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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3808-3816
Simple Collagen-Like Peptides Support Platelet Adhesion Under Static
But Not Under Flow Conditions: Interaction Via  2 1 and von
Willebrand Factor With Specific Sequences in Native Collagen Is a
Requirement to Resist Shear Forces
By
Marilyn W. Verkleij,
Laurence F. Morton,
C. Graham Knight,
Philip G. de Groot,
Michael J. Barnes, and
Jan J. Sixma
From the Postgradual School of Biomembranes, Department of
Haematology, University Hospital Utrecht, Utrecht, The Netherlands; and
the Strangeways Research Laboratory, Worts Causeway, Cambridge, UK.
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ABSTRACT |
The aim of this study was to define the need for specific collagen
sequences and the role of their conformation in platelet adhesion to
collagen under both static and flow conditions. We recently reported
that simple triple-helical collagen-related peptides (CRPs),
GCP*(GPP*)10GCP*G and GKP*(GPP*)10GKP*G
(single-letter amino acid code, P* = hydroxyproline; Morton et al,
Biochem J 306:337, 1995) were potent stimulators of platelet
activation and were able to support the adhesion of gel-filtered
platelets examined under static conditions. The present study
investigated whether these same peptides were able to support platelet
adhesion under more physiologic conditions by examining static adhesion with platelet-rich plasma (PRP) and adhesion under flow conditions. In
the static adhesion assay, we observed 20% surface coverage with
platelet aggregates. In marked contrast, there was a total lack of
adhesion under flow conditions examined at shear rates of 50 and 300 s 1. Thus, the interaction of platelets with the CRPs is
a low-affinity interaction unable on its own to withstand shear forces.
However, the addition of CRPs to whole blood, in the presence of 200 µmol/L D-arginyl-glycyl-L-aspartyl-L-tryptophan (dRGDW) to prevent
platelet aggregation, caused an inhibition of about 50% of platelet
adhesion to collagens I and III under flow. These results suggest that the collagen triple helix per se, as defined by these simple collagen sequences, plays an important contributory role in the overall process
of adhesion to collagen under flow. The monoclonal antibody (MoAb)
176D7, directed against the  2 subunit of the integrin 2 1, was
found to inhibit static platelet adhesion to monomeric but not
fibrillar collagens I and III. However, under flow conditions, anti-2 MoAbs (176D7 anf 6F1) inhibited adhesion to both monomeric and fibrillar collagens, indicating that 21 is essential for adhesion to collagen under flow, independent of collagen conformation, whether monomeric or polymeric. To obtain further insight into the
nature of the different adhesive properties of CRPs and native collagen, we investigated the relative importance of von Willebrand factor (vWF) and the integrin 21 in platelet adhesion to collagen types I and III, using the same shear rate (300 s1) as
used when testing CRPs under flow conditions. Our results, together
with recent data of others, support a two-step mechanism of platelet
interaction with collagen under flow conditions. The first step
involves adhesion via both the indirect interaction of platelet
glycoprotein (GP) Ib with collagen mediated by vWF binding to specific
vWF-recognition sites in collagen and the direct interaction between
platelet 21 and specific 21-recognition sites in collagen.
This suffices to hold platelets at the collagen surface. The second
step occurs via another collagen receptor (thought to be GPVI) that
binds to simple collagen sequences, required essentially to delineate
the collagen triple helix. Recognition of the triple helix leads to
strengthening of attachment and platelet activation.
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INTRODUCTION |
THE INTERACTION OF platelets with
collagens in the vessel wall is one of the first and basic underlying
mechanisms in a normal hemostatic response. Collagen is a unique ligand
for platelets in that it both supports adhesion and causes platelet
activation leading to platelet aggregate formation. Identification and
characterization of adhesive domains in the collagen molecule and their
interaction with specific receptors on blood platelets is of great
importance for understanding the hemostatic process.
The mechanism of platelet-collagen interaction can be supported by a
direct (primary) interaction mediated by collagen receptors with
collagen and an indirect (secondary) interaction mediated by bridging
molecules that bind both to platelet membrane receptors and to
collagen. The 21 integrin (glycoprotein [GP] Ia/IIa, VLA 2, or
CD49b/CD29), GPIV (GPIIIb or CD36), and GPVI (P62) have been proposed
as direct collagen receptors.1 Among these direct receptors, the 21 integrin meets several of the criteria as a
major collagen receptor for different collagen types (reviewed by
Santoro et al,2 Sixma et al,3 and Sixma et
al4). First, platelet adhesion under flow conditions to
collagens type I, III, IV, and VI with blood from a patient whose
platelets are deficient in the 2 subunit of the integrin
215 has been shown to be severely
impaired.6,7 Second, using the monoclonal antibody (MoAb)
176D7 antibody against the 2 subunit, Saelman et al8
showed that 21 is the main integrin involved in platelet adhesion
under flow conditions to collagens types I through VIII. Two important
studies investigating the role of GPIV indicate that this receptor is
only of minor importance for platelet adhesion under flow
conditions.9,10 It is now becoming clear that GPVI is of
minor importance in the first phase of adhesion but plays a major role
in the second phase, involving platelet activation leading to the
formation of platelet aggregates.11 Both fibronectin and
von Willebrand factor (vWF) are bridging molecules that are important
for platelet adhesion to collagen.12-14 vWF is essential
for conferring resistance of platelets to shear, with a more pronounced
role under high shear forces.14,15
Fragmentation of collagen with cyanogen bromide suggested the existence
of separate receptors for adhesion and platelet
activation,16-21 a view supported by the effects of
chemical modification of collagen on its platelet
reactivity.22 Synthetic, triple-helical peptides have also
been used to try to identify platelet-reactive sequences in collagen in
view of the evidence that the collagen helical conformation is
essential for its recognition of platelets and other
cells.17,23-25 To examine the platelet reactivity of short, defined collagen sequences in triple-helical form, the sequence in
question has been synthesized with additional Gly-Pro-Hyp triplets present to stabilize the helical structure.26 Helical
peptides have also been synthesized covalently bonded at the C-terminus to facilitate correct alignment of the three constituent chains within
the helix.27 Using this approach, platelet-reactivity has
been found in a number of peptides based on the platelet-reactive collagen III fragmeny 1(III)CB426,28 and in a peptide
containing residues 1263-1277 of the collagen IV 1(IV)
chain.29 An 21-recognition sequence has been ascribed to residues 522-528 of the 1(III) collagen chain.28
These studies with synthetic peptides have been concerned with
identifying highly specific platelet-reactive sequences in collagen.
However, we have recently examined the platelet reactivity of very
simple collagen-like peptides composed basically of only a repeat
Gly-Pro-Hyp tripeptide structure and therefore ostensibly lacking
highly specific collagen sequences. These peptides assumed spontaneously a very stable triple-helical conformation and, after cross-linking to yield a quaternary structure, were found, against expectation, to be extremely platelet-reactive.30 Their
platelet reactivity was totally unaffected by anti-21 MoAbs.
These antibodies were without effect on adhesion measured under static
conditions at room temperature, using gel-filtered platelets, and
showed no inhibition of aggregation, measured at 37°C with
platelet-rich plasma (PRP). These results suggested that the basic
triple-helical structure of collagen may possess an intrinsic platelet
reactivity independent of the presence of highly specific sequences.
The aim of the current study was to determine whether the structure of
these simple collagen-related peptides (CRPs) was sufficient to support
platelet adhesion under flow conditions. We first studied adhesion to
the peptides under static conditions using more physiologic conditions
than before (using PRP at 37°C) and then under physiologic flow
conditions with whole blood. As an alternative approach to investigate
whether recognition of the helix per se, independent of specific
sequences, might contribute to adhesion to collagen under flow, we
added the peptides to whole blood before perfusion over collagen. As a
corollary to these studies, we also examined the relative vWF and
21 dependence of adhesion under low shear flow conditions to
collagens I and III as monomers and fibers. In contrast to high shear
conditions, we found that 21-dependent platelet adhesion to
(monomeric) collagens I and III under low shear conditions can occur in
the absence of vWF. Although adhesion to collagen as monomer is known
to involve 21 under both static and flow conditions, adhesion to
fibers has been reported under static conditions to be largely 21
independent.17 We consider it important, therefore, to
establish the dependence on 21 of adhesion under flow to the
collagens as fibers to confirm the essential role of this integrin as
an adhesive receptor under flow conditions.
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MATERIALS AND METHODS |
Blood.
Whole blood, obtained from healthy volunteer donors who denied having
taken aspirin or other platelet function inhibitors in the preceding
week, was anticoagulated with 1/10 vol of 200 U/mL low molecular weight
heparin in 0.15 mol/L NaCl (LMWH-blood; LMWH: Fragmin; Kabi Pharmacia,
Stockholm, Sweden). In experiments in which the effect of the addition
of D-arginyl-glycyl-L-aspartyl-L-tryptophan (dRGDW) and CRPs during
perfusion was studied, whole blood was anticoagulated with 1/10 vol of
400 U/mL LMWH in 0.15 mol/L NaCl to prevent fibrin formation on the
collagen surface.
Peptides/antibodies.
The peptides [Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly
(CRP 1) and Gly-Lys-Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly
(CRP 2); of which Hyp = hydroxyproline] were synthesized as described
previously.30 Peptides were cross-linked [CRP 1 via the
cysteinyl residues using N-succinimidyl 3-(2-pyridyldithio) propionate
and CRP 2 via the lysyl residues using glutaraldehyde] as
described.30
The dRGDW peptide was kindly provided by Dr J. Bouchaudon
(Rhône-Poulenc-Rorer, Chemistry Department, Centre de Recherche de Vitry, Vitry sur Seine, France).31 Where indicated, 200 µmol/L dRGDW was added during perfusion experiments to prevent
platelet aggregation in whole blood. dRGDW was added 15 minutes and
CRPs (free peptide or cross-linked) 10 minutes before perfusion to the
whole blood perfusate and incubated at 37°C. The CRPs were solubilized in 10 mmol/L acetic acid. Control perfusion experiments were performed in which the effect of the presence or absence of
vehicle buffer on platelet adhesion to collagen type I and III was
compared. No significant differences were observed. Furthermore, the
addition of 100 µg/mL CRPs in this vehicle to whole blood showed no
effect on the pH.
MoAb 176D7 and MoAb 6F1, directed against 2 subunit of the integrin
21, were kindly provided by Dr H.R. Gralnick (Department of
Health and Human Services, National Institutes of Health, Bethseda, MD)
and by Dr B. Coller (Mount Sinai Hospital, New York, NY), respectively.
Both MoAbs inhibit adhesion under static conditions,32,33 and the 176D7 MoAb has also been shown to inhibit adhesion under flow
conditions.8 MoAb AK-2, directed against GPIb, was a
generous gift of Dr M. Berndt (Baker Medical Research Institute,
Prahan, Australia).34 In perfusion experiments, a 1:1,000
dilution was used.
Surfaces.
Glass coverslips (18 × 18 mm; Menzel Gläser, Braunschweig,
Germany) and circular glass coverslips (Knittel Gläser,
Braunschweig, Germany; surface area, 1.1 cm2) were soaked
overnight in chromosulfuric acid, rinsed thoroughly with deionized
water, and air-dried. Thermanox coverslips (Nunc, Inc, Napervillle, IL;
surface area, 1.2 cm2) were soaked overnight in 80%
ethanol, rinsed thoroughly with deionized water, and air-dried.
Human placental collagens type I and III (Sigma, St Louis, MO) were
solubilized in 0.05 mol/L acetic acid (1 mg/mL) and sprayed on glass
coverslips at a final density of 30 µg/cm2 with a
retouching airbrush (Badger model 100; Badger Brush Co, Franklin Park, IL). Fibrillar collagens type I and III were prepared by
dialysing the solubilized human placenta collagens type I and III twice
for 24 hours at 4°C against a sodium phosphate buffer (20 mmol/L
Na2HPO4), pH 7.4.14,35 Fibrillar
collagens type I and III were also sprayed on glass coverslips at a
density of 30 µg/cm2. CRP 1 or 2 (free peptide or
cross-linked) were sprayed on Thermanox coverslips at a density of 10 µg/cm2. When the micro-spray method was used, a small
surface area (0.13 cm2) in the center of a glass coverslip
was sprayed with 300 µg/cm2 of CRP.
After coating or spraying, the coverslips were blocked for 90 minutes
at room temperature with 1% human albumin solution (Behring, Marburg,
Germany) in HEPES-buffered saline (HBS; 10 mmol/L HEPES, 150 mmol/L
NaCl, pH 7.35) when used in a perfusion experiment or with 3% bovine
serum albumin (BSA) in HBS when used in a static adhesion assay.
Electron microscopy.
Drops of collagen preparations before and after dialysis were applied
to 400 mesh specimen grids covered with collodion films lightly coated
with carbon. After 5 minutes, the drops were removed with filter paper;
the preparations were either negatively stained with sodium
phoshotungstate (pH 7.2) or positively stained with phosphotungstic
acid (pH 3.4) and contrasted with uranyl acetate (1%).36
The preparations were examined in a Philips EM 420 electron microscope
(Philips, Eindhoven, The Netherlands) operated at 60 kV. Electron
microscopy indicated the absence of micro fibrils or particulate
structures in the acid-soluble monomeric collagen preparations (before
dialysis).
Static adhesion assay.
Static adhesion experiments were performed in 24-well plates (Costar,
Cambridge, MA) using circular coverslips (surface area, 1.1 cm2) or small Thermanox coverslips (surface area, 1.2 cm2). PRP was prepared by centrifugation of LMWH-blood at
120g for 10 minutes at 22°C. The platelet count was
adjusted to 200,000 platelets/µL with platelet-poor plasma (PPP).
Five hundred microliters of PRP were added per well and platelets were
allowed to adhere for 1 hour at 37°C. Subsequently, the coverslips
were removed, rinsed, and stained as described below.
When tested in the assay, the MoAb 176D7 was added to PRP 30 minutes
before the assay was performed and incubated at 37°C.
Perfusions.
Perfusions were routinely performed in a single pass perfusion chamber
of which the characteristics have been described by Henrita G. van
Zanten et al.37 This single pass perfusion
chamber has a modified parallel plate perfusion chamber with a slit
height of 0.1 mm and a slit width of 2 mm,37,38
corresponding with a flow rate of 60 µL/min (shear rate,
300 s1). Blood was drawn through the perfusion
chamber by a Harvard infusion pump (pump 22, model 2400-004;
Harvard, Natick, MA). The perfusion time was 5 minutes.
Some perfusions were repeated with the parallel plate plate perfusion
chamber. The characteristics of this perfusion chamber have been
described by Sakariassen et al.39 Duplicate coverslips were
inserted in the chamber. Fifteen milliliters of whole blood was
prewarmed at 37°C for 5 minutes and then recirculated through the
chamber for 5 minutes at a wall shear rate of 300 s1. Then, 15 mL of prewarmed HBS was drawn through
the system to wash the coverslips. No significant differences in the
extent of platelet adhesion were observed between the two perfusion
chambers.
Evaluation.
After a static adhesion assay or after perfusion, the coverslips were
removed, rinsed with HBS, fixed with 0.05% glutaraldehyde, dehydrated
with methanol, and stained with May-Grünwald/Giemsa as previously
described.40 Platelet adhesion was quantitated with a light
microscope (1,000× magnification) connected to a computerized
image analyser (AMS 40-10, Saffron Walden, UK). In experiments
performed with the parallel plate perfusion chamber, three lines
perpendicular to the flow direction were evaluated: one line in the
center of the coverslip and two lines 3 mm upstream or downstream of
this line, respectively. Platelet adhesion was expressed as the
percentage of the surface covered with platelets.
Statistical analysis.
Results were expressed as the mean ± standard error of the mean
(SEM) for data obtained from different experiments or as the mean ± standard deviation (SD) for data obtained from coverslips within one
experiment. The Student's t-test was used to test for significance of differences between groups. P values less than .05 were considered significant.
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RESULTS |
Platelet adhesion to CRPs under static conditions.
Under static conditions, platelet adhesion, at least as good as that to
collagen type I both monomeric and fibrillar, was observed to both CRPs
as free peptide or cross-linked (Table 1). Platelet adhesion to the CRPs consisted solely of platelet aggregates ranging from intermediate to large in size. The morphology of the
platelet aggregates was similar in all cases. Platelet aggregates adhering to cross-linked (-XL) CRP 1 are shown in
Fig 1 by way of example. Adhesion to CRP
1-XL was significantly higher than to CRP 1. Adhesion to CRP 2-XL,
which was more variable than to CRP 1-XL, was also higher than to the
equivalent free peptide (CRP 2), but not significantly so. Addition of
the platelet activation inhibitor indomethacin (30 µmol/L) had no
effect on the extent of static platelet adhesion to CRP 1-XL or
collagen type I (data not shown).
In a previous study, we observed that static adhesion of gel-filtered
platelets to the CRPs was not inhibited by a MoAb (6F1) against the
2 subunit of the integrin 21.30 We studied here the effect of another MoAb against 21 (176D7; 20 µg/mL) in the present static adhesion assay in which plasma proteins are present. Again, there was no effect of the antibody on static platelet adhesion
to either CRPs or CRP-XLs (Table 1). Furthermore, the antibody clearly
inhibited static platelet adhesion to monomeric but not to fibrillar
collagen type I.
Platelet adhesion to CRPs under flow conditions.
In contrast to platelet deposition under static conditions, there was
no adhesion to CRPs under flow conditions. No platelet deposition was
observed on either peptide (free or cross-linked) at the two shear
rates tested (50 and 300 s1). Under the same
conditions, adhesion to human collagen type I was 15.2% ± 2.3% at
the higher and 4.1% ± 1.5% at the lower shear rate
(Table 2). To ensure that the lack of
adhesion to the peptides was not due to their removal during perfusion,
a number of control experiments were performed. Denhardt-coating of
glass coverslips, which has been described to retain collagen fragments
on coverslips under flow conditions,19 was performed before
spraying of the CRPs. Using this treatment, again no platelet deposition occurred during perfusion. Using a micro-spray method in
which a very high concentration of peptide (300 µg/cm2)
is sprayed onto a very small surface area, sufficient peptide was
absorbed to allow its detection by May-G rünwald/Giemsa staining. However, platelet adhesion was again absent. When coverslips that had
been used in a perfusion experiment were washed with HBS, and then were
used to test static adhesion with blood (PRP) from the same donor, the
same level and morphology of platelet adhesion was observed as on
coverslips that had been used only to measure static adhesion (data not
shown). The lack of adhesion under flow in this experiment was
confirmed by staining parallel coverslips. In conclusion, these
experiments confirm that CRPs and CRP-XLs do not support adhesion under
flow conditions.
Effect of CRPs and CRP-XLs on platelet deposition on human collagen
type I and III under flow.
As an alternative approach, to investigate whether the adhesive
properties of the CRPs contribute to adhesion to collagen under flow,
we added these peptides to whole blood before perfusion over collagen.
Because the cross-linked CRPs are highly platelet reactive30 and, when added to whole blood, will cause
platelet aggregate formation that will affect platelet adhesion under
flow conditions, we included the synthetic RGD-containing peptide, dRGDW (200 µmol/L), to prevent this. This adhesive recognition sequence for platelet receptor GPIIb/IIIa, the integrin
IIb3, has been shown to inhibit aggregate
formation induced by collagen without inhibiting adhesion to
collagen.41-43 The absence of aggregate formation after the
addition of CRP 1 or 2 (free peptide or cross-linked, 100 µg/mL) was
routinely measured by determining the platelet count before and after
addition. Addition of CRP 1, CRP 1-XL, and CRP 2-XL resulted in a
substantial decrease in platelet number that was prevented by
preincubation with dRGDW.
Platelet adhesion under flow conditions (shear rate, 300 s1) to human collagens type I and III in the
presence of dRGDW resulted in a substantial increase in platelet
deposition (from 18.4% ± 2.8% to 33.9% ± 7.2% [n = 4] in
the case of collagen type I and from 20.3% ± 3.6% to 43.7% ± 0.5% [n = 3] for collagen type III). The same effect has been
reported by Saelman et al31 and de Groot and
Sixma.38 The addition of CRP 1, CRP 1-XL, or CRP 2-XL (100 µg/mL) together with dRGDW during perfusion resulted in an inhibition
of platelet adhesion compared with a parallel perfusion to which only
dRGDW was added (Table 3). Because the
addition of 100 µg/mL of CRP is the highest possible experimental
concentration achievable in the small perfusion system, this
concentration was used in all experiments. The adhesion to collagen
type I and III was diminished by 32.9% and 41.8%, respectively, after
the addition of CRP 1 and by 53.2% and 51.7%, respectively, after the
addition of CRP 1-XL. The latter is significantly more active than CRP 1 in inhibiting adhesion to collagen type III under flow conditions. Adhesion to collagen type I was only slightly inhibited and that to
collagen type III only inhibited 33.6% after the addition of CRP 2, but the addition of CRP 2-XL caused inhibition of 56.9% and 68.4%,
respectively.
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Table 3.
Inhibition of Platelet Adhesion to Human Collagen Type I
and III by CRPs in the Presence of dRGDW Under Flow Conditions
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To exclude the possibility that the diminished level of adhesion in the
presence of the CRPs is an indirect effect caused by the activating
properties of the CRPs, platelets were stimulated with 15 µmol/L
thrombin-receptor activating peptide (TRAP) in the presence of 200 µmol/L dRGDW. This had no effect on platelet adhesion to human
collagen type III under flow conditions. Furthermore, FACS experiments,
using a panel of MoAbs against various platelet membrane GPs (GPIb, CD
62, CD 63, GMP 33, CD31, and GP IIIA),44 showed that 100 µg/mL CRP-XL 1 and 15 µmol/L TRAP result in a similar level of
membrane surface expression of these markers for platelet activation
(data not shown).
Role for vWF and the integrin 21 in
interactions with specific collagen sequences.
Our results as described here show that CRPs, simple collagen-like
peptides, are not able to mediate platelet adhesion under flow.
However, when added to whole blood, they cause a significant decrease
in platelet adhesion to collagen types I and III under flow conditions.
These data imply that the CRPs are involved in the adhesive process but
are unable to mediate adhesion on their own. Apparently, they lack
specific sequences that are present in native collagen and are
necessary for primary adhesion under flow. The relative importance of
the GPIb-bound vWF and integrin 21 in mediating platelet adhesion
to native collagen under low shear conditions (300 s1) has not been investigated in detail. To
investigate the relative importance of vWF- and 21-recognition
sequences in collagens I and III in mediating adhesion under these
conditions, we undertook perfusion experiments using specific MoAbs
directed against GPIb or 21.
The MoAb AK-2 (1:1,000) against the vWF binding site on GPIb reduced
the adhesion to collagen type I by 52.0% and to collagen type III by
62.7% (Table 4). In both cases, a
considerable extent of platelet deposition was still present. The
addition together of both MoAb AK-2 and MoAb 176D7 (against the
2-subunit of 21; 20 µg/mL) resulted in a further inhibition
of adhesion (80.7% and 88.6%, respectively; Table 4). The results
suggest that the integrin 21 interaction with collagen is capable
of partly mediating platelet adhesion to collagen in the absence of vWF
under low shear conditions. This conclusion is supported by previous
results obtained in perfusion experiments at a shear rate of 490 s1 using von Willebrand disease (vWD)
blood.14 We now repeated this latter experiment at a shear
rate of 300 s1 and furthermore tested the effect of
addition of MoAb 176D7 to vWD blood during perfusion (n = 2 vWD type 3 patients). Again, substantial platelet adhesion was observed, of which
the extent of adhesion of the vWD patient was almost as high as that to
the control. The addition of MoAb 176D7 during perfusion with vWD blood
resulted in 50% inhibtion of adhesion (data not shown).
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Table 4.
Inhibitory Effect of Anti-GPIb MoAb Alone or in
Combination With Anti-21 MoAb on Platelet Adhesion to
Monomeric Collagen Type I and III Under Flow Conditions
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vWF is an essential bridging molecule in mediating platelet adhesion to
both monomeric and fibrillar collagen under high shear.14 It is also known that adhesion to monomeric collagen under flow involves integrin 218 (compatible with our results
described above under relatively low shear conditions). However, it is
unclear to what extent adhesion under flow to collagen fibers is also
21-dependent. This is an important question, because, of course,
collagen occurs in the vessel wall and elsewhere in vivo as fibers. We
therefore analyzed the dependency on integrin 21 of platelet
adhesion to both monomeric and fibrillar collagen type I and III in our static assay and under flow conditions.
Our results in the static adhesion assay confirmed the results of
Morton et al17 that adhesion to fibrillar collagen type I
(Table 1) and III (data not shown) is not inhibited with a MoAb (176D7)
against the 2 subunit of the integrin 21. However, static
platelet deposition to monomeric collagen type I and III is inhibited
by 90.0% ± 2.0% (n = 7) and 63.9% ± 10.2% (n = 10), respectively. In contrast, under flow conditions, the MoAbs 176D7 and
6F1 (both against the 2 subunit of the integrin 21) cause a
large decrease in platelet adhesion to both monomeric and fibrillar collagen types I and III (Table 5). These
data confirm the important role of 21 under flow conditions
irrespective of whether collagen is monomeric or fibrous.
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Table 5.
Inhibitory Effect of Anti-21 MoAbs on Platelet
Adhesion to Monomeric and Fibrillar Collagen Type I and III Under
Flow Conditions
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Because structural differences may exist between native collagen
fibrils and those formed in vitro,45,46 we analyzed the fibrils formed in this study under the electron microscope. Fibrils were observed with a banding pattern, visualized either via positive or
nagative staining, comparable to that observed by others using collagen
obtained by a limited pepsin digestion,46 with a banding pattern similar but not identical to native collagen fibrils and indicating the presence of a 67-nm periodicity as in native collagen fibrils (Fig 2). The electron microscopical
data and the similar behavior of our fibrils in static adhesion assay
as that of intact native collagen type I fibrils (Morton et
al17) strongly suggest that the fibrils used in this study
closely resemble native collagen type I fibrils.
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| Fig 2.
(A) Transmission electronic micrographs of fibrils formed
in vitro of human collagen type I. (A) and (B) were stained with phosphotungstate and (C) was stained with phoshotungstic acid and
uranyl acetate. Bar in (A) = 0.5 µm. Bar in (B) and (C) = 0.1 µm.
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DISCUSSION |
Earlier studies suggested that collagen-platelet interaction may be a
two-step process involving the sequential recognition of separate
platelet receptors, one mediating the initial adhesion and the other
necessary for activation, each recognizing different structural
elements in the collagen molecule.16,20-22 Several platelet
surface proteins have been implicated in collagen-platelet interaction,
including integrin 21, CD36, GPVI,1 and a 65-kD collagen species specific for collagen type.47
We demonstrated previously that simple CRPs, composed basically of a
Gly-Pro-Hyp repeat sequence, were potent stimulators of platelet
activation.30 The peptides were triple-helical and associated to form ordered aggregates that could be stabilized by
cross-linking. As in the case for collagen, the triple-helical conformation and polymeric conformation structures were essential for
CRP-induced platelet activation. The suggestion was made that the
collagen triple helix per se may be platelet reactive and that collagen
tertiary and quaternary structures may be sufficient alone to meet the
structural requirements of collagen for platelet activation, without
the need for highly specific collagen sequences. CRPs appear to
activate platelets by the same mechanism as collagen, because both
elicit the same specific signals.48-50 CRPs may be very
useful therefore to better understand collagen-platelet interaction. Because activation by CRPs is not prevented by anti-integrin 21 MoAbs,30 it may be argued that this integrin is not
involved directly in the activation mechanism. In accord with this, we have noted previously30 the relative ineffectiveness of the MoAb 6F1 in preventing collagen-induced platelet aggregation. In fact,
there is now a considerable body of evidence that GPVI may be the
activatory collagen-receptor,51-55 and this is probably the
receptor recognized by the CRPs, because like collagen, CRPs do not
activate GP VI-deficient platelets (Ichinohe et al55 and
Kehrel et al56).57
CRPs immobilized on plastic support the static adhesion of gel-filtered
platelets30 and, as shown here, the adhesion also of
platelets in the presence of plasma proteins (PRP). Adhesion in both
cases is 21-independent. We have found that adhesion to
fibrillar, as opposed to monomeric, collagen under these conditions is
also 21-independent. The receptor involved in this
21-independent static adhesion is not known for certain, although
both CD369 and GPVI11,51,53 have been reported
to play a role in platelet adhesion to collagen. This adhesion to CRPs
might suggest that collagen tertiary and quaternary structures are
sufficient alone to support the primary adhesion step as well as the
subsequent activation. However, a crucial observation in the current
work is that there is a complete absence of adhesion to
the CRPs under flow. This suggests that CRP structure is not adequate
to permit shear-resistant platelet adhesion and that the primary
adhesion receptor responsible for the arrest of platelets on the
collagen surface under flow must interact with specific, likely
high-affinity, binding sites in collagen.
Interestingly, whereas CRPs did not themselves support adhesion under
flow, they inhibited adhesion to collagen when added to whole blood
before perfusion over collagen. In the latter case, both CRPs and
platelets are in the fluid phase and the interaction between them is
therefore not subjected to shear forces and the CRPs can presumably
bind to a platelet receptor (eg, GPVI) under low-affinity conditions.
Inhibition was 50% at 100 µg/mL, the highest concentration of
peptide we could conveniently test (to conserve the use of peptide).
Inhibition was less at lower CRP levels and it is possible that the
inhibition may have been greater at peptide concentrations higher than
100 µg/mL. Surprisingly, the inhibitory activity appeared to require
the cross-linked peptide. Thus, there was little if any inhibition by
CRP 2 as free peptide. Whereas CRP 1 showed some inhibition, this was
less than that by CRP 1-XL, and we believe the activity of CRP 1 may be
due to some degree of cross-linking arising from spontaneous disulphide bond formation. This cannot occur with CRP 2, which has lysyl residues
in place of cysteinyl residues in CRP 1. Presumably, CRP must bind to
platelets by a multivalent mechanism requiring peptide in quaternary
form. The exact mechanism of the inhibition is unclear but could
indicate that the receptor binding CRP-XL is involved somehow in the
overall adhesion process to collagen. It seems unlikely that the
inhibition can be due to low-affinity binding of CRP to 21,
because anti-21 MoAbs do not prevent static adhesion to CRP. We
believe it more likely that inhibition arises because of binding of CRP
to GPVI, indicating that CRP sequences in collagen through their
recognition of this receptor are directly or indirectly contributory to
overall adhesion.
Our data show that CRPs support static adhesion but not adhesion under
flow, indicating that this is a low-affinity interaction. This further
implies that CRPs lack specific sequences that are present in native
collagen and are necessary under flow conditions. Both integrin
21 and GPIb are known to be required for platelet adhesion to
collagen under flow, the latter through its recognition of vWF bound to
collagen.12-15 We have found no specific binding of vWF to
CRPs (our unpublished data). Furthermore, using anti-GPIb and anti-21 MoAbs and vWD blood used in perfusion studies we discovered the relative importance of vWF in supporting platelet adhesion to collagen under flow. However, at low shear
conditions, some 21-dependent platelet adhesion to collagens I
and III could occur in the absence of vWF. Concerning the relative
importance of the integrin 21, there is no doubt of its
requirement for platelet adhesion to monomeric collagen under
flow.8 However, given that static adhesion to collagen
fibers is 21-independent, a crucial question is the need for the
integrin for adhesion to fibers under flow. Our finding that the
integrin is essential for adhesion to both monomeric and fibrillar
collagen confirms the view that this receptor is a critical adhesive
receptor. Clearly, the failure of CRPs to support adhesion under flow
can be partly attributed to the absence in the molecule of vWF binding
sites and of specific 21-recognition sequences such as those
tentatively identified in collagen.28
In conclusion, our results, together with recent results by
others,11,56,58 support a two-step mechanism
of collagen platelet interaction as speculated
earlier.16,20-22 This mechanism is presented in
diagrammatic form in Fig 3. The first step
is the arrest of platelets. Under high shear conditions, the
interaction of platelet GPIb with vWF12-15 bound to
collagen through high-affinity vWF-binding sites in collagen is
essential. This results in a marked decrease in velocity of
platelets,58 which enables the direct interaction between
collagen and the platelet integrin 21,5-8 again
through specific recognition sites in collagen. The precise function of 21 in the process of activation is unclear, but an assisting role
has been proposed.59,60 The second step, associated
primarily with activation, involves low-affinity binding between
platelets and collagen, which can occur because the platelets are held
fast at the collagen surface. This low-affinity interaction involves a
different receptor, likely GPVI, and simple (nonspecific) collagen sequences associated basically with generation of the collagen triple-helical structure. This second step leads to full activation and
a concomitant increase in adhesion.
View larger version (15K):
[in this window]
[in a new window]
| Fig 3.
Model of platelet interaction with collagen under flow
conditions. The first step is the arrest of blood platelets on collagen (A + B). The interaction between GPIb-vWF and specific and
high-affinity vWF-binding sites in collagen results in a marked loss of
velocity of the blood platelet (A). The actual arrest of platelets
occurs after 21-collagen interaction (B). This may also result in
some degree of activation (*). Both vWF and 21 bind to specific
adhesive sites on collagen that are depicted as solid rectangles. The
second step is the binding of other collagen receptors (Col-R; eg, GP VI) to simple collagen sequences (delineating the collagen triple helix) that are depicted as open rectangles. This results in full platelet activation (**), firm attachment, and GPIIb/IIIa activation.
|
|
| |
FOOTNOTES |
Submitted August 4, 1997;
accepted January 12, 1998.
Supported in part by the Thrombosis Foundation, The Netherlands (Grant
No. 93.002). M.J.B., C.G.K., and L.F.M. are grateful to Medical
Research Council for financial support.
Address reprint requests to Marilyn W. Verkleij, PhD,
Department of Haematology, University Hospital Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Martin J.W. IJsseldijk and Glenda J. Heijnen-Snyder
for their technical assistance. We further thank Eric G. Huizinga for
critical advice on the manuscript and Gertrude Bunt for the electron
micrographs.
| |
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D. Bouvard, C. Brakebusch, E. Gustafsson, A. Aszodi, T. Bengtsson, A. Berna, and R. Fassler
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B. Nieswandt, W. Bergmeier, A. Eckly, V. Schulte, P. Ohlmann, J.-P. Cazenave, H. Zirngibl, S. Offermanns, and C. Gachet
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M. Roest, J. D. Banga, D. E. Grobbee, P. G. de Groot, J. J. Sixma, M. J. Tempelman, and Y. T. van der Schouw
Homozygosity for 807 T Polymorphism in {alpha}2 Subunit of Platelet {alpha}2{beta}1 Is Associated With Increased Risk of Cardiovascular Mortality in High-Risk Women
Circulation,
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M. Jandrot-Perrus, S. Busfield, A.-H. Lagrue, X. Xiong, N. Debili, T. Chickering, J.-P. L. Couedic, A. Goodearl, B. Dussault, C. Fraser, et al.
Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily
Blood,
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M. Roest, J. J. Sixma, Y.-P. Wu, M. J. W. Ijsseldijk, M. Tempelman, P. J. Slootweg, P. G. de Groot, and G. H. van Zanten
Platelet adhesion to collagen in healthy volunteers is influenced by variation of both alpha 2beta 1 density and von Willebrand factor
Blood,
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N. von Beckerath, W. Koch, J. Mehilli, C. Bottiger, A. Schomig, and A. Kastrati
Glycoprotein Ia gene C807T polymorphism and risk for major adverse cardiac events within the first 30 days after coronary artery stenting
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E. Monnet and F. Fauvel-Lafeve
A New Platelet Receptor Specific to Type III Collagen. TYPE III COLLAGEN-BINDING PROTEIN
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C. Smith, D. Estavillo, J. Emsley, L. A. Bankston, R. C. Liddington, and M. A. Cruz
Mapping the Collagen-binding Site in the I Domain of the Glycoprotein Ia/IIa (Integrin alpha 2beta 1)
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D. Estavillo, A. Ritchie, T. G. Diacovo, and M. A. Cruz
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P. Siljander and R. Lassila
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T. Nakamura, J.-i. Kambayashi, M. Okuma, and N. N. Tandon
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D. J. Onley, C. G. Knight, D. S. Tuckwell, M. J. Barnes, and R. W. Farndale
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K. Suzuki-Inoue, Y. Ozaki, M. Kainoh, Y. Shin, Y. Wu, Y. Yatomi, T. Ohmori, T. Tanaka, K. Satoh, and T. Morita
Rhodocytin Induces Platelet Aggregation by Interacting with Glycoprotein Ia/IIa (GPIa/IIa, Integrin alpha 2beta 1). INVOLVEMENT OF GPIa/IIa-ASSOCIATED Src AND PROTEIN TYROSINE PHOSPHORYLATION
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M. Achison, C. M. Elton, P. G. Hargreaves, C. G. Knight, M. J. Barnes, and R. W. Farndale
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Abstract
Introduction
Abstract
