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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 968-975
Real-Time Analysis of Mural Thrombus Formation in Various Platelet
Aggregation Disorders: Distinct Shear-Dependent Roles of Platelet
Receptors and Adhesive Proteins Under Flow
By
Shizuko Tsuji,
Mitsuhiko Sugimoto,
Shigeki Miyata,
Mitsuhiro Kuwahara,
Seiji Kinoshita, and
Akira Yoshioka
From the Department of Pediatrics, Nara Medical University,
Kashihara, Nara, Japan; and Higashi-Osaka General Hospital,
Higashi-Osaka, Osaka, Japan.
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ABSTRACT |
We evaluated real-time processes of platelet thrombus formation on a
collagen surface in a flow chamber with whole blood from patients with
various platelet aggregation disorders, such as Bernard-Soulier
syndrome (BSS), Glanzmann's thrombasthenia (GTA), type 3 von
Willebrand disease (vWD), and congenital afibrinogenemia (Af), who lack
platelet glycoprotein (GP) Ib-IX complex, GP IIb-IIIa, von Willebrand
factor (vWF), and fibrinogen, respectively. Blood from GTA patients
showed impaired thrombus growth but significant initial
platelet-surface interaction under all shear conditions tested (50 to
1,500 s 1). By contrast, blood from patients with BSS
or type 3 vWD showed no platelet-surface interaction under high shear
( 1,210 s1) but normal thrombus formation under low
shear ( 340 s1). When shear rate was increased
stepwise to 1,500 s1 during perfusion, the thrombus
growth observed in type 3 vWD or BSS under low shear was arrested,
whereas that in control blood was sharply accelerated as a function of
shear rate. Overall thrombus formation in Af appeared indistinguishable
from that of a control under shear rates between 50 and 1,500 s1. However, Af thrombi formed under such conditions
collapsed immediately when shear rate was further increased to 4,500 s1, whereas thrombi of type 3 vWD or BSS formed under
low shear were stable even when shear rate was elevated to 9,000 s1 during perfusion. These findings suggest that
distinct molecular mechanisms underlie the pathologic bleeding in these
diseases and point to the distinct roles of two major adhesive
proteins, vWF and fibrinogen. In mural thrombus formation
under flow conditions, vWF, perhaps mainly through its interaction with
GP Ib-IX, acts as an "initiator and promoter," whereas
fibrinogen, via its binding to GP IIb-IIIa, acts as a
"stabilizer" against heightened shear forces that could lead to
peeling off of platelets from the surface.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PLATELET PLUG FORMATION in vivo at sites
of vessel wall damage is critical in ensuring blood flow to vital
organs, but may also contribute to the generation of pathologic
intravascular thrombosis.1-4 Many platelet membrane
receptors and plasma adhesive proteins are thought to be involved in
this crucial event, although the precise mechanisms involved are not
fully understood, particularly under flow conditions. Indeed, most
previous studies of platelet function were performed in a static or
closed stirring experimental system. More recent platelet functional
studies that take blood flow into consideration indicate the
significant effect of shear rate on platelet aggregation
mechanisms.5-7 Indeed, studies that use a
cone-and-plate-type viscometer have shown that platelet aggregation
induced by experimental high shear stress (80 dyne/cm2)
in a soluble phase is a consequence of the interaction between von
Willebrand factor (vWF) and platelet glycoprotein (GP) Ib-IX and GP
IIb-IIIa, whereas platelet aggregation under low shear stress (6 to 12 dyne/cm2) is mediated by the interaction between
fibrinogen and GP IIb-IIIa.8,9 However, the mechanisms of
platelet aggregate accumulation on a thrombogenic surface (mural
thrombus formation) under flow conditions, ie, mechanisms relevant for
in vivo hemostatic plug formation at sites of vascular injury, may be
more complex or different from those of soluble-phase shear-induced
platelet aggregation. Recent studies using experimental flow systems to
analyze mural thrombus formation on several types of collagen, a major
component of vascular subendothelium, have demonstrated that at least
four platelet receptors, GP Ib-IX, Ia-IIa, IIb-IIIa, and VI play a role
in this event.10-15 Although recent studies suggest the
critical role of the interaction between plasma vWF immobilized to
collagen and platelet GP Ib in initial platelet adhesion to the
surface,15,16 the precise mechanisms of overall mural
thrombus formation on a collagen surface, especially shear-specific
functions of platelet receptors and adhesive proteins, remain to be addressed.
In the present study, we observed the real-time process of mural
thrombogenesis on a type I collagen-coated surface under flow
conditions with various shear rates in the blood of patients with major
congenital platelet aggregation disorders. Our experimental approach
indicates distinct pathogenic mechanisms relevant for the life-long
bleeding symptoms in these diseases and illustrates in detail the
shear-specific and time-course-dependent functions of major platelet
receptors and adhesive proteins in platelet thrombus formation under
dynamic blood flow conditions.
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MATERIALS AND METHODS |
Patient profiles.
Two unrelated patients with Bernard-Soulier syndrome (BSS) fulfilled
the criteria of this disease, namely, giant platelets and mild
thrombocytopenia (120 × 103/µL and
160 × 103/µL, respectively), with a typical lack of
ristocetin- (1.2 mg/mL) induced platelet aggregation. Flow cytometric
analysis confirmed no measurable  -chain of GP Ib, with trace amounts
of GP IX on the surface of platelets from these patients. Blood of a
patient with May-Hegglin anormaly, whose platelets were similar to
those of the BSS patients in population size and counts, served as a control for BSS.
Three independent Glanzmann's thrombasthenia (GTA) patients were all
categorized as type I GTA with a defect of clot retraction, complete
lack of platelet aggregation by adenosine diphosphate or collagen, and
normal platelet counts (200 to 350 × 103/µL), on the
surface of which only 1% to 2.2% of GP IIb-IIIa complex were
expressed, as judged by flow cytometry. Normal plasma levels of vWF
(75% to 130%) and fibrinogen (250 to 350 mg/dL) were confirmed in
patients with BSS and GTA by enzyme-linked immunosorbent assay for vWF
and electroimmunoassay for fibrinogen, respectively.17-19
One patient with type 3 von Willebrand disease (vWD) had no detectable
vWF antigen in plasma or platelets, as confirmed by multimer analysis
with sodium dodecyl sulfate (SDS) (1.5%) agarose gel
electrophoresis.20,21 Thus, patient exhibited complete lack
of ristocetin-induced platelet aggregation, with normal ranges of
platelet counts and plasma fibrinogen.
Plasma fibrinogen levels of two unrelated afibrinogenemia (Af) patients
were below the detection limit by electroimmunoassay and no significant
fibrinogen in their platelets was confirmed by SDS-polyacrylamide gel
electrophoresis, followed by immunoblotting. These Af patients showed
complete lack of platelet aggregation by adenosine diphosphate or
collagen, with normal platelet counts and plasma vWF antigen levels. In
all patients with congenital platelet aggregation disorders, bleeding
time was markedly prolonged (>20 minutes). Thrombus formation in 10 nonsmoking healthy volunteers, who were not taking any medications for
the previous 2 weeks and whose platelet counts were all between 200 to
350 × 103/µL, was analyzed as a normal control. Blood
donors, including patients in this study, showed no significant anemia,
with hematocrit values always greater than 35%.
Blood collection and platelet labeling.
Blood was collected by using the specific thrombin inhibitor argatroban
(MD-805, final concentration 0.125 mg/mL; Mitsubishi Chemical, Corp,
Tokyo, Japan) as an anticoagulant to maintain physiologic
concentrations of divalent cations in blood. This argatroban
concentration was used to ensure elimination of a possible thrombin
generation during the entire processes of platelet thrombogenesis. Indeed, preliminary experiments confirmed no observable fibrin clot
formation for at least 3 hours after blood collection at this
argatroban concentration. Anticoagulated whole blood was kept at 37°C
and used in perfusion studies within 1 hour after blood collection.
Before perfusion, the fluorescent dye mepacrine (quinacrine
dihydrochloride; final concentration 0.01 mmol/L, Sigma-Aldrich Co,
Tokyo, Japan), was added to the blood to label platelets, allowing
visualization of platelet-surface interaction with epifluorescence
videomicroscopy. At the concentration used, mepacrine, which
immediately accumulates in the dense granules of platelets, does not
interfere with normal platelet function.13,16,22
Preparation of collagen solution and collagen-coated coverslips.
Suspensions of type I acid-insoluble collagen fibrils (2.1 mg/mL) were
prepared from bovine Achilles tendon (Sigma-Aldrich Co, Tokyo, Japan)
in 0.5 mol/L acetic acid (pH 2.8) as described.13,22,23 Glass coverslips (24 × 50 mm; Matsunami Glass Co Ltd, Japan) were coated with 200 µL of the collagen solution, placed in a humid environment for 60 minutes, rinsed with 10 mL of 50 mmol/L phosphate buffered saline (pH 7.35) to remove nonadherent collagen, and placed in
a flow chamber (see below).
Flow chamber and epifluorescence videomicroscopy.
A flow chamber that varies shear rate in a linear manner was assembled
and mounted on a microscope (BX60; Olympus Co, Tokyo, Japan) equipped
for epifluorescent illumination (BX-FLA; Olympus Co) and charge-coupled
device (CCD) camera system (U-VPT-N; Olympus Co) as
described.22-24 A whole blood sample was
aspirated through the flow chamber and across the collagen-coated
coverslip at a constant flow rate via a syringe pump (Model 935;
Harvard Apparatus, South Natick, MA). Unless otherwise indicated, the
entire thrombus generation process, from initial platelet-surface
interaction to platelet aggregate accumulation on the surface, was
observed in real time at positions of the flow chamber corresponding to 50 s1 and 1,500 s1 and recorded with a
video cassette recorder (Hi8 VIEWCAM; Sharp Co, Ltd, Osaka, Japan).
These two shear rates were selected to represent a typical low and high
shear rate, respectively, based on recent studies indicating that
platelet functions and the mechanisms of mural thrombus formation under
these two shear rates differ from each other.16,22,23
Evaluation of platelet thrombi generated on the surface.
Time-course images of thrombus formation in a videotape were digitized
by a frame grabber (DIG98; DITECT Co, Tokyo, Japan) and subjected to
computer-assisted analysis with an image processing application (Win
ROOF; Mitani Corp, Fukui, Japan). This program allows evaluation of
platelet thrombi generated on a surface at a defined area in each
image. First, the average fluorescence intensity corresponding to a
single platelet was calculated in each perfusion, with the image
representing the earliest period of platelet-surface interaction. At
this phase, platelets are not yet visibly cohered on the surface and
are clearly distinguishable from one another. This value was used to
set the threshold for background subtraction from each image to
standardize image intensity. Platelet thrombi generated on the surface
in time-course images obtained in the analogous perfusion were then
evaluated based on the integration patterns of fluorescence
(fluorescence intensity multiplied by the area). Total fluorescence of
platelet thrombi in each image, expressed as an arbitrary "pixel
unit," is the sum of fluorescence of each thrombus present in a
defined area in each image. Although this value may not directly
represent the actual number of adherent platelets, it does reflect at
least the extent of platelet thrombus generation on the surface and can
be used for relative comparison of thrombus growth among various diseases.
Evaluation of the extent of platelet immobilization to the surface.
The extent of platelet immobilization to the surface was evaluated in
frames at a defined observation period by using the method of Savage et
al,16 with minor modifications. Consecutive images with
0.033-s intervals for 2 s (total 60 frames) were digitized and
binarized after subtracting background as described above. The
overlapping area of platelets (the logical AND) of the initial two
consecutive frames was derived by superimposing, and the next logical
AND of the initial three consecutive frames was also calculated with
Win ROOF computer software (Mitani). This process was
repeated for a total of 60 frames. The value of logical AND is expected to decrease as a function of time when platelets attached to the surface are reversibly adhered and moving and to remain unchanged when
platelets are firmly immobilized. Thus, a "platelet mobility index," calculated as (1  each logical AND/total area value of platelets in the first frame) × 100, was used to express the extent of platelet immobilization to the surface during a defined observation period. According to this formula, the platelet mobility index must
equal 100 when all platelets in the first frame move by a distance
greater than its diameter and 0 when all platelets in the first frame
are firmly immobilized to the surface. Although this experimental
approach may not follow the precise movement of an individual platelet
on the surface, especially when platelet cohesion is extensive at late
stages of perfusion, this index does reflect the extent of platelet
immobilization on the surface.
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RESULTS |
Thrombus generation on a collagen-coated surface by normal control
blood perfused under low or high shear rate.
Platelets were gradually immobilized to the surface at either 50 s1 or 1,500 s1. The image obtained at 1 minute after the beginning of perfusion, taken as an early phase of
platelet-surface interaction, represents "platelet adhesion" in
which platelets were adhered to the surface superficially, although
small platelet aggregates were already observed in some areas (Fig
1). At either 50 s1 or 1,500 s1, platelets adhering to the surface gradually
assembled to form mural thrombi as a function of time, so that the
image obtained at 7 minutes of perfusion, taken as a late phase of
platelet-surface interaction, represents "thrombus growth" (Fig
1). Mural thrombi formed under high shear, however, tended to cover
more surface area than those under low shear. Consistent with the
platelet surface coverage visually recognized, the amount of platelets adhering to the surface evaluated by computer-assisted analysis increased in a time-dependent manner (Fig 1).
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| Fig 1.
Thrombus generation on a collagen-coated surface by
normal control blood perfused under high or low shear rate. Upper
panels: time-course images (taken at 1, 3, 5, and 7 minutes of
perfusion) of platelet-surface interaction at 50 s1 and
1,500 s1, displayed as accumulated fluorescence, are
representative of 10 independent perfusions with blood from 10 individual donors. At either shear rate, the images at 1 minute after
the beginning of perfusion indicate superficial platelet adhesion.
Platelets adhering to the surface gradually assembled to form mural
thrombi as a function of time. Mural thrombi formed under a shear rate
of 1,500 s1 appear to cover much more surface area than
those found under 50 s1. Lower panel: computer-assisted
evaluation of amount of platelets adhering to the surface,
corresponding to the images displayed above.
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Thrombus generation on a collagen-coated surface by blood from
patients with various congenital platelet aggregation disorders
perfused under low or high shear rate.
In blood perfusion of BSS, type 3 vWD, and Af under a shear rate of
50 s1, the extent of initial platelet-surface
interaction, as judged by the images and based on the values of
platelets adhering to the surface at 1 minute after the beginning of
perfusion, as well as thrombus growth, as judged by the results at 7 minutes of perfusion, were comparable with those of a normal control
(Fig 2A and Table 1). In
perfusion of GTA blood, significant initial platelet interaction comparable with that of the control was observed, but the results at 7 minutes of perfusion remained nearly unchanged from those at 1 minute
(Fig 2A and Table 1), indicating the lack of thrombus growth in this
disease.
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| Fig 2.
Thrombus generation on a collagen-coated surface by blood
from patients with various congenital platelet aggregation disorders
perfused under (A) low or (B) high shear rate. BSS, Bernard-Soulier
syndrome; GTA, Glanzmann's thrombasthenia; vWD, von Willebrand
disease; Af, congenital afibrinogenemia. Images were taken at 1 minute
and 7 minutes after the beginning of perfusion of blood from one
patient with each disease (numbered as "1"; see Table 1);
control images are identical to those in Fig 1. (A) Note the nearly
unchanged image of thrombus formation in GTA blood at 7 minutes of
perfusion; all other images at both 1 minute and 7 minutes of perfusion
are comparable with the control. (B) Images correspond to blood of
patients in (A). Note the absence of platelet-surface interaction in
BSS and type 3 vWD even at 7 minutes of perfusion. Note also that the
extent of platelet-surface interaction in GTA blood is comparable with
that of the normal control at 1 minute of perfusion, while thrombus
growth is absent in the GTA patient even at 7 minutes of perfusion.
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Table 1.
Evaluation of Platelets Adhering to a Collagen-Coated
Surface at 1 Minute and 7 Minutes of Perfusion Under Low or High Shear
Rate Conditions in Various Congenital Platelet Aggregation
Disorders
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In blood perfusion of BSS and type 3 vWD under a shear rate of 1,500 s1, no platelet-surface interaction was observed even at
7 minutes of perfusion (Fig 2B and Table 1). In the case of BSS, this
result is assumed to reflect the lack of GP Ib and not the presence of giant platelets or mild thrombocytopenia, because thrombus growth in
blood of a May-Hegglin patient, whose platelets were similar to those
of BSS patients in population size and counts, was comparable with
normal under such flow conditions (data not shown). In contrast, thrombus generation in Af blood appeared indistinguishable from that of
the control (Fig 2B and Table 1). In the case of GTA blood, the initial
platelet-surface interaction appeared comparable with that of the
control, although thrombus growth was undetectable at 7 minutes of
perfusion (Fig 2B and Table 1).
Shear-dependency of thrombus generation on a collagen-coated surface
in BSS, GTA, and type 3 vWD.
To further confirm the discrepant thrombus generation at high versus
low shear rate conditions in BSS and type 3 vWD, the amount of
platelets adhering to the surface at 7 minutes of perfusion was
analyzed at various shear rates. In the control, the amount of platelet
adhering to the surface basically increased as a function of shear
rate, whereas maximal values at 340 s1 in BSS and type 3 vWD decreased as a function of shear rate, and no platelet adhesion was
detected at shear rates greater than 1,210 s1 (Fig
3). No thrombus growth was detected
under any shear rates examined for GTA blood (Fig 3).
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| Fig 3.
Amount of platelets adhering to a collagen-coated surface
in BSS, GTA, and type 3 vWD at various shear rates. Data represent the
amount of adherent platelets on the surface, expressed as an arbitrary
units, at 7 minutes of perfusion of blood from each patient (numbered
as "1"; see Table 1) with BSS ( ), type 3 vWD ( ), and
GTA ( ) at the indicated shear rates. In BSS and type 3 vWD,
the maxmal value at 340 s1 decreased as a
function of shear rate, with no detectable platelet adhesion at shear
rates greater than 1,210 s1, whereas adhesion in control
blood ( ) increased as a function of shear rate. Thrombus
growth in GTA blood was absent at all shear rates.
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Evaluation of the extent of platelet immobilization to a
collagen-coated surface in GTA.
To characterize in detail the impaired thrombus growth in GTA blood
under all the shear rates examined, we evaluated the extent of platelet
immobilization to a collagen surface. Under a shear rate of 1,500 s1, the platelet mobility index with a plateau value of
about 60 at 1 minute of perfusion in the control decreased to 12 at 7 minutes of perfusion (Fig 4), suggesting
that firm platelet adhesion progressed as a function of perfusion time.
In contrast, the plateau value of the index in GTA blood at 1 minute of
perfusion (86) decreased only slightly at 7 minutes of perfusion (77)
(Fig 4), indicating the reversible interaction of GTA platelets with
the surface even at a late stage of perfusion. A similar tendency was
also observed under a shear rate of 50 s1 (Fig 4),
indicating that firm adhesion of GTA platelets was incomplete even
under flow conditions insufficient to peel off platelets from the
surface.
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| Fig 4.
Extent of platelet immobilization to a collagen-coated
surface in GTA. The platelet mobility index (Materials and Methods)
during 2-second observation period was calculated at 1 minute (upper
panels) and 7 minutes (lower panels) of perfusion of blood from a
patient (numbered as "1") with GTA () under a shear rate of
50 s1 (left panels) and 1,500 s1 (right
panels). At either shear rate, the relatively high
platelet mobility index of a normal control () at 1 minute of
perfusion decreased significantly at 7 minutes of perfusion, indicating
that firm platelet adhesion progressed as a function of time. Note that
the platelet mobility index for GTA, which was higher than that of
normal at 1 minute of perfusion, decreased only slightly at 7 minutes
of perfusion, indicating only limited firm adhesion of platelets in GTA
during perfusion.
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Strength of thrombi against increasing shear rates in BSS or type 3 vWD.
Although platelet thrombi were found to form to an extent comparable
with normal in BSS and vWD type 3 under low shear rate conditions (Figs
2A and 3 and Table 1), the quality of these thrombi generated in the
absence of vWF or GP Ib, especially at high shear rates, is uncertain.
Therefore, we observed the changes in thrombi of BSS and type 3 vWD
during stepwise increases in shear rate during perfusion. Based on the
amount of platelets adhering to the surface, the time-dependent
thrombus growth during the 7-minute perfusion of BSS or type 3 vWD
blood with a shear rate of 340 s1 was arrested when the
applied shear rate was raised to 1,500 s1, although
thrombus growth in control blood was greatly accelerated in a
shear-dependent manner (Fig 5). Moreover,
real-time observations of thrombus growth processes (not shown here)
and the platelet mobility index (Table 2)
confirmed that thrombi of BSS or type 3 vWD generated under low shear,
like those of control, were stable and firmly fixed during stepwise
increases of shear rates to 9,000 s1. These results
indicate that vWF and GP Ib, in addition to their critical contribution
to initial platelet adhesion, play a determining role in thrombus
growth under high shear rate conditions. Further, the
resistance of thrombi formed in the abence of vWF or GP Ib against very high shear rates, a force that could peel off
platelets from the surface, suggests that neither vWF nor GP Ib
plays a substantial role in thrombus stability.
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| Fig 5.
Changes in platelet thrombi generated under low shear
rate conditions by stepwise increase of shear rate during perfusion in
BSS or type 3 vWD. Blood from control (), BSS (patient No. 1) (),
or type 3 vWD () was perfused over a collagen surface under 340 s1 for 7 minutes. The shear rate applied was then
increased stepwise to 1,500 s1 for 2 minutes and
subsequently to 4,500 s1 for 1 minute and to 9,000 s1 for 1 minute. Based on the amount of platelets
adhering to the surface, the time-dependent thrombus growth in BSS or
type 3 vWD was arrested when the applied shear rate was raised to 1,500 s1, although thrombus growth in control blood was
greatly accelerated in a shear-dependent manner.
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Table 2.
Changes in the Platelet Mobility Index Around the
Transitional Time Point From 1,500 s1 to 4,500 s1 During Blood Perfusion of a BSS, vWD Type 3, and Af
Patient*
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Strength of thrombi against very high shear rates in congenital Af.
The overall process of mural thrombus formation in Af appeared to be
comparable with normal under either low or high shear rate conditions
(Fig 2 and Table 1). However, all prior experiments suggested that
increasing shear rates induce a decrease in thrombus formation in most
of the bleeding diseases while apparently upregulating thrombus growth
in the normal control (Fig 3). Therefore, we examined thrombus
formation by Af blood under higher shear rate conditions. Real-time
observations showed progressive loosening of thrombi formed under a
1,500 s1 shear rate, with gradual collapse after a
stepwise increase of the shear rate to 4,500 s1 (Fig
6B). Based on the values of platelets
adhering to the surface, thrombus growth observed under a shear rate of
1,500 s1 in Af blood perfusion was arrested when shear
rate was shifted to 4,500 s1, whereas thrombus formation
in control blood was sharply accelerated under 4,500 s1
conditions (Fig 6A). This result (Fig 6A) is similar to those in the
case of BSS and vWD type 3 (Fig 5) and does not directly reflect the
gradual collapse of Af thrombi, because flowing collapsed pieces of
thrombi continuously came into the observed field from the outside of
frame. Besides, the initiation of platelet adhesion and aggregation
(although not much growing), concomitantly with the collapse, occurred
newly and repeatedly as a function of vWF, not resulting in a drastic
decrease in the amount of platelets in a examined frame. However, the
platelet mobility index of Af blood around the transitional time point
from 1,500 s1 to 4,500 s1 greatly
increased from 9 to 42.3 (Table 2), apparently because of the
alteration of shear rate, demonstrating the breakdown of platelet
immobilization to the surface once established under lower shear rates.
The control index, as well as BSS and vWD index showed no such increase
(Table 2). Moreover, consecutive images obtained under a shear rate of
4,500 s1 and at 2-second intervals provided a direct
evidence of the gradual collapse of the thrombi formed in Af that have
never been observed in the case of vWD (Fig 6B). These results indicate
the critical involvement of fibrinogen in maintaining thrombus strength
against heightened shearing forces.
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| Fig 6.
Evaluation of thrombi formed in Af blood at 1,500 s1 against a very high shear rate. (A) Control () or
Af (patient No. 1) ( ) blood was perfused over a collagen-coated
surface under 1,500 s1 for 3 minutes. Based on the
amount of platelets adhering to the surface, thrombi of Af formed more
rapidly than those of the control. When the shear rate applied was
increased stepwise to 4,500 s1 at 3 minutes after the
beginning of perfusion, the time-dependent thrombus growth in Af blood
was arrested, while thrombus growth in control blood was accelerated.
(B) Consecutive images of thrombi in Af blood collapsing under a shear
rate of 4,500 s1. The images at 2-second intervals were
captured immediately after the shear rate transition to 4,500 s1. Note that thrombi in Af blood collapse as a function
of time, especially around the areas indicated ( ).
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DISCUSSION |
Recent flow studies using a vWF-coated glass surface showed that the
interaction of surface-immobilized vWF with GP Ib on flowing platelets
mediates tethering and translocation of platelets along the
surface.16 This initial transient attachment of platelets is assumed to be an activating signal for platelets, leading to firm
platelet adhesion through binding of activated GP IIb-IIIa to
surface-immobilized vWF.16,25 Although an analogous event on a collagen-coated surface, to which plasma vWF bound first in the
early stage of blood perfusion, was recently
confirmed,14,15 the overall mechanisms of platelet
thrombogenesis on a collagen surface could be more complex than those
that underlie the event on a purified vWF surface because of
involvement of a variety of adhesive proteins and platelet membrane
receptors on this thrombogenic surface.
To gain insight into platelet thrombogenesis on a collagen surface
under physiologic flow conditions, we observed real-time processes of
platelet thrombus formation on this surface in several congenital
platelet aggregation disorders. Although several previous studies have
analyzed the thrombogenicity of bleeding diseases under flow
conditions,26-29 ours is the first to simultaneously monitor the entire process of thrombus formation in real time under
identical flow experimental conditions, in platelet aggregation disorders that respectively lack a single component of two major platelet receptors (GP Ib-IX and IIb-IIIa), or two major soluble adhesive proteins (vWF and fibrinogen).
With the exception of GTA, the platelet aggregation disorders examined
under low shear rate conditions showed thrombus generation processes
comparable with those of a normal control, indicating compensation for
the lack of a single component by other adhesive proteins or platelet
receptors under conditions in which platelets flow at a relatively low
speed. This conclusion contrasts with previous studies of soluble-phase
shear-induced platelet aggregation, in which platelet GP IIb-IIIa
strictly required fibrinogen, not vWF, as an adhesive ligand for
platelet cohesion under low shear stress conditions.8,9
However, among the factors examined in our study, only GP IIb-IIIa
appeared to be indispensable in establishing firm platelet adhesion
even under low shear rate conditions. Although a recent flow study
indicated the critical involvement of GP Ia-IIa in firm platelet
adhesion to a collagen-surface,30 the contribution of GP
Ia-IIa might be insufficient in the complete absence of GP IIb-IIIa.
Consistent with previous reports,28,29 the interaction of
surface-fixed vWF with GP Ib is absolutely required for initiation of
the platelet-surface interaction, because neither BSS nor type 3 vWD
showed evidence of platelet-surface interaction under high shear rate
conditions. When platelets flow much faster than under low shear rates,
the extremely high association rate of bond formation between vWF and
GP Ib is critical in mediating the initial communication of flowing
platelets with the surface, even on a collagen-coated surface where the
known platelet collagen receptors GP Ia-IIa, GP IV, and GP VI may play
a role. Further, our results confirmed that the interaction of vWF with
GP Ib is also crucial for thrombus growth under high shear rate
conditions. Although it remains unclear exactly how the vWF-GP Ib
interaction functions in thrombus growth, the requirement of this
interaction in platelet communication with the surface under high shear
rates suggests a scenario whereby vWF flowing at high speed can be
captured only through its transient interaction with GP Ib on platelets
adhering to and immobilized on the surface, followed by the
irreversible binding of vWF transiently trapped by GP Ib to neighboring
activated GP IIb-IIIa. Captured vWF on platelets adhering to the
surface might then mediate the second layer platelet adhesion via the
vWF-GP Ib interaction and via the subsequent vWF-GP IIb-IIIa
interaction, in a mode similar to the initial platelet adhesion on the
surface. Thus, basic mechanisms of mural platelet aggregate
accumulation on the surface might represent repeated cycles of these
events under high shear rate conditions. Based on this interpretation,
the interaction of vWF with GP Ib, regardless of whether vWF is in
solution or immobilized, is an absolute prerequisite for the overall
thrombus generation process. Indeed, a recent inhibition study, using
function-blocking antibodies and confocal laser microscopy in
combination with a flow chamber system analogous to ours, showed that
the interaction of vWF with GP Ib was essential for mural thrombus
growth on a type I collagen-coated surface.31
Fibrinogen is probably not involved in fundamental mechanisms of mural
thrombogenesis under high shear flow conditions. Indeed, mural thrombi
of Af blood formed in a manner indistinguishable from that of the
normal control at the high shear rate (1,500 s1) in
this study. However, the observation that platelet thrombi, once formed
under such flow conditions, began to collapse when the shear rate was
greatly elevated suggests that fibrinogen is needed to maintain the
strength of thrombi under heightened hemodynamic shearing forces that
could peel off platelets from the surface. Thus, unlike the initial
phase of platelet-surface interaction, blood flow situations above the
surface may become heterogeneous during mural thrombogenesis,
especially at sites around thrombi, which, by their bulk and ensuing
collisions with blood components, create local low shear rate
situations that favor binding of fibrinogen to GP IIb-IIIa. Flowing
plasma fibrinogen is, therefore, thought to be gradually integrated as
a function of time into thrombi, which are composed of vWF and
platelets in the early phase of the process, through binding to GP
IIb-IIIa even under high shear rates. Interestingly, thrombi formed in
the absence of vWF or GP Ib, unlike those formed without fibrinogen,
showed sufficient strength against very high shear rates, suggesting no
substantial role for vWF as an adhesive ligand in thrombus stability.
Together, results clearly show the distinct roles of two major adhesive proteins, vWF and fibrinogen, in mural thrombus formation under flow
conditions with high shear rates; vWF, perhaps mainly through its
interaction with GP Ib-IX, acts as an "initiator and
promoter," whereas fibrinogen, via its binding to GP IIb-IIIa,
acts as a "stabilizer" against heightened shearing forces that
could lead to peeling off of platelets from the surface.
In conclusion, the present study shows that distinct pathogenic
mechanisms underlie the life long bleeding symptoms associated with
prolonged bleeding time in these diseases. Impaired mural thrombus
generation was observed to some extent in all diseases under high shear
rate conditions and those under low shear were mostly normal except for
GTA, implying the physiologic relevance of platelet functions under
high shear blood flow situations. The shear specific and
time-course-dependent functions of GP Ib, GP IIb-IIIa, vWF, and
fibrinogen in our study might shed light on the complex mechanisms
involved in mural thrombogenesis under physiologic flow conditions and
might provide the groundwork for therapeutic strategies against
pathologic intravascular thrombosis formed under high shear stress,
such as coronary occlusive diseases.
| |
ACKNOWLEDGMENT |
We thank Marina Hoffman for editorial assistance.
| |
FOOTNOTES |
Submitted January 28, 1999; accepted March 31, 1999.
Part of this work was presented at the American Society of Hematology
Meeting in San Diego, CA, December 5-9, 1997 (abstract No. 92), and in
Miami Beach, FL, December 4-8, 1998 (abstract No. 1422).
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 Mitsuhiko Sugimoto MD, Department of
Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara
634-8522, Japan; e-mail: sugi-ped{at}nmu-gw.cc.naramed-u.ac.jp.
| |
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ABSTRACT
INTRODUCTION