© 1997 American Heart Association, Inc.
Articles |
Shear-Induced Platelet Activation and Platelet Microparticle Formation at Blood Flow Conditions as in Arteries With a Severe Stenosis
From the Research Institute for Internal Medicine, Rikshospitalet, University of Oslo (P.A.H., N.O.S., F.R.B.), and Nycomed Pharma AS, Oslo (U.Ø., M.J.A.G.H., R.M.B., K.S.S.), Norway.
Correspondence to Pål André Holme, MD, Research Institute for Internal Medicine, Rikshospitalet, Pilestredet 32, 0027 Oslo, Norway. E-mail: p.a.holme{at}klinmed.uio.no
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Abstract In the present study, we investigated whether high arterial shear stresses at various exposure times or a sudden increase in shear stress introduced by a stenosis affect platelet activation and platelet microparticle formation in native human blood. We used a parallel-plate perfusion chamber device through which nonanticoagulated human blood was drawn (10 mL/min) by a pump directly from an antecubital vein through the flow channel of a perfusion chamber at wall shear rates of 420, 2600, and 10 500 s-1. In another set of experiments, an eccentric stenosis was introduced into the flow channel. Wall shear rates of 2600 or 10 500 s-1 at the stenosis apex were maintained at the same flow rate. The wall shear rate upstream and downstream of these stenoses was 420 s-1. A shear rate of 420 s-1 is within the range of those encountered in healthy small coronary arteries, whereas those of 2600 and 10 500 s-1 are representative for vessels with various degrees of stenotic lesions. The blood was exposed to these shear rates for periods varying from 0.075 to 3.045 seconds. Platelet activation was assessed as activated glycoprotein (GP) IIb/IIIa by FITC-labeled monoclonal antibody (MAb) PAC-1 and aminophospholipid translocation by FITC-labeled annexin V. Microparticle formation was quantified by FITC-labeled MAb Y2/51 directed against GP IIIa. Significant platelet activation and formation of microparticles were observed at 10 500 s-1 only (P<.008). This shear-induced platelet activation and microparticle formation were enhanced by introduction of a thrombus-promoting surface consisting of type III human collagen fibrils. Introduction of the most severe stenosis at 10 500 s-1 further increased platelet activation (P<.017). The collagen-induced thrombus formation increased the platelet thrombus volume at 10 500 s-1 from 16.5 to 33.8 µm3/µm2 (P<.003) on the stenosis apex when the most severe stenosis was used. A correlation (P<.0001) between platelet thrombus volume and platelet microparticle formation was observed in the presence of the eccentric stenoses. Apparently, high shear stress (315 dynes/cm2 at 10 500 s-1), as encountered in severe atherosclerotic arteries, activated platelets and triggered platelet microparticle formation. In contrast, no significant platelet activation or formation of platelet microparticles was observed at physiological shear (420 s-1) or at the shear condition simulating shear in arteries with a less severe stenosis (2600 s-1). The data imply that platelets are activated and form microparticles in native blood at very high shear stresses. These events are potentiated by prolonged exposure to the high shear or by a sudden change of increasing shear due to the stenosis. The latter situation apparently enhances platelet thrombus formation at the stenosis.
Key Words: platelet activation • microparticles • shear rate • stenosis • flow cytometry
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Introduction |
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Atherosclerotic plaques that develop into stenotic lesions have profound impact on the local blood flow behavior.1 2 3 Wall shear rate and wall shear stress may increase by one or two orders of magnitude within a fraction of a second at such lesions. Indeed, wall shear rates exceeding 40 000 s-1 and wall shear stresses >300 dynes/cm2 have been reported1 2 for mechanically constricted epicardial arteries in dogs.1 In comparison, the physiological ranges of these physical flow parameters vary from 20 to 2000 s-1 and from 1.4 to 60 dynes/cm2,4 respectively. Sustained high shear conditions are not maintained at focal lesions but may prevail in vessels with diffuse lesions. It is apparent that the shape and degree of luminal occlusion introduced by such stenoses are determining the blood flow characteristics at the lesion.
Rapid and dramatic changes in blood flow behavior may activate passing blood platelets. Whether shear-induced platelet activation occurs in vivo remains to be established, and if so, its consequences for thrombus formation should be elucidated. This is of great importance, since arterial thrombus formation is generally triggered by plaque rupture at stenoses, where high shear or disturbed blood flow may prevail. Nevertheless, in vitro experiments have shown platelet activation elicited by shear stresses and wall shear rates as low as 50 dynes/cm2 5 and 100 s-1,6 respectively. Furthermore, the exposure time to the shear is important, since at least 7 ms is required to trigger platelet activation by a shear stress of 170 dynes/cm2 in a viscometer.7
Platelet-derived microparticles are formed from the surface membrane by an exocytotic budding process19 on platelet activation by agonists such as thrombin or collagen.8 9 10 11 Microparticles have an average diameter of 0.1 µm.10 They express negatively charged phospholipids12 13 and factor Va14 and Xa activity15 and thus possess procoagulant surface properties.8 9 10 11 16 17 18 Their potential significance in hemostasis and thrombosis remains unknown, although they have been detected in blood from patients with activated coagulation and fibrinolysis,20 with autoimmune thrombocytopenias,21 22 and after cardiopulmonary bypass.23 Recently, it was reported that microparticle formation correlates well with exposure of the platelet procoagulant surface11 and that activation of glycoprotein (GP) IIb/IIIa may be of some importance for this process with certain agonists.11 24
The aim of the present work was to study platelet activation and microparticle formation at different shear conditions as encountered in healthy and atherosclerotic arteries. This was performed by an ex vivo flow model using nonanticoagulated human blood and a panel of parallel-plate perfusion chambers with or without the presence of a cosine-shaped eccentric stenosis in the flow channel.25 26 27 Platelet activation and formation of microparticles were studied by flow cytometry. The wall shear rates used ranged from a physiological value of 420 s-1 to values above the physiological range of 2600 and up to 10 500 s-1. The latter two shear conditions are representative of those in atherosclerotic vessels, thus simulating flow conditions in vessels with various degrees of stenotic lesions. Platelet activation was assessed as activated GP IIb/IIIa complexes by binding of a monoclonal antibody (MAb, PAC-1, and by expression of negatively charged phospholipids on the surface as measured by annexin V. Platelet-derived microparticles were quantified by an MAb against GP IIIa. These parameters were assessed in the presence or in the absence of a thrombus-promoting surface (placed at the apex of the stenoses) consisting of human type III collagen fibrils.25 27
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Reagents
FITC was purchased from Molecular Probes, Inc. Recombinant annexin V was a generous gift from Bender Wien (Vienna, Austria).
Antibodies
The MAb PAC-1 (IgM), which recognizes an epitope on
activated GP IIb/IIIa, was purchased from The Cell Center,
University of Pennsylvania.28
The FITC-conjugated monoclonal antibody Y2/51 directed against GP IIIa, and X927, a FITC-conjugated negative control of the same subtype (IgG1), were purchased from DAKO A/S.
FITC Labeling of Antibodies and Proteins
FITC conjugation of annexin V and PAC-1 was performed according
to Goding29 and as previously described in
detail.11
Parallel-Plate Perfusion Chambers With or Without an Eccentric
Stenosis
Two types of parallel-plate perfusion chambers were used (Table 1
). These included four perfusion chambers with unobstructed blood
flow25 26 30 and two perfusion chambers with an eccentric
stenosis in the flow channel.27 32
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The chambers with unobstructed flow have been well characterized
regarding blood flow behavior and thrombus formation.25 26
The blood flow is laminar in these chambers. The chambers selected for
this investigation had wall shear rates of 420, 2600, and 10 500
s-1 at a flow rate of 10 mL/min and a Reynolds
number of 20. Geometrical dimensions of the flow channels as well as
corresponding shear rates and stresses are summarized in Table 1
.
A miniature chamber with a rectangular flow channel 18.0 mm long
was constructed for the present study. The width and height of the
flow channel were chosen such that the wall shear rate was 10 500
s-1 at a flow rate of 10 mL/min. The length of
the flow channel was identical to the axial length of the
stenosis apex of the perfusion chamber, with an eccentric
stenosis having an apex wall shear rate of 10 500
s-1.27 31 32 Thus, the exposure
time to this wall shear rate was identical in the two chambers. The
exposure time to the respective shears is given in Table 1.
The two chambers with a stenosis in the flow channel were
previously characterized.27 31 32 Briefly, a cosine-shaped
eccentric stenosis is introduced into the rectangular flow
channel of the original parallel-plate perfusion
chamber25 26 with a wall shear rate of 420
s-1. The cosine-shaped stenosis step
has an axial length of 0.5 mm, whereas the axial length of the
stenosis is 18.0 mm. The protrusion of the
stenosis into the flow channel determines the wall shear rate
at the apex, which in the present investigation was 2600 and
10 500 s-1 at a flow rate of 10 mL/min. The
corresponding stenotic occlusions of the flow channels were
60% and 80%, respectively. The exposure time to these shear
conditions corresponds to 0.075 and 0.151 second, respectively (Table 1). The wall shear rate upstream and downstream of the stenoses
is 420 s-1. However, shear overshoots of 2200
and 4000 s-1 at the rear end of the upstream
half-cosine–shaped step of the 60% and 80% occluding
stenoses, respectively, were established by numerical
analysis with a finite-element program developed by Fluid
Dynamics International Inc (FIDAP).27 31 32 The perfusion
chambers with these two stenoses are intended to simulate blood
flow in coronary arteries with "advanced" single
eccentric stenosis with a long axial dimension. Blood perfusion
experiments were performed at 2600 and 10 500
s-1 with exposure to purified human type
III collagen fibrils spray-coated on Thermanox coverslips (Miles
Laboratories).25 Uncoated Thermanox coverslips were
exposed to blood in all perfusion chambers and thus at all shear
conditions. The Thermanox surface interacts poorly with flowing blood,
whereas the collagen fibrils trigger rapid and pronounced thrombus
formation.25
Preparation of the Collagen Surface
Type III collagen was purified from human placenta by pepsin
digestion and selective salt precipitation.33 Collagen
fibrils were prepared by dialysis against 20 mmol/L
Na2HPO4, pH 7.5, for 48 hours at 4°C and
coated on Thermanox plastic coverslips as previously
described.25 27 The collagen fibrils do not
activate coagulation,25 and the coating gives a
maximal thrombogenic stimulus at densities of 
10
µg/cm2.
Blood Donors and Blood Sampling
Each of the blood donors gave informed consent to donate 55 mL
blood per perfusion experiment. According to their statements, none of
the individuals had taken aspirin or other drugs for at least 14 days
before the perfusion experiments. Immediately before each perfusion
experiment, 4 mL blood was collected into EDTA-containing tubes for
determination of hemoglobin, hematocrit, and white cell and
platelet counts (Auto Counter AC 920, Swelab Instruments).
Individual hemoglobin, hematocrit, platelet, and leukocyte values
were within the normal range for all subjects studied.
Human Ex Vivo Perfusions, Fixation, and Embedding
Ex vivo perfusion experiments were performed at 37°C with
parallel-plate perfusion chambers34 with or without a
stenosis in the flow channel and in the presence of a
collagen-coated or an uncoated Thermanox coverslip.
Venipuncture was performed with a No. 19 butterfly infusion
set (Abbott Laboratories). Nonanticoagulated blood from 6 to 17 healthy
volunteers was drawn through each perfusion chamber. The blood flow
rate was maintained at 10 mL/min for 5 minutes by an occlusive roller
pump (Minipuls 3, Gilson) placed distal to the chamber. Blood
perfusions were immediately followed by a 20-second perfusion (10
mL/min) with a buffer containing (in mmol/L) NaCl 130, KCl 2,
NaHCO3 12, CaCl2 2.5, and MgCl2 0.9
at pH 7.4, 37°C and by a 40-second perfusion with fixation solution
(2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer,
pH 7.4, 22°C). Subsequently, the coverslip was removed from the
chamber and kept in freshly prepared fixation solution for 1 hour.
Specimens were stored in 7% sucrose/0.1 mol/L cacodylate at 4°C
until they were embedded in epoxy resin.
Morphometry
Evaluation of thrombotic deposits was performed on epoxy
resin–embedded semithin sections (1 µm) prepared perpendicular
to the direction of the blood flow and 1 mm downstream from the
upstream end of the coverslip.34 35 Platelet thrombus
volume (µm3/µm2) was derived from the
sectional thrombus area measured by computer-assisted morphometry
(Kontron Vidas, Eching).35
Platelet Isolation
For flow cytometry analysis, 1 mL of blood
anticoagulated with 0.1 vol 0.129 mol/L trisodium citrate was sampled
distal to the perfusion chamber after 4.5 minutes of perfusion.
Platelet-rich plasma was then immediately prepared by
centrifugation for 5 minutes at 160g at
20°C. Subsequently, platelets were fixed by addition of
paraformaldehyde in PBS to 0.33%. Fixation could not
be used for studies of annexin V binding, because this was strongly
impaired by the fixation procedure.
Preparation of Platelet Samples for Flow Cytometry
Platelets (1x106) were added to
polystyrene tubes containing filtered PBS (pH 7.4) at a final volume of
100 µL after addition of fluorescent probes. The various
FITC-labeled probes were added in final concentrations of 5 µg/mL
FITC-Y2/51, 6 µg/mL FITC–X-927, 39 µg/mL FITC–PAC-1, or 25
µg/mL FITC–annexin V. The mixtures were incubated in the dark at
4°C for 30 minutes. Subsequently, they were diluted with 1 mL
filtered PBS.
Flow Cytometry
Platelets labeled with the FITC-conjugated probes were
analyzed in a FACScan flow cytometer (Becton Dickinson)
equipped with a 15-mW air-cooled 488-nm argon laser as previously
described.11 The light scatter and fluorescence
channels were set at logarithmic gain. The platelets were
analyzed by the FACScan in two ways. To study the amounts of
the probes (annexin V and PAC-1) bound to platelets after
perfusion, platelets were gated on the basis of the forward- and
side-scatter properties. The percentages of platelets
activated during perfusion were calculated from the
fluorescence intensity of the platelets before and after
perfusion through the chamber. A fluorescence threshold was set
at the upper limit of the prechamber sample, and cells with a
fluorescence intensity exceeding this threshold level after
perfusion through the chamber were considered positive.
To study platelet microparticle formation and to resolve platelet-derived microparticles from background light scatter, acquisition was gated so as to include only positive events for antibody bound to GP IIIa (Y2/51). Consequently, a fluorescence threshold was set to analyze only platelets and microparticles.
Microparticles and platelets were separated analytically on the basis of their characteristics in forward and side scatter. To quantify and discriminate between platelets and microparticles, the lower limit of the platelet gate was set at the left border of the forward scatter profile of unperfused platelets. The number of microparticles present was expressed as the number of particles below this limit in percent of the total number of fluorescent particles counted (ie, platelets plus microparticles). Altogether, 10 000 positive events were analyzed each time, and the Cellquest program (Becton Dickinson) was used for data processing on an Apple computer.
Statistical Analysis
The Mann-Whitney test was used for statistical analysis.
The data shown represent mean±SEM. Correlations between
parameters were studied by linear regression
analysis.
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Results |
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Microparticle Formation in the Absence of Stenosis
When the platelet-specific monoclonal antibody Y2/51 against GP IIIa was used to detect platelets and microparticles by flow cytometry, the difference in microparticle concentration before and at 4.5 minutes of perfusion was quantified. Fewer than 1.5% microparticles were detected in blood samples collected from the arm vein. No significant increase in platelet-derived microparticles was detected during perfusion at shear rates of 420 and 2600 s-1, not even during collagen-induced thrombus formation (Fig 1A
). However, a significant increase in
the number of microparticles was measured at 10 500
s-1 (P<.008), both with
collagen-coated coverslips (2.8±1.0%) and with noncoated coverslips
(1.6±0.3%) (Fig 1A). The difference in formation of microparticles
between collagen-coated and noncoated coverslips was not
significant.
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Microparticle Formation in the Presence of Stenosis
Introduction of an eccentric stenosis in the flow channel
did not increase the microparticle formation at 2600
s-1 (60% stenosis occlusion).
However, at 10 500 s-1 (80% stenosis
occlusion), the microparticle formation was increased
(P<.006) even in the absence of collagen-induced thrombus
formation (Fig 1B). However, more microparticles were formed during the
collagen-induced thrombus (P<.018) (Fig 1B). Microparticle
formation at 10 500 s-1 was larger in the
perfusion chamber without a stenosis. However, the exposure
time to the high shear, or the time it takes for platelets to pass
the length of the flow channel, or the stenosis apex at the
shear rate of 10 500 s-1, was different in
the two chambers (Table 1). The exposure time was calculated to 0.609
second for the chamber without the stenosis and 0.075 second
for the perfusion chamber with the eccentric stenosis. Taking
this difference into consideration, it was estimated that 
15 times
more microparticles were produced per unit time in the presence of the
stenosis compared with the chamber without the
stenosis, indicating that the stenosis itself and/or
the stenosis geometry was much more efficient in producing
microparticles than the shear rate itself. To confirm this hypothesis,
a novel perfusion chamber was constructed. This chamber had no
stenosis, but the blood exposure time to 10 500
s-1 was kept similar to that in the chamber
with the stenosis by shortening the length of the flow channel
(Table 1). The shear conditions in this parallel-plate perfusion
chamber (noncoated Thermanox coverslip) elicited only a slight increase
in microparticle formation during perfusion, not statistically
different from the low levels measured at 420 and 2600
s-1 (Fig 1A).
Activation of GP IIb/IIIa in the Absence of Stenosis
Activation of GP IIb/IIIa was studied by flow cytometry using the
MAb PAC-1 directed against activated GP IIb/IIIa and the same
blood samples as used for quantification of microparticles (see above).
GP IIb/IIIa activation paralleled the formation of microparticles.
No significant increase in GP IIb/IIIa activation was observed at 420
or 2600 s-1. However, at 10 500
s-1, increased activation (P<.03)
was measured (Fig 2A). About 9% to 16% of the
platelets were activated whether perfused over noncoated or
collagen-coated coverslips (Fig 2A). Although no significant
differences in activation were observed between collagen-coated and
noncoated coverslips, there was a tendency toward more activation in
the presence of collagen-induced thrombus formation.
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Activation of GP IIb/IIIa in the Presence of Stenosis
The same set of experiments as reported above was repeated in the
presence of eccentric stenoses of 60% (2600
s-1) or 80% (10 500
s-1) occlusion. No significant GP IIb/IIIa
activation was detected at 2600 s-1 (Fig 2B).
However, pronounced GP IIb/IIIa activation (P<.003) was
measured at 10 500 s-1, thus at a flow
condition simulating flow in a severely stenosed artery. Although more
activation was measured during collagen-induced thrombus formation
(10.7±2.5%) than in the absence of collagen (6.7±2.9%), the
difference was not statistically significant. Considering the
difference in shear exposure time as described above, approximately
five times more activated platelets were measured in the
presence of the stenosis than in its absence. Experiments with
the miniature perfusion chamber, in which the 10 500
s-1 shear exposure time was identical to the
exposure time at the stenosis, showed a tendency of increased
PAC-1 binding; however, this was not significantly different from the
values measured in blood samples collected from the arm vein
(P<.06) (Fig 2A).
Annexin V Binding in the Absence of Stenosis
Translocation of aminophospholipids was measured as binding of
FITC-labeled annexin V to the surface of the activated
platelets by flow cytometry.36 37 Significant annexin
V binding was not detected during perfusions at 420 or 2600
s-1. Collagen-induced thrombus formation did
not affect the binding of annexin V to the perfused platelets.
However, at 10 500 s-1, the annexin V binding
increased (P<.01) (Fig 3A), being 3.8%
and 2.8% in the presence and absence of collagen-induced thrombus
formation, respectively.
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Annexin V Binding in the Presence of Stenosis
No significant annexin V binding was measured at 2600
s-1 either in the presence or in the absence
of collagen-induced thrombus formation. However, significant annexin V
binding was measured at 10 500 s-1
(P<.006). The binding was 3.8±0.8% in the presence of
collagen-induced thrombus formation and 3.7±0.8% in the absence of
collagen (Fig 3B). In evaluation of the annexin V binding with regard
to the 10 500 s-1 shear exposure time, it was
found that 10-fold more activation per unit time occurred with the
stenosis than without the stenosis. Again, the results
imply that the stenosis itself and/or its geometry was much
more important for platelet activation than the shear rate itself.
To confirm this hypothesis, perfusion studies with the miniature
chamber at 10 500 s-1 were performed. A
tendency toward increased annexin V labeling during perfusion was
detected; however, this was not significantly different from the values
measured in blood samples collected from the arm vein
(P<.07) (Fig 3A).
Platelet Thrombus Volume in the Absence of Stenosis
Platelet thrombus volume on collagen and Thermanox was
determined by computer-assisted morphometry.35 Virtually
no thrombotic material was detected on noncoated Thermanox coverslips
at 420, 2600, or 10 500 s-1 (Fig 4A). In contrast, pronounced thrombus formation on
collagen was observed at 2600 and 10 500 s-1.
The platelet thrombus volumes averaged 16.5±2.5 and 20.9±4.0
µm3/µm2, respectively.
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Platelet Thrombus Volume in the Presence of Stenosis
Platelet thrombus volume on collagen-coated coverslips at the
80% stenosis (10 500 s-1) was
significantly higher (33.8±2.4
µm3/µm2) (P<.0002) than at the
60% stenosis (2600 s-1)
(11.7±2.5 µm3/µm2) (Fig 4B). Also,
with noncoated coverslips, a statistically significant increase in
thrombus volume was detected at 10 500 s-1
(12.6±4.6 µm3/µm2) compared with the
volume at 2600 s-1 (2.0±1.2
µm3/µm2) (P<.04).
It is interesting to note that the thrombus volume in the collagen-induced thrombus formation at 10 500 s-1 was twofold increased on the stenosis apex relative to the volume measured in the absence of stenosis (P<.003).
Linear regression analysis was used to see whether statistical
correlations existed between the platelet activation markers
measured and the platelet thrombus volume on collagen-coated and
noncoated coverslips. The thrombus volume was correlated to the
formation of microparticles after perfusion through the two perfusion
chambers with the eccentric stenosis (r=.66,
P<.0001) (Fig 5). No such correlation was
observed in the absence of the stenosis, nor was there any
significant correlation between PAC-1 or annexin V labeling of the
platelets and the thrombus volume. Significant correlation was
found both for annexin V and PAC-1 labeling compared with the formation
of microparticles in the different perfusion chambers
(r=.38, P<.003 and r=.42,
P<.002, respectively).
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The present investigation provides the first evidence that human platelets in native blood are activated and form microparticles by complex blood flow behavior, as may be encountered in atherosclerotic vessels. It is apparent that high shear and a sudden increase in shear leads to activation of the platelets, with the exposure time to high shear also being important. These factors may act simultaneously and synergistically on the platelet in diseased arteries.
It appeared that flow conditions that activate platelets are nonphysiological. Evidence of platelet activation at physiological shear was not detected, not even at a wall shear rate of 2600 s-1, which is slightly above the physiological range. Also, inclusion of a thrombus-promoting surface in the form of collagen did not trigger detectable platelet activation at these shear conditions. However, a nonphysiological wall shear rate of 10 500 s-1 triggered significant platelet activation and microparticle formation, and more so in the presence of collagen-induced thrombus formation.
It was not possible to differentiate between chemical activation by substances released from platelets in collagen-attached thrombi, by local thrombin formation at the stenosis, or by shear-induced activation introduced by the stenosis and the gradually increasing shear imposed by the growing thrombi at the stenosis apex. The possibility of high and low shear in close proximity to the thrombotic deposits, and thus the potential sudden change from low to high shear in and around the growing thrombi, may support activation and microparticle formation. Also, flow channels through the platelet-rich thrombi at 10 500 s-1 may represent regions of very high shear27 that may activate passing platelets. On the other hand, activation of platelets and coagulation by the butterfly infusion set is less likely, since the coagulation activation markers F 1+2, thrombin-antithrombin, and fibrinopeptide A and the platelet activation marker ß-thromboglobulin are still within the normal range within 5 minutes of perfusion.38 However, prolonging the perfusion time more than 5 minutes results in significant chemical activation of both coagulation and platelets and should thus be avoided.
The significant platelet activation observed at 10 500 s-1 in the presence or in the absence of the eccentric stenosis coincides with the insensitivity of aspirin to thrombus formation at this shear condition.39 Shear-induced platelet activation is apparently insensitive to aspirin, which previously was shown in in vitro studies.40 41 42 This is in accordance with the observation that aspirin significantly reduces thrombus formation at 2600 s-1, both in the presence and absence of the stenosis,39 43 thus at flow situations in which platelet activation and microparticle formation were not detected in the present study. Clinical angiographic studies of reocclusion after coronary thrombolysis is in accordance with our observations as well, since aspirin administration is reported not to be effective in preventing reocclusion of lesions with >90% stenosis, whereas protection was observed at less severe stenosis.44
Whereas no platelet activation or microparticle formation was
observed at 420 or 2600 s-1 or with the 60%
stenosis at 2600 s-1, a significant
increase in microparticles and PAC-1 and annexin V labeling was
detected at 10 500 s-1 both in the presence
and in the absence of the eccentric stenosis. There was no
apparent difference in platelet activation whether the
stenosis was present or not. However, because the exposure
time to the 10 500 s-1 shear rate was 8.2
times longer (Table 1) in the absence of the stenosis, the
ratio between the degree of activation and the exposure time was
calculated. These calculations showed the presence of 5 to 15 times
more activated platelets per unit time when calculated for
the binding of PAC-1 or annexin V or as formation of microparticles at
the eccentric stenosis. The experiments with the miniature
perfusion chamber, in which the time of shear exposure was identical to
the exposure time at the stenosis, confirmed these
calculations, since only a slight platelet activation or
microparticle formation was detected. These results confirm that
shear-dependent platelet activation is coupled to the exposure time
in native blood, as was shown in anticoagulated blood by
Konstantopoulos et al.42 The data further imply that the
stenosis geometry is important for the degree of platelet
activation. The sudden change in shear stress that occurs over a very
short distance at the stenosis (in this model, over an axial
length of 0.5 mm), including the transient shear overshoot of 4000
s-1 at the stenosis inlet, appears to
be more important for the activation and microparticle formation than
the wall shear rate itself.
It is plausible to suggest that the twofold-increased thrombus volume on the stenosis apex at 10 500 s-1 was due to the sudden increase in shear introduced by the stenosis and thus due to the resulting platelet activation. This observation suggests that the geometry of the stenosis, and not only the shear rate itself, must be considered in the evaluation of the resulting platelet activation and thrombus formation. It is well known that geometric alterations in the flow path, such as those produced in the present study at the respective stenoses, can produce wide variations in local shear levels, which are further complexed as thrombus formation proceeds, especially since initial deposits seem to favor the apex area.45
A relatively low percentage of the platelets exiting from the flow chamber was activated, implying that most platelets were still unactivated after the passage through the perfusion chamber. However, the number of platelets actually activated may be higher, because some of these platelets are incorporated into the growing thrombi. Obviously, these activated platelets would not be included in the flow cytometric analysis. In contrast, platelets transiently in contact with the growing thrombi without being incorporated into the thrombus but being chemically and/or shear-activated would be detected by this method.
A significant correlation was found between the thrombus volume on Thermanox or collagen at the eccentric stenosis and the number of microparticles found (r=.66, P<.0001). No such correlation was observed between PAC-1 or annexin V labeling and the thrombus volume. This may indicate that most of the microparticles were released from activated platelets that were incorporated into growing thrombi, whereas the binding of PAC-1 or annexin V to platelets represented platelets that generally had been activated in the streaming blood.
We conclude that high shear stress in the parallel-plate perfusion chambers simulating those encountered in severely stenosed arteries activates platelets and produces platelet-derived microparticles and that the sudden change in shear stress induced by the stenosis triggers more platelet activation and formation of microparticles than the shear itself. It is apparent that much remains to be learned about the relationship of intravascular platelet activation and thrombus formation in atherosclerotic vessels. Nevertheless, this study has provided new knowledge about flow conditions promoting platelet activation, microparticle formation, and concomitant enhanced thrombus formation in native human blood.
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Acknowledgments |
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This study was supported by The Research Council of Norway, Nycomed Pharma AS, The Norwegian Council on Cardiovascular Diseases, the Anders Jahres fund, and the Professor Paul A. Owrens fund.
Received June 6, 1996; accepted July 15, 1996.
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