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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 172-178
Contribution of Distinct Adhesive Interactions to Platelet
Aggregation in Flowing Blood
By
Zaverio M. Ruggeri,
Judith A. Dent, and
Enrique Saldívar
From the Roon Research Center for Arteriosclerosis and Thrombosis,
the Division of Experimental Hemostasis and Thrombosis, the Department
of Molecular and Experimental Medicine, and the Department of Vascular
Biology, The Scripps Research Institute, La Jolla, CA.
 |
ABSTRACT |
Aggregation of blood platelets contributes to the arrest of bleeding
at sites of vascular injury, but it can occlude atherosclerotic arteries and precipitate diseases such as myocardial infarction. The
bonds that link platelets under flow conditions were identified using
confocal videomicroscopy in real time. Glycoprotein (GP) Ib and von
Willebrand factor (vWF) acted in synergy with
IIb 3 and fibrinogen to sustain platelet
accrual at the apex of thrombi where three-dimensional growth resulted
in increasing shear rates. The specific function of distinct adhesion
pathways in response to changing hemodynamic conditions helps to
explain hemostatic and thrombotic processes.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE CONTROL OF HEMORRHAGE at wound sites
requires platelet adhesion and aggregation,1,2 particularly
when bleeding involves arterioles, where rapid blood flow results in
high wall shear rates.3,4 In pathologic conditions, such as
in atherosclerotic arteries, ruptured plaques and elevated shear rates
at sites of stenosis5 may induce formation of platelet-rich
thrombi that become life-threatening when they occlude the vascular
lumen blocking blood flow.6,7 The first response to
vascular injury, whether in normal hemostasis or pathologic thrombosis,
consists of platelet adhesion to the damaged vessel wall or to exposed
tissue components, and is mediated by interactions that have a key
influence on subsequent thrombus growth.8 At high shear
rates, binding of the membrane glycoprotein (GP) Ib to immobilized
von Willebrand factor (vWF)9 is the necessary initial step
tethering platelets to reactive surfaces.10,11 However,
stable attachment also requires the synergistic binding of integrins
21 and IIb3
(GP IIb-IIIa complex) to their respective substrates.8,12
Notwithstanding the importance of initiating events, a thrombus is
constituted essentially of platelets linked to one another, not
directly to subendothelial components; thus, its development is
strictly dependent on the formation of interplatelet bonds. At present,
it is generally accepted that fibrinogen bound to activated
IIb3 is the predominant adhesive bridge
between aggregating platelets.13 Only at abnormally
elevated shear rates, typically greater than 5,000 s 1, is vWF thought to participate in the process by
binding to IIb3, in addition to GP Ib,
in alternative to fibrinogen.14-16 However, these concepts
have been suggested by the results of experiments with platelets in
suspension not interacting with a reactive substrate,15-17 thus under fluid dynamic conditions different from those that exist
when aggregating platelets are attached to a surface exposed to flowing
blood. To address this issue, we have studied the bonds that link
platelets during thrombus development in real time, using collagen type
I fibrils as a model substrate8 and confocal videomicroscopy for the three-dimensional quantitative evaluation of
aggregation in a flow chamber. Our findings demonstrate that vWF and
fibrinogen have distinct but synergistic roles in promoting interplatelet bonds at all but low venous shear rates. This novel understanding of the adhesive mechanisms involved in platelet aggregation during hemostatic and thrombotic processes suggests new
strategies for antithrombotic intervention.
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MATERIALS AND METHODS |
Preparation of blood specimens and perfusion studies.
Blood was collected from healthy volunteers using as anticoagulant
D-phenylalanyl-L-prolyl-L-arginine
chloromethyl ketone dihydrochloride (PPACK) at a final concentration of
80 µmol/L.8 Glass coverslips forming the bottom of the
perfusion chamber exposed to blood were coated with acid-insoluble
fibrillar type I collagen from bovine Achilles tendon (Sigma, St Louis,
MO) prepared in 0.5 mol/L acetic acid, pH 2.8, as previously
described.18 A silicone rubber gasket maintained a flow
path height of 254 µm,8,19 and flow rates were selected
to obtain the desired shear rates near the inlet of a variable linear
shear stress chamber.20 Blood was treated with mepacrine
(quinacrine dihydrochloride) at a final concentration of 10 µmol/L to
render platelets fluorescent, and was aspirated with a syringe pump
through the chamber mounted on the stage of a Zeiss
Axiovert 135M/LSM 410 invert laser scan confocal microscope (Carl
Zeiss, Oberkochen, Germany). The chamber was maintained at 37°C
with controlled airflow. When indicated, the platelet membrane
receptors GP Ib and IIb3 were blocked with the previously characterized monoclonal antibodies LJ-Ib1 and
LJ-CP8, respectively.9,21,22 IgG was purified from ascitic fluid.23 Confocal optical sections were obtained with a
488-nm laser and a scanning time of 2 seconds on an area of 102,236 µm2 during blood flow, with a 1-µm distance between
adjacent sections throughout the height of thrombi. All experiments
were recorded in real time.
Preparation of washed blood cells.
Blood cells were washed free of plasma constituents by repeated
centrifugation and resuspension in buffer using a method previously described in detail,21 but with two modifications:
(1) cells were sedimented at 2,100g for 15 minutes at
22°C to 25°C; and (2) the final suspension was prepared with
all blood cells at counts within 5% of the values in the original
specimen using HEPES-Tyrode buffer, pH 7.4, containing 50 mg/mL bovine
serum albumin, as well as 1.0 mmol/L Ca2+, 0.5 mmol/L
Mg2+, and 0.02 mmol/L Mn2+.
Fibrinogen22 and vWF24 were purified as
previously described.
Image analysis and volume measurement.
Images were analyzed with a commercial software package (Metamorph;
Universal Imaging Corp, West Chester, PA). A
threshold was applied to distinguish platelets from the
background, and the same value was then used for analyzing all the
stacks of confocal images collected for a given experiment. Thrombus
volume was calculated as the summation of partial volumes measured from
the area occupied by platelets in each optical plane multiplied by
height, ie, the z interval (1 µm) between adjacent
planes.8
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RESULTS |
Role of two membrane receptors in platelet aggregation at different
shear rates.
Platelets interacted with collagen within seconds from the beginning of
blood perfusion, and then rapidly with one another, accumulating into
thrombi firmly attached to the surface. In agreement with the results
obtained in stirred platelet suspensions,17,25 aggregation
in blood flowing with wall shear rate of 100 s1, a
value within the range that may be found in veins,3 was dependent on the function of integrin
IIb3, but not of GP Ib (Fig
1). The participation of
IIb3 in aggregation was still apparent
when the shear rate was increased to 300 s1, but
selective inhibition of GP Ib under these conditions limited the
height reached by thrombi in spite of normal initial growth (Fig 1). It
is known8 that vWF is not required to initiate platelet
adhesion to collagen fibrils at wall shear rates as high as 500 s1, which suggests that the effect of blocking
GP Ib on thrombus development at 300 s1 was not
likely to be the consequence of impaired platelet-surface interactions.
With further increase of the wall shear rate to 1,500 s1, a value within the range found in
arterioles,3,4 individual blockade of either GP Ib or
IIb3 completely hindered aggregation on
the surface (Fig 1). However, under these conditions, vWF and GP Ib
are absolutely required to initiate adhesion8; thus, conclusions on their role in supporting platelet aggregation cannot be
drawn from such results.
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| Fig 1.
(A) Structure of thrombi formed at different shear rates
in the absence (Control) or presence of monoclonal antibodies blocking
GP Ib (LJ-Ib1, 100 µg/ml) or IIb3
(LJ-CP8, 100 µg/ml), visualized by confocal optical sections at
1-µm intervals in the z-axis. Each plane shows an area of
52,345 µm2. For clarity, the z scale is expanded
and only sections at selected distances from the surface are presented.
Confocal sections were obtained after 840 seconds of flow at either 100 or 300 s1, or after 420 seconds at 1,500 s1. The inset in the middle panel displays the partial
reproduction with less size reduction of a thrombus formed at wall
shear rate of 300 s1, demonstrating the resolution of
single platelets in the confocal sections. The two insets in the bottom
panel show with greater magnification part of the surfaces exposed to
antibody-containing blood flowing with wall shear rate of 1,500 s1. No thrombi were formed, but single platelets could
easily be detected. Note that the confocal sections shown here are
intended to give a global view of thrombus formation and not to
visualize single platelets. (B) Total volume of thrombi calculated from
confocal sections. Representative experiment of two that gave
comparable results. Experiments at the shear rate of 1,500 s1 were terminated after collecting z sections at 420 seconds when thrombi had already reached a large volume. Prolonging
perfusion at this shear rate resulted in markedly abnormal flow
patterns owing to nearly complete occlusion of the chamber.
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An experimental approach was devised to analyze the role of adhesion
receptors in mediating interplatelet contact without interference from
possible effects on initial surface interactions influencing subsequent
aggregation.26,27 For this purpose, untreated blood was
perfused over collagen fibrils for 100 seconds at the wall shear rate
of 1,500 s1, and was then followed by blood that
contained function-blocking antibodies, or buffer as a control, at
either 1,500 or 300 s1. Thrombi, therefore, started
to form with normal anchorage to the surface (Fig
2), such that any consequence of receptor
blockade could only result from effects on platelet-platelet
interactions. Continuing perfusion with untreated blood at 300 or 1,500 s1 resulted in progressive thrombus growth (Fig 2).
As seen in Fig 2A for one experiment at 300 s1, this
growth in volume resulted from increasing height, as well as increasing
cross-sectional area in planes at a distance from the surface. On the
other hand, the base of thrombi did not change appreciably from the
development attained at the end of the initial perfusion at 1,500 s1 for 100 seconds (compare the bottom sections in
stacks A and B of the control experiment in Fig 2A), which implies that
surface coverage was already maximal or nearly maximal at that time. In contrast to these results, selective blockade of either GP Ib or
IIb3 reduced the continuing accrual of
platelets by 50% and 80%, respectively, when blood was perfused at
300 s1 (Fig 2). It should be noted that this effect
resulted from limiting the increase in height, as well as the
cross-sectional area of thrombi above a threshold distance from the
surface (Fig 2), with both factors contributing to reduced volume
increase. Thrombus growth was completely prevented when blood that
contained either antibody was perfused at 1,500 s1
(Fig 2), which confirms the role of both receptors in linking platelets
to one another.
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| Fig 2.
Progression of thrombus growth in 2-stage experiments,
first with perfusion of untreated blood over collagen type I fibrils at
1,500 s1 for 100 seconds, then continuing without
interruption at 300 or 1,500 s1 with blood containing
either buffer (Control) or specific monoclonal antibodies, as
indicated. Antibodies were incubated in blood for 10 minutes before
initiating perfusion. (A) Stacks of z sections labeled
A show thrombi formed after 100 seconds of perfusion with
blood containing no antibody at wall shear rate of 1,500 s1; those labeled B show the growth of thrombi
after an additional 740 seconds of perfusion at 300 s1
with blood containing either buffer or the indicated antibodies (total
perfusion time, 840 seconds). The effect of the antibodies in limiting
increase in thrombus height, as clearly seen, is representative of the
results observed in all experiments. (B) Thrombus volume was measured
at the indicated cumulative perfusion times in these two-stage
experiments. Bars are identified according to the type of inhibitor
used in the second stage, but perfusion for the first 100 seconds was
always performed with untreated blood. The results are expressed as
mean ± SEM of between 4 and 10 experiments for the different
conditions tested. Note that experiments at the shear rate of 1,500 s1 were terminated after collecting z sections at 420 seconds, since thrombi had already reached a large volume in control
blood. Experiments with the combination of anti-GP Ib and
anti-IIb3 monoclonal antibodies were not
performed at 1,500 s1 since at this shear rate each
individual antibody completely prevented thrombus growth as compared to
the volume attained after the initial perfusion for 100 seconds with
untreated blood.
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Synergistic interplatelet adhesive functions of fibrinogen and vWF at
arterial shear rates.
The demonstration that GP Ib, a vWF receptor,9
participates in platelet aggregation along with
IIb3, a fibrinogen as well as vWF
receptor,24 prompted an investigation on the roles of these
ligands in linking platelets to one another. At all shear rates tested,
only small thrombi formed in the absence of plasma proteins. Addition
of fibrinogen alone considerably increased thrombus volume at 300 s1, whereas vWF by itself was minimally effective at
this shear rate (Fig 3A). When the two
proteins were present together, the total volume of aggregated
platelets on the surface was similar to that seen with fibrinogen alone
(Fig 3A). Different results were observed at 1,500 s1, since not only did fibrinogen fail to support
platelet aggregation, but also vWF by itself promoted the development
of large thrombi in spite of increased shear stress (Fig 3A). The
apparent enhancement of vWF-mediated platelet cohesion with increasing
shear rate is consistent with previous findings demonstrating that vWF
binding to platelets, involving both GP Ib and
IIb3, is positively modulated by shear
stress.21 However, the platelet aggregates supported by vWF
were unstable, reaching their peak volume between 1 and 3 minutes and
then decreasing in size (Fig 3A and B). Real-time monitoring and
three-dimensional analysis showed that the reduction in volume after
formation of large thrombi in the presence of vWF at 1,500 s1 resulted from progressive detachment of single
platelets or small aggregates, causing decreasing height but leaving a
platelet monolayer on the collagen substrate (Fig
4). Only the concurrent presence of both
fibrinogen and vWF resulted in thrombi that resisted the forces
generated by rapid flow for at least 7 minutes (Figs 3 and 4). These
findings demonstrate that fibrinogen is required for stable platelet
aggregation even when hemodynamic conditions prevent the molecule from
exerting its adhesive functions directly. The efficient, albeit
transient, interplatelet cohesion initiated by vWF provides the
synergistic mechanism enabling fibrinogen to play its role at high
shear rates.
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| Fig 3.
(A) Time course of platelet thrombus development at wall
shear rate of 300 s1 (top) or 1,500 s1
(middle), obtained using washed blood cells with or without the
addition of fibrinogen (Fg) or vWF, either individually or concurrently
as indicated. The concentration of fibrinogen was 2 mg/mL,
corresponding to the lower limit in normal plasma, but in excess of the
amount needed to saturate IIb3; that of
vWF was 20 µg/mL, approximately twofold higher than in normal plasma
to compensate for the loss of larger multimers during purification. All
results are reported as mean ± SEM of between four and six
experiments for the different conditions analyzed. Open triangles in
the middle panel indicate the results obtained with washed cells plus
vWF in the presence of 100 µg/mL of the
anti-IIb3 monoclonal antibody, LJ-CP8.
(B) The results at wall shear rate of 1,500 s1 shown in
(A), but expressed as the mean ± SEM of the rate of change in
thrombus volume during successive time intervals.
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| Fig 4.
Color rendition of the shape and height of thrombi formed
on type I collagen fibrils at different wall shear rates. Washed blood
cells and adhesive proteins were used as described in the legend to Fig
3. Note that both at 300 s1 and 1,500 s1,
larger thrombi were present at later perfusion times in the presence of
vWF and fibrinogen (Fb) combined than either of the two ligands alone.
To obtain these pseudocolor images, stacks of confocal sections taken
throughout the height of thrombi at 1-µm intervals in the
z-axis were processed after fixing brightness and contrast.
Noise was reduced by applying a 3 × 3 median filter, and images were
binarized so that the gray scale value of all the pixels above a set
threshold was 255. The value of each pixel composing individual images
in a shear rate group was then multiplied by a factor, p/N, where p is
the plane number (distance from the surface in µm) corresponding to
the image, and N is the number of images in the stack including the
thrombus of maximum height in that group. After this operation, the
value of each pixel in a stack of confocal sections was proportional to
the position in the z axis, ie, to height, and could be used to
generate single pseudocolor images using for each pixel the maximum
calculated intensity value in the stack. Thrombi were larger in the
experiments performed at 300 s1 than at 1,500 s1 owing to longer perfusion times; thus, distinct
height calibration color scales were calculated for the two groups, as
shown.
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The ability of vWF to form bridges across platelets at wall shear rate
of 1,500 s1 depended not only on GP Ib (Figs 1
and 2), but also on IIb3, as shown by the
effect of a function-blocking antibody (Fig 3A) and in agreement with
the notion that both receptors bind this ligand concurrently under
flow.14,21 The rate of platelet aggregation during the
first 90 seconds of perfusion was slower when fibrinogen was added to
vWF in excess molar concentration to approximate the situation in
normal plasma (Figs 3A and B, and 4). The latter finding reflects the
known competition between the two ligands for
IIb3 occupancy,28 and
confirms the lesser efficacy of fibrinogen in promoting platelet
aggregation at high shear rates. At the physiologic concentrations in
normal blood, therefore, fibrinogen may limit the amount of vWF bound
to IIb3, but the consequent effect of
reducing the initial rate of thrombus growth is compensated by
increased stability in time. On the other hand, vWF contributed to
platelet aggregation in flowing blood even at the relatively low wall
shear rates, such as 300 s1, at which its
presence did not increase total thrombus volume (Fig 3A). Indeed, a
greater number of larger individual thrombi, rather than more numerous
and smaller ones, formed at this shear rate when vWF was present in
addition to fibrinogen (Figs 4 and 5). This
result reflects enhanced efficiency of adhesive bonds, since platelets
must oppose greater hydrodynamic forces (drag) to aggregate into
thrombi of increasing size with higher shear rates at the growing edge.
A similar function may be supported by vWF released from the
-granules of activated platelets,29 which explains why
at the shear rate of 300 s1 blocking GP Ib was
more inhibitory of thrombus formation (Figs 1 and 2) than removing vWF
from the fluid phase (Fig 3A).
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| Fig 5.
Size distribution of thrombi formed on type I collagen
fibrils at different wall shear rates. Washed blood cells were perfused
after suspension in buffer only (Control) or with the addition of
fibrinogen (Fg) or vWF, individually or concurrently as indicated. The
concentration of fibrinogen was 2 mg/mL, and the concentration of vWF
was 20 µg/ml (see Fig 3). At the indicated time points, a single
image was obtained from the arithmetic sum of all those in a confocal
series, and the area of thrombi thus projected on the surface was
determined. Thrombus size was arbitrarily classified as small when the
latter value was less than 300 µm2, and large when it was
greater than 300 µm2. Height and shape were not
considered in these definitions; thus, the classification is not
directly related to volume. The results shown are the mean ± SEM of
four to seven experiments performed for each condition tested.
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DISCUSSION |
The results of these studies elucidate a novel interpretation of the
mechanisms that mediate homotypic platelet aggregation during thrombus
development in blood flowing at arterial shear rates. In fact, the
long-held contention that fibrinogen and
IIb3 have an exclusive role in linking
platelets to one another appears to be applicable only to the
hemodynamic environment of veins in which, however, these processes may
be less directly relevant for hemostasis.1,2,30 Moreover,
our findings modify two additional concepts currently held as valid,
namely, that vWF may have a role in platelet aggregation but limited to
pathologically extreme shear rates,31 and that the
functional involvement of fibrinogen decreases as that of vWF becomes
manifest.16 On the contrary, our results demonstrate that
the two adhesive proteins are complementary and synergistic, as they
are both needed to support thrombus development at all levels of
arterial flow. This proposed interpretation of the mechanisms of
aggregation provides a plausible explanation for the altered hemostatic
properties of platelets from patients with isolated congenital
deficiency of either fibrinogen32 or vWF.33
Because neither protein by itself can sustain the full development of
stable thrombi within the range of pathophysiologically relevant flow
conditions, hemostasis cannot be normal unless both are present and functional.
The synergy of fibrinogen and vWF in supporting platelet aggregation
depends on the recognized ability of each of these molecules to
establish bonds with distinct adhesive properties. The initial function
of vWF may depend primarily on its rapid rate of association with GP
Ib.8,10 This is particularly important in hemodynamic conditions characterized by high flow rates that increase the velocity
differential between adjacent platelets. Subsequent interaction of
multimeric vWF with both GP Ib and activated
IIb314,21 may favor optimal
propagation of interplatelet contacts, allowing permanent bridging
mediated by fibrinogen binding across activated platelet membranes. The
latter interaction appears to be crucial owing to the intrinsic
stability of the association between fibrinogen and
IIb3. In fact, the same distinctive
characteristics of adhesive bonds are thought to explain the efficient
but transient platelet tethering to immobilized vWF at high shear rates
as opposed to the immediate arrest onto fibrinogen (or fibrin) that,
however, involves fewer platelets with increasing flow
velocity.10 In this respect, therefore, adhesive
interactions between the platelet membrane and a thrombogenic surface
or between two platelets may depend essentially on the same mechanisms
to oppose fluid dynamic forces generated by blood flow. Thus, the
threshold shear stress value above which the functions of vWF and GP
Ib become absolutely required for thrombus development may be
similar for both heterotypic adhesion of platelets to extracellular
matrix components and homotypic aggregation. In fact, the unique
biomechanical properties of this bond may support the estimation of
approximate shear rate values in areas of blood flow depending on
whether blocking vWF binding to GP Ib results in inhibition of
platelet adhesion or aggregation. The results obtained by measuring
single platelet adhesion in flow fields with minimal
distortion8,10 suggest that a positive finding in this
regard may indicate a shear rate value of 1,500 s1
or higher.
Such considerations may explain why blocking GP Ib binding to vWF
negatively affects thrombus growth even when blood is perfused with
shear rates that do not prevent normal initiation of platelet adhesion
and aggregation.8 Computing shear rate values around thrombi is complicated by distortions in the flow field caused by the
growing mass of aggregating platelets. Nevertheless, it is intuitive
that in areas at the edge of a thrombus the shear rate will increase as
a function of distance from the wall because narrowing of the lumen
will cause increased local flow velocity for a constant volumetric
flow. In fact, the experimental results shown in Fig 1A indicate that
blocking GP Ib when the initial wall shear rate was 300 s1 started to impair platelet aggregation at the tip
of thrombi 5 to 8 µm high. By analogy with the value at which vWF
function is needed to initiate interactions of single platelets on a
substrate,8,10 it seems reasonable to assume as a first
approximation that inhibition of thrombus growth happened when the
local shear rate reached the value of 1,500 s1.
The results presented here support the conclusion that only the
functional integration of bonds with distinct biomechanical properties
may provide efficient and stable platelet adhesion with subsequent
thrombus development through interplatelet cohesion at normal and
pathologic arterial shear rates. These findings, moreover, identify the
vWF-GP Ib interaction as a target for the selective inhibition of
platelet aggregation aimed at preventing and treating arterial
thrombotic diseases.
| |
ACKNOWLEDGMENT |
The authors thank James R. Roberts and Benjamin Gutierrez for their
assistance with the productions and characterization of monoclonal
antibodies; Richard A. McClintock for the purification and
characterization of proteins; and Rachel A. Braithwaite for expert
secretarial help.
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FOOTNOTES |
Submitted December 3, 1998; accepted March 2, 1999.
Supported in part by Grants No. HL-31950, HL-42846, and HL-48728 from
the National Institutes of Health (NIH). Additional support provided by
NIH 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 Zaverio M. Ruggeri, MD, The Scripps
Research Institute, MEM-175, 10550 N Torrey Pines Rd, La Jolla, CA
92037; e-mail: ruggeri{at}scripps.edu.
| |
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TOP
ABSTRACT
INTRODUCTION