From the Servizio Immunotrasfusionale e Analisi
Cliniche and § Divisione di Oncologia Sperimentale 2, Centro
di Riferimento Oncologico, Instituto Nazionale Tumori Centroeuropeo,
Aviano (PN) 33081 Italy, ¶ Department of Pediatrics Nara Medical
University, 840 Shijo-cho, Kashihara City Nara 634 Japan,
** Dipartimento di Scienze e Tecnologie Biomediche University of Udine,
Udine 33100 Italy, and
Department of Evolutionary and
Functional Biology, University of Parma, Parma, 43100 Italy
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ABSTRACT |
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We have identified type VI collagen
(Col VI) as a primary subendothelial extracellular matrix component
responsible for von Willebrand factor (vWF)-dependent
platelet adhesion and aggregation under high tensile strength. Intact
tetrameric Col VI was the form of the collagen found to be capable of
promoting vWF-mediated platelet adhesion/aggregation under this shear
condition, whereas removal of the predominant portion of the terminal
globules by pepsin treatment abrogated its activity. The inability of
the pepsin-digested Col VI to support any platelet interaction at high
flow was because of the failure of the A3(vWF) domain to bind to this
form of collagen, suggesting a stringent requirement of a
tridimensional conformation or of intactness of its macromolecular structure. In contrast, the A1(vWF) domain bound to both intact and
pepsin-digested Col VI tetramers but, in accordance with the cooperating function of the two vWF domains, failed to support platelet
adhesion/aggregation under high shear onto Col VI by itself. The
putative A1(vWF) binding site resided within the A7(VI) module
(residues 413-613) of the globular amino-terminal portion of the
Platelet adhesion to the exposed vascular subendothelial matrix
proteins at the injury site is a crucial step in initiating the
hemostatic and thrombotic processes. Hemodynamic forces play a
significant role in the process of thrombus formation. There is a high
shear stress opposing platelets adhesion, and they are forced toward
the vascular lining surface and adhere at sites of vascular damage.
Rapid formation of the initial platelet layer involves bridging between
collagens and maybe other components (1-8) of the subendothelium on
one side and platelet membrane receptors on the other side (GPIb The prevailing importance assigned to collagens in the initial steps of
the vWF-mediated thrombogenic events has led to the pinpointing of two
collagen binding sites in vWF: one located in the Al(vWF) and one in
the A3(vWF) domains
(15-17).2 However, the
binding site contained within the A3(vWF) domain, rather than that of
the A1(vWF) domain, has been proposed to be the predominant collagen
binding site, at least for collagen type III (15, 18, 19).
Possibly, this is a consequence of the fact that the A1(vWF) domain may
also be engaged in the binding to the GPIb Our results are consistent with a pivotal role of Col VI in mediating
vWF-dependent platelet cohesion at high shear forces and
shed light on the complex mechanisms of Col VI-vWF-platelet interaction
by unraveling the occurrence of a cooperative binding of the Al(vWF)
and A3(vWF) domains of vWF to multiple sites of Col VI. Among these,
one located within the A7(VI) vWFA module at the amino-terminal portion
of the Antibodies--
Specificities of the mouse monoclonal antibodies
(mAbs) used in this study were as follows. mAbs LJ-Ib1 (24) and LJ-Ibl0 (25) are directed against the platelet GPIb Purification of Col VI and Other Extracellular Matrix
Molecules--
Intact tetramers of Col VI were purified from embryonic
chick gizzard, human placenta, and adult bovine aorta by extraction in
Tris-HCI, pH 7.6, with 6 M urea and protease inhibitors and separated by gel filtration chromatography on Sepharose CL-4B columns
as described previously (32, 33). These Col VI preparations were
analyzed by SDS-agarose gel electrophoresis and were found to be
composed mainly of tetramers, with an estimated
Mr of >2,000 kDa. Chick Col VI tetramers
deprived of their amino-terminal globular domains and the predominant
portion of the carboxyl-terminal domains were produced by treatment of
the purified intact Col VI with 1% (w/v) pepsin as described
previously (34-36). Intact and pepsin-digested tetramers purified from
human placenta following extraction with 6 M guanidine HCl
(GuHCl) (34, 35) were kindly received from Huey-Ju Kuo (The Shriners
Hospital for Crippled Children, Portland, OR). Human vWF was purified
from plasma obtained from healthy donors after informed consent by gel
filtration chromatography on Sepharose CL-4B columns (Amersham
Pharmacia Biotech) as described previously (37). The ristocetin
cofactor activity in this purified vWF was determined to be 155 units/mg (38).
Preparation of Recombinant Bacterial Polypeptides--
Bacterial
recombinant polypeptides corresponding to selected vWFA modules of the
Solid-phase Binding Assays--
96-well plates (Costar) were
coated with 100 µl (0.25-30 µg/ml, final concentration) of human,
bovine, or chick Col VI in 0.05 M bicarbonate buffer, pH
9.6, for 24 h at room temperature, washed twice in imidazole
buffer (0.12 M NaCl, 0.02 M imidazole, 0.005 M citric acid, pH 7.3, 0.1% BSA), and then saturated with 5% BSA for l h at room temperature in imidazole buffer. After removal
of excess BSA, plates were incubated with a final concentration of
1-40 µg/ml purified vWF in imidazole buffer, pH 7.3, containing 1%
BSA. After a 2-h incubation at room temperature, plates were extensively washed and incubated with a rabbit polyclonal anti-human vWF antiserum (DAKO) at 1:1,000 dilution for 30 min at room
temperature, followed by horseradish peroxidase-conjugated secondary
goat anti-rabbit antibodies (Bio-Rad) at 1:3,000 dilution for 30 min at
room temperature. Plates were then extensively washed and further
incubated with 0.4 mg/ml o-phenylenediamine (SIGMA) in 0.02 M
Na3C6H5O7, 0.05 M NaH2PO4, pH 5.0, and 0.02%
H202, and the absorbance was read at 492 nm in
an Autoreader III (Ortho Diagnostic System). Intact Col VI tetramers
from the three different species/tissues yielded comparable
dose-dependent bindings. The affinities of the interaction between vWF and Col VI were the following: intact Col VI/vWF
(EC50 Preparation of Col VI and vWF Substrates and Flow Chamber
Assembly--
Intact and pepsin-digested Col VI tetramers were
dissolved in 0.05 M bicarbonate buffer, pH 9.6, to 5-100
µg/ml and coated onto a central area of glass coverslip (24 × 50 mm, 100 µl of Col VI solution/coverslip), that was delimited by a
15-mm silicon ring (Flexiperm-Disc Heraeus Instruments). The amount of
immobilized collagen (either chick or human) was estimated by
independent coating with 125I-labeled Col VI and was
determined to range between 0.15 and 4.36 µg/cm2 for the
coating concentrations used. In experiments in which vWF was used in
immobilized form, coverslips were coated with 100 µl of vWF at 100 µg/ml in 0.04 M phosphate buffer, pH 7.4, containing 0.15 M NaCl. Coated coverslips were placed in a humid chamber at
4 °C overnight, followed by washings in phosphate-buffered saline
and saturation with 1% BSA in 20 mM Tris-HCI, pH 7.4, with 0.15 M NaCl for 60 min at room temperature. Saturated
coverslips were then assembled in a parallel-plate flow chamber
(modified Richardson's flow chamber) (11), which was then filled with isotonic saline. A syringe pump (Harvard Apparatus, Boston, MA) was
used to aspirate the fluid through the chamber at a constant flow rate
for 1-10 min before being perfused with platelet-containing solutions.
Flow rates utilized were 0.13, 0.26, 0.53, 2.66, 5.33, 7.99, and 10.66 ml/min, which produced 25, 50, 100, 500, 1,000, 1,500, and 2,000 s-1 wall shear rates, respectively. Before being tested,
blood samples were incubated at 37 °C for 30 min so as to
re-equilibrate the system to physiological temperature.
Blood Sampling--
Blood from healthy volunteers and from a
patient affected by a severe form of vWD (less than 1% of ristocetin
cofactor and undetectable or barely visible vWF multimers after
SDS-polyacrylamide gel electrophoresis) was obtained after informed
consent. All donors denied ingestion of drugs known to interfere with
platelet function for a period of at least 2 weeks before blood
sampling. Blood was collected from the antecubital vein through a
18-gauge needle into syringes containing 400 units/ml (final
concentration) thrombin inhibitor hirudin (Iketon, Italy) as anticoagulant.
Preparation of Platelets--
Platelets were prepared by a
modification of a previously described method (41). To prevent unwanted
platelet activation, apyrase III (Sigma), an ADP scavenger, was added
to the blood samples at a final concentration of 10 units/ml. Blood
samples were divided into 5-ml aliquots and centrifuged at 800 × g for 14 min, the plasma was removed, and the sedimented
cells, including platelets and leukocytes residing at the top of the
erythrocyte cushion, were resuspended in 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, containing 5 units/ml apyrase
(final concentration) and recentrifuged at 800 g for additional 14 min. This procedure was repeated four times, and after the last
centrifugation cycle, the pellet was resuspended in HEPES-Tyrode buffer
containing 2 mM CaCl2, 2 mM
MgCl2, and 1% BSA (BSA buffer). In some experiments, 10 µM PGE1 (Sigma) was added to the platelet suspension to prevent platelet activation. Platelets and leukocytes counts ranged from 1-1.5 × 108/ml and from 4-6 × 106/ml, respectively, and the hematocrit ranged from
44-48%.
Platelet Aggregation Assay--
Platelet-rich plasma from whole
blood, obtained from healthy donors after informed consent, was
prepared by dilution 1:6 (v/v) into citric acid/citrate/dextrose, pH
4.5, and differential centrifugation. Platelets were washed free of
plasma constituents using a previously described modification of the
albumin density gradient protocol and employed at a final count of
2.5 × 108/ml in Tyrode buffer containing 2% BSA.
Platelet aliquots of 350 µl were placed in glass cuvettes and stirred
with a Teflon-coated magnetic bar at 37 °C and 1,200 rpm in an
aggregometer (Chrono-Log Corp.). Purified vWF was added to the platelet
suspension at a final concentration of 5 µg/ml, with and without
prior incubation (15 min at 37 °C) with 100 µg/ml recombinant
A7(VI) polypeptide. Immediately after, 1 mg/ml of ristocetin
(Chrono-Log) was added to the platelet suspension, and the extent of
platelet aggregation was assessed by changes in light transmittance
(37).
Perfusion Experiments--
The flow chamber was mounted on an
inverted microscope equipped with epifluorescent illumination
(Diaphot-TMD; Nikon) and an intensified CCD video camera (C-2400-87,
Hamamatsu Photonics). To allow visualization, platelets were labeled by
direct incubation with 5-8 µM fluorescent dye mepacrine
(Sigma). To prevent platelet photoactivation by the irradiating UV
light, the neutral blocking filters ND32 and ND8 were used
simultaneously. The total area of one optical field corresponded to
about 0.037 mm2. To assure flowing of comparable blood
volumes and hence, a comparable number of platelets to be perfused for
each shear rate, the assessment of platelet surface coverage was
normalized to 1 min of flow at a shear rate of 1,000 s-1.
Each time point corresponds to a single frame of video tape recording
with a time resolution of 0.04 s/frame. Images were captured by a
personal computer equipped with a TARGA-2000 PLUS board (Truevision),
either in real time during perfusion or from stored video frames that
had been recorded at a sampling rate of 25 frames/s during the
experiment. To classify and estimate the number of attached particles,
to determine their coordinates on the analyzed substrate area, and to
assess the size of the surface area covered, images of predefined
optical fields were elaborated using the Microimage (image-processing
software; CASTI Imaging, Venice, Italy). The final size of the formed
platelet aggregates was estimated assuming an average platelet diameter of 2.4 µm (1.6-4-µm range), and the observed particles were
arbitrarily classified as: single platelets, 2-13 µm2;
micro-aggregates, 14-50 mm2; small aggregates, 51-200
µm2; and large aggregates > 200 µm2.
Shear Rate-dependent Platelet Interaction with Intact
Col VI Tetramers--
In pilot experiments we noticed that intact Col
VI tetramers extracted with 6 M GuHCl (34-36) were not
capable of promoting platelet adhesion at high shear rates although
being fully active in stimulating adhesion and migration of a number of
cell types (Refs. 36, 42, 43 and data not shown). In contrast,
urea-extracted Col VI from late embryonic chick gizzard, bovine aorta,
and human placenta efficiently promoted platelet adhesion (see below).
Immobilized intact Col VI tetramers efficiently and
dose-dependently supported platelet adhesion at a shear
rate of 1,000 s-1, which is accepted to exceed the
threshold rate at which platelet adhesion/aggregation may exclusively
occur via vWF. These experiments also established the optimal quantity
of Col VI to be immobilized onto the substrate to promote maximal
surface coverage by the flowing platelets in our system. Extrapolation
of the 50% surface platelet coverage observed after 1 min of flow was
reached at 0.44 µg/cm2 immobilized Col VI (Fig.
1A). In a time course analysis
of platelet adhesion and aggregation at two different shear rates, it
was found that a plateau of surface coverage was attained very rapidly (i.e. maximal coverage within the optical field analyzed was
reached after 30 s at 2,000 s-1 and after 60 s at
1,000 s-1) (Fig. 1B). Platelets adhered to the
same extent onto tetrameric Col VI of either chick, bovine, or human
origin, whereas almost no binding was detected on the pepsin form (Fig.
1C).
Platelet Receptors Responsible for the Interaction with Col VI at
High Shear Rates--
Under conditions of high shear rate, functional
inhibition of the platelet
To further define the shear rate-related involvement of soluble vWF in
cases when flowing platelets were confronted with an underlying Col VI
substrate, platelets were perfused at different shear forces, ranging
from those found in larger veins (e.g 25 s-1) to those
found in small arterioles (e.g. 1,500-2,000
s-1), in the presence or absence of the anti-GPIb
Because there was no significant change in the surface coverage above
1,000 s-1, all subsequent high shear rate experiments were
carried out under these conditions. The results obtained after blockage
of the vWF-GPIb Identification of the vWF Domains Responsible for Platelets
Interaction with Col VI and of the vWF Binding Sites on Col VI--
To
identify the vWF domains responsible for platelet adhesion and
aggregation on Col VI and to determine their relative importance, we
utilized mAbs directed against the vWF domains Al(vWF), i.e. mAb NMC-4, and A3(vWF), i.e. mAb MR5. In solid-phase binding
assays (i.e. static conditions) with no platelets, mAbs
NMC-4 and MR5 inhibited the molecular interaction of vWF to intact Col
VI tetramers to about 75 and 60%, respectively, whereas when added
together, the inhibitory action was complete (Fig.
4A). Conversely, the addition
of the anti-Al(vWF) and anti-A3(vWF) mAbs to platelets perfused at high
shear rates abrogated adhesion, irrespectively of the mAb added,
whereas addition of the functionally unrelated mAb LJ-C3 did not
disturb platelet adhesion (Fig. 4B). These findings indicated that binding of both A1(vWF) and A3(vWF) to Col VI was a
prerequisite for optimal vWF-mediated platelet-Col VI interaction at
high shear rates. Because mAb NMC-4 also interferes with the GPIb
To identify the vWF binding sites within the globular regions of Col
VI, which could account for the binding activity exceeding that given
by the collagenous region, we employed amino-terminal Functional Identification of the vWF Binding Sites on Col
VI--
The complete blocking effect exerted either by the
anti-A1(vWF) or anti-A3(vWF) antibodies on the
vWF-dependent platelet adhesion to intact Col VI tetramers
at high shear rates suggested that both vWF domains could be equally
important in mediating the molecular interaction (Fig. 4B).
However, the bivalency of the blocking effect caused by the
anti-A1(vWF) mAb NMC-4, which affects both the binding to GPIb In this study we provide definite evidence that Col VI may be a
primary subendothelial ligand for vWF mediating the high shear rate-induced platelet adhesion and aggregation at sites of vascular injury. Binding of both the A1(vWF) and A3(vWF) domains to immobilized urea-extracted Col VI tetramers is essential for initiating the platelet adhesion/aggregation cascade, implying that none of the individual domains alone is capable of supporting platelet tethering and aggregation under high tensile strength. Although the A3(vWF) domain solely binds to the Col VI tetramers in which the terminal globular domains have been retained intact, the A1(vWF) domain interacts with both the triple-helical region and the amino terminus of
the 3(VI) chain. Soluble recombinant A7(VI) polypeptide strongly perturbed the vWF-mediated platelet adhesion to Col VI under high shear
rates, without affecting the binding of the vWF platelet receptor
glycoprotein Ib
to its cognate ligand A1(vWF). The findings provide
evidence for a concerted action of the A1(vWF) and A3(vWF) domains in
inducing platelet arrest on Col VI. This is accomplished via an
interaction of the A1(vWF) domain with a site contained in the
3
chain A7(VI) domain and via a conformation-dependent interaction of the A3(vWF) domain with the intact tetrameric collagen. The data further emphasize that Col VI microfilaments linking the
subendothelial basement membrane to the interstitial collagenous network may play a pivotal role in the hemostatic process triggered upon damage of the blood vessel wall.
INTRODUCTION
Top
Abstract
Introduction
References
and
IIb
3). A pivotal role in this hemodynamic process is played by
von Willebrand factor (vWF)1,
which is the major adhesive protein mediating the bridging interaction as a function of shear forces. vWF, recognized by the platelet surface
glycoprotein GPIb
, is particularly efficient in capturing platelets
under high flow rates (9), and the participation of vWF in hemostasis
is fundamental for thrombus formation under high shear stress
conditions such as those present in small arteries or in pathological
vessel affections such as stenosed arteries in artheriosclerosis. As
extracellular matrix-assembled vWF of the subendothelium may not always
be freely accessible to platelets in areas of rapid flow (10), the
circulating vWF pool may be rather important in initiating platelet
cohesion (11). Consistent with this idea is the observation that
soluble plasma vWF binds rapidly and tightly to an underlying
extracellular matrix, even when this latter is produced by fibroblasts
(12). Moreover, high blood flow rates have been suggested to modulate
the vWF release from the endothelium such as to provide additional
available vWF for platelet interaction (13). High shear forces also
induce a conformational transition in vWF, which converts it from a
globular state to an extended chain structure. This structural
transition is believed to result in the exposure of the intramolecular
domains and in a reorientation of the polymers in the direction of the stress field (14).
, glycosaminoglycans, and
sulfatides (16, 20). On the other hand, the Al(vWF) domain has been
recently proposed to mediate vWF interaction with Col VI (21).
Accordingly, in addition to the main fibrillar collagens types I, III,
and V, previous studies have suggested that the microfibrillar Col VI could similarly be effective in stimulating a vWF-dependent
platelet aggregation at low but not at high shear rates (6, 22,
23).
3(VI) chain (amino acids 413-613) was found to be a critical
A1(vWF) binding site required for optimal platelet adhesion/aggregation
at high shear rates.
EXPERIMENTAL PROCEDURES
; mAbs LJ-CP8 (26) and
LJ-P5 (27) are against the
IIb
3 integrin complex. mAb LJ-Ibl
blocks binding of GPIb
to vWF, whereas mAb LJ-Ibl0 displays only a
minimal inhibitory effect on the binding of GPIb
to vWF but
completely obliterates the
-thrombin binding to the same receptor.
mAb LJ-CP8 blocks the binding of the
IIb
3 integrin to both vWF
and fibrinogen, whereas mAb LJ-P5 selectively inhibits the binding of
the
IIb
3 integrin to soluble vWF but not to fibrinogen. mAbs
NMC-4 and MR5 (kindly provided by Dr. L. W. Hoyer, Holland Laboratory, American Red Cross, Rockville, MD) react with the Al(vWF)
domain (28) and the A3(vWF) domain (29, 30), respectively. mAb LJ-C3 is
directed against the amino-terminal portion of vWF corresponding to
residues 1-272 of the mature subunit, and it is known to inhibit the
interaction of vWF with coagulation factor VIII (31). Purified IgG and
F(ab')2 were prepared as described previously (27). All
mAbs were used at saturating concentrations determined in pilot
dose-dependent experiments.
3(VI) chain were generated from original chick
3(VI) chain
cDNA clones (39) by 6× His-tagging and purification on nickel
nitrilotriacetic acid resins (Diagen GmbH) as described (33).
Recombinant polypeptides corresponding to the single human Col VI
modules A6(VI), A7(VI), A8(VI), and A9(VI) were produced from reverse
transcription-polymerase chain reaction amplificates according to the
same protocol. Polypeptide pB10 was derived from the corresponding
3.3-kilobase pair-long cDNA and embodies the A8-4 modules and a
substantial portion of the A3 module (39). A recombinant polypeptide
encompassing the GPIb
binding domain of vWF residing within the
Al(vWF) domain (rvWF(445-733)) (40) was kindly provided by Dr. Z. M. Ruggeri (The Scripps Research Institute, La Jolla, CA). The
recombinant polypeptides were more than 90% pure as judged by
SDS-polyacrylamide gel electrophoresis.
2 nM); pepsin Col VI/vWF
(EC50
3 nM)
RESULTS
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Fig. 1.
Platelet adhesion to intact Col VI
tetramers. A, dose-dependent platelet
adhesion to intact tetramers of Col VI in flow conditions. Whole blood
containing recombinant hirudin as anticoagulant and treated with the
fluorescent dye mepacrine to label flowing platelets was perfused
through a parallel flow chamber at 37 °C under the indicated shear
rates. Data points report the percentages of surface coverage by
platelets in an area of 0.037 mm2 after 1 min of perfusion
in a representative case. B, effect of wall shear rate on platelet
adhesion to intact Col VI. Percentage of surface coverage as a function
of perfusion time is shown at 1,000 s-1 (gray
circles) and 2,000 s-1 (black squares)
shear rates. These data are representative of three separate
experiments that gave similar results and were obtained with the same
blood sample employed for experiments described in A.
C, real time observation of platelet adhesion and
aggregation onto Col VI tetramers: effect of large globular domain
destruction. Representative single-frame images showing direct
comparisons of platelet adhesion and aggregation onto intact and
pepsin-digested tetramers of Col VI under conditions of high and low
shear rate and when perfused in whole blood.
IIb
3 integrin receptor by the mAbs
LJ-P5 (not shown) and LJ-CP8 (Figs.
2A) caused a substantial
abrogation of platelet arrest and the subsequent tethering and
aggregation. However, interference with
IIb
3 activity did not
impede the vWF-induced transient contact of platelets with the
substrate, which was noticed to be of variable duration. Thus, at each
given time point of analysis, a certain coverage of the substrate area under consideration was observed. In accordance with the prerequisite of a platelet GPIb
-vWF interaction to bring through thrombus formation under high tensile strength (9, 11, 44), platelet adhesion/aggregation was completely blocked by mAb LJ-Ibl (Fig. 2A), known to perturb the GPIb
binding to vWF. mAb
LJ-Ib10 specifically interfering with the GPIb
binding to
-thrombin did not have any effect, ascertaining the specificity of
the GPIb
interaction with vWF in the system. In analogy, no platelet
adhesion was observed with vWF-deficient blood from a patient with
severe vWD (Fig. 2A) or when GPIb
function was inhibited
with 5 µM rvWF(445-733) polypeptide, a recombinant
containing A1(vWF) domain responsible for GPIb
binding site of vWF
(Fig. 2A).
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Fig. 2.
Inhibition of platelet adhesion to intact Col
VI tetramers. A, inhibition of platelet adhesion to intact
Col VI under conditions of high shear rate by the addition of 100 µg/ml (final concentration) F(ab')2 of antibodies to
GPIb (mAb LJ-Ib1),
IIb
3 (mAb LJ-CP8), the recombinant A1(vWF)
polypeptide rvWF(445-733) (5 µM, final concentration) to
whole healthy blood, and lack of adhesion of platelets perfused in
whole blood obtained from an individual afflicted by the severe form of
vWF disease vWD. The data represent the mean of six experiments
performed with blood from different donors and are reported as the
total surface covered by platelets (left y axis)
and relative surface coverage of the optical field in percentage +S.E.
(right y axis). B, shear
rate-dependent inhibition of platelet adhesion to intact
Col VI in the presence of optimal amounts of mAb LJ-Ib1. Antibody
LJ-Ib10, known to interfere only with the
-thrombin binding to
GPIb
, was used at an equivalent concentration as reference.
antibody LJ-Ibl. These experiments demonstrated that, contrary to what
was previously reported for collagen type I (and possibly also type
III), Col VI supported vWF-dependent platelet
adhesion/aggregation down to a shear force of about 100 s-1 (Fig. 2B). Shear rates of 50 s-1 or lower still
promoted some platelet adhesion and aggregation, which, however, was
independent of the participation of vWF (Fig. 2B).
interaction underscored that vWF was an active
component in the system but did not rule out a possible cooperation
between vWF and other blood factors. We therefore perfused washed
platelets in a BSA-containing Tyrode buffer suspension of red and white cells in which increasing amounts of purified vWF were added. In these
cases, a substantial platelet adhesion and aggregation on Col VI was
noted in a manner that was both dose-dependent and saturable (Fig. 3). Depending upon the
amount of vWF added, microaggregates, small and large aggregates (see
"Experimental Procedures") could be observed, with the formation of
the large aggregates starting at a vWF concentration of 2.5 µg/ml
(Fig. 3). Apart from promoting platelet tethering to a molecular or
cellular substrate, vWF has also been suggested to mediate the
subsequent platelet-platelet interaction leading to
IIb
3
integrin-dependent aggregate formation. To establish
whether this was the case also on a Col VI substrate, washed platelets
were similarly perfused in the presence of mAb LJ-P5, known to inhibit
only the
IIb
3 integrin binding to vWF. In such conditions, single
platelets could still be detected on the Col VI substrate, but no
aggregates formed (not shown).
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Fig. 3.
Real time observation of platelet adhesion to
intact Col VI tetramers at high shear rate; effect of vWF
concentration. Representative single-frame images (derived from
three independent experiments) of platelet adhesion to intact Col VI
captured under flowing at high shear rates in the presence of
increasing concentrations of soluble vWF (0-20 µg/ml, final
concentration). Washed platelets were resuspended together with red
cells and white cells in a Tyrode buffer containing BSA and divalent
cations, mixed with the increasing concentrations of purified vWF, and
perfused. Top panels show single-frame images taken after 1 min of perfusion for each vWF concentration. Bottom panels
show the corresponding percentage frequency (relative surface
distribution) of single platelets (A), microaggregates
(B), small aggregates (C), and large aggregates
(D; see "Experimental Procedures").
binding to the Al(vWF) domain, the complete blockage of platelet
adhesion/aggregation observed after addition of this mAb could be
attributed to a dual blockade of the Col VI and GPIb
receptor
binding to the Al(vWF). Thus, these observations indicated that the
vWF-dependent platelet interaction with Col VI at high shear rates required integrity of its terminal globular domains. Intriguingly, however, when the molecular interaction of vWF with pepsin-digested Col VI tetramers was examined in solid-phase binding assays under the inhibitory influence of the anti-A(vWF) domain antibodies, it was found that only the anti-A1(vWF) mAb NMC-4 was
capable of abolishing the interaction (Fig. 4A). This
finding demonstrated that despite the reported ability of the A3(vWF) domain to interact with interstitial collagens, this domain failed to
contribute to binding of vWF to the triple-helical region of Col
VI.
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Fig. 4.
Inhibitory effect of anti-A1(vWF) and
anti-A3(vWF) mAbs. A, solid-phase binding assays showing the
interaction of vWF with intact (black bars) and
pepsin-digested tetramers (gray bars) in the presence or
absence of anti-A1(vWF) and anti-A3(vWF) antibodies used at their
maximal inhibitory concentration of 50 µg/ml (final concentration).
mAb LJ-C3 against the amino-terminal region of vWF (factor VIII binding
domain) was used as a control antibody. The percentage bound vWF was
calculated on the basis of vWF to either form of the Col VI in the
absence of antibody. Data represent the mean +S.E. of five different
experiments. B, platelet adhesion and aggregation onto
intact Col VI substrates in the presence of the same antibodies as in
A, when perfused at the indicated shear rate in whole blood.
Data represent mean +S.E. from three independent experiments.
3(VI) chain
recombinant polypeptides (Fig.
5A) both in static and dynamic
phase. In static phase, about 70% of the maximal vWF binding activity
displayed by the intact tetramers was detected for the recombinant Col
VI polypeptide pB10, encompassing almost the entire amino-terminal
portion of the
3(VI) chain, i.e. the A8-A3(VI) modules.
The prevailing binding activity of polypeptide pB10 could further be
pinpointed to the A7(VI) module, as determined by direct binding of
purified vWF to the polypeptide and the ability of this polypeptide,
but not other A(VI) polypeptides, to compete for the binding of vWF to
intact Col VI tetramers (Fig. 5B). The competition ability
of soluble A7(VI) was dose-dependent, and the residual
binding could be completely abrogated by simultaneous addition of the
anti-A3(vWF) mAb MR5 (Fig. 5C). Further evidence for a
specific role of A(vWF) domain(s) in binding to the amino-terminal globular domain of Col VI was provided by the elected ability of the
anti-A1(vWF) mAb NMC-4 to block the binding of vWF to pB10 and A7(VI)
(Fig. 5D). In contrast to the A1(vWF) domain, the A3(vWF) failed to interact with the isolated region of the
3(VI) chain, as
demonstrated by the lack of inhibition of the vWF binding to
3
recombinant polypeptides in the presence of anti-A3(vWF) antibodies (Fig. 5D). This finding suggested that the interaction
site(s) of the A3(vWF) domain within the Col VI globular region was
conformation-dependent and most likely required intactness
of the quaternary structure of the fully assembled tetramer.
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Fig. 5.
vWF binding to Col VI recombinant
polypeptides of 3(VI) chain. A, schematic diagram
illustrating the module organization of the
3(VI) chain, adopting
the nomenclature of gene chick polypeptide (39), and the relative
extension of the recombinant polypeptides produced from the
corresponding human cDNAs. B, binding of vWF to
immobilized recombinant Col VI polypeptides (vWF bound), when assessed
as the percentage of bound protein in comparison to the amount bound to
intact Col VI tetramers and binding of vWF to immobilized intact
tetramers in the presence of soluble recombinant Col VI polypeptides
(vWF displaced). In this latter case, values refer to percentage
inhibition of vWF binding to Col VI by the various soluble competitors.
Fibrinogen was used in both immobilized and soluble phase as a control
protein. C, dose-dependent inhibition of vWF
binding to immobilized intact tetramers by soluble A7(VI) recombinant
polypeptide. At saturation of inhibition, further addition of the
anti-A3(vWF) antibody MR5 completely abrogated the residual binding.
D, binding of vWF to immobilized recombinant Col VI
polypeptides in the presence of antibodies as in Fig. 4. Data represent
mean +S.E. from three independent experiments.
and
to Col VI, precluded the possibility of determining the reciprocal role
of the two vWF domains in these experiments. We therefore analyzed the
ability of the soluble A7(VI) polypeptide to prevent platelet adhesion
to intact Col VI tetramers under high shear rates in the presence of
soluble or immobilized vWF. In experiments in which washed platelets
were perfused over intact Col VI tetramers at high shear rate in the presence of soluble vWF, the time-dependent platelet
adhesion to the collagen substrate was markedly perturbed by the
additional presence of competing soluble A7(VI), whereas the A6(VI)
polypeptide had only a marginal effect (Fig.
6A). However, soluble A7(VI) polypeptide did not affect the ristocetin-induced platelet
agglutination-aggregation in the simultaneous presence of soluble vWF
(Fig. 6B). In another set of analogous experiments in which
washed platelets were similarly perfused at high shear rates on
substrates of intact tetramers in the presence of soluble vWF or on
substrates of immobilized vWF in the absence of soluble vWF, we
examined the effect of anti-vWF antibodies and competing recombinant
polypeptides on the extent of platelet adhesion at a given time point.
In both experimental situations, the prostaglandin PGE1 was
included to prevent platelet activation. Platelets perfused over Col VI
substrates in the presence of soluble vWF adhered well to the collagen
substrate in the simultaneous presence of the control antibody against
the factor VIII binding domain of vWF (mAb LJ-C3) or the recombinant
A6(VI) polypeptide (Fig. 7). In contrast,
the addition of either of the anti-A1(vWF) and anti-A3(vWF) antibodies
(mAbs NMC-4 and MR5, respectively) or the A7(VI) recombinant
polypeptide strongly perturbed platelet adhesion to Col VI (Fig. 7). In
the experimental situation in which platelets were perfused over
immobilized vWF, solely the anti-A1(vWF) antibody NMC-4 was capable of
blocking the platelet-vWF interaction. This finding indicated that
neither the anti-A3(vWF) antibody nor the A7(VI) interfered with the
GPIb
-A1(vWF) interaction, and hence the inhibitory effect was
exerted exclusively on vWF-Col VI interaction.
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Fig. 6.
Inhibitory effect of the A7(VI) recombinant
polypeptide. A, time course of platelet adhesion and
aggregation onto intact Col VI tetramers, perfused under high
shear rate conditions (1,000 s-1) in Tyrode buffer in the
presence of 5 µg/ml (final concentration) of vWF and the presence or
absence of soluble recombinant Col VI polypeptides (100 µg/ml, final
concentration). Circles, control; squares,
A6(VI); triangles, A7(VI). B, aggregation of
washed platelets in the presence of vWF (5 µg/ml, final
concentration) with or without 100 µg/ml (final concentration) of
A7(VI) polypeptide, as induced by ristocetin (1 mg/ml, final
concentration).
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Fig. 7.
Real time observation of platelet adhesion
onto Col VI tetramers and vWF at high shear rate; effect of
anti-A1(vWF) and anti-A3(vWF) inhibitors. Representative
single-frame images showing representative platelet adhesions to either
immobilized intact Col VI tetramers and soluble vWF (5 µg/ml, final
concentration) or immobilized vWF in the presence of anti-A(vWF)
antibodies or soluble recombinant Col VI polypeptides (added at their
maximal inhibitory concentration). Washed platelets were perfused at
high shear rate in Tyrode buffer containing divalent cations and 10 µM PGE1, final concentration.
DISCUSSION
3(VI) chain, and its binding is independent of the quaternary structure of the collagen (Fig. 8). A
primary binding site of the A1(vWF) domain could be identified within
the constitutively expressed A7 vWFA module of the
3(VI) chain (33)
and shown to be distinct from that involved in the binding of the
A1(vWF) domain to the platelet receptor GPIb
(Fig. 8).
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Fig. 8.
Schematized model of the vWF-mediated
platelet adhesion onto Col VI at high shear rate. Upon damage of
the arterial wall, the subendothelial Col VI-containing extracellular
matrix becomes exposed to the circulating globular vWF, which interacts
with it and, at high shear rates, assumes an extended chain
conformation (14). The cooperating A1 and A3 domains of the vWF are in
concert implicated in the linking of the molecule to the underlying Col
VI microfilaments by binding to both the triple-helical region
(dark blue) and the globular domains (red) of the
tetrameric blocks of the microfilaments. A primary site responsible for
the A1(vWF)-mediated vWF interaction with the globular domains of Col
VI resides within the A7(VI) module of the amino terminus of the 3
chain. In contrast, the A3(vWF) domain recognizes an apparently
structurally complex site residing exclusively within the globular
regions of the collagen. Under high flow, the immobilized vWF becomes a
filamentous ligand for the GPIb-IX-V complex of the platelet surface
(14) through recognition of the A1(vWF) domain, such as to allow for
the initial, reversible tethering of the platelets to the substrate.
Subsequently, the platelet is activated, and its
IIb
3 integrin is
converted from an inaccessible to an accessible state on the cell
surface (56). This permits its engagement in the platelet-vWF
interaction through recognition of the RGD motif. At this point, the
platelet is arrested onto the substrate and may bind to other platelets
to initiate aggregate formation.
Although the participation of vWF in the high shear rate-induced platelet aggregation upon damage of the blood vessel wall is a critical step in aggregate formation, the ligands that favor optimal immobilization of the circulating vWF (45) remain to be identified. A vaste number of previous studies have suggested that interstitial collagen types I and III (and to a lesser extent V) could be the candidate ligands. However, their localization in the connective tissues significantly below the zone interfacing the subendothelial basement membrane and the interstitium raises some doubts about their physiological relevance in the early phases of vascular repair. Instead, the intimate association of Col VI microfilaments with the basement membrane on one hand (46) and the interstitial collagen fibrils on the other (47) strongly supports an important role for this collagen in binding the circulating vWF at the sites of vascular injury. Furthermore, the colocalization of Col VI and vWF in the vascular subendothelium (48, 49), recently confirmed at the ultrastructural level (50), strongly points to the possibility that Col VI may represent a central component of the subendothelial matrix contributing to platelet aggregation upon rupture of the blood vessel wall. However, previous studies utilizing pepsin-digested and/or guanidine HCl-extracted Col VI tetramers have failed to demonstrate a significant role for this collagen under conditions of high shear rates (23). At present, the nature of the discrepancy between urea- versus GuHCl-extracted Col VI is not clear, nor was it investigated in detail here, but several explanations may be considered. The loss of platelet aggregation-promoting activity of the GuHCl-extracted collagen may be attributed to a more severe unfolding of the molecule by exposure to GuHCl, or inhibitory contaminants may be present in the Col VI preparation based on GuHCl extraction. One such possible contaminant may be the proteoglycan decorin noted to remain associated with Col VI tetramers under such purification procedures (47). Finally, it remains possible that urea but not GuHCl extraction brings out a reactivity that does not occur in the native structure.
The mode and extent of platelet adhesion/aggregation onto Col VI at
high shear rates was found to be similar to that reported for collagen
type I (15, 51) and was strictly proportional to the amount of vWF that
associated with the collagen substrate. Our findings are in
disagreement with those previously reported and implicating the
2
1 integrin in the direct arrest and tethering of platelets on
Col VI under high shear rates (6, 52) but do not preclude the
possibility that both this receptor and glycoprotein VI (53) may be
responsible for the transduction of specific intracellular signals
essential for further activation of platelets, following the initial
vWF/GPIb
-dependent contact with the collagen substrate.
Crystallographic analysis of the wild type and point-mutated A3(vWF)
domain reveals that, opposite to the I domain of the 2
1 collagen
binding integrin, disruption of the vestigial MIDAS motif of the
A3(vWF) domain does not affect collagen binding (54, 55). Because the
2
1 integrin has an elected preference for the triple helix of
collagens, the above finding is overtly in accord with our observations
that the A3(vWF) domain is not involved in the vWF interaction with the
triple-helical region of Col VI. Accordingly, the Col VI interaction
site of the A3(vWF) domain may reside within the globular domains of
the collagen and/or be dependent upon a specific macromolecular
structure assumed by the intact, but not the pepsin-digested tetramer
deprived of globular domains. This conclusion is in accordance with the
A3(vWF)-mediated collagen type I-vWF interaction for which there is a
strict requirement of a higher order-organized fibrillar structure to
achieve maximal promotion of platelet aggregation (8). Nonetheless, the
marked difference in macromolecular configuration between interstitial collagens and the microfilamentous Col VI indicates that the structural nature of the A3(vWF) binding sites must differ in these molecules. Yet, the versatility of this binding site is highlighted by the indiscriminate ability of the A3(vWF) domain to interact with fibrillar
collagen type I, monomeric type III, and tetrameric Col VI. The A3(vWF)
domain has recently been shown to contain a binding site(s) for both
interstitial collagens (15, 19, 21, 54) and the basement membrane
collagen type IV (21) but has been proposed to lack binding affinity
for Col VI, for which a pivotal binding role has been attributed to the
classical collagen-binding A1(vWF) domain (21). Our experiments, in
static phase using intact Col VI tetramers purified from three
different tissues avoiding the use of GuHCl, revealed that the
A1(vWF)-Col VI binding accounted for about 60% of the interaction.
Moreover, blockade of either the A1(vWF)-Col VI or the A3(vWF)-Col VI
interaction demonstrated an essential role for both A(vWF) domains in
promoting the high shear rate-induced platelet adhesion/aggregation.
Thus, our results are in disagreement with those of Hoylaerts et
al. (21) and may reconduce to subtle structural alterations in the Col VI tetramers deriving from diverse extraction/purification procedures. These alterations may similarly be central for the differential loss of the platelet adhesion-promoting activity at high
shear rates (23).
Mapping studies based upon the combined usage of pepsin-digested Col VI
tetramers, recombinant polypeptides corresponding to the noncollagenous
globular regions of the 3(VI) chain, and anti-Al(vWF)/A3(vWF)
antibodies were instrumental in localizing the main A1(vWF) binding
site within the globular portion of the Col VI molecule. This site
could be identified within the A7(VI) module. This module inhibited
platelet adhesion to the collagen under high flow conditions and, in
contrast to the recombinant polypeptide rvWF(445-733) and to the
anti-A1(vWF) antibody NMC-4, did neither affect the GPIb
-vWF binding
nor interfere with the A3(vWF)-Col VI interaction. It has previously
been reported that in the acquired autoimmune disease vWD, circulating
autoantibodies reacting with both the A1(vWF) and A3(vWF) domains may
be present that inhibit the vWF-collagen interaction without affecting
the ristocetin-induced GPIb
-vWF interaction (57). Similarly, a number of experiments using recombinant mutated vWF polypeptides as
well as studies on individuals carrying genetic mutations that result
in substitutions of amino acids critical for the GPIb
-A1(vWF) interaction have also suggested that these two A1(vWF) binding functions can be attributed to disparate binding motifs (15, 18,
58-60). Recent crystallographic analysis of A1(vWF) (61) and
A1(vWF)-NMC-4 Fab complex (62) provide structural evidence for a
distinct localization of GPIb
and other functional sites in the
crystal. These findings and our data suggest that the A1(vWF) binding
sites for the platelet receptor GPIb
and collagens might be distinct.
In conclusion, we propose that to achieve maximal efficiency of vWF in
initiating platelet adhesion onto Col VI under conditions of high shear
rates, both A1(vWF) and A3(vWF) are necessary. Their concerted
participation would provide the biomechanical conditions supporting
reversible GPIb-dependent platelet adhesion,
IIb
3 activation and its binding to the RGD sequence of vWF, and platelet arrest onto the Col VI. In the setting of vascular lesions,
irreversible platelet adhesion to Col VI might enhance the platelet
activation response and aggregate formation followed by the activity of
IIb
3,
2
1, and GP VI (63). Thus, our findings provide new
prospects for the understanding of the molecular mechanism responsible
for platelet adhesion phenomena and open new avenues for exploring the
possible relevance of functional defects in the A1(vWF) and A3(vWF)
domains of vWD and for developing new antithrombotic drugs.
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. Zaverio Ruggeri and L. W. Hoyer for providing monoclonal antibodies and recombinant vWF polypeptides and for helpful suggestions. Dr. Roberto Doliana and Bruna Wassermann are thanked for their assistance in the preparation of recombinant Col VI polypeptides, and Maria Teresa Mucignat is thanked for her supporting technical assistance.
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FOOTNOTES |
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* This work was supported by AIRC (to A. C.) and by Progetti Ricerca Finalizzata IRCCS 1994 (to L. D. M. and A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed at Servizio
Immunotrasfusionale e Analisi cliniche, Centro di Riferimento
Oncologico, Via Pedemontana Occidentale, 12, 33081 Aviano (PN), Italy.
Tel.: ++390-434-659 360; Fax: ++390-434-659 427; E-mail:
ldemarco{at}ets.it.
The abbreviations used are: vWF, von Willebrand factor; vWD, von Willebrand's disease; rvWF, recombinant polypeptide of vWF; Col VI, type VI collagen; mAb, monoclonal antibody; GP, glycoprotein; BSA, bovine serum albumin; GuHCl, guanidine HCl.
2 According to the nomenclature proposed by Bork and Koonin (64) for the vWF type A domains (65).
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REFERENCES |
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