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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2704-2715
Influence of Fibrillar Collagen Structure on the Mechanisms of Platelet
Thrombus Formation Under Flow
By
Brian Savage,
Mark H. Ginsberg, and
Zaverio M. Ruggeri
From the Roon Research Center for Arteriosclerosis and Thrombosis,
Division of Experimental Hemostasis and Thrombosis, Departments of
Molecular and Experimental Medicine and of Vascular Biology, The
Scripps Research Institute, La Jolla, CA.
 |
ABSTRACT |
We have used real-time video microscopy to study the mechanisms of
platelet adhesion to type I collagen fibrils of distinct structure
exposed to flowing blood. Electron microscopy analysis by surface
replication demonstrated morphological differences between
acid-insoluble fibrils, displaying a regularly repeating striated
pattern (banded collagen), and acid-soluble fibrils generated by pepsin
treatment of insoluble collagen, smaller in size with a helical
configuration (nonbanded collagen). These structural differences proved
to be related to the role of platelet integrin  2 1 in stabilizing adhesion to collagen
under a variety of flow conditions. Blocking
21 function with a monoclonal antibody had no effect on platelet adhesion to insoluble type I collagen coated
at high density on a glass surface, whereas there was an absolute
dependence of 21 function for the initial
permanent arrest of platelets and subsequent thrombus formation on
pepsin-solubilized type I collagen under the same conditions. In
contrast, reconstituted, banded fibrils prepared from
pepsin-solubilized type I collagen supported platelet adhesion and
thrombus development even when platelet
21 function was blocked, a process that
was greatly accelerated by pre-exposure of this substrate to autologous
plasma under flow. These results implicate a collagen receptor(s) on platelets other than 21 that can
selectively engage domains in banded, but not nonbanded type I collagen
when 21 function is blocked. In addition,
collagen structure may regulate the extent and affinity of the binding
under flow of plasma components such as von Willebrand factor and/or
other IIb3 ligands.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PLATELET ADHESION TO exposed components
of the subendothelium at sites of vascular injury is an essential step
in hemostatic and thrombotic processes. Collagen has been recognized as
a key thrombogenic component of the vessel wall, in particular types I,
III, and VI.1-3 Several collagen-binding proteins are
expressed on the platelet surface that may mediate collagen-induced
platelet activation and/or platelet adhesion under flow, including the integrin  21,4-6 glycoprotein
(GP) VI,7,8 GP IV (GP IIIa, CD-36),9-11 and a
nonintegrin 65-kD protein specific for type I collagen.12
Although multiple putative collagen receptors exist on platelets, the
relative contribution of each in collagen-induced platelet activation
and adhesion under flow is ill-defined. One reason for this is that the
effects of distinct collagen fibril morphologies on platelet reactivity
are poorly understood. For example, numerous studies of platelet
adhesion under both stationary and flow conditions have used type I
collagen solubilized by pepsin, which cleaves collagen in the nontriple
helical regions where the covalent cross-links responsible for the
typical banded structure and collagen insolubility are found. The
triple helix itself is resistant to most proteases except collagenases.
Because collagen monomers obtained from pepsin-digested collagen have
altered nonhelical extremities,13 they may polymerize to
form fibrils with helical configurations distinct from that found in
native collagen fibrils.14 Furthermore, the morphology of
collagen fibrils in vivo may be heterogeneous. Vascular collagen with a
spiraled appearance (composed of an assembly of fibrils in a helical
configuration) has been demonstrated by electron microscopic and
immunohistochemical studies in both normal and pathological
conditions.15 In these studies, spiraled collagen was more
abundant in veins as compared with arteries and was particularly noted
in the left anterior descending coronary artery beneath a myocardial
bridge that had been free from atherosclerosis. Such collagens were
also more evident in normal saphenous veins as compared with
corresponding phlebosclerotic vessels in diseased patients.
Immunohistochemical examination of matrix metalloproteinases (MMP-1,
-2, and -3) showed significant expression of MMP-1 in smooth muscle
cells of the vascular wall in which spiral collagens were
abundant.15 Thus, spiral collagen may be formed
preferentially in normal blood vessels through a physiologic
degradation of normal collagen fibrils. How such structural modifications impact on the mechanisms of platelet adhesion under flow
has not been previously documented. The purpose of this study was to
examine the molecular mechanisms of collagen-induced platelet adhesion
and thrombus formation in flowing whole blood by measuring in
real-time the dynamics of platelet interaction with type I collagen
fibrils having distinct morphologies.
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MATERIALS AND METHODS |
Blood donors.
Blood was collected through a 19-gauge needle from the antecubital vein
of healthy adult donors with normal cell count profiles. The syringes
contained the anticoagulant D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone dihydrochloride (PPACK; 40 µmol/L final
concentration) that inhibited thrombin activation. Blood was
supplemented with additional PPACK, where necessary, at approximately
2-hour intervals to ensure thrombin inactivation. All donors claimed to
have abstained from taking aspirin, or other drugs known to affect
platelet function, in the preceding 10 days.
Preparation of insoluble fibrillar collagen-coated coverslips.
Acid-insoluble fibrillar type I collagen from bovine achilles tendon
(Sigma, St Louis, MO) in 0.5 mol/L acetic acid, pH 2.8, at
a concentration of 2.5 mg/mL, was prepared as previously
described16 and 200 µL was applied evenly over a
horizontal glass coverslip (Corning, Inc, Corning, NY;
24 × 50 mm) covering all but the first 10 mm that
remained uncoated to facilitate handling. Coated coverslips were then
placed in a humid environment at room temperature (22°C to
25°C) for 60 minutes. Excess, unbound collagen was removed by 4 sequential rinses with phosphate-buffered saline (PBS), pH 7.4, and the
coverslip was assembled in the flow chamber described below. Uncoated
coverslips did not support platelet adhesion under the flow conditions
used in these studies, and saturating the surface with bovine serum
albumin (coating at 0.1 mg/mL) did not affect initial platelet adhesion
or subsequent thrombus formation (unpublished observations).
Preparation of pepsin-solubilized collagen-coated coverslips.
Acid-soluble type I collagen from human placenta (Sigma) was dissolved
in 0.1 mol/L acetic acid to give a stock solution at a final
concentration of 2.5 mg/mL. The stock solution was diluted with PBS, pH
7.4, to a final concentration of 200 µg/mL for coating on glass
coverslips as described above. Under these conditions, the surface was
fully saturated with collagen; increasing the coating concentration up
to 2.5 mg/mL had no effect on the extent of platelet adhesion or
thrombus formation under all flow conditions examined (unpublished
observations). These preparations were used in all flow
studies except those experiments using a substrate derived from pepsin
treatment of achilles tendon-insoluble collagen. In these studies, a
suspension of acid-insoluble type I collagen from bovine achilles
tendon (Sigma) at a concentration of 2.5 mg/mL was incubated with
pepsin (Sigma) at an enzyme to substrate ratio of 1:1,000 (wt/wt), at
4°C for 18 hours. All insoluble material was sedimented by
centrifugation at a relative centrifugal force of 100,000g for
3 hours, and the supernatant was removed. Untreated collagen suspension
were also sedimented under the same conditions, and the supernatant was
removed for control studies.
Preparation of reconstituted collagen fibrils from
pepsin-solubilized collagen.
Fibrils of human type I collagen were formed by dialyzing acid-soluble
type I collagen (from human placenta; Sigma) dissolved in 0.1 mol/L
acetic acid at a concentration of 2.5 mg/mL against 4 changes of 4 L of
PBS, pH 7.4, for 96 hours at 4°C. The concentration of
reconstituted collagen was essentially unchanged from that of the
starting material. This procedure has been used to prepare banded
collagen fibers from pepsin-solubilized collagen and is based on the
observations that purified solutions of collagen under physiological
conditions in vitro assemble spontaneously into typical cross-striated
fibrils.17,18 The resulting preparation was applied over a
glass coverslip and kept in a humid environment for 60 minutes at
22°C to 24°C. Excess, unbound collagen was removed by rinsing
the coverslip with PBS, pH 7.4, which was then assembled in the flow chamber.
Inhibition of platelet receptor function and binding of von
Willebrand factor (vWF) to collagen.
The monoclonal antibodies used to inhibit the function of platelet
receptors IIb3,
21, and GP Ib were purified from
murine ascites using protein A (Sigma) chromatography.19
These antibodies have been extensively characterized and their ability
to inhibit receptor function has been well-documented. LJ-Ib1
(IgG1) reacts with the amino-terminal 45-kD domain of GP
Ib containing the vWF binding site20-22 and inhibits
fully the ligation of platelet GP Ib with immobilized vWF under a
variety of flow conditions.23 LJ-CP8 (IgG1) is
specific for the integrin IIb3 (GP
IIb-IIIa complex) and blocks the activation-dependent binding of
soluble ligands to this receptor24,25 as well as platelet
aggregation and thrombus formation.25 MR-5
(IgG1) binds to the A3 domain of vWF and prevents the
binding of vWF to collagen under a variety of experimental
conditions.24,25 Antibody R2-7E4 (IgG1) was obtained from the same fusion as the previously reported antibody R2-8C826 that is specific for the 2 integrin
subunit and prevents the adhesion of Chinese hamster ovary cells
transfected with 21 to type I collagen
under stationary conditions. Antibody 12F1 (IgG2a) binds to
platelet 21, but does not inhibit
receptor function.27 All antibodies were used at a final
concentration of 100 µg/mL in whole blood. This concentration was
shown to produce a maximal specific effect.
Real-time epifluorescence videomicroscopy and flow chamber.
A modification of the Hele-Shaw perfusion chamber, described in detail
elsewhere,28,29 was used to study the interaction of
platelets in flowing blood with immobilized collagen under a variety of
flow conditions. A flow path height of 254 µm was determined by a
silicone rubber gasket placed on a collagen-coated coverslip that
formed the lower surface of the chamber. The flow chamber was assembled
and filled with PBS, pH 7.4. A syringe pump (Harvard Apparatus
Inc, Holliston, MA) was used to draw blood through the
flow chamber. Flow rates of 1.94, 0.65, and 0.13 mL/min produced
initial wall shear rates of 1,500 s 1, 500 s1, and 100 s1, respectively, at
the inlet of the flow chamber. Measurements of platelet adhesion and
thrombus formation were made at a position proximal to the inlet of the
chamber, thereby avoiding pre-exposure of flowing platelets to either
the substrate or to preformed platelet thrombi. The fluorescent dye
mepacrine (quinacrine dihydrochloride; final concentration, 10 µmol/L) was used to label platelets in whole blood. Fluorescently
labeled leukocytes were distinguished from platelets by their
relatively large size, nuclear morphology, and sparsity; moreover,
permanent leukocyte attachment to collagen was negligible at wall shear
rates greater than 500 s1. At a wall shear rate of
100 s1, the contribution of adherent leukocytes to
the total thrombus volume was typically less than 10% (unpublished
observations). Erythrocytes were completely opaque to
fluorescence detection and visualization due to fluorescence quenching
by hemoglobin. Mepacrine concentrates in the dense granules of
platelets and has been shown to have no effect on normal platelet
function at the concentration used in these studies.30
Platelet secretion after adhesion does not prevent their visualization,
and mepacrine does not interfere with platelet adhesion.29
The perfusion chamber was positioned on the motorized stage of an
Axiovert 135M/LSM 410 inverted epifluorescence/laser scan confocal
microscope (Carl Zeiss Inc, Oberkochen, Germany). The
platelet adhesion process was visualized in real time and recorded on
tape with a videocassette recorder (Magnavox; Philips, Eindhoven, The Netherlands).
Measurement of thrombus volumes.
The cumulative volume occupied by thrombi in an area of 102,236 µm2 was calculated from confocal sections, obtained at
1.0-µm intervals in the z axis while blood was flowing using
an excitation laser wavelength of 488 nm and a scanning time of 2 seconds per section. The microscope settings, including contrast,
brightness, magnification, and pinhole aperture, were maintained at
constant values to facilitate comparisons between different
experiments. Confocal sections were analyzed using the Metamorph
software package (Universal Imaging Corp, West Chester,
PA). A threshold was applied to the image stack to
distinguish thrombus sections from the background; this value was then
used in all subsequent analyses of confocal sections for a given
experiment. The area occupied by all thrombi in a given cross-section
was calculated and the volume of a 1.0-µm-thick section was
estimated by multiplying the area by the height of the section
(1.0 µm). The cumulative volume occupied by all thrombi was
then estimated as the sum total of the sectional volumes.
Measurement of platelet motion.
Platelets were defined as moving on the surface when they were
displaced by a distance exceeding their diameter.29 A
series of images from the recorded experiment was digitized at a
sampling rate of 6 frames per second using a computer controlled VCR
(Sony 9500; Sony Corp, New York, NY) and a frame grabber
(Matrox Image LC; Matrox Electronic Systems Ltd, Dorval,
Quebec, Canada). A threshold was applied to the images to distinguish
platelets from the background, and the images were then binarized. Time
was calculated by referring to the frame number. Image analysis was
performed using the Metamorph software package (version 2.76; Universal Imaging Corp). The first 2 consecutive frames in the series were then
superimposed (by using the logical AND function) so that the resultant
image represented only the overlapping areas of the platelet at 2 different times. The new image was then superimposed to the next frame
and the process was continued until the overlapping area was equal to
0. When this occurred, a platelet had moved by a distance greater than
its diameter; if this did not occur, a platelet was considered firmly
attached during the period of observation. The time for individual
platelet motion was computed and the process was repeated until all the
platelets in the microscopic field of study moved or for a preselected
time interval, whichever occurred first.
Measurement of platelet surface coverage.
Single frame images were captured from videotapes at various times
after the onset of blood flow and a threshold was applied to
distinguish both single platelets and platelet thrombi from the
background; this value was then used in all subsequent image analyses
of surface area coverage. The area occupied by all platelets and
thrombi in an image was measured using the Metamorph software package
(version 2.76; Universal Imaging Corp).
Platelet aggregation assay.
Aggregation induced by fibrillar type I collagen was studied in a dual
channel aggregometer (Chrono-Log Corp, Havertown, PA) using 3 × 108 platelets per milliliter of autologous
plasma containing citrate as anticoagulant. Turbidity changes were
recorded after the addition of agonist. The pH of the collagen
suspension was adjusted to 7.4 before it was added to the platelet suspension.
Gel electrophoresis.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed using standard procedures.31
Electron microscopy analysis by surface replication.
Frozen samples of immobilized collagen were placed in a Balzers freeze
fracture machine (BAF-400T; Balzers AG, Liechtenstein) and were shadowed at an angle of 45° with platinum. The resulting replica was supported with a backing of carbon. The replica was subsequently cleaned with bleach, picked up on carbon-coated mesh grids, and examined on a Hitachi HU600 electron
microscope (Hitachi Instruments Inc, San Jose, CA).
Representative areas of specific interest were documented photographically.
| |
RESULTS |
Structural features of native insoluble and pepsin-solubilized type I
collagen fibrils.
Surface replication images of surface-bound insoluble fibrillar type I
collagen (from bovine achilles tendon) and microfibrils derived from
pepsin-solubilized type I collagen (from human placenta) showed
distinct structural features (Fig 1). A
characteristic striated pattern was seen with insoluble collagen due to
the regular quarter-staggering of collagen monomers with a 67 nm
periodicity,32 yielding regions of alternate maximal
electron density regardless of the thickness of the fiber (Fig 1A). In
contrast, this banded pattern was noticeably absent from the
microfibrils derived from pepsin-solubilized collagen (Fig 1B). Here,
collagen monomers polymerize to form thinner microfibrils with a
clearly discernible spiraled structure comprising 2 to 3 fibers in a
twisted configuration. Gel electrophoresis of pepsin-solubilized type I
collagen under denaturing and nonreducing conditions showed the
characteristic 1(I) and 2(I) bands (in the predicted ratio of
2:1) as well as high molecular weight multimers that did not enter the
gel (Fig 1). The substrates shown in Fig 1 were prepared in an
identical manner to those used for evaluating platelet adhesion and
thrombus formation under flow described below and are therefore
representative of the surfaces presented to flowing blood in these
studies. Based on these structural features, the immobilized substrates
prepared from insoluble and soluble type I collagen are referred to as banded and nonbanded collagens, respectively, throughout the text. Note
also that preparations of nonbanded collagen may contain monomeric
collagen in addition to polymerized helical fibrils.
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| Fig 1.
Structural features of native insoluble and
plasmin-solubilized type I collagen as a representative example.
Native, insoluble fibrillar type I collagen (from bovine achilles
tendon) and pepsin-solubilized type I collagen (from human placenta)
were coated on glass coverslips as described in Materials and Methods.
Surface replication analysis of insoluble type I collagen (A) showed a
characteristic banded pattern that was noticeably absent from the
smaller fibrils derived from pepsin-solubilized type I collagen (B),
where microfibril assemblies displayed a nonbanded spiraled
configuration. Polyacrylamide gel electrophoresis under denaturing and
nonreducing conditions showed the presence of the 1(I) and 2(I)
type I collagen subunits as well as high molecular weight multimers
(right).
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Role of platelet integrin 21 in
platelet adhesion and thrombus formation on type I collagen under flow.
Platelets in flowing whole blood adhered to both banded and nonbanded
type I collagens at wall shear rates ranging from 100 s1 to 1,500 s1
(Fig 2). Banded collagen presented a more
thrombogenic surface than nonbanded collagen to flowing control blood
at 1,500 s1, as reflected in the higher total
thrombus volumes measured after 3 minutes perfusion, whereas the
cumulative thrombus volumes measured with control blood at shear rates
of 500 s1 and 100 s1 were
essentially equivalent for both banded and nonbanded collagens (Fig 2).
The concentrations of collagen used to coat the coverslips in these
experiments produced maximal platelet adhesion: increasing the coating
concentration of nonbanded collagen (up to 2.5 mg/mL) had no
significant effect on the extent of platelet adhesion and thrombus
development (data not shown). Furthermore, decreasing the coating
concentration of banded collagen by up to 10-fold had no measurable
effect on thrombus formation at the shear rates indicated.33 Therefore, differences in thrombogenicity seen at 1,500 s1 with these substrates were not due to
differences in the extent of surface saturation with collagen.
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| Fig 2.
Role of platelet integrin
21 in platelet adhesion and thrombus
formation under flow on type I collagens with distinct structural
specificities. Blood containing PPACK as anticoagulant and treated with
the fluorescent dye mepacrine for platelet visualization was perfused
at 37°C over distinct type I collagen preparations, in a parallel
plate flow chamber. The flow rate was set to produce a wall shear rate
of 1,500 s1, 500 s1, or 100 s1 at the inlet of the flow chamber and each experiment
was recorded on tape using a videomicroscopy system. The figure shows
single frame images of the surface, each corresponding to an area of
65,536 µm2, obtained after 3 minutes of perfusion. Blood
was either untreated (control) or treated with a monoclonal antibody
selectively inhibiting the platelet integrin
21 function. Note that functional
inhibition of platelet 21 completely
abolished stable platelet attachment and thrombus formation on
pepsin-solubilized (nonbanded), but not on native insoluble collagen
(banded) or collagen reconstituted from the same pepsin-solubilized
collagen (from human placenta) used in these studies. These images are
representative of the results obtained in at least 6 separate
experiments with blood from different donors. After 3 minutes of
perfusion, the total volume of platelet thrombi present in an area of
102,236 µm2 was measured by confocal sectioning at
1.0-µm intervals, as described in Materials and Methods. Volume
measurements represent the mean ± standard error of the mean of 4 separate experiments with different blood donors. The size of single
platelets can be appreciated in the panel showing the results obtained
in the presence of the anti-21 antibody
with pepsin-solubilized collagen or in Figs 3 and 7, in which platelet
to platelet interactions have been prevented by blocking
IIb3 function.
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In marked contrast to banded collagen, platelet adhesion and thrombus
development on nonbanded type I collagen required competent 21 function, because functional
inhibition of this integrin completely abolished stable platelet
attachment at all shear rates tested (Fig 2), resulting in a transient
surface translocation of platelets mediated by platelet GP Ib and
the A1 domain of collagen-bound vWF absorbed from plasma. A nonfunction
blocking anti-21 monoclonal antibody had
no effect on the extent of platelet adhesion and thrombus formation
with either banded or nonbanded collagen (data not shown). Blocking
platelet 21 function had no significant
effect on the extent of thrombus formation on banded collagen at any
shear rate tested (P > .1 for all shear rates; Fig 2).
Inhibition of plasma vWF A3 domain binding to collagen, or functional
blockade of platelet GP Ib, completely abolished platelet
interaction with both banded and nonbanded collagens at 1,500 s1, but not at 500 s1 or 100 s1 (data not shown), in a manner similar to that
previously described.33
Properties of reconstituted type I collagen derived from
pepsin-solubilized collagen.
The differential role of platelet 21 in
platelet adhesion to type I collagen fibrils having distinct structural
features was confirmed with reconstituted type I collagen, produced
from pepsin-solubilized type I collagen (from human placenta) by
extensive dialysis of the latter against phosphate buffer, pH 7.4, at
4°C. These conditions have been shown to produce banded collagen
fibers from pepsin-solubilized collagen and are based on the
observation that purified solutions of collagen under physiological
conditions in vitro assemble spontaneously into typical cross-striated
fibrils.17,18 Reconstituted type I collagen prepared in
this way supported platelet adhesion and thrombus formation in a manner
that did not require competent 21
function; blocking platelet 21 function
with a monoclonal antibody resulted in only a slight, albeit
significant (P < .05) decrease in the total thrombus volume
after 3 minutes of perfusion at 1,500 s1 compared
with controls (Fig 2). Furthermore, at wall shear rates of 100 s1 and 500 s1, inhibition of
21 function had no significant effect on
the extent of thrombus formation compared with control blood (P > .1 at both shear rates; Fig 2). These results contrast sharply those seen with nonbanded type I collagen, in which inhibition of
21 function completely abolished stable
platelet attachment and thrombus formation at all shear rates. Thus,
reconstituted type I collagen showed distinct functional properties
compared with the nonbanded type I collagen from which it was derived, demonstrating that structural characteristics of fibril assembly have a
profound effect on the mechanisms of platelet interaction with this
substrate under flow.
Further confirmation of the differential role of platelet
21 in platelet adhesion to different type
I collagen preparations was obtained by controlled pepsin-digestion of
native, banded type I collagen obtained from achilles tendon. The
supernatant obtained after sedimenting pepsin-treated banded type I
collagen, when immobilized on glass, supported stable platelet
attachment and thrombus formation at shear rates ranging from 100 s1 to 1,500 s1 in a manner that
required intact 21 function; blocking
21 function resulted in a continuous
turnover of transiently attached platelets with no further accrual of
surface associated platelets, even after prolonged perfusion of blood
(data not shown). Thus, pepsin-solubilized type I collagen prepared
from either achilles tendon or placenta required competent
21 function for irreversible platelet
attachment and thrombus formation.
Time course of platelet adhesion and thrombus development.
The role of platelet 21 was further
studied by measuring the extent of platelet surface coverage as a
function of time after the onset of blood flow. Platelets adhered most
rapidly to banded collagen in a manner that was essentially independent
of 21 function
(Fig 3A). Under these conditions, half
maximal surface coverage was achieved after approximately 30 seconds.
In contrast, the rate at which the surface became covered with
platelets was lower on nonbanded collagen exposed to the same control
blood at this shear rate (half maximal surface coverage was achieved after ~1.5 minutes), and blocking 21
function resulted in the complete inhibition of stable platelet
attachment and subsequent lack of surface accumulation of platelets at
any time after the onset of blood flow (Fig 3A). Furthermore,
reconstituted collagen presented a surface that differed substantially
from both the nonbanded collagen from which it was derived and from
native, banded collagen. Blocking platelet
21 function with this substrate resulted
in a prolonged lag phase before the accrual of firmly attached
platelets (Fig 3A), although after 3 minutes of perfusion, there was
significant platelet adhesion and thrombus formation, as shown
previously (Fig 2). Although the anti-21
antibody was clearly effective in blocking
21 function, as evidenced by its inhibitory effect at early time points after the onset of blood flow
over this substrate, prolonged perfusion of blood resulted in platelet
adhesion and thrombus formation in a manner that was independent of
21 function (Fig 3A).
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| Fig 3.
Time course of platelet adhesion on type I collagens with
distinct structural specificities at a wall shear rate of 1,500 s1. (A) Control blood (containing no antibody) or blood
treated with a function blocking anti-21
monoclonal antibody was perfused through a parallel plate chamber as
described in the legend to Fig 2, and the flow rate was adjusted to
produce a wall shear rate of 1,500 s1 at the inlet of
the chamber. Single frame images corresponding to an area of 65,536 µm2 were captured from videotapes at various times after
the onset of blood flow and analyzed for the area occupied by surface
covered platelets (indicated as surface coverage). Note the
differential effects of inhibiting platelet
21 function with type I collagens having
distinct structural features, particularly the differences seen between
reconstituted collagen and pepsin-solubilized collagen (nonbanded) from
which it was derived. Surface coverage measurements represent the mean ± standard error of the mean of 4 separate experiments with different
blood donors. (B) Blood containing an
anti-21 monoclonal antibody, either alone
or concurrently with an anti-IIb3
monoclonal antibody, was perfused over reconstituted type I collagen at
a wall shear rate of 1,500 s1. After 3 minutes of
perfusion (corresponding to time 0), consecutive images were captured
from videotapes and analyzed for platelet movement (for definition, see
Materials and Methods). The percentage of platelets displaced from
their initial position was calculated as a function of time relative to
the total number of platelets attached to the surface in the first
image analyzed. Note that approximately 80% of all platelets
(indicated as platelets displaced) moved on the surface within 3 seconds of observation when 21 and
IIb3 were inhibited concurrently,
compared with greater than 60% of the platelets remaining stationary
after selective inhibition of IIb3. The 2 single frame images on the right, each representing an area of 65,536 µm2, depict surface coverage after 3 minutes of perfusion
with selective IIb3 inhibition (lower) or
combined IIb3 and
21 inhibition (upper). Note the
differences in surface coverage by platelets, reflecting differences in
the stability of platelet attachment.
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In a previous study using native banded type I collagen,33
the efficacy of the anti-21 monoclonal
antibody was confirmed by concurrently blocking platelet
IIb3 function with a well-characterized anti-IIb3 monoclonal
antibody.24,25 Because IIb3
function is required for platelet aggregate and thrombus formation, its selective inhibition allowed direct evaluation of individual platelet surface interactions. In the present study, we have used a similar strategy to demonstrate the inhibitory efficacy of the
anti-21 antibody using reconstituted type
I collagen. Platelets adhered to a surface coated with this substrate
in a predominantly stable manner after 3 minutes at a wall shear rate
of 1,500 s1, when IIb3
function alone was inhibited (Fig 3B); only 30% to 35% of attached
platelets were displaced on the surface by a distance greater than
their respective diameters over a period of 6 seconds. In contrast,
when platelet 21 and
IIb3 function were blocked concurrently,
a predominantly transient platelet attachment was seen, with
approximately 85% of platelets displaced over the same time period
(Fig 3B). A control, nonfunction blocking anti-21 monoclonal antibody had no effect
on the stability of platelet attachment when
IIb3 function was blocked (data not shown). These differences in the stability of platelet attachment were
reflected in the number of surface-attached platelets at 3 minutes
after the onset of blood flow, as shown visually in Fig 3B. Thus, when
only IIb3 function was blocked, the
majority of platelets were irreversibly adherent through bonds
involving the ligation of 21 with domains
in collagen (after the initial tethering of platelets, mediated by
platelet GP Ib engagement of collagen-bound vWF, derived from
plasma, as described below). These results confirm that the
anti-21 monoclonal antibody used in
these studies was of sufficient inhibitory efficacy to prevent ligation
of 21 with domains in reconstituted
collagen, despite its inability to prevent stable platelet attachment
and thrombus development on this substrate when
IIb3 function was not concurrently blocked, as shown in Figs 2, 3, and 4.
Influence of collagen-bound plasma components on the rate of platelet
accrual under flow.
The lag phase before the onset of stable platelet attachment to
reconstituted collagen when 21 function
was inhibited may reflect the time-dependent binding of plasma
components to collagen, including vWF or other
IIb3 ligands such as fibronectin, which can then mediate stable platelet attachment and thrombus development in
a manner that does not require the participation of
21. To test this hypothesis, autologous
plasma was perfused over reconstituted collagen at a wall shear rate of
1,500 s1 for 5 minutes before perfusing with either
control whole blood or blood containing the
anti-21 antibody. When
21 function was not blocked, the rate of
surface coverage was similar, regardless of whether the substrate was
previously exposed to plasma under flow or to purified vWF under
stationary conditions (Fig 4A). In marked
contrast, when 21 function was inhibited,
the initial lag phase was substantially reduced when the substrate was
pre-exposed to flowing plasma or precoated with purified vWF under
stationary conditions (Fig 4A). In this regard, reconstituted type I
collagen differed substantially from the nonbanded collagen from which it was derived, because pre-exposing nonbanded collagen to plasma under
flow or to purified vWF did not ameliorate the inhibitory effect of
blocking 21 (Fig 4B). In fact,
the extent of surface coverage when nonbanded collagen was precoated
with vWF, and 21 function was blocked,
was similar to that seen with vWF coated directly onto a glass surface
without collagen (data not shown). When
21 function was blocked, cumulative
thrombus volumes measured after 2.5 minutes of perfusion were
significantly higher when reconstituted collagen was pre-exposed to
plasma under flow or to purified vWF under stationary conditions
(Fig 4A).
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| Fig 4.
Effect of exposing collagen to plasma under flow or
vWF before perfusion with whole blood at a wall shear rate of 1,500 s1. Blood containing no antibody or blood containing an
anti-21 monoclonal antibody was perfused
over reconstituted type I collagen (A) or over pepsin-solubilized
(nonbanded) collagen from which it was derived (B) in a parallel plate
flow chamber as described in the legend to Fig 2, and the flow rate was
adjusted to produce a wall shear rate of 1,500 s1 at the
inlet of the chamber. (A) Upper figures: The time course of platelet
adhesion on reconstituted collagen was measured as described in
the legend to Fig 3. The collagen substrate was pre-exposed to
autologous plasma at a wall shear rate of 1,500 s1 for 5 minutes ( ) or to purified vWF under stationary conditions for 30 minutes ( ) before perfusing with blood. Control blood ( ) was
perfused directly over reconstituted collagen without prior exposure to
plasma or vWF. (A) Lower figures: After 2.5 minutes of perfusion, the
total volume of platelet thrombi present in an area of 102,236 µm2 was measured by confocal sectioning at 1.0-µm
intervals, as described in Materials and Methods. Measurements
represent the mean ± standard error of the mean of 4 separate
experiments with different blood donors. The single frame images, each
representing an area of 65,536 µm2, depict surface
coverage of platelet thrombi after 3 minutes of perfusion under the
conditions indicated. (B) The time course of platelet adhesion on
pepsin-solubilized (nonbanded) collagen. The collagen substrate was
pre-exposed to autologous plasma at a wall shear rate of 1,500 s1 for 5 minutes () or to purified vWF under
stationary conditions for 30 minutes () before perfusing with blood.
Control blood () was perfused directly over pepsin-solubilized
(nonbanded) collagen without prior exposure to plasma or vWF.
Measurements represent the mean ± standard error of the mean of 4 separate experiments with different blood donors.
|
|
Collagen-induced platelet activation and aggregation.
Insoluble banded collagen, when added to platelets in plasma with
citrate as anticoagulant, caused irreversible platelet aggregation in a
dose-dependent manner; maximal platelet aggregation response was seen
at an agonist concentration of 20 µg/mL
(Fig 5). In contrast, nonbanded
collagen was less efficient at inducing platelet aggregation; a 50-fold
increase in concentration compared with banded collagen was required
for a full platelet response, and even then, there was a prolonged lag
phase before the onset of full aggregation (Fig 5). Blocking platelet
21 function had no effect on the lag
phase or extent of platelet aggregation with banded collagen at a
concentration of 20 µg/mL, whereas, at suboptimal concentrations of
this agonist (2.0 µg/mL), there was a partial inhibition of platelet
aggregation; increasing the antibody concentration did not produce
further inhibition under these conditions (data not shown), indicating
the presence of an activating collagen receptor on platelets other than
21. In fact, simple collagen-like
peptides have been synthesized that are potent platelet agonists whose activity is totally
21-independent.34 In
contrast, blocking platelet 21 function
completely inhibited platelet aggregation induced by nonbanded collagen
(Fig 5), indicating that this integrin is essential to mediate platelet
activation induced by this agonist. However, reconstituted collagen
prepared from nonbanded collagen was a potent platelet agonist,
promoting platelet activation and aggregation in a manner that was
independent of 21 function when used at a
concentration of 10.0 µg/mL (Fig 5).
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| Fig 5.
Platelet aggregation induced by type I collagen fibrils
with distinct morphologies. Aggregation at 37°C is shown as an
increase in light transmittance. The arrows indicate addition of
agonist at the final concentrations indicated. The pH of all collagen
preparations was adjusted to 7.4 immediately before adding to
platelet-rich plasma. The anti-21
monoclonal antibody was added as indicated at a final concentration of
100 µg/mL, which was shown to produce a maximum specific effect.
|
|
Distinct mechanisms of platelet attachment to banded and nonbanded
collagen.
Blocking the function of platelet integrins
IIb3 and 21
concurrently with inhibition of the binding of plasma vWF completely abolished platelet interaction, including initial tethering, with nonbanded collagen at wall shear rates ranging from 100 s1 to 1,500 s1
(Fig 6). Thus, with this substrate,
platelet adhesion and thrombus formation under flow require the
functional integration of 3 platelet receptors at a wall shear rate of
1,500 s1, namely GP Ib,
21, and
IIb3. In contrast,
21 is not required with native banded
collagen when coated at high density under the conditions used in the
present study (Figs 2 and 3), although blockade of
21 caused significant inhibition of
thrombus formation at low surface densities of this
substrate.33 In contrast with the results obtained with
nonbanded collagen, reconstituted collagen supported stable platelet
attachment at 100 s1 and 500 s1
when vWF binding to collagen was blocked concurrently with functional inhibition of both 21 and
IIb3 (Fig 6), implicating the
participation of another collagen receptor(s) other than
21, possibly GP VI,7,8 GP
IV,9-11 or the recently identified 65-kD collagen-binding
protein.12 Results similar to those seen for reconstituted
collagen were obtained with native, banded collagen (data not shown).
Stable platelet attachment under these conditions was completely
inhibited when divalent cations were chelated with EDTA (data not
shown). Therefore, platelet adhesion to banded (native or
reconstituted) collagen type I at shear rates less than 500 s1, although cation-dependent, does not require vWF
or the platelet integrins 21 or
IIb3, whereas requisite
21-collagen pairing leading to stable
platelet attachment to nonbanded collagen was always observed at shear
rates ranging from 100 s1 to 1,500 s1 (Fig 2). Consistent with the present findings are
recent binding studies showing that platelets interact with soluble and
insoluble collagens through different mechanisms, implying that
21 and another collagen receptor mediate
collagen-platelet interactions with different
specificities.35
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| Fig 6.
Role of distinct collagen receptors in mediating platelet
attachment under flow to reconstituted (banded) and pepsin-solubilized
(nonbanded) type I collagen. Blood was perfused through the parallel
plate chamber as described in the legend to Fig 2. Control blood or
blood containing monoclonal antibodies to block specifically the
function of platelet 21 and
IIb3 concurrently with inhibition of
plasma vWF binding to collagen was perfused over pepsin-solubilized
(nonbanded) type I collagen or reconstituted collagen derived from the
former, as indicated. Note the complete inhibition of platelet
interaction with pepsin-solubilized (nonbanded) collagen when platelet
21 and IIb3
function were blocked concurrently with inhibition of plasma vWF
binding to collagen, compared with the stable attachment of single
platelets at 100 s1 and 500 s1 under the
same conditions with reconstituted type I collagen. Representative
images of 6 separate experiments with blood from different donors are
shown.
|
|
Shear rate dependence of 21-mediated
platelet attachment to nonbanded collagen.
Simultaneous inhibition of platelet IIb3
function and the binding of plasma vWF to collagen resulted in the
stable attachment of single platelets at shear rates up to
approximately 1,000 s1 when blood was perfused over
nonbanded collagen (Fig 7). The extent of
surface coverage was inversely related to the wall shear rate, with
virtually no platelet-surface interactions at 1,500 s1. Under these conditions, platelet attachment was
contingent upon ligation of collagen domains with
21 on platelets; blocking 21 function under these conditions
completely prevented platelet interaction with this substrate (Fig 6).
The biomechanical properties of the
21-collagen bond appear to be similar to
those observed for IIb3-fibrinogen
interactions, in which IIb3-mediated
platelet adhesion to surface-bound fibrinogen cannot occur above a
limit wall shear rate.29 Functional inhibition of
21 by chelation of divalent cations with
EDTA completely abolished platelet interaction under these conditions
(data not shown).
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| Fig 7.
Flow-dependent characteristics of
21-mediated platelet attachment to
pepsin-solubilized (nonbanded) type I collagen. Blood containing
function blocking anti-IIb3 and anti-vWF
A3 domain monoclonal antibodies (the latter to inhibit plasma vWF
binding to collagen) was perfused over immobilized pepsin-solubilized
(nonbanded) type I collagen in the parallel plate flow chamber as
described in the legend to Fig 2. Note that, for stable platelet
attachment under these conditions, there was an absolute requirement
for 21-collagen interactions, because
additional functional inhibition of 21
completely inhibited platelet interaction with this substrate (as shown
in Fig 6). The number of single platelets attached to the surface after
3 minutes of perfusion was measured as a function of the wall shear
rate.
|
|
| |
DISCUSSION |
The nonbanded collagen structures seen with the substrate derived from
pepsin-solubilized type I collagen (Fig 1B) may be a distinctive
feature of fibrils generated by enzymatic cleavage of collagen
precursors,14 because monomers obtained from pepsin-treated collagen have altered nonhelical extremities.13 Presumably, monomers prepared in this manner lack crucial domains required for the
assembly of lateral aggregates present in native fibrillar type I
collagen, but can generate spiraled structures by lateral packing of
subfibrils. These structural differences had a profound effect on the
mechanisms of platelet adhesion and thrombus formation under flow:
platelet 21 function was essential for
the permanent arrest of platelets on nonbanded collagen, whereas native
banded type I collagen supported platelet adhesion and thrombus growth even when 21 function was compromised.
Studies of patients with a deficiency of platelet 2
subunit or with autoantibodies directed against this protein attest to
the involvement of 21 in
collagen-platelet interactions in vivo, because platelet adhesion to
collagen and collagen-induced aggregation are both
impaired.36-39 However, simple collagen-like peptides
consisting essentially of a repeating Gly-Pro-Hyp sequence have been
synthesized that are potent platelet agonists whose activity is totally
21-independent.34 The
platelet reactivity of these peptides could be attributed solely to
their tertiary (triple helical) and quaternary (polymeric,
cross-linked) structures. Thus, platelet-collagen interactions in vivo
are likely to involve the integration of at least 2 distinct
receptor-collagen interactions acting in concert: one involving
21 as an adhesion/signaling receptor and
the other involving the recognition of collagen structural features by
a receptor other than 21. A likely
candidate for the latter function is GP VI which appears to play a
major role in collagen-induced platelet activation.8
Recent studies involving the adhesion of GP VI-deficient platelets to
collagen under flow testify to the importance of GP VI as a signaling
receptor required for the activation of platelet IIb3.8 Moreover, GP
VI-deficient platelets were unresponsive to the collagen-related
peptides comprising a repeating Gly-Pro-Hyp motif that mimic the
collagen tertiary and quaternary structure.40 GP VI
therefore appears to act as a signaling receptor after
recognition/coupling with distinct structural components of collagen.
In this context, collagen-induced activation of the protein-tyrosine
kinase Syk was severely compromised in GP VI-deficient
platelets.41 In a recent report, the activation of
IIb3 induced by platelet adhesion to
collagen was found to be mediated by both
21 and GP VI.42 In this
study, platelet adhesion to insoluble fibrillar type I collagen under
static conditions was mediated by both 21 and GP VI, whereas platelet adhesion to monomeric type I collagen was
exclusively mediated by 21. This report
is entirely consistent with the present study in which we demonstrate
that, under physiologically relevant flow conditions, platelet adhesion
and subsequent thrombus formation on nonbanded collagen requires
competent 21 function, whereas with
banded collagen another receptor other than
21, possibly GP VI, is involved and can
mediate platelet adhesion and thrombus formation even when
21 function is blocked. However, it
should be noted that, although the
anti-21 antibody blocks adhesion to
nonbanded collagen, this does not exclude the role for a second
collagen receptor with this substrate, which may act in concert or
synergistically with 21 such that
participation of both receptors may be required for stable platelet
attachment. Such a mechanism would be analogous to that previously
described for the irreversible adhesion of platelets to immobilized vWF that requires the functional integration of 2 receptors, namely GP Ib
and IIb3.29,43
Furthermore, a conformational change triggered by collagen binding to
21 may induce an increased affinity of
the receptor for its ligand.44
Reconstituted type I collagen showed distinct functional properties
compared with the nonbanded collagen from which it was derived. First,
platelet adhesion and thrombus growth under flow did not require
competent 21 function, although when this
receptor was blocked, there was a prolonged lag phase before the
accrual of firmly attached platelets compared with that seen with
native banded collagen (Fig 3A). The delayed response before the onset of 21-independent platelet attachment
proved to be related to the time-dependent binding of plasma
components, as evidenced by the significant increase in the total
thrombus volume measured after 2.5 minutes of perfusion when
reconstituted collagen was pre-exposed to plasma under flow or to
purified vWF under stationary conditions (Fig 4A). Thus, the rate of
surface coverage by platelets and the development of platelet thrombi
on this substrate are processes that are greatly accelerated by the
presence of collagen-bound vWF and/or other collagen-bound plasma
components when 21 function is
compromised. The present findings indicate that collagen structural characteristics may regulate the extent and/or affinity of the binding
of components, including vWF or other
IIb3 ligands such as fibronectin, present
in plasma. In this context, activated IIb3 promotes platelet arrest and
subsequent thrombus growth on surface-bound vWF by interacting with the
Arg-Gly-Asp sequence in the C1 domain near the carboxyl terminus of the
vWF subunit.29 The extent to which this process occurs when
plasma vWF or other IIb3 ligands are
immobilized onto collagen may therefore be regulated by the morphology
of the collagen fibrils. Despite differences with regard to the role of
21 in mediating the adhesion of platelets flowing over banded and nonbanded collagens, binding of plasma vWF was
requisite at 1,500 s1, but not at 100 s1, for both substrates.
Although platelet 21 has generally been
considered to be constitutively active,45 recent studies
suggest that 21 on nonactivated platelets
does not bind soluble type III collagen, whereas activation of
platelets transforms it to a state with higher affinity for soluble
type III collagen.35 It can be inferred from the present
study that soluble type I collagen binds to
21 on resting platelets, because it
induces platelet aggregation in an
21-dependent manner (Fig 5). Furthermore,
when soluble type I collagen was immobilized on a surface, blocking
platelet activation with prostaglandin E1
(PGE1) did not prevent
21-dependent platelet attachment under
flow (data not shown). It therefore appears that
21 on nonactivated platelets can
recognize and ligate domains in soluble type I collagen when the latter
is immobilized on a surface. We have also demonstrated the inhibitory
efficacy of the anti-21 antibody under
conditions in which platelets are known to be activated (eg, under high
shear flow when IIb3 function is blocked
to prevent platelet aggregation; Fig 3B). Thus, the
anti-21 antibody used in these studies
blocks 21 function on both resting and
activated platelets.
Because there are multiple potential collagen receptors on platelets,
their relative contribution in adhesion/signaling induced by collagen
is difficult to discern. As shown in the present study, multiple
collagen receptors can participate in adhesion to banded (native or
reconstituted) type I collagen under flow, whereas 21 is absolutely required for the initial
attachment of platelets to nonbanded collagen fibrils. Furthermore,
conditions can be obtained (by concurrent blocking of GP Ib and
IIb3) where competent 21 function is requisite for platelet
attachment to this substrate under flow. Such conditions could
therefore be exploited to compare distinct signaling pathways
associated with 21-dependent and 21-independent adhesion to immobilized
collagen under flow.
Although at least 7 genetically distinct collagens (types I, III, IV,
V, VI, VIII, and XIII) have been identified as constituents of the
vessel wall,46 their relative contribution to the
thrombogenicity of the extracellular matrix after vascular injury by
tissue trauma or by inflammatory and degenerative processes has not
been clearly defined. Structural differences in platelet binding to
these different collagen types under flow have been
reported.2,47 For example, pepsin-digested collagen type
VI-coated surfaces were found to support platelet adhesion only at
relatively low wall shear rates (100 s1), although
these surfaces were found to be more reactive than collagen type I
surfaces under the same low shear conditions.47 Such
studies imply that the relevance of different collagen types in
hemostasis and thrombosis may depend on their relative distribution in
the vascular extracelluar matrix as well as the prevailing flow
conditions in different regions of the vasculature. However, more
recent studies have demonstrated that intact tetrameric collagen type
VI supports platelet adhesion and aggregation at high shear rates in a
manner similar to that seen for collagen type I,48 suggesting a stringent requirement for an intact conformational macromolecular structure. Whereas collagen types III, IV, and VI may be
more relevant in normal blood vessels, collagen type I may be important
in fibrous atherosclerotic plaques that contain a higher proportion of
collagen type I compared with normal arterial vessel
walls49 and in deep vascular injuries where it is abundant in the media and adventitia of blood vessel.50 Physiologic
degradation of collagen fibrils by interstitial enzymes such as matrix
metalloproteinases is known to occur at extracellular spaces; their
assembly to form spiral, nonbanded collagen may represent a process
that regulates platelet responses to vascular injury at sites of
vascular wound repair during remodeling of the vasculature.
In conclusion, our results show that type I collagens with different
structural characteristics elicit platelet adhesion and aggregation
through distinct mechanisms as evidenced by the differential role of
platelet 21 under various flow
conditions. We also demonstrate that a collagen receptor(s) other than
21 can selectively engage domains in
banded, but not nonbanded type I collagen when
21 function is blocked and that the state
of collagen fibril assembly may regulate the extent and affinity of the
binding of plasma vWF as well as other plasma components, crucial for a
full thrombotic response under high shear flow.
| |
ACKNOWLEDGMENT |
The authors thank James R. Roberts and Benjamin Gutierrez for preparing
monoclonal antibodies; Rolf Habermann, Judith Dent, and Dr Enrique
Saldivar for help with videomicroscopy, computation, and image
analysis; Dr Malcolm Wood and George Klier (deceased) for assistance
with surface replication analysis; and Dr Virgil Woods for providing
the nonfunction blocking anti-21
monoclonal antibody, 12F1.
| |
FOOTNOTES |
Submitted January 18, 1999; accepted June 10, 1999.
Supported in part by Grants No. HL-31950, HL-42846, and HL-48728 from
the National Institutes of Health. Additional support was provided by
National Institutes of Health Grant No. RR0833 to the General Clinical
Research Center of Scripps Clinic and Research Foundation and by the
Stein Endowment Fund.
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.
Address reprint requests to Brian Savage, PhD, Department
of Molecular and Experimental Medicine, MEM-175, The Scripps Research
Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037; e-mail:
brian{at}scripps.edu.
| |
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ABSTRACT
INTRODUCTION