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INTRODUCTION |
The A1 domain of von Willebrand factor
(vWF)1 immobilized onto
exposed surfaces at sites of vascular injury initiates platelet adhesion and thrombus formation by interacting with the glycoprotein (GP) Ib
receptor (1-3). This function is absolutely required for
hemostasis in vessels such as arterioles or arterial capillaries, where
rapid blood flow creates high shear rates (4, 5), and may also
precipitate thrombosis in larger arteries, for example the coronary
arteries of the heart, particularly at sites of stenosis caused by
atherosclerotic lesions (6-8). The efficient interaction between GP
Ib
and immobilized vWF is in apparent contrast to the lack of
measurable binding of soluble plasma vWF (9). This observation has led
to the generally accepted concept that conformational changes induced
by surface adsorption regulate A1 domain function. Such an effect is
thought to be caused in vivo by binding to collagen (10-12)
or other subendothelial structures (13, 14), whereas in
vitro it may be mimicked by interaction with modulators such as
ristocetin (15, 16) or botrocetin (17-19). Indeed, surface-bound vWF
may change shape under the influence of high shear stress, appearing as
an elongated filament (20) rather than the loosely coiled structure
predominantly seen under static conditions (21). Extended multimers
expose repeating functional sites, thus supporting multiple and more
efficient adhesive interactions. Whether the change in molecular shape
parallels specific conformational transitions in the A1 domain is
unknown at present. Nevertheless, physicochemical modifications of the
isolated A1 domain in solution can result in heightened interaction
with GP Ib
(22), in agreement with the notion that the native
conformation is functionally unfavorable for receptor recognition but
can be positively modulated. Consequently, it is generally assumed that
the mechanisms leading to soluble vWF binding to GP Ib
reflect
conditions that endow the A1 domain with the ability to initiate
platelet adhesion.
The physiologic characteristics of the interaction between
surface-immobilized vWF A1 domain and GP Ib
have been well
established under relevant flow conditions, with the demonstration that
the process supports efficient tethering even at high shear rates. Platelets kept in contact with the surface by this ligand receptor pairing, however, are not irreversibly adherent; rather, they translocate constantly in the direction of flow albeit at a markedly reduced velocity relative to freely flowing platelets (1). This is
sufficient to allow the formation of additional bonds, mediated by
receptors other than GP Ib
, resulting in irreversible attachment and
subsequent thrombus formation (3). We have now studied the GP
Ib
-binding function of isolated recombinant A1 domain fragments of
distinct conformation to evaluate how adhesive properties under flow
correlate with the ability to bind to the receptor in solution. We
found that an immobilized fragment with refolded conformation supported
platelet tethering at high shear rates as efficiently as native vWF but
had the lowest GP Ib
binding capacity in solution. In contrast,
disruption of the tertiary structure (22) resulted in a fragment that
exhibited markedly enhanced binding to the receptor in solution, as
judged by the ability to block its function, but defective support of
platelet adhesion when immobilized onto a surface, particularly at high shear rates. Moreover, platelets became irreversibly attached to the
fragment with disrupted conformation, rather than translocating through
transient interactions as seen with native refolded fragments and
multimeric vWF. After forming a complex with botrocetin, however, even
A1 domain fragments with native conformation supported irreversible adhesion. Our findings indicate that the affinity regulation of vWF A1
domain binding to GP Ib
is a complex event since distinct and, in
most instances, mutually exclusive structural characteristics control
the ability to establish stable bonds or to initiate platelet tethering
opposing elevated shear forces.
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EXPERIMENTAL PROCEDURES |
Preparation of Reconstituted Blood--
Blood was collected from
healthy and medication-free donors into polypropylene syringes
containing as anticoagulant the
-thrombin inhibitor
D-phenylalanyl-L-prolyl-L-arginine
chloromethyl ketone dihydrochloride (PPACK) at the final concentration
of 50 µM. All human subjects participating in these
studies were aware of the experimental nature of the research and gave
their informed consent in accordance with the Declaration of Helsinki.
In order to eliminate potential effects of vWF and/or other plasma
proteins in the experiments to be performed, washed blood cells
suspended in perfusion buffer were prepared as follows. After adding
the ADP scavenger apyrase and prostaglandin E1 to prevent
platelet activation (10 units/ml and 10 µM, respectively,
final concentration), blood was centrifuged at 2200 × g for 15 min at room temperature (22-25 °C), and the resultant supernatant plasma was removed from the sedimented cells, including platelets and leukocytes on top of the erythrocyte cushion. After adding an equivalent volume of divalent cation-free Hepes/Tyrode buffer (10 mM Hepes, 140 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 10 mM NaHCO3, and 5 mM dextrose),
pH 6.5, and mixing gently, the cell suspension was centrifuged again
and the supernatant fluid was removed. This procedure was repeated
three additional times, and after the final centrifugation the cells
were suspended in divalent cation-free Hepes/Tyrode buffer, pH 7.4, containing 50 mg/ml bovine serum albumin. Final cell counts were within
normal blood limits. In some experiments, platelet-depleted
reconstituted blood was prepared by centrifuging the final cell
suspension at 150 × g for 15 min, removing the
resulting platelet-rich supernatant fluid, and replacing it with an
equivalent volume of Hepes/Tyrode buffer, pH 7.4, containing 50 mg/ml
bovine serum albumin. After counting the platelet number in both the
whole cell suspension and the platelet-rich suspension, appropriate
volumes of the latter were added into the former to obtain the target
platelet count in the reconstituted blood, at the same time maintaining
a normal hematocrit. In some experiments, whole blood containing PPACK as anticoagulant and prostaglandin E1 to prevent platelet
activation (see above) was used instead of reconstituted blood.
Preparation of Purified Native vWF and Recombinant Fragments
Containing the vWF A1 Domain--
Native multimeric vWF was purified
as previously reported (23). Two recombinant polypeptides coding for
residues 508-704 and 445-733 of the mature vWF subunit, designated
rvWF508-704 and
rvWF445-733, respectively, were expressed in
host Escherichia coli BL21-DE3 using plasmids containing the
T7 RNA polymerase promoter (24, 25) and induction with
isopropyl-
-D-thiogalactopyranoside, as described
previously in detail (22, 26, 27). To prevent formation of random
aggregates during purification, five codons for Cys residues at
positions 459, 462, 464, 471, and 474 in the rvWF445-733 construct were replaced with Gly
codons by site-directed mutagenesis. Expressed fragments were purified
by reverse-phase high pressure liquid chromatography and subjected
either to reduction and alkylation (S-carboxyamidomethylation) or oxidization of the two Cys
residues at positions 509 and 695, according to previously described
procedures (22). Oxidization created the intramolecular disulfide bond that exists in native plasma vWF (28). Purified recombinant fragments
were dialyzed against 2 mM acetic acid titrated to pH 3.5 with HCl and stored at
70 °C. As reported in detail elsewhere (22), refolding of the oxidized recombinant fragments from the denatured state following exposure to acidic pH was achieved by slow
dialysis with incremental pH increase in steps of 0.5 units each up to
a final value of 7.0 for rvWF508-704 or 5.0 for
rvWF445-733. This was obtained by dialyzing the
samples at 4 °C against 2 mM acetic acid to which an
appropriate amount of ammonium hydroxide was added every 8 h. Limiting the pH for refolding
rvWF445-733 was necessary because this
fragment tended to form aggregates at pH values above 5.0. Reduced and
alkylated rvWF508-704 was not refolded slowly,
rather it was rapidly returned to neutral pH just before use by direct
mixing with an appropriate buffer. Protein concentration was determined
with the micro BCA assay (Pierce) according to the instructions of the manufacturer.
Purification of Two-chain Botrocetin--
This modulator of vWF
A1 domain binding to platelet GP Ib
was purified from the venom of
the snake Bothrops jararaca as previously reported in detail
(29, 30). It was stored in Hepes buffer (10 mM Hepes, 140 mM NaCl), pH 7.4, at
70 °C until used.
Monoclonal Antibodies--
The anti-vWF, anti-GP Ib
, and
anti-
IIb
3 (GP IIb-IIIa) monoclonal
antibodies used in these studies have been characterized in previous
publications. Pooled M13 monoclonal antibodies (31, 32) recognize
epitopes in the M13 cyanogen bromide fragment of vWF comprising
residues 631-710 of the mature subunit (33). NMC-4 (34) recognizes a
defined epitope in the A1 domain of vWF, as demonstrated by atomic
structure resolution (35). LJ-Ib1 reacts with the amino-terminal 45-kDa
domain of GP Ib
(36, 37) and is a competitive inhibitor of vWF
binding to this platelet receptor. LJ-CP8 is a complex specific
antibody against the integrin
IIb
3 and
completely blocks binding of all ligands to this receptor (38, 39). All
monoclonal antibodies were mouse IgG1 and were purified by
protein A-Sepharose (Amersham Pharmacia Biotech) chromatography according to published procedures (40). The inhibitory effect of
specific monoclonal antibodies on platelet interaction with immobilized
native vWF or recombinant fragments was assessed by incubating purified
IgG with reconstituted blood at room temperature for 20 min before
perfusion through the chamber.
Assessment of the Interaction between Soluble Recombinant vWF
Fragments and GP Ib
as Measured by Inhibition of the Anti-GP Ib
Monoclonal Antibody LJ-Ib1 Binding to Platelets--
This assay, an
indirect measurement of the binding to platelet GP Ib
of recombinant
vWF fragments containing the A1 domain, has been described in detail in
a previous publication (22). Platelet-rich plasma was prepared by
centrifugation of blood containing PPACK at 150 × g
for 15 min at room temperature and then diluted in 10 mM
Hepes buffer, pH 7.4, to give a final platelet count of 1 × 108/ml. In other experiments, washed cells were prepared as
described above, and a plasma-free suspension of platelets was obtained by centrifuging the final cell suspension at 150 × g
for 15 min yielding a platelet-rich supernatant fluid that could be
separated from the sedimented red and white blood cells. A constant
volume (equal to or less than 12.5 µl) of various concentrations of
each recombinant vWF fragment was added to the platelet suspension, followed by 10 µg/ml 125I-labeled LJ-Ib1 (a concentration
of antibody resulting in half-maximum binding to platelets) diluted in
10 mM Hepes buffer, pH 7.4. When indicated, botrocetin was
also added to a final concentration of 5 µg/ml (0.17 µM); in this case, washed platelets (41) were used rather
than platelet-rich plasma. The final volume of each experimental
mixture was 125 µl, and all indicated concentrations were final.
After incubation at room temperature for 30 min, platelets were
separated by centrifugation though a layer of 20% sucrose, and bound
radioactivity was measured in a
-scintillation counter. Nonspecific
binding was estimated by adding a 100-fold excess of nonlabeled LJ-Ib1,
and the corresponding value (always less than 10% of the total counts
added) was subtracted from all data points. Binding was expressed as
percentage of that measured in a control mixture containing, instead of
a recombinant vWF fragment, 12.5 µl of 10 mM ammonium
acetate with the same pH.
Preparation of Glass Coverslips Coated with Immobilized
Protein--
Recombinant vWF fragments and native vWF were diluted to
a final concentration of 100-130 µg/ml with 2 mM
ammonium acetate, pH 7.0. A glass coverslip (number 1, 24 × 50 mm; Corning) was coated evenly with 200 µl of protein solution and
placed in a humid environment at room temperature for 1 h. In some
experiments, larger volumes (300-600 µl) of solutions with lower
protein concentration were used. Just before assembly of the flow
chamber, unbound protein was removed by rinsing the surface of the
coverslip with 0.04 M phosphate buffer, pH 7.4, containing
0.15 M NaCl (PBS, phosphate-buffered saline composed of
0.04 M monosodium phosphate and 0.04 M disodium phosphate with 0.15 M NaCl, pH 7.4). In some cases, after
coating with the desired protein, the glass surface was saturated with a solution of 50 mg/ml bovine serum albumin in PBS for 1 h at room
temperature before use. To calculate the amount of protein adsorbed
onto the glass surface, the supernatant containing unbound protein was
carefully recovered after coating as were three successive 200-µl
aliquots of PBS used to rinse the coverslip (washing solution). The
amount of adsorbed protein was calculated as the difference between the
total amount added for coating and that recovered in the coating plus
washing solutions.
Flow Chamber and Perfusion Studies--
Platelet interaction
with immobilized recombinant fragments or native vWF under flow
conditions was observed in real-time by means of epifluorescence
videomicroscopy in a modified Hele-Shaw flow chamber (42), as described
previously (1, 3). The bottom of the chamber was formed by the
protein-coated surface of a glass coverslip, and a flow path height of
254 µm was determined by a silicon rubber gasket designed with a
shape that resulted in a linear variable wall shear rate from 1500 s
1 at the inlet to 50 s
1 near the outlet
when the flow rate was maintained at 2 ml/min. The entire flow path of
the chamber, mounted on the stage of an inverted epifluorescence
microscope (Axiovert 135 M, Carl Zeiss Inc.), was kept at
37 °C with a thermostatic air bath. Platelets were visualized by
adding mepacrine (quinacrine dihydrochloride; 10 µM final
concentration), a fluorescent dye that becomes concentrated in the
dense granules and has no effects on function at the concentration used
(43). When native vWF was immobilized on the surface, reconstituted blood also contained the anti-
IIb
3
monoclonal antibody, LJ-CP8, at the final concentration of 50 µg/ml
in order to eliminate any irreversible interaction between platelets
and the RGD sequence in vWF (1, 3). In some experiments, recombinant
vWF fragments were mixed with the reconstituted blood to evaluate their
capacity to inhibit, when in solution, the interaction of platelets
with immobilized native vWF or recombinant fragments adsorbed onto the
surface. Reconstituted blood, considered to have the same viscosity of
4 centipoise as native blood, was aspirated through the chamber,
initially filled with PBS, by a syringe pump (Harvard Apparatus Inc.)
for the desired time, and all experiments were continuously recorded on
videotape using a video cassette recorder (VCR, Magnavox).
Analysis of Platelet Interaction with Native vWF and Recombinant
vWF Fragment Immobilized onto a Glass Surface--
The number of
individual platelets interacting at any given time with immobilized
native vWF or recombinant fragments was measured on images obtained at
different positions in the flow path of the chamber, corresponding to
selected wall shear rates. Each image corresponded to a single frame
from the real time (30 frames/s) videotape recording, digitized and
processed by computer analysis using a Sony 9500 VCR, a Matrox Image LC
frame grabber, and the MetaMorph software package (Universal Imaging
Corp.). The motion of platelets not irreversibly attached to the
surface was analyzed as previously reported (1, 3). To evaluate the
number of platelets displaced from the point of initial interaction, a
series of images at 1/3-1/15-s intervals from real time recording was
digitized and binarized after application of a threshold to distinguish
platelets from background. Movement was defined as spatial displacement
on the surface greater than one platelet diameter. The first two
consecutive frames in the series were superimposed by the logical AND
function, so that the resultant image represented only overlapping
areas of an individual platelet at two different times. This computed
image was then superimposed to next frame in the time series, and the
same logical AND function was applied. The process was repeated for a
number of frames corresponding to a preselected time interval, or until
the area of overlap was equal to 0. When the latter occurred, the
platelet had moved by a distance greater than its diameter; if this did
not occur, the platelet was considered firmly attached during the
period of observation. To measure the velocity of translocation onto
the surface, the centroid of individual platelets on each image was
assigned a set of x and y coordinate values.
Centroid displacement was then followed as a function of time,
typically on a total of 60 frames for each analyzed position in the
chamber corresponding to 4-20 s. The rate of frame acquisition from
real time recording was selected in relation to the speed of
translocation. The velocity of individual platelets was calculated as
the distance traveled by the centroid divided by the time interval (1,
3).
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RESULTS |
Coating of vWF A1 Domain Fragments onto a Glass
Surface--
Preliminary experiments established that platelet
adhesion to immobilized native vWF or recombinant fragments was maximal after coating glass with a solution at the concentration of 100 µg/ml
for 1 h at room temperature (22-25 °C). The corresponding amount of bound protein was in the range of 8.7-15.2 µg per glass slide for all recombinant fragments as well as purified native vWF
(Table I). The number of platelets
interacting with the coated surface was directly correlated to the
amount of adsorbed protein, but considerably less native multimeric vWF
than recombinant fragment was required for maximum effect (Fig.
1).
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Table I
Amount of native multimeric vWF and recombinant vWF fragments absorbed
onto the surface of glass coverslips
The amount of protein adsorbed onto the surface of glass coverslips was
determined by subtracting from the total initial amount in the coating
solution the sum of that recovered in the supernatant solution after
coating and in all washing solutions.
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Fig. 1.
Effect of immobilized ligand density on the
number of surface interacting platelets. Reconstituted blood with
mepacrine-labeled platelets was perfused at 37 °C through a
Hele-Shaw chamber at a constant flow rate such that the indicated wall
shear rates were attained at set positions on the x-y axes
(see "Experimental Procedures"). The bottom of the chamber was
formed by a glass coverslip coated with either native multimeric vWF
(left panel) or rvWF445-733 cyclic
(right panel). The amount of surface-immobilized ligand per
glass coverslip (surface area of 1200 mm2) was measured as
described in Table I and is indicated in parentheses after
the corresponding concentration in the coating solution for each
experiment performed. The amount of immobilized ligand at the lowest
vWF coating concentration was too low to be determined accurately. The
number of surface interacting platelets was measured after 2 min from
the beginning of flow on single frames from real time recording and
represents instantaneous (1/30th of a second) surface coverage
including both transiently interacting and firmly attached platelets in
an area of 65,536 µm2.
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Platelet Interaction with Immobilized A1 Domain Fragments of
Distinct Conformation--
Platelet interaction with immobilized
recombinant fragments as well as native multimeric vWF was evaluated in
real time at wall shear rates between 50 and 6000 s
1.
Fragments of different conformation exhibited considerable variation in
their ability to support platelet attachment (Fig.
2). Immobilized rvWF445-733, refolded after oxidation of the
Cys509-Cys695 intrachain disulfide bond,
interacted with platelets at all shear rates tested in a manner
indistinguishable from multimeric vWF. As with the native molecule, the
surface coated with this fragment became saturated with interacting
platelets within seconds from the initiation of perfusion. In contrast,
the shorter rvWF508-704 with oxidized
intrachain disulfide bond was efficient in interacting with platelets
at shear rates up to 1500 s
1 but exhibited progressive
loss of function with increasing shear and was essentially inactive at
6000 s
1 (Fig. 2). The same
rvWF508-704, but with reduced and alkylated Cys
residues and brought from acidic to physiologic pH just before coating
onto glass, supported the attachment of fewer platelets and only at the
lower shear rates tested (Fig. 2). Real time images of platelets
interacting with different immobilized substrates are shown in Fig.
3.

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Fig. 2.
Effect of wall shear rate on platelet
interaction with immobilized native vWF and different recombinant vWF
fragments containing the A1 domain. Reconstituted blood was
perfused in a Hele-Shaw chamber, as described in the legend to Fig. 1,
over glass coverslips coated with saturating amounts (100 µg/ml in
the coating solution) of the indicated substrates (R/A
indicates the fragment with reduced and alkylated Cys residues).
Two different flow rates were used, one to evaluate wall shear rates
between 50 and 1500 s 1 and the other between 2,000 and
6,000 s 1. After 2 min of perfusion monitored in real time
and recorded on video tape, single frame images from different
positions in the chamber corresponding to the indicated wall shear
rates were analyzed to measure the number of platelets interacting with
the surface in an area of 65,536 µm2. Each experimental
point represents the mean ± S.D. of three separate
experiments.
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Fig. 3.
Images of surface-interacting platelets.
These images of single frames from real time recording show single
platelets, appearing as individual bright spots, either permanently
attached or transiently interacting with glass surfaces coated with the
indicated substrates (see Fig. 2). Each frame represents an
instantaneous (1/30th of a second) view of a 65,536-µm2
surface area at three different wall shear rates.
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Analysis of Platelet Motion on Immobilized Native vWF and A1 Domain
Fragments--
The distinctive features of GP Ib
-mediated platelet
interaction with immobilized vWF A1 domain are efficient initial
tethering even at elevated wall shear rates (Fig. 2) and continuous
surface translocation mediated by rapidly forming but transient bonds (Fig. 4). Motion on the surface, defined
as displacement from the point of initial contact greater than one
platelet diameter, occurred with nearly identical time course on native
vWF and rvWF445-733 cyclic at both relatively
high (1500 s
1) or low (340 s
1) wall shear
rates (Fig. 4). Within less than 5 s, essentially all the
platelets that became tethered to either substrate were displaced from
the point of first contact, but many remained attached to the surface
for as long as they could be visualized, moving at a velocity dependent
to some extent on the flow rate. Nevertheless, these platelets failed
to arrest permanently as long as their interaction with the surface was
mediated only by GP Ib
binding to native vWF A1 domain (Fig. 4). In
remarkable contrast, when platelets were perfused over immobilized
rvWF508-704 with reduced and alkylated Cys
residues (R/A), initial tethering resulted in arrest at the site of
first contact in greater than 70% of events at either relatively high
or low shear rates (Fig. 4). Note that only for this fragment the
highest shear rate for evaluation of surface translocation was 1200 s
1, since initial tethering was extremely reduced at
higher values (Fig. 2). The functional properties of
rvWF508-704 were considerably different when
Cys509 and Cys695 were linked in intrachain
bond, and the protein was refolded before use. On such a substrate,
greater than 70% of initially tethered platelets attached irreversibly
at the site of first contact at the lower shear rate of 340 s
1, but only approximately 25% did so at 1500 s
1, and the remaining 75% showed surface translocation
(Fig. 4). Thus, it is apparent that conformational changes influenced
by the presence or absence of the Cys509-Cys695
disulfide bond in the vWF A1 domain are reflected both in the ability
to support initial platelet-surface contacts, particularly at higher
shear rates (Fig. 2), and in the stability of binding to GP Ib
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Fig. 4.
Analysis of platelet motion mediated by
interaction with immobilized native vWF or different recombinant vWF
fragments containing the A1 domain. Platelet interaction with
immobilized native vWF or the indicated recombinant fragments was
observed and recorded in real time as described in the legend to Fig. 1
(C = fragment with oxidized Cys residues;
R/A = fragment with reduced and alkylated Cys
residues). After 2 min of perfusion, platelet motion on the surface was
analyzed as described under "Experimental Procedures." Platelets
whose centroid moved from the point of first contact by more than their
own diameter were considered to be displaced and are reported as
percentage of the total number of individual platelets that could be
visualized on the surface during the observation period. Platelets that
were not displaced according to this definition for more than 20 s
were considered to be permanently adherent. Upper panel,
perfusion at 1500 s 1, except for the fragment
rvWF508-704 with reduced and alkylated Cys
residues that was tested at the highest shear rate of 1200 s 1 owing to its decreased ability to initiate platelet
tethering to the surface (see Fig. 2). Lower panel,
perfusion at 340 s 1.
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Inhibition of Platelet Interaction with Immobilized Recombinant vWF
Fragments by Homologous Species in Solution--
Platelet adhesion to
immobilized vWF fragments containing the A1 domain was inhibited to
variable extent by homologous species in solution. Notably,
rvWF455-733 cyclic, the fragment that exhibited
function similar to native vWF with respect to mediating platelet
tethering and continuous translocation onto a surface (see Figs. 2 and
4), had essentially no ability to inhibit these processes even when
present in excess amounts in solution (Fig.
5). In contrast,
rvWF508-704 with reduced and alkylated Cys
residues, the fragment with markedly reduced ability to mediate initial
platelet tethering but capable of supporting stable adhesion rather
than translocation of platelets (see Figs. 2 and 4), could completely
inhibit platelet interaction with both immobilized
rvWF455-733 and
rvWF508-704 cyclic (Fig. 5). The latter, when
in solution, could inhibit platelet adhesion to the identical fragment
immobilized onto a surface but only at the higher shear rates and
concentrations tested (Fig. 5). The fact that these fragments in
solution had different ability to bind to GP Ib
was confirmed by
measuring the capacity to block binding to platelets of the monoclonal
anti-GP Ib
antibody, LJ-Ib1 (Table
II). Thus, it is apparent that
conformational changes within the A1 domain of vWF can enhance its
capacity to block GP Ib
in solution but at the expense of a decrease
in the normal function of tethering platelets to a surface under high flow conditions. These results suggest that structural modifications reducing the dissociation rate of the interaction between vWF A1 domain
and GP Ib
, a feature required for good inhibitory function in
solution, may result in a slower association rate with consequent decreased efficiency in initiating platelet adhesion.

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Fig. 5.
Platelet adhesion to immobilized cyclic
(C) vWF fragments containing the A1 domain and
inhibition by homologous fragments in solution either cyclic or with
reduced and alkylated (R/A) Cys residues. The indicated
soluble recombinant vWF fragments were added into reconstituted blood
at room temperature for 20 min before perfusion through the chamber.
The different final concentrations tested are identified by distinct
symbols (control = no soluble fragment added).
Inhibitory activity was evidenced by a decrease in the number of
platelets interacting with the immobilized fragments on the surface.
Measurements were performed over an area of 65,536 µm2,
regardless of whether platelets were moving or firmly attached. Note
(upper left panel) that even soluble
rvWF445-733, a noninhibitory fragment by
itself, could completely prevent platelet adhesion after forming a
complex with 0.5 µM botrocetin in solution.
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Table II
Inhibitory effect of different recombinant vWF fragments on the binding
of the monoclonal antibody LJ-Ib1 to platelet glycoprotein Ib
The anti-GP Ib monoclonal antibody, LJ-Ib1, and vWF are competitive
inhibitors for binding to platelets (22). Thus, the concentration (in
µmol/liter) of recombinant vWF fragments containing the A1 domain
required to inhibit by 50% the binding to platelets of the antibody
LJ-Ib1 (IC50) is inversely correlated to the affinity for GP
Ib . The reported values are the mean ± S.D. of 4-7 assays for
each fragment tested.
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Effect of Complex Formation with Botrocetin on the Function of the
A1 Domain Containing rvWF445-733 Cyclic--
The GP
Ib
-binding activity of the vWF fragment
rvWF445-733 with oxidized
Cys509-Cys695 intrachain disulfide bond was
modulated by botrocetin. As judged by competitive inhibition of
monoclonal antibody LJ-Ib1 binding to platelets in the absence of flow,
the fragment in solution exhibited a 100-fold greater affinity for GP
Ib
in the presence than in the absence of the modulator (Fig.
6). In perfusion studies, immobilized
fragment in complex with botrocetin mediated adhesion of an increased
number of platelets as compared with the fragment alone at all shear
rates tested, and in either case the interaction was inhibited by
blocking GP Ib
(Fig. 7). In striking
contrast to the results obtained in the absence of botrocetin, however, addition of increasing amounts of the modulator to
rvWF445-733 before immobilization onto glass
resulted in progressively more stable platelet adhesion. Thus, instead
of continuous surface translocation, the majority of platelets
interacting with rvWF445-733-botrocetin complex
in equimolar proportion became irreversibly attached at the point of
first contact (Fig. 8). This effect of botrocetin resembles that seen after altering the structure of rvWF508-704 by reducing and alkylating Cys
residues (Fig. 4), but with the notable difference that the
rvWF445-733-botrocetin complex was efficient in
tethering platelets even at high shear rates (Fig. 7).

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Fig. 6.
Effect of botrocetin on the inhibition of
monoclonal antibody LJ-Ib1 binding to GP Ib by
soluble rvWF445-733 cyclic.
Interaction of the recombinant vWF fragment with GP Ib was evaluated
indirectly by the ability to inhibit platelet binding of the specific
monoclonal antibody LJ-Ib1. Platelet-rich plasma was mixed with
refolded rvWF445-733 cyclic, 10 µg/ml
125I-labeled LJ-Ib1 IgG, and 10 mM HEPES buffer
with 140 mM NaCl to give a final platelet count of
100,000/µl and the indicated final concentrations of reagents in a
volume of 125 µl. When indicated, botrocetin was also added to a
final concentration of 5 µg/ml (0.17 µM), but in this
case washed platelets were used instead of platelet-rich plasma. After
incubation at room temperature for 30 min, platelets were separated by
centrifugation through a layer of 20% sucrose, and the radioactivity
of bound LJ-Ib1 was measured in a -scintillation counter.
Nonspecific binding was estimated with the addition of a 100-fold
excess of nonlabeled antibody and was always <10% of total binding;
the corresponding value was subtracted from all data points. Results
are expressed as percentage of binding relative to a control mixture
containing 125I-labeled LJ-Ib1 IgG but no vWF fragment and
are presented as mean ± S.D. (n = 4 for fragment
alone; n = 2 for fragment + botrocetin). Addition of
botrocetin without recombinant vWF fragment had no effect on antibody
binding to platelets (not shown).
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Fig. 7.
Platelet adhesion to rvWF445-733
cyclic immobilized by itself or in complex with botrocetin. Blood
was perfused over glass coverslips coated with either
rvWF445-733 cyclic by itself (3 µM) or mixed with equimolar amounts of botrocetin. When
indicated, the function blocking anti-GP Ib monoclonal antibody
LJ-Ib1 was added to the blood at the final concentration of 100 µg/ml
(experiments without botrocetin) or 400 µg/ml (experiments with
botrocetin). The higher concentration of antibody in the latter
situation was necessary to achieve complete inhibition, owing to the
fact that botrocetin enhances the apparent affinity of interaction
between vWF A1 domain and GP Ib , and LJ-Ib1 acts as a competitive
inhibitor of ligand binding to the receptor. Perfusion at the indicated
shear rates and evaluation of platelet adhesion was performed as
described in the legend to Fig. 2.
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Fig. 8.
Analysis of the stability of platelet
adhesion mediated by rvWF445-733 cyclic immobilized by
itself or in complex with botrocetin in various molar
proportions. The recombinant vWF fragment (3 µM) was mixed with botrocetin in the indicated molar
proportions for 30 min at room temperature to allow for complex
formation. The solution was then used to coat glass coverslips
representing the reactive surface in perfusion experiments at the wall
shear rate of 1,500 s 1. Consecutive images from video
tapes recorded in real time were captured at intervals of 1/3rd to
1/10th of a second, and the movement of individual platelets was
followed as a function of time. Platelets showing spatial displacement
on the surface greater than their own diameter were considered to be
moving and are reported as percentage of the total number of individual
platelets that could be visualized on the surface during the
observation period. The two insets show platelets adhering
to rvWF445-733 cyclic alone (left)
or in complex with botrocetin (right). Each image
corresponds to a surface of 15,000 µm2 and was created by
superimposing 30 consecutive frames taken at intervals of one-third of
a second from experiments recorded in real time (video rate of 30 frames per second); thus, a 3-s observation period is shown. The
strings of fluorescent objects on the left result from the
motion of single platelet tethered to the surface; the single round
objects on the right reflect superimposition of platelets
that did not move from the point of initial adhesion throughout the
period of observation.
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DISCUSSION |
The interaction between vWF A1 domain and GP Ib
is key for
initiating platelet thrombus formation at sites of vascular injury exposed to fast flowing blood (1, 3). Our results indicate that this
function is independent of stable ligand-receptor binding, since the
latter event may take place after induction of distinct A1 domain
conformations that are not compatible with efficient platelet
recruitment at high shear rates. The tethering of fast flowing GP Ib
to surface-immobilized vWF A1 domain requires rapid interaction,
whereas stable binding to immobilized or soluble vWF is favored by slow
dissociation of formed bonds. The findings reported here imply that the
association rate with GP Ib
is the crucial parameter for A1 domain
function under conditions of high shear stress, whereas prolonged
stability is not a relevant nor a sufficient feature of the bond. Such
a concept is in agreement with the evidence that permanent platelet
adhesion is mediated by integrins, but these receptors bind to the
respective ligands with relatively slow association rates and can be
effective at high shear rates only after GP Ib
tethering to vWF (3).
Platelet adhesion, therefore, results from the synergistic and
coordinated function of several distinct interactions. In this context,
critical to the role of vWF and GP Ib
is the balance of their
"on" and "off" binding rates that must be tuned to support
initial platelet attachment and transient arrest but without the need
to establish stable bonds that are provided by other receptors (3).
This appears to be best achieved by A1 domain conformations that allow rapid GP Ib
binding but retain intrinsically high dissociation rates. In fact, the two key parameters regulating the interaction seem
to be modulated so that fast association and stable binding are
mutually exclusive.
At present, it is generally assumed that the lack of measurable
interaction between soluble plasma vWF and GP Ib
on circulating platelets is the expression of regulatory mechanisms needed to maintain
ligand and receptor in the same environment without adverse consequences. Hence the notion that plasma vWF has a
"nonfunctional" ("nonadhesive") conformation that must be
switched to "functional" in order to initiate platelet adhesion,
leading to the commonly accepted concept that a specific transition
occurs upon adsorption of vWF onto appropriate substrates such as
collagen. The results presented here, along with other published
findings (3), suggest the possibility of a different scenario, since it
is now apparent that A1 domain function in platelet adhesion does not
necessarily associate with the ability to form stable interactions with
GP Ib
. Thus, native vWF may potentially enable platelet tethering at
sites of vascular injury even when no measurable binding to GP Ib
can be detected. Consequently, it may no longer be necessary to invoke
a conformational change to explain the induction of vWF adhesive
function. In this regard, attention has been paid to the effect of
shear forces on the shape of vWF molecules. Transition from coiled
forms in solution (21) to extended filaments bound to substrates under
fluid shear stress (20) may correlate to enhanced adhesive potential of
vWF multimers, but a similar mechanism is unlikely to be relevant for
isolated A1 domain fragments with tight globular shape (35). Yet, our
results show an almost identical function of surface-bound vWF
multimers and refolded A1 domain fragments in promoting efficient
platelet tethering and rolling at high shear rates, as well as a
similar inability of the soluble counterparts to interact significantly
with GP Ib
. Thus, without the need for induction, the native A1
domain conformation may be set for receptor binding with high
association and dissociation rates, a functional regulation that allows
the molecule to support rapid platelet tethering at wound sites and, at
the same time, to be nonreactive in blood.
The understanding that the functionality of vWF binding to GP Ib
depends on rapidity of bond formation but not stability in time (1, 3)
provides new perspectives for defining the relevant aspects of A1
domain structure and function. An immediate and unrestricted response
to vascular injury is clearly favored by the fact that plasma vWF is
competent for binding to GP Ib
promptly after immobilization,
without the need for induction by specific modulators. Such a feature,
however, implies high dissociation rate from GP Ib
as a necessary
property to allow the coexistence of platelets and vWF in blood, so
that occasional interactions have limited lifetime and cause no
functional consequences in the absence of other thrombogenic stimuli.
The possibility of ligand-receptor contacts in blood is also minimized
by the low plasma vWF concentration. Considering that each subunit
contains one A1 domain, even a molar concentration 10-fold above the
normal average would be well below the amount necessary to measure
binding to GP Ib
in the absence of exogenous modulators (see Fig.
6). Moreover, the coiled shape of vWF in solution (21), shielding A1
domain sites, may further reduce the reactivity of the molecule. At
least in this respect, therefore, molecular shape (20) is likely to
have a role in regulating the consequences of vWF contact with
platelets. In fact, multivalency contributes to efficient vWF
interaction with GP Ib
, since a single multimer with extended shape
is a cluster of A1 domains with the potential of forming multiple bonds
concurrently. This explains why the surface density of isolated A1
domain must be considerably higher than that of the native multimeric
ligand to support comparable interaction with platelets under flow.
The native conformation expressing the adhesive properties of intact
vWF is maintained in a fragment with the carboxyl-terminal portion of
domain D3 preceding domain A1. After oxidation of the Cys509-Cys695 intrachain disulfide bond and
refolding, such a molecule supports binding to GP Ib
with high
association and dissociation rates, resulting in rapid platelet
tethering at high shear rates but with continuous translocation and no
permanent attachment. The native A1 domain conformation can be
modulated to support stable platelet adhesion, reflecting decreased
dissociation from GP Ib
, but acquisition of this property seems to
be necessarily accompanied by less efficient platelet tethering at high
shear rates, evidence for a concomitant decrease in the association
rate. The functional transition occurs in molecules expressed without
the portion of domain D3 preceding A1 and more markedly after reduction
of the Cys509-Cys695 disulfide bridge,
suggesting that a less constrained conformation favors stability in A1
domain-GP Ib
bonds. In this regard, the consequences of complex
formation with botrocetin (18, 44) are unique, in that irreversible
binding to GP Ib
supporting permanent platelet adhesion is achieved
without affecting recruitment at high shear rates. By analogy with the
mechanism of platelet interaction with vWF bound to collagen or
subendothelial matrix (3), the effect of botrocetin may be interpreted
as the result of a contribution to the stability of binding, not of
direct modulation of A1 domain activity. According to this alternative
view, the two components of the vWF-botrocetin complex would act in
sequence, initiating binding to GP Ib
through the intrinsically fast
A1 domain association rate and providing additional contacts through botrocetin that essentially obliterate dissociation. Notable in this
case is that both initial platelet tethering and permanent adhesion may
be mediated by GP Ib
. A precedent suggesting the possibility of
botrocetin participation in receptor binding can be found in the
function of the highly homologous Jararaca GP Ib-BP (binding protein),
a snake venom molecule with high affinity for GP Ib
(45). The
significant difference may be that botrocetin cannot bind by itself but
only contribute to the stability of the interaction initiated by the
vWF A1 domain. It has been suggested that molecules functioning like
botrocetin exist in the vessel wall (14), but mechanism of action and
physiologic relevance of such a potential pathway of platelet thrombus
formation remain to be established.
A relevant conclusion supported by the studies presented here is that
A1 domain fragments with native conformation can mediate initial
platelet-surface contacts as efficiently as multimeric vWF but have
essentially no activity as soluble inhibitors of GP Ib
function.
However, conditions have been defined to express modified A1 domain
fragments that effectively block platelet adhesion to vWF at high shear
rates. These results demonstrate the existence of mutually exclusive A1
domain conformations, one that can tether platelets in rapidly flowing
blood but cannot form a lasting bond with GP Ib
, and one that
mediates stable binding to the receptor but is poorly adhesive at high
shear rates. Such considerations are relevant for a successful
development of anti-thrombotic molecules interfering with the GP
Ib
-vWF interaction. Indeed, an ideal candidate inhibitor should
effectively block the platelet receptor with sufficient duration in
time but should not become a possible substrate for platelet
recruitment if adsorbed by subendothelial components exposed at sites
of vascular injury. Achieving this goal may be facilitated by a
detailed understanding of the structural bases regulating vWF A1 domain
binding to GP Ib
, in particular by defining the residues
specifically involved in determining association and dissociation rates
of the interaction.