(Received for publication, September 6, 1995; and in revised form, January 6, 1996)
From the
The interaction between von Willebrand factor (vWF) A1 domain
and platelet glycoprotein Ib occurs in the presence of high shear
stress or when vWF becomes immobilized onto a surface but not
appreciably in the normal circulation. To investigate the structural
properties regulating A1 domain function, we have used recombinant
fragments prepared either in cyclic form with oxidized
Cys
-Cys
disulfide bond or reduced and
alkylated. Interaction with glycoprotein Ib
was assessed by
testing inhibition of monoclonal antibody LJ-Ib1 binding to platelets
and inhibition of shear-induced platelet aggregation mediated by native
vWF. Fragments exposed to pH between 2.5 and 3.5 adopted the molten
globule conformation with loosened tertiary structure intermediate
between native and completely unordered state. Maximal receptor binding
activity was observed when fragments kept at acidic pH, particularly
after reduction of the Cys
-Cys
disulfide bond, were subjected to quick refolding by rapid pH
increase. In contrast, slow refolding by incremental pH change over
several hours resulted in at least 20-fold lower activity. A specific
single point mutation (I546V) resulted in enhanced receptor binding,
whereas another mutation (S561G) caused markedly reduced binding. These
results provide experimental evidence that conformational transitions
can modulate function of the vWF A1 domain in solution.
Platelet adhesion and thrombus formation at sites of vascular
injury depend on the function of von Willebrand factor (vWF), ()particularly in areas of the circulation where blood flow
creates high shear stress conditions(1) . In these processes,
essential for normal hemostasis but also important in the development
of arterial thrombosis(2, 3, 4) , the
interaction between a specific domain of vWF (5) and platelet
glycoprotein (GP) Ib
(6) , a component of the GP Ib-IX-V
complex, is the necessary initial event(7, 8) . The GP
Ib
-binding domain of vWF was initially identified in a monomeric,
reduced and alkylated tryptic fragment comprising residues
449-728 of the mature subunit (5) and corresponding
essentially to the A1 domain of the molecule(9) . It contains
both intrachain and interchain disulfide bonds and can be isolated
either as a monomer (10) or a dimer (11) after limited
proteolytic digestion of vWF; it can also be expressed recombinantly in
either form(12, 13, 14) .
Native vWF in
blood does not show detectable binding to platelets. The interaction
can be induced by exogenous nonphysiologic modulators, such as
ristocetin (15, 16) and
botrocetin(17, 18) , and by fluid shear stress (19) or can be the consequence of selected mutations in the A1
domain(20) ; moreover, platelets adhere to vWF immobilized onto
a surface(7) . It has generally been assumed that the
modulation of GP Ib binding activity involves conformational
changes in vWF with exposure of functional sites normally cryptic when
the molecule is in solution. In vivo, this may occur when vWF
interacts with subendothelial components, like collagen(21) ,
where integrity of the vessel wall is disrupted.
The present studies
have been undertaken to test the hypothesis that conformational changes
may influence the GP Ib binding function of vWF. Our results
demonstrate that quick refolding following loosening of tertiary
structure may lead to increased binding affinity and that single point
mutations can either enhance or decrease A1 domain interaction with GP
Ib in the absence of exogenous modulators. These findings provide
initial information on the mechanisms involved in the regulation of
receptor recognition sites in the A1 domain of vWF.
Figure 1:
Schematic representation of the
recombinant vWF fragments. The amino- and carboxyl-terminal residue in
each sequence are shown with the number corresponding to the position
in the sequence of the mature vWF subunit(49) . In the E.
coli-derived fragments, however, amino-terminal sequence analysis
demonstrated that the first predicted vWF residue is preceded by the
initiating N-formyl Met not removed by bacterial processing of
the expressed protein. Note that in the mammalian cell expressed rvWFC462A, Cys
was
mutated to Ala to avoid dimerization(14) ; of the remaining six
Cys residues, four are known to pair in native vWF as indicated
(471-474 and 509-695), whereas the remaining two are likely
to be paired in this fragment but may be involved in different bonds in
native vWF. Note also that this fragment is expected to contain eight O-linked (open circles) and one N-linked (filled circles) carbohydrate chains by analogy with native
vWF (49) and in agreement with its mobility in SDS-PAGE (see Fig. 2). The E. coli-derived rvWF
was expressed with five Cys
Gly mutations (at positions 459, 462, 464, 471, and 474) to
reduce disordered disulfide-dependent aggregation during purification.
All fragments were obtained either with intact
Cys
-Cys
intramolecular disulfide bond
(cyclic form) or with reduced and S-carboxyamidomethylated Cys
residues (reduced and alkylated; R/A). The occurrence of S-carboxyamidomethylation of Cys residues was demonstrated
unequivocally by the expected 116-Da mass increase as measured by mass
spectroscopy following reduction and alkylation. Because of limited
availability, this assay was not performed with the mammalian
cell-derived fragment. Amino acids are identified with single-letter
notation in the figure.
Figure 2:
SDS-PAGE and dot blot analysis of the
recombinant vWF fragments. A, dot blot analysis. An aliquot of
each fragment was applied onto a nitrocellulose membrane in a circular
area. After drying, the membrane was treated with a solution of the
anti-vWF monoclonal antibody NMC-4 (5 µg/ml) followed by
fluorographic detection of the bound antibody using an alkaline
phosphatase-conjugated rabbit anti-mouse IgG. Note that the antibody
reacts well with the cyclic fragments containing the intact
Cys-Cys
disulfide bond (C)
but only minimally after reduction and alkylation of Cys residues (R/A). B, SDS-PAGE analysis. Fragments were analyzed
in a 10% gel under nonreducing conditions. Proteins in the gel were
stained with Coomassie Brilliant Blue. The numbers to the right indicate the molecular masses of standard proteins (in
kDa). Note the slower migration and apparent size heterogeneity of the
mammalian cell derived rvWF
C462A as
compared with the corresponding E. coli derived rvWF
, composed of approximately the
same number of residues. These findings are compatible with the
presence of variable carbohydrate side chains in the mammalian cell
derived molecule, as in native
vWF(5, 13) .
Figure 3:
Intrinsic fluorescence spectra of rvWFC462A, cyclic or reduced and
alkylated, at acidic pH or after slow refolding at neutral pH. The
fragment was tested at concentrations between 0.5 and 1 µM in either 2 mM acetic acid titrated with HCl to pH 2.5 or
2 mM ammonium acetate, pH 7.0. Excitation wavelength of either
280 or 295 nm was used; the latter minimizes the contribution of Tyr
emission.
Both cyclic and reduced and alkylated rvWFC462A maintained either at
physiologic or acidic pH showed similar CD spectra, indicative of the
presence of
helix as well as
strand secondary structures (Fig. 4). A slight difference in mean residue ellipticity at
<205 nm in the spectrum of the fragments kept at pH 2.5,
particularly after reduction and alkylation, indicated some increase in
the content of unordered structure. Overall, the CD and intrinsic
fluorescence spectra were typical of the molten globule intermediate
between the native and the completely unfolded states(42) .
Figure 4:
Circular dichroism spectra of rvWFC462A, cyclic or reduced and
alkylated, at acidic pH or after slow refolding at neutral pH. The
fragment was tested at concentrations between 0.5 and 1 µM in either 2 mM acetic acid titrated with HCl to pH 2.5 or
2 mM ammonium acetate, pH 7.0. Far ultraviolet circular
dichroism spectra were recorded at wavelength intervals of 0.1 nm
ranging from 260 to 185 nm. Each measurement was repeated 20 times. The
results were averaged and then corrected for spurious signals generated
by the solvent. The spectra were converted to mean residue
ellipticities.
Figure 5:
Inhibition of anti-GP Ib monoclonal
antibody LJ-Ib1 binding to platelets by rvWF
cyclic or reduced and alkylated. Platelet-rich plasma was mixed
with 10 mM HEPES buffer, pH 7.4, a constant volume (always
<12.5 µl) of various concentrations of rvWF fragment
and 10 µg/ml of
I-labeled monoclonal LJ-Ib1 IgG in 10
mM HEPES buffer, pH 7.4. The fragment was either added into
the experimental mixture from a solution in 2 mM acetic acid
at pH 3.5 (this was called ``quick'' refolding because the
fragment was brought to pH 7.0-7.4 by direct mixing with the
other test reagents) or refolded before testing by slow pH change from
acidic to 7.0 (``slow'' refolding). The control mixture
contained either 2 mM acetic acid titrated to pH 3.5 with HCl
or 2 mM ammonium acetate, pH 7.0, instead of vWF fragment at
the corresponding pH. The total volume of each mixture was 125 µl,
and the final platelet count was 1
10
/ml; after
adding all reagents, the pH of each mixture was between 7.0 and 7.4.
After incubation at room temperature for 30 min, platelets were
separated by centrifugation though a layer of 20% sucrose, and the
radioactivity of LJ-Ib1 bound to platelets was measured in a
-scintillation counter. Nonspecific binding of
I-labeled LJ-Ib1 was estimated with the addition of a
100-fold excess of unlabeled LJ-Ib1 and was always <10% of total
binding; the corresponding value was subtracted from all data points.
The results are expressed as the mean ± S.E. of percent residual
binding relative to the corresponding control mixture (n = 7 for cyclic fragments and n = 6 for
reduced/alkylated fragments).
The
results obtained by measuring inhibition of LJ-Ib1 binding to platelets
were confirmed by testing directly the ability of the rvWF
fragments to inhibit shear-induced aggregation, a phenomenon that is
strictly dependent on native vWF binding to GP Ib. Also in this
case, quickly refolded fragments kept at acidic pH until just before
the assay were more effective inhibitors than the corresponding slowly
refolded ones, and the greatest inhibitory effect was obtained with
reduced and alkylated fragments. Results similar to the ones shown here
for E. coli-derived rvWF
(Fig. 6) were obtained with all the fragments prepared for
this study.
Figure 6:
Inhibition of shear-induced platelet
aggregation by rvWF cyclic or reduced
and alkylated. Each mixture (final volume, 400 µl) contained washed
platelets to give a final count of 3
10
/ml, 1
mM CaCl
, 5 mg/ml bovine serum albumin, 15
µg/ml purified vWF, and the indicated concentration of rvWF fragment, prepared as described in the legend to Fig. 5, or equivalent volume of the corresponding buffer in the
control mixtures. Each sample was exposed to a shear rate of 120
s
for 5 s, during which the light transmittance of
the platelet-rich suspension (0% aggregation) was recorded; the shear
rate was then linearly increased to 10,800 s
in 15 s
and maintained at that level for 340 s at room temperature (22-25
°C). The results are shown as extent of platelet aggregation,
calculated by recording the values of laser light transmittance through
the platelet suspension at 1-s intervals and applying the
Lambert-Beer's equation (41) .
Figure 7:
Inhibition of anti-GP Ib monoclonal
antibody LJ-Ib1 binding to platelets by normal or mutant rvWF
fragments. These experiments were
performed as described in the legend to Fig. 5using either
normal or mutant rvWF
containing the
amino acid substitutions identified in two patients with type 2B von
Willebrand disease (I546V or P574L) or in subtype B of type 2M (G561S).
All fragments were tested in either cyclic (left panels) or
reduced and alkylated form (R/A; right panels) with
slow (upper panels) or quick refolding (lower panels)
after exposure to pH 3.5. The results are expressed as the mean
± S.E. of the percentage of residual binding relative to the
corresponding control mixture (n = 3 for cyclic
fragments and n = 2 for R/A fragments).
The interaction between vWF A1 domain and GP Ib has been
characterized previously with the use of ristocetin (15, 16) and botrocetin(17, 18) , two
molecules without known physiologic counterpart that modulate activity
but have no defined effects on vWF structure. Here, we show that slowly
refolded recombinant fragments with intact
Cys
-Cys
disulfide loop behave like
the A1 domain of native plasma vWF, which typically cannot interact
with GP Ib
in the absence of modulators or mechanical shear
forces. In particular, both rvWF
C462A
and rvWF
inhibited antibody LJ-Ib1
binding to platelets with IC
well above the 20 nM approximate plasma concentration of vWF subunit, tantamount to the
lack of measurable interaction shown by the latter. In contrast,
reduced and alkylated fragments refolded quickly after exposure to
acidic pH attained an apparent affinity for GP Ib
that, if
exhibited by circulating vWF, would result in binding to platelets. Our
findings, therefore, although based on experimental conditions that
cannot be directly relevant in vivo, establish a correlation
between specific conformational characteristics of vWF A1 domain and
function.
It has been suggested previously that isolated recombinant
fragments expressed in bacteria or mammalian cells reflect the
biological activities of native
vWF(12, 13, 20, 24, 43) .
This concept is further supported by the demonstration provided here
that mutations causing enhanced or reduced interaction of native vWF
with GP Ib produce similar functional alterations when expressed
in monomeric rvWF
. It is of interest
that only one of the two type 2B mutations tested resulted in increased
receptor binding activity in the absence of modulators, suggesting that
different amino acid substitutions known to be associated with type 2B
von Willebrand disease (29) have distinct structural and
functional consequences. Indeed, it is not known to date whether all
type 2B mutations result in enhanced multimeric vWF binding to
platelets in the absence of modulators and/or shear
stress(44) , and, in at least one specific case, it has been
shown that expression of a dimeric A1 domain fragment in mammalian
cells may be necessary to demonstrate heightened activity (20) . Equally noteworthy is the fact that expression of the
type 2M subtype B mutation in monomeric rvWF
demonstrates markedly decreased function in the absence of
modulators, whereas native or recombinant multimeric vWF with the same
mutation shows reduced activity with ristocetin but normal with
botrocetin(30) . Our results provide experimental evidence that
the defect, in this case, is intrinsically related to structural
changes affecting A1 domain receptor binding function and not
necessarily modulator-dependent. Interestingly, GP Ib
binding
activity, although well below normal, could be elicited in the subtype
B mutant rvWF
by quick refolding after
denaturation, suggesting that the procedure leads to exposure of
``hidden'' functional sites similar to what may be caused by
complex formation with botrocetin.
The segment of sequence
containing Trp has been implicated in interacting with
the modulator botrocetin(18) , thus, indirectly, in the control
of binding to GP Ib
, a role also suggested by the observation that
a Trp
Cys mutation causes enhanced interaction
with the receptor in type 2B vWF(20) . Our results now
demonstrate that perturbations in the position of the side chain of
Trp
correlate with an increase of between 1 and 2 orders
of magnitude in GP Ib
binding affinity, presumably consequent to
surface exposure of a previously masked active site. Transition from
low to high affinity appears to require loosening of native tertiary
structure followed by quick refolding. During this process, exposed
hydrophobic regions in the molten globule conformation created by
partial unfolding at acidic pH (42) may form initially
``incorrect'' intramolecular associations, leading to a
transient non-native structure with unmasked GP Ib
interaction
sites. This state may be followed by rearrangement of the transient
hydrophobic interfaces during slow refolding, resulting in the final
native structure with masked interaction sites. In this regard, the
Cys
-Cys
disulfide bond can be viewed
as a fixed tertiary structural element that tends to maintain native
intramolecular interactions even after exposure to low pH; hence the
impossibility for the cyclic fragments to achieve maximal GP Ib
binding activity. Because quick refolding, in the context of these
studies, was initiated by a change from acidic to neutral pH coincident
with functional measurements, formation of the intermediate and active
conformation may occur rapidly and be relatively short lived. Whether
refolding under these conditions was also affected by the presence of
the receptor on platelets acting as a template for inducing optimal
binding conformations cannot be determined at present. If this
happened, the fragments used for CD and intrinsic fluorescence
measurements, refolded quickly in solution in the absence of platelets,
may have been structurally different from those used in functional
tests.
The present study, in agreement with previous results from
our laboratory (5, 24) but in apparent contrast with
conclusions reached by others(45) , clearly indicates that the
Cys-Cys
disulfide loop is not
strictly required to preserve A1 domain interaction with GP Ib
.
Disruption of the bond was proven unequivocally by molecular mass
determinations, thus eliminating any concern about its possible
resistance to the reducing procedures utilized(45) . Rather,
methodological differences with respect to the use of exogenous
modulators are the most likely cause of the conflicting findings. It
has been reported that full-length recombinant vWF multimers containing
Cys
Ser mutations at positions 509 and 695 fail to bind to
platelets in the presence of ristocetin(45) , suggesting that
the Cys
-Cys
disulfide bond may be
needed for the expression of activity induced by this modulator.
However, loss of function in this case may also result from major
conformational alterations within the A1 domain, possibly caused by
abnormal folding of the large vWF subunit in the absence of an
important stabilizing structural element, and/or as a consequence of
disulfide bond rearrangement. These factors appear to be irrelevant in
the case of isolated A1 domain fragments, providing a more direct
assessment of the involvement of the
Cys
-Cys
loop in the expression of
function.
The identification of GP Ib interaction sites in the
A1 domain of vWF is a topic of ongoing research. A recent study based
on the mutation of charged residues to Ala has highlighted the role of
the segment of sequence 596-645 (46) in receptor binding;
experiments with synthetic peptides have lead to similar conclusions
for residues 474-488 and 694-708 in one study (33) or residues 514-542 in another (47) . The
reasons for these conflicting results are not clear at present, but all
these findings are likely to be only partially informative because each
method may identify functionally important segments of sequence but
cannot exclude the potential involvement of others. The issue is
further complicated by the still undefined effects of the modulators
used, because the interaction sites induced in the presence of
ristocetin or botrocetin have never been proven to be identical and are
likely to be at least partially distinct(48) . Relevant to the
present studies, E. coli-expressed fragments terminating at
residue 696 but still comprising the
Cys
-Cys
disulfide loop have
previously been found to exhibit GP Ib
binding function similar to rvWF
when oxidized but to have no
measurable activity after disulfide bond reduction(24) . These
results imply that residues on the carboxyl-terminal side of
Cys
are necessary for GP Ib
binding but only after
disruption of the intramolecular disulfide loop(24) . Thus, the
GP Ib
interaction sites expressed by cyclic or reduced and
alkylated A1 domain fragments in the absence of exogenous modulators
may be partially different, and residues in the sequence 697-704
may be responsible for the enhanced binding activity of reduced and
alkylated fragments. Incidentally, it has also been shown that
recombinant vWF fragments truncated at residue 696 fail to interact
with the monoclonal antibody NMC-4(24) , even though they
contain all the residues indicated by others as necessary for
expression of the corresponding epitope within the
Cys
-Cys
loop(46) . This is
further indication that residues 697-704 may participate in
molecular recognition events involving the A1 domain of vWF. From the
sum of all results available to date, it appears that A1 domain
interaction with GP Ib
is complex and likely to involve
discontinuous sites that individually may play functional roles of
varying relevance depending on conformational conditions and/or the
presence of exogenous modulators.
It has recently been proposed that
vWF binding to GP Ib is influenced by three segments of sequence
(residues 497-511, 540-578, and 687-698) that
``inhibit'' the interaction by repressing an endogenous
``botrocetin-like'' modulator site corresponding to residues
520-534 and 626(46) . Our results agree with the concept
that the sequence 540-578, comprising Trp
, may
control A1 domain function and that regions flanking the
Cys
-Cys
loop, particularly on the
amino-terminal side, may act as negative control elements and cause
decreased affinity in receptor binding. Furthermore, carbohydrate
chains in the mammalian cell-derived fragment, also located on both
sides of the A1 loop(49) , appear to provide additional
negative regulation of function. Of note, reduction of the intrachain
disulfide bond in association with partial denaturation/quick refolding
had the most pronounced effect in enhancing the activity of
``long'' fragments with flanking regions surrounding the
disulfide loop, although their GP Ib
binding affinity remained
approximately 1 order of magnitude lower than that of a
``short'' fragment composed essentially of the loop itself.
This indicates that selected sequences within and/or in proximity of
the A1 loop may conformationally constrain and/or sterically hinder
functional receptor recognition sites. Forthcoming information on the
three-dimensional structure of the vWF A1 domain will allow
verification of these hypothetical interpretations of our findings.