb-IIIa activation. Consistent with a role for cytoskeletal attachment in GP
Ib-IX-V functions, recent studies show that even small C-terminal truncations of GP Ib greatly increase the mobility of the complex within the plane of the plasma membrane and decrease its ability to
bind vWF.102
A second possible mechanism by which the GP Ib-IX-V complex may
transmit signals derives from the recent finding that its cytoplasmic
domain contains binding sites for the isoform of 14-3-3.103,104 Although platelet 14-3-3 was originally
reported to have phospholipase A2 activity,105 this
enzymatic activity was not found in other studies.106
Rather, 14-3-3 proteins have recently been shown to regulate the
activity and assembly of key signaling molecules that, in turn,
regulate such diverse processes as mitogenesis, cell cycling, vesicular
transport, and apoptosis. Proteins reported to bind 14-3-3 include the
cell death agonist BAD, raf-1, bcr, cbl, PKC , PKC , the cdc25a and
cdc25b phosphatases, the p85 subunit of PI 3-kinase, tyrosine
hydroxylase, tryptophan hydroxylase, and ADP
ribosyltransferase.107-109 The 14-3-3 protein family
consists of a number of closely related isoforms with subunit molecular
weights of approximately 30 kD that form highly stable homodimers and
heterodimers.107 This latter property allows them to bridge
and assemble cytoplasmic proteins containing 14-3-3 recognition motifs.
The 14-3-3 isoform most commonly identified as binding signaling
molecules is 14-3-3.107
Recent analysis of 14-3-3 binding to raf-1 has identified 2 nonoverlapping binding sites for 14-3-3 within raf-1.108
Both sites contain serines within a conserved R S X S X P
motif, a motif also found in other 14-3-3 binding proteins, including
PKC, cdc25b, bcr, and BAD. The binding of 14-3-3 to these sites is regulated by phosphorylation, with the presence of phosphate on the
serine favoring binding. Within the GP Ib-IX-V complex, a major binding
site for 14-3-3 corresponds to the 4 C-terminal amino acids of GP
Ib, Gly-His-Ser-Leu.104,110 Additional binding sites
have been identified by analysis of overlapping peptides corresponding
to the cytoplasmic sequences of GP Ib, GP Ib, GP IX, and GP
V.110 These include the central region of the GP Ib
cytoplasmic domain (Arg557-Gly575) and the entire cytoplasmic tail of
GP V. Another binding site for 14-3-3 encompasses the PKA
phosphorylation site in GP Ib. Serine phosphorylation of a synthetic
peptide containing this sequence increased its affinity for 14-3-3.
This effect of phosphorylation on a 14-3-3-binding sequence in GP
Ib suggests an additional effect of PKA-dependent phosphorylation on
regulating platelet activation. Because GP Ib phosphorylation
specifically inhibits actin polymerization,40 the increased
affinity for 14-3-3 is consistent with a role for this protein in
the control of this process. Whether 14-3-3 is involved in mediating
the assembly of signaling complexes in response to vWF ligation of the
GP Ib-IX-V complex remains to be determined.
| |
SYNTHESIS OF THE GP Ib-IX-V COMPLEX |
GP Ib, Ib, and IX exist in equal numbers on the surfaces of
platelets17 and cells transfected with the cDNAs encoding the 3 polypeptides.111 Only half as many molecules of GP V
are found on platelets,18,19 although the preciseness of
this molar relationship with the rest of the complex requires further
characterization. Based on studies using both transfected
cells47,111-113 and the platelets of BSS patients with
different mutations,114-116 it appears that maintenance of
this stoichiometry relies primarily on the relative instability of
partial complexes and single polypeptides. For example, in studies of
GP Ib surface expression in transfected cells, it was shown that
this polypeptide is expressed on the surface of the cells most
efficiently when both GP Ib and GP IX were
cotransfected.111 Cotransfection with GP Ib of less than
the full complement of the other 2 polypeptides did not completely prevent GP Ib expression, but did decrease it
substantially. None of these 3 polypeptides is expressed
efficiently on the cell surface unless expression in the cells is
increased by manipulations such as gene
amplification.112,117 Combinations of 2 polypeptides are
more efficient in reaching the cell surface than single polypeptides if
the 2 polypeptides interact with each other directly.112 GP V is not necessary for efficient expression of the rest of the complex
and has only a minor effect, at most, on the expression of GP
Ib-IX.47,118,119 It is the only 1 of the 4 complex
polypeptides that can be efficiently expressed alone on the surfaces of
transfected cells, although its surface expression is increased in the
presence of the rest of the complex.47 From these studies,
it was suggested that BSS could be caused by mutations of either GP
Ib, GP Ib, or GP IX, but the typical syndrome was unlikely to be
caused by mutations of GP V.47,111,112
The molecular defects characterized thus far in patients with BSS
support the findings from these in vitro studies in that mutations
responsible for BSS have only been shown to involve the genes for GP
Ib, GP Ib, and GP IX
(Table
1). Mutations of the latter 2 polypeptides apparently cause the
disorder by decreasing surface expression of GP
Ib.114,115,121 In several of the cases described,
residual quantities of the unaffected polypeptides are still found in
the platelets.114,116,122
Studies in transfected cells have also proved useful for determining
how the polypeptides interact with each other. From such studies, it
has been demonstrated that GP Ib and GP Ib are able to interact
in the absence of the other polypeptides, as are GP Ib and GP
IX.112 Thus, GP Ib is the polypeptide bridging the interaction between GP Ib and GP IX, at least initially, because no
interaction between the later 2 polypeptides could be detected in the
absence of GP Ib. In contrast, antibody inhibition studies of
platelet lysates and purified GP Ib-IX complex suggest that GP IX is
more strongly associated with GP Ib than with GP
Ib.123 Confocal microscopy and expression studies
indicate that the interaction of GP V with GP Ib-IX is through a direct
link with GP Ib.47 This association has a direct
functional consequence, because expression of GP V in cultured cells is
required for the complex to bind thrombin with high affinity, even
though the site of thrombin binding is on GP Ib.82
The polypeptides of the complex all associate soon after their
synthesis and insertion into the membrane of the endoplasmic reticulum.124 Before the complex reaches the cell surface,
which in cultured cells takes approximately 3 hours,124 its
polypeptides undergo a number of posttranslational modifications,
including the addition of both N- and O-linked
carbohydrate, modification of the intracytoplasmic cysteines of GP
Ib and GP IX by acylation with fatty acids, and sulfation of
tyrosines in the ligand-binding domain of GP Ib. These modifications
are all likely to influence the functions of the complex, and it is
probable that mutations that disrupt any of the posttranslational
modifications in vivo will result in variant forms of BSS.
| |
GENES ENCODING THE GP Ib-IX-V POLYPEPTIDES |
A separate gene encodes each component of the GP Ib-IX-V complex
receptor. Like the polypeptides of this complex, the genes share a
number of structural features (Fig 3). All
except the gene for GP Ib contain the entire coding sequence within
one exon45,125,126; the GP Ib gene contains an intron 10 bases after the start of the coding sequence.25 All are
also relatively devoid of introns, with only the GP IX gene containing
more than 1 (it contains 2).126 These genes share this
compact structure and paucity of introns with other genes of the
leucine-rich repeat family, the best example being the gene for
oligodendrocyte-myelin glycoprotein, which contains one small intron in
its 5 untranslated region and the entire coding region in 1 exon.127 Despite their structural similarity, the genes
encoding the GP Ib-IX-V polypeptides are not clustered in 1 region of
the human genome. The GP Ib gene is located on the short arm of
chromosome 17,23 the GP Ib gene is on the long arm of
chromosome 22,24 and the GP IX and GP V genes are located
on the long arm of chromosome 327 (3q21 and 3q29,
respectively; Fig 3).
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| Fig 3.
Structures of the genes encoding the 4 polypeptides of
the GP Ib-IX-V complex with exons shown as boxes, introns as the lines between boxes, and open reading frames in black. The position of the
ATG start codon is also indicated.
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Expression of the GP Ib-IX-V complex is limited to a very small number
of tissues, the only major constitutive expression being in
megakaryocytes and platelets. This complex may also be expressed in
endothelial cells, although this is a matter of controversy. There have
been reports of low level expression of GP Ib in endothelial cells,128,129 expression that can be enhanced by the
inflammatory cytokine, tumor necrosis factor-.130,131
Further evidence for expression of GP Ib in endothelium was obtained
by the cloning of a GP Ib cDNA from an endothelial cell
library.131 This cDNA was virtually identical to the
original GP Ib cDNA cloned from a HEL cell library.31
More recently, Wu et al20 have provided evidence that
endothelial cells, in culture and in vivo, express the full GP Ib-IX-V
complex. One difference with the platelet complex is in the nature of
GP Ib. Kelly et al24 found a polypeptide in endothelial
cells that reacted with GP Ib antisera, but that migrated at a
higher molecular mass (~50 kD) than the platelet polypeptide (~25
kD). They also cloned a cDNA that encoded a polypeptide with an amino
terminus unrelated to platelet GP Ib but fused in frame with the
platelet sequence such that the new polypeptide also contained
essentially all of the platelet sequence. This interpretation of the
data has since been challenged by Zieger et al,132 who also
cloned a cDNA containing the GP Ib sequence. They identified a new
gene immediately 5 to the GP Ib gene that produced 2 transcripts, 1 containing the GP Ib sequence. The latter transcript
presumably arose because the 5 gene contains a suboptimal
polyadenylation sequence. Hence, the transcription machinery sometimes
reads through it and into the GP Ib gene, eventually using the GP
Ib polyadenylation sequence. The resulting transcript thus also
contains the GP Ib sequence, albiet out of frame, a finding at odds
with that of Kelly et al.24
One potential function for the complex expressed in endothelial cells
derives from the work of Beacham et al,133 who suggested that the complex can mediate attachment of endothelial cells to vWF.
Bombeli et al134 also recently proposed a role for
endothelial cell GP Ib in adhesion of activated platelets to
umbilical vein endothelial cells. Others have not been able to
demonstrate GP Ib-IX-V-mediated attachment of endothelial cells to vWF
and have even called into question whether these cells have significant levels of complex expression.135 If, how, and when the GP
Ib-IX-V complex is expressed in endothelial cells are thus still open questions in need of more investigation. Such expression of the GP
Ib-IX-V complex in endothelium in vivo may depend on such variables as
regional shear stresses, the presence of inflammatory cytokines, and
the particular vascular bed from which the cells are derived.
At least part of the restricted expression of the GP Ib-IX-V complex
can be ascribed to the unusual structure of the promoter regions of its
genes. None of the promoters contain functional TATA or CAAT boxes,
consensus transcription factor-binding sequences found in a high
percentage of eukaryotic genes (GP V does contain 2 potential TATA
boxes, but primer-elongation studies did not show transcripts of the
expected sizes45). Instead, these promoters contain binding
sites for the GATA and ETS families of transcription factors, a feature
shared with other genes expressed in cells of megakaryocytic and
erythroid lineages.136-142 Neither GATA nor ETS is specific
for megakaryocytes; it has been suggested that particular combinations
and relative levels of the GATA and ETS families are what determine
megakaryocyte specificity.138,140 This specificity may also
be related to transcriptional cofactors. Recently, a transcription
factor named FOG (Friend of GATA-1) was described, which acts as a
cofactor for GATA-1 during both erythroid and megakaryocytic cell
differentiation.143 Together, the 2 transcription factors
may stimulate transcription in a context-specific manner.144
The importance of these factors for transcription of the GP Ib-IX-V
genes is demonstrated by both synthetic and natural mutations. Mutations of both the GATA and ETS binding sequences in the promoters of GP Ib and GP IX have been shown to reduce or abolish reporter gene expression in human erythroleukemia cells.141,142
Likewise, a single-base mutation of the GATA-1 site in the GP Ib
promoter markedly reduced expression of GP Ib and caused BSS in a
patient with deletion of the other GP Ib allele and
velo-cardio-facial syndrome.115
| |
POLYMORPHISMS AFFECTING THE GENES AND POLYPEPTIDES OF THE GP
Ib-IX-V COMPLEX |
Several polymorphisms of the GP Ib-IX-V complex have been described,
affecting primarily the GP Ib gene. In addition to potentially affecting the structure and functions of the complex, these
polymorphisms serve as useful linkage markers for the genes affected.
The first described polymorphism of the complex was a variable number
of tandem repeats (VNTR) polymorphism affecting the region encoding the
GP Ib macroglycopeptide.36,37,145,146 The 4 alleles vary
in the number of tandem repeats of a 39-nucleotide sequence, which is
present either 1, 2, 3, or 4 times in the different alleles.36,37 The resulting polypeptides specified by these alleles contain different numbers of 13-amino acid repeats in their
macroglycopeptide region. Each repeat contains 5 potential sites for
O-glycosylation, a modification predicted to add approximately 6 kD to the mass of the macroglycopeptide and 32 Å to its
length.36 This VNTR polymorphism is the most informative as
a genetic marker because of the high frequency of heterozygosity at
this locus (25% to 30% in most populations).36 The
frequencies of the different alleles vary widely in different ethnic
populations, although the variant with 2 repeats (C variant) is the
most common in all populations studied.22
Another polymorphism of GP Ib results in dimorphism at residue 145, with either Thr or Met occupying this position. The allele frequencies
have been reported to be 90% and 10%, respectively, for the Thr and
Met codons in both European and Japanese
populations.147,148 This marker is closely linked to the
VNTR polymorphism, with Met at position 145 being found only associated
with the 3 largest size variants.37,149,150 Thus, this
marker might be of use in determining heterozygosity in someone
homozygous for the larger VNTR alleles. This marker has the additional
advantage that the products of its alleles can be recognized on
platelets with antisera, because this polymorphism accounts for the
HPA-2 (or Ko) alloantigen system.147,148
Recently, 2 more polymorphisms of the GP Ib locus were described,
the RS system, its alleles specifying either C or T at position
 5 from the ATG start codon,151 and a nucleotide
dimorphism (A or G) of the third base of the codon for
Arg358.151,152 The degree of association between these
markers and the other GP Ib polymorphisms has yet to be determined.
To analyze for possible linkage of the BSS phenotype with the GP Ib
locus, markers used in the analysis of the Di George and
velo-cardio-facial syndromes can be used.153-156 As yet, no
markers are available for the GP IX or GP V genes.
| |
BSS: CLINICAL MANIFESTATIONS, DIAGNOSIS, AND THERAPY |
BSS is extremely rare. In the populations of Europe, North America, and
Japan, which have been studied most intensively, a prevalence of less
than 1 in 1,000,000 can be estimated from cases reported in the
literature. No doubt, this is an underestimate due to misdiagnosis and
underreporting, but the low frequency of reported cases nevertheless is
an indication of the rarity of the disorder. Perhaps one reason for
this low prevalence is that, despite the potential for the disorder to
be caused by mutation of any of 3 genes (and perhaps 4), the
compactness of these genes decreases the frequency at which they are
subject to random mutation. The lack of introns interrupting the coding
sequence also greatly decreases the possibility that missplicing will
cause deficiency of the encoded polypeptides. The low frequency of
mutation at these loci is reflected also in the fact that the majority
of the reported cases are homozygous for the same allele, having inherited 2 mutant alleles from parents who are blood relatives. The
clinical features of the BSS patients reported to date are summarized
in Table 1. Based on this relatively small number of reported cases,
there appears to be no gender preference for BSS (47 of 88 patients
described in Table 1 are female), as one would expect from an autosomal
disorder. Of the patients in Table 1 for whom ethnicity was reported,
49 are Caucasian, 13 are Japanese, and 4 are of other ethnic groups.
Inheritance.
Inheritance of the BSS is usually autosomal recessive and is often
associated with consanguinity (Table 1). Heterozygous family members
may show about half the normal levels of platelet GP Ib-IX-V
expression, but with no bleeding diatheses or only mild bleeding.
Autosomal dominant inheritance has been reported in only 1 family.75
Clinical manifestations.
BSS is characterized clinically by a prolonged skin bleeding time,
morphologically enlarged platelets, and thrombocytopenia (Table 1 and
reviewed in Dunlop et al157). Clinical manifestations commonly include frequent episodes of epistaxis, gingival and cutaneous
bleeding, and hemorrhage associated with trauma. Although these
characteristics are typical, comparisons of the clinical profiles of
BSS patients reveal considerable variation between individuals.
Platelet counts may range from very low (<30,000/µL) to marginally
low or normal (~200,000/µL) and in individual patients may
fluctuate considerably over a period of years. Skin bleeding times may
range from only marginally prolonged (5 to 10 minutes) to greater than
20 minutes. Bleeding tendencies associated with BSS are usually evident
from early childhood. However, the severity of symptoms may
progressively worsen or become alleviated throughout puberty and adult
life. Most often, severe bleeding episodes are associated with
tonsillectomy, appendectomy, splenectomy, other surgical procedures,
dental extractions, menses and pregnancies, or accidents. Ecchymoses
without significant trauma are relatively common, as are episodes of
spontaneous epistaxis and gingival and gastrointestinal bleeding.
Menorrhagia in premenopausal women is of variable severity and may be
controlled in some cases by oral contraceptives.13,158,159
Pregnancy in BSS patients may be relatively uneventful or may present
complications of varying severity.116,121,122,158,160-166
Bleeding associated with childbirth is generally supported by blood
and/or platelet transfusions and may necessitate hysterectomy
to control bleeding.161 Multiple childbirth is not
uncommon.116,121
Diagnosis.
Congenital platelet disorders related to platelet adhesion, activation,
secretion, aggregation, or number and various coagulopathies are often
not distinguishable from their clinical manifestations alone,
presenting a challenge to diagnosis that often requires specialized
tests or biochemical analyses. For example, BSS has frequently been
misdiagnosed as idiopathic thrombocytopenic purpura (ITP),116,121,167-170 based on a prolonged bleeding time
and thrombocytopenia, and often is treated unsuccessfully with steroids
or splenectomy. The initial laboratory assessment of BSS should involve
measurement of blood cell counts and examination of a blood smear for
thrombocytopenia and morphological abnormalities of platelets. BSS can
usually be differentiated experimentally from other bleeding disorders by functional analysis of stirred platelet suspensions in an
aggregometer. The characteristic abnormality in BSS is an isolated
defect in ristocetin-induced agglutination. Unlike the defect in von
Willebrand disease, this abnormality is not corrected by the addition
of normal plasma. Platelet aggregation in response to other agonists, such as collagen and ADP, as well as clot retraction, is usually normal. The provisional diagnosis based on aggregometry should be
confirmed biochemically (reviewed in Dunlop et al157). This may involve assessment of platelet surface glycoprotein expression by
flow cytometry, surface-labeling of washed platelets followed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography,
or immunoblotting of platelet lysates with specific antiplatelet
glycoprotein antibodies. Finally, establishing an abnormal genotype by
molecular studies may allow precise definition of the abnormality
causing the platelet defect, as discussed below.
Therapy.
The therapeutic approaches to the management of patients with BSS
involve both general supportive measures and specific treatment of
bleeding episodes. General measures include educating the patients about their bleeding diathesis and the importance of avoiding even
relatively minor trauma and advising them against the use of
antiplatelet medications such as aspirin. Adequate dental hygiene should be maintained to prevent gingival disease and to minimize dental
procedures. Iron deficiency may result from chronic gingival bleeding
or menorrhagia and should be treated. In some cases, splenectomy has
apparently been beneficial in moderating thrombocytopenia and the
severity of clinical symptoms,158,168,171,172 although this
treatment should be avoided because of the high risk for perisurgical
hemorrhage and the lack of controlled data to support its use. Control
of bleeding episodes or prophylaxis for prevention of bleeding during
surgical procedures usually requires transfusion of blood
and/or platelets, despite the risk that these patients will
develop antiplatelet and/or antierythrocyte
alloantibodies.158,168,171,172 General anesthesia has been
successful in a BSS patient, although anesthetics such as halothane or
dibucaine that compromise platelet reactivity should be
avoided.173 The use of antifibrinolytic drugs, such as
-aminocaproic acid or tranexamic acid, may or may not be
beneficial.169,170,172 DDAVP may shorten the bleeding time
in some168,171,174 but not all159,169 BSS
patients. The different responses of individual patients to these
latter measures may reflect differences in the underlying disease, with
those with milder forms of the disease more likely to respond to these therapies.
Because of the relative ease with which the molecular lesions can be
determined and given the simplicity of the affected genes, BSS seems an
ideal candidate disease for gene therapy. Such therapy would presumably
involve transduction of a hematopoietic stem cell with a working copy
of the defective gene, under the control of its own promoter or of
another platelet-specific promoter. Among the questions to be answered
before such therapy becomes reality is whether reconstituting the blood
with only a relatively small proportion of normal platelets will be
sufficient to ameliorate the bleeding diathesis associated with BSS.
| |
BSS: CLASSIFICATION |
The genetic defects underlying BSS so far determined (Table 1) are
clearly heterogeneous, but may be broadly categorized in 2 ways. First,
the abnormality may be either (1) a biosynthetic defect affecting
synthesis, processing, or expression of the GP Ib-IX-V complex; or (2)
a functional defect in which GP Ib is expressed in a dysfunctional
form that fails to bind ligand. Second, the genetic lesion may be
localized to (1) the GP Ib gene (chromosome 17pter-p12), (2) the GP
Ib gene (chromosome 22q11.2), (3) the GP IX gene (chromosome 3q21),
or possibly (4) the GP V gene (chromosome 3q29). The syndrome may
conveniently be classified, therefore, as type 1a to indicate a defect
of the GP Ib gene that results in a biosynthetic defect, type 1b for
a synthetic defect of the GP Ib gene, etc. The molecular defects
thus far reported arise from missense, nonsense, or deletion mutations
of the GP Ib, GP Ib, or GP IX genes (Table 1) that produce
truncated, unstable, or dysfunctional polypeptides.
| |
INFORMATIVE MUTATIONS IN BSS AND PLATELET-TYPE VON WILLEBRAND DISEASE |
Clearly, much remains to be learned about the cause of several
phenotypic features of the BSS. Nevertheless, elucidation of the
molecular basis of BSS and platelet-type von Willebrand disease has
provided several very valuable insights into the synthesis and
functions of the complex. A few of the more informative mutations will
be reviewed in this section.
GP Ib mutations.
Several mutations of GP Ib provide interesting information regarding
the functions of the GP Ib-IX-V complex. The Bolzano variant of BSS,
caused by a substitution of Val for Ala at position 156 of GP Ib,
produces mutant complexes that appear on the cell surface essentially
at normal levels.76 This mutant is unable to bind vWF
normally, but binds thrombin with a similar affinity to that of the
wild-type complex.175 The platelets of the patient with the
Bolzano variant of BSS thus do not have the defect in the response to
low concentrations of thrombin that most BSS platelets do. This finding
suggests that the leucine-rich repeats have an important role in
binding vWF, but not thrombin. Another interesting feature of this
mutant is that it has no mutations of its cytoplasmic region that would
be predicted to influence the association of the complex with the
platelet cytoskeleton, yet the platelets of the affected patient are
much larger than normal, indicating that the large platelets in BSS
cannot be explained simply by a defective membrane-cytoskeletal
association.
Only one instance of autosomal-dominant transmission of BSS has been
described. The responsible mutation, Leu57Phe, like the
Bolzano mutation, affects the leucine-rich repeat region of GP Ib
and encodes a mutant polypeptide that appears on the cell surface, but,
once there, is apparently abnormally susceptible to cleavage by plasma
proteases.75 The dominant nature of this mutation suggests
that the product of the mutant allele interferes with the functions of
the wild-type polypeptide, giving further support for the existence of
a vWF receptor containing more than 1 GP Ib polypeptide.
Also of interest is the recently described deletion of the last 2 bases
of GP Ib codon 492, which results in a reading frame-shift within
the region encoding the GP Ib membrane-spanning segment, with the
addition of 81 novel amino acids before the polypeptide reaches a
premature stop. Two unrelated patients homozygous for this mutation
were described simultaneously; both had a considerable amount of GP
Ib or a degradation product in their plasma, indicating that GP
Ib was synthesized normally but failed to be anchored in the plasma
membrane.120,176 In addition to carrying the same mutation,
these 2 patients had an identical haplotype, with identical sequences
at 3 other polymorphic sites. The ancestors of both of these patients
emigrated to the United States from Germany. Interestingly, a Finnish
patient carrying an identical mutant haplotype was recently reported in
abstract form.177 This mutant haplotype probably arose at
least several centuries ago in the northern European population and
will likely be a common mutation associated with the disorder in
patients of northern European ancestry.
Platelet-type von Willebrand disease is another bleeding disorder
caused by mutations affecting the GP Ib-IX-V complex, but in this case
resulting in a dominant gain-of-function phenotype.60,61 The resultant mutants bind vWF with high affinity and the paradoxical presence of a bleeding predisposition is due to clearance of the hemostatically most active large vWF multimers. The mutations described
in this disorder (Gly233Val59 and
Met239Val58,178,179) are found within a short
linear sequence encompassing residues 233 to 239 of GP Ib, a region
that lies within the loop formed by a disulfide bond between Cys209 and
Cys248 of GP Ib (Fig 2). Molecular modeling studies of this region
suggest that the mutations produce an active conformation of GP Ib
that is competent to bind vWF in the absence of
modulators.180,181 These mutants thus provide clues as to
changes that the receptor undergoes when platelets are exposed to shear
or ristocetin.
GP Ib.
Two BSS variants that result from mutations of GP Ib are
particularly informative. The first was described in a patient with velo-cardio-facial syndrome, a developmental disorder caused by deletion of the chromosomal region 22.11.2, which contains the gene for
GP Ib.182 This patient's remaining GP Ib gene did not contain any mutations in the polypeptide coding region, but a
single point mutation was found in the promoter region within a binding
sequence for the GATA-1 transcription factor.115
Transcription studies performed in cell lines demonstrated that the
mutation decreased the transcription of a reporter gene sixfold. So
far, this is the only reported case of BSS not caused by mutations of
polypeptide coding regions.
Another interesting variant of BSS caused by mutations of the GP Ib
gene was described in a Japanese patient with a very mild propensity
for bleeding.183 This patient was a compound heterozygote
for 2 mutations (Tyr88Cys and Ala108Pro). The
platelets were not defective for agglutination by either ristocetin or
botrocetin |
Introduction
References