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The Journalof
The American Society of Hematology
BLOOD
VOL 88, N O S
SEPTEMBER 1,1996
REVIEW ARTICLE
Platelets and Shear Stress
By Michael H. Kroll, J. David Hellums, Larry V. Mclntire, Andrew 1. Schafer, and Joel L. Moake
C
LINICAL BLEEDING or thrombosis results from a
disturbance in the balance between a complex network
of procoagulant and anticoagulant factors. This network involves three primary interactions, first described by the eminent pathologist Rudolph Virchow during the previous century, between blood (soluble and cellular constituents), the
blood vessel (including fixed and dynamic responses), and
blood flow. This review examines one increasingly recognized feature of this interplay that appears to affect both
hemostasis and thrombosis. This feature is the effect on
blood platelets of the mechanical forces of shear generated
by flowing blood. Knowledge of the link between platelets
and shear stress provides clues to general mechanisms governing physiologic and pathologic processes by re-emphasizing the importance of physical forces in regulating cellular
function in vivo.
Platelets have long been regarded as the preeminent cell
involved in physiologic hemostasis and pathologic thrombosis. In both cases, the plasma protein von Willebrand factor
(vWF) and the blood platelet work together to effect the
biologic response. The versatility in biologic responses mediated by a single receptor-ligand coupling has been, and
remains, one of the challenging conundrums placed before
clinical investigators and practicing hematologists. It is all
the more intriguing when one considers that mixing vWF
with platelets in a static or stirred suspension evokes no
response. The discovery that the mechanism of toxicity of
the antibiotic ristocetin is its capacity to induce plasma vWF
to bind to platelet glycoprotein (GP) Ib’ stimulated numerous
important discoveries about the structure and function of
vWF and its platelet receptors, including the nosology of
von Willebrand (vWD) disease.’-4 However, elucidation of
mechanisms of hemostasis and thrombosis in vivo required
the convergence of classical pathology with modem molecular biology. One hundred and forty years after it was proposed, Rudolf Virchow’s concept of the pathogenesis of
thrombosis holds the key to understanding the relationship
between platelets and shear stress.
Virchow’s triad is a concept that is old but does not recidivate; rather, it confronts observations and their interpretation
within a context of the interrelated pathophysiologic elements of blood, blood vessel, and blood flow. It is the application of Virchow’s principles to the puzzle of vWF-mediated platelet aggregation that ultimately led to the discovery
Blood, Vol 88,No 5 (September I), 1996:pp
1525
1525-1541
that rheological factors mediate the binding of vWF to platelets in vivo, and that shear stress appears to be the physiologic, or pathophysiologic, equivalent of ri~tocetin.’.~
This
review will attempt to summarize our current understanding
of the molecular basis of Virchow’s triad operating in platelet-dependent arterial thrombosis, and to explore the mechanismsby which rheologic and vascular factors interact to
effect platelet-dependent hemostasis.
The scope of the problem of arterial thrombosis is staggering: at least 5 million adults in the United States alone
suffer from related symptoms. About 50% of the annual
nonaccident deaths in the United States are caused by
thrombi predominantly composed of platelets in coronary or
cerebral arteries. In addition, thrombosis of these and the
peripheral arteries causes extensive morbidity. Antiplatelet
agents presently available in clinical practice, which were
originally tested predominantly in static or stirred systems,
are only moderately effective in preventing the development
or recurrence of thrombotic diseases of the coronary and
cerebrovascular circulation.’ To improve the therapy of arterial thrombotic disorders, therefore, it is imperative to establish basic mechanisms of platelet thrombus formation under
conditions of arterial blood flow. In vitro systems capable
of modeling flow-mediated platelet adhesion and aggregation
have been developed to investigate the mechanisms by which
mechanical forces affect platelet thrombus formation. The
reconstitution in vitro of the components of Virchow’s triad
operating in vivo in pathologically constricted arteries is
likely to provide improved methods for the evaluation of
From theVA Medical Center, Buylor College of Medicine and
Rice University, Houston, TX.
Submitted August 7, 1995; accepted March I , 1996.
Supported by Public Health Service Grants No. HL02311,
HL54169, HL18584, HL36045, HL18672,HL 32200, and NS23327;
the Research Service of the Department of Veterans’ Affairs; The
Robert A. Welch Foundation; and the Advanced Technology Program of the State of Texas. This work was undertaken during the
tenure of an American Heart Association Established Investigator
Award to M.H.K.
Address reprint requests to Michael H. Kroll,MD, Section of
Hematology-Oncology (IIIH), Houston VA Medical Center, 2002
Holcombe Blvd, Houston, TX 77030.
0 1996 by The American Society of Hematology.
0006-4971/96/8805-08$3.00/0
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KROLL ET AL
1526
new antithrombotic agents. Modem investigations of mechanisms of platelet adhesion and aggregation must go beyond
static conditions and begin to account for rheologic variables
that affect physiologic and pathologic responses.
Table 1. Typical Ranges of Wall Shear Rates and Wall Shear
Stresses, Assuming That the Viscosity of Whole
Blood Is 0.038 Poise
Wall Shear Rate
Blood Vessel
PRINCIPLES OF RHEOLOGY
In the past several years, engineering principles have been
used to design model systems that simulate flowing conditions in injured, narrowed, or branched arteries.*These models are used to generate controlled mechanical forces in vitro
similar to those produced by hemodynamic forces in vivo.
The mechanical forces produced in blood vessels may result
from (1) luminal pressure changes that cause bloodflow
(which yields shear stress), or ( 2 ) transmural pressure
changes that cause circumferential deformation of the layers
of the vessel wall during the cardiac cycle (caused by tensile
stress). Fluid shear stress (in dynes per square centimeter)
is the force per unit area generated byflowof
a viscous
liquid.
Tensile stress produces strain, which is a measure of the
percent change in vessel circumference generated bythe
expansion and contraction of the luminal diameter. Strain is
the force which would tear a cell, or layer, from its normal
position within the vessel wall. One effect of tensile stress,
and the strain it produces, is to stretch individual cells that
are anchored.
The mechanical force most relevant to platelet-mediated
hemostasisand thrombosis is shear stress. Shear stress is
defined as “the force per unit area between laminae”; and
blood flow can bedescribed as an “infinite number of infinitesimal laminae sliding across one another, each lamina suffering some frictional interaction with its neighbors.”’ Liquid shear stress in a tubular blood vessel is the force per
unit area applied to thebloodwithintheflowing
stream.
Wall shear stress is the force per unit area applied by the
flowing viscous blood to the vessel surface (Fig 1).
In the relatively highflow environment of the arterial
circuit, blood is a suspension that approximately behaves as
a Newtonianfluid.’ This meansthat shear stress ( T ) in a
tubular chamber having radius r and flow direction z can be
represented by the mathematical formula: T = -p(dv,/dr),
(or shear rate) and
with dvJdr the local velocity gradient
Velocity vz
vz = 0 at the wall
Shear rateyz
= 0 at centerline
y
Yw
Fig 1. Blood flow in a tubular chamber generates a parabolicflow
velocity profile. This results in maximal velocity and minimal shear
at the center of the blood flow stream, and minimal velocity and
maximal shear at the vessel wall. It is the difference in velocity between laminae of flowing blood that generates shearing forces.
Large arteries
Arterioles
Veins
Stenotic vessels
(/S)
300-800
500-1,600
20-200
800-10,000
Wall Shear Stress
(dynes/crn21
11.4-30.4
19.0-60.8
0.76-7.6
30.4-380
Reprinted with permission from AlevriadouBR, Mclntire LV: Rheology, i n Loscalzo J, Schafer AI (eds): Thrombosis and Hemorrhage.
1995, p p 369-384.’86Reprinted by permission of Blackwell Science,
Inc.
viscosity ( p ) the proportionality constant. This formula for
shear stress holds throughout the fluid, including the immediate vicinity of the tube wall, where it yields the wall shear
stress. In Fig 1, the shear rate dv,/dr is designated as y . As
illustrated in the figure, the shear rate (and hence, the shear
stress) varies continuously through the fluid from zero at the
centerline to a maximum at the wall. For a Newtonian fluid,
the relationship between shear stress and shear rate [T =
-p(dv,/dr)] allows one to convert simply from shear stress
to shear rate: shear rate(incm/s per cm or S”) = shear
stress/viscosity. For example, in whole blood witha viscosity
of 0.038 poise, a wall shear stress (T,) of 90 dynes/cm2
results in a wall shear rate of 2,368 S”. Wall shear stress of
Newtonian fluids for flow intubular vessels can be calculated
as a function of volumetric flow rate: T , = 4pQ/77r3, where
p is viscosity, Q is the volumetric flow rate, and r = radial
distance of the tubular chamber. This formula has been used
to calculate shear forces in vivo (Table 1).
Steady Newtonian flowthrough a cylinder generates a
parabolic velocity profile and a linear distribution of shear
stresses, with liquid components (eg, platelets) at the periphery of the cylinder (the wall) subjected to maximal shear
stress and liquid components at the center of the cylinder
subjected to zero shear stress (Fig 1). Time-averaged blood
flow through the arterial circuit is often modeled using this
simplified approach, andwall shear stress data basedon
this model form the foundation for much of the information
discussed in this review. However, blood flow in vivo is far
more complicated, and rheologic variables related to eddy
formation, pulsatility, and dynamic constriction may predominate under many physiologic and pathologic conditions.
Fluid shear stresses in the circulation are imposed on the
surfaces of blood cells and blood vessels. Within a liquid
stream, freely moving particles rotate in the shear field and
are therefore subjected to time-varying (sinusoidal) shear
stress. The time-average of this shear stress is the liquid
shear stress applied by viscometers (see below). However,
the time-average of shear stresses sensed by platelets is not
identical to that sensed by an ideal spherical particle. This
is because platelets have an irregular shape (unactivated
platelets are discoid and activated platelets become rounded
with many irregular tendrils) that creates a surface subjected
to tangential shearing forces even near the center of a flowing
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PLATELETS AND SHEAR
stream at some distance from the vessel wall. The precise
level of shear stress on platelets inflowingblood
is not
known. However, it is known that platelets in whole blood
are pushed toward the periphery of flow nearer to the maximal shear stresses generated at the vessel wall.
It should also be emphasized that thrombus formation is
a dynamic process, and the components of Virchow’s triad
operating within the developing thrombus continually
change over time. For example, suspended platelets passing
through stenotic arteries are transiently subjected to elevated
shear stresses,” whereas adherent platelets and platelets accumulating in the developing thrombus are continuously subjected to elevated shear stresses until flow is decreased by
the occlusive thrombus.” Flow probes placed in arteries and
arterioles in animal models of occlusive vascular disease
have been used to provide data on the complex rheologic
forces operating in vivo.’o.”
DEVELOPMENT OFTHEHYPOTHESIS THAT SHEAR
AFFECTSPLATELET FUNCTION
There was little research in arterial thrombosis and relatively little understanding of platelet function before 1960.
In 1962, BornI2 devised a simple method for studying the
response of platelets to chemical agonists. The method involved recording changes in light transmission of a stirred
platelet suspension as platelet aggregates are forming. This
“platelet aggregometer” had a profound effect on the study
of platelet reactions, and it rapidly came into routine and
widespread use. Because platelets are stirred to maintain a
suspension, platelet-platelet interactions in the platelet aggregometer take place under the influence of fluid mechanical shear stress. However, the shear stress is low and it
varies throughout the platelet suspension in a complex and
uncharacterized way. Therefore, the aggregometer is inadequate for studying platelet responses within the context of
Virchow’s triad.
By the middle of the next decade, quantitative rheologic
methods began to be used to study platelet reactions under
controlled shear stress, and several workers established the
importance of the shear field itself as a determinant of plateletreactions.’”’’ In 1975 Brown et all’first reported the
direct effect of shear stress on platelets in a cone-plate viscometer. These experiments were undertaken within a research program to try to develop an artificial heart, and the
hypothesis under investigation was that shear stress generated by intracardiac devices leads to mechanical platelet
damage. These investigators observed that pathologic levels
of shear stress (>50 dynes/cm2) applied to platelet-rich
plasma (PRP) induced changes in platelet morphology, along
with secretion and aggregation. The cause of these changes,
vis a vis platelet activation versus platelet lysis, was uncertain, although lysis was minimalat shear stresses below
250 dynes/cm2. In 1978 Weiss et
reported the first
experiments to demonstrate that platelet adhesion to a vascular surface isboth shear rate and vWF-dependent. These
investigators used a concentric cylinder flow system over
everted rabbit aorta, and citrate- and non-anticoagulated
blood from patients with vWD (in which the ligand, vWF,
1527
-!-
+l
,Cone
le
Fig 2. One type of viscometer used to generate shear stress involves a cone rotating on a plate, with thelevel of shear stress proportional to thecone angle (@l and the rotation rate (01. In this device,
the shear stress within a suspension of platelets, or at the plate
surface. is constant and uniform.
is deficient or defective) and Bernard-Soulier syndrome (in
which the platelet GpIbAXN receptor complex is deficient).l5.IhShear forces at low (venous) levels had no measurable effect on platelets, but as the magnitude and duration
of shear forces were increased, measurable changes developed in the three indices of platelet response (adhesion, secretion, and aggregation). These observations suggested that
shear stress, like chemical agonists, shows consistent
“dose’’-and time-response characteristics. Based on these
early experiments, it was concluded that the threshold stress
for any specific response depends on two factors: ( I ) plateletsolid surface interactions and (2) the duration that platelets
are subjected to the shear stress. These prescient studies not
only led the field of platelet physiology to develop a novel
paradigm of stimulus-response coupling within Virchow’s
triad, but they formed the basis for the new and fertile field
of biorheology.
To investigate the mechanism of the direct effects of shear
on platelets it therefore became necessary to attempt to eliminate platelet-surface interactions from the experimental systems. Although platelet reactions with the solid surfaces of
damaged blood vessels are essential for hemostasis and
thrombosis, in vitro studies of the direct effects of shear
stress on platelets (ie, effects independent of platelet-surface
interactions) require that surface interactions be minimized
or eliminated. Eliminating platelet-surface interactions is important notonly because it allows one to investigate the
direct effects of bulk fluid shear stress on platelets in vitro,
but also because it provides a model in which to investigate
mechanisms of thrombosis under shear stress conditions that
mimic arterial stenosis in the presence of an intact vascular
endothelium.
The instrument of choice for experiments to investigate
the importance of bulk shear stress versus surface effects
came to be a rotational viscometer. There are many rotational
viscometer prototypes, the mechanical designs of which have
been described in
Some have been
modified
to
apply pulsatile shear stress,24and others have been adapted
to allow real-time optical measurements of platelet aggregation and calcium flux.” The two most used designs are (1)
a stationary bob placed within a rotating concentric cylinder
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KROLL ET AL
1528
an artifact of cell lysis, but rather depends on the presence
of plasma vWF and functional platelet receptor complexes
GpIbIIXN and GpIIb-IIIa. There is indirect evidence that
fibrinogen may be the bridging ligand effecting platelet aggregation at shear stresses below 12 dynes/cm2 however,
at shear stresses greater than 10 to 12 dynedcm’. platelet
secretion and aggregation depend on vWF and platelet GpIb/
I X N and GpIIb-IIIa, and are independent of plasma or platelet fibrin~gen.~’”’
vWF is a multivalent, multimeric plasma protein that is
essential for platelet adhesion to thesubendothelium of damvessel^.^ vWF has binding sites for platelet
agedblood
GpIba and GpIIb-IIIa, and for various subendothelial constituents, including collagen types I, 111, and VI. vWF bridging the platelet GpIbIIXN complex to the subendothelium
leads to adhesion in vivo, and vWF bridging GpIb/LX/V on
adjacent platelets leads to the cohesive inter-platelet interactions induced by ristocetin in vitro. Under shear stresses in
vitro and in vivo, vWF binding to the GpIbIIXN complex
is critically important for platelet adhesion andaggregation.’x
In the plasma milieu under static conditions, vWF binding
to GpIIb-IIIa is minimal,” but when high shear stresses are
applied to platelets, vWF binds to GpIIb-IIIa as well as to
the GpIbIIXN complex, and this binding contributes substantially to direct shear-induced platelet aggregate formation.4”,4’Larger vWF multimers support shear-induced platelet aggregation more effectively than smaller multimers,”
but the minimal vWF valency requirements for shear-induced platelet aggregation have not been established. ADP,
whether released from platelets or derived from red blood
cells, contributes substantially to shear stress-induced platelet responses.3’ When platelets are pretreated with epinephrine before shearing, a synergistic aggregation response is
ob~erved.~’.~’
These results indicate that the effect of shear
forces on platelet thrombus formation within the complex in
vivo milieu is modified by chemical agonists. This may be
one reason why coronary artery occlusion occurs in patients
with relatively low-grade stenoses.
Shear-induced,vWF-mediated platelet aggregation requires functional vWF binding sites on both the platelet
GpIb/IX/V complex and GpIIb-IIIa.’h~37
It does not require
ristocetin, other exogenous agents, or chemical modification
of vWF. Evidence accumulated to date indicates thatthe
predominant effect of elevated fluid shear stress is on the
MECHANISMS OF SHEAR STRESS-INDUCED
platelet vWF receptors, rather than on plasma vWF, affecting
PLATELET AGGREGATION
an ephemeral state of platelet glycoprotein receptivity for
vWF.’” These shear-sensitive platelet membrane components
Much of the early literature about the effects of bulk shear
may be a part of the GPIb/IX/V ~ o r n p l e x The
. ~ ~mecha~~~~~
stresssuggestedthatshearstressdirectlyactivatesplatelets,
although there was little information about mechanisms.”~’4~’7 nism by which this occurs is unknown, but it has been hypothesized that the initial interaction betweenmultimeric
Some workers believed that shear stress didnot directly
vWF and platelet GpIba serves as a nidus for a growing
cause platelet activation, but rather caused platelet lysis,
irregular clump of platelets in an evolving aggregate that is
thereby yielding extracellular concentrations of stored platesubject to increasing liquid shearing forces.41This growing
let agonists, principally adenosine diphosphate (ADP),
irregular clump may also serve as a microenvironment that
which subsequently activated the remaining platelets.29Howconcentrates various platelet agonists, including epinephrine
ever, in 1986 Moake et
applying information derived
and ADP, resulting in the amplification ofan initial shearfrom other laboratories using flow systems that model platerelated stimulus. The initial shear-induced triggering event
let-surface interaction^,^'.^^ determined that platelet aggregahas so far been elusive, suggesting that it is a very subtle
tion in response to pathologically elevated shear stress is not
(the Couette viscometer); and (2) a rotating cone whose
center is placed in proximity (-25 pm) to the center of a
flat plate (the cone-plate viscometer). Both designs generate
a constant and uniform shear stress within the liquid suspension. The cone-plate viscometer offers the advantage of an
open suspension, allowing for fairly rapid removal of liquid
for analyses (Fig 2). Cone-plate viscometers can also be used
to analyze platelet-surface reactions by anchoring cells a n d
or extracellular material on the plate.2h-28
The basic geometry
of the cone-plate viscometer does not change with the specific device, and experimental results are remarkably consistentusing a variety of cone-plate viscometers. The shear
rate in this system is nearly constant throughout the volume
between the cone and the plate. It is directly proportional to
the rotations per minute of the cone, and inversely proportional to the gap angle between the cone and the plate. The
gap angle used to generate arterial-level shear forces is typically in the range of 0.30 to 1 .OO degrees, and the viscosity
(in poise) of a suspension of cells is constant through the
range of shear stresses applied experimentally. Thus, a constant shear stress can be applied to a suspension or a monolayer of cells placed into this rotational viscometer by maintaining a constant revolutions-per-minute of the cone on the
plate. This type of rotational viscometer is capable of generating shear stresses from less than 2 dynes/cm2(venous level)
to 20 to 30 dynes/cm2 (arterial) to greater than 200 dynes/
cm2 (as occurs in stenosed coronary, peripheral, or cerebral
arteries).
To evaluate directly the effects of bulk fluid shear stress
on platelets, it is first necessary to eliminate as thoroughly
as possible all platelet-surface interactions in the cone-plate
viscometer. Platelet-surface interactions can be minimized
by coating the shear stress-generating surfaces with silicone
or another nonthrombogenic material. This results in platelet
responses to shear remaining nearly constant over a large
range of surface to volume ratio^.'^,'"'^.*' The silicone-coated
cone-plate viscometer therefore provides researchers with a
reliable in vitro system to apply shear stress to cell suspensions for the analysis of bulk phase platelet responses. These
devices are used to investigate mechanisms of platelet responses to shear stress, as well as to explore the physiologic
or pathologic significance of shear-induced platelet activation and aggregation.
-
’‘;
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LATELETS AND SHEAR
ffect that is difficult to “trap” for study by extant experinental techniques.
Elevations of platelet cyclic AMP or cyclic GMP inhibit
hear stress-induced platelet adhesion and aggregati0n.4~”~
n contrast, inhibition of cycloxygenase by acetysalicylic
cid has little effect on the initiation of aggregation in reponse to shear stress35;this may account for the relative
ack of potency of aspirin as antithrombotic therapy for some
rterial thrombotic disorders. Except for EDTA (see below),
nost anticoagulants have little affect on shear stress-inb e d platelet responses. For example, one study shows that
he extent of shear-induced platelet aggregation in a cone)late viscometer is similar in PRP anticoagulated with solium citrate, recombinant hirudin, unfractionated heparin,
md low-molecular-weight heparin (LMWH).48However, re,ults from other studies suggest that there may be a small
nhibitory effect of unfractionated heparin on shear-induced
Aatelet a g g r e g a t i ~ nand
~ ~ a modest inhibitory effect of com)ined hirudin and LMWH on shear-dependent platelet
hrombus formation on ex vivo perfused diseased vessel^.^'
ihear stress-induced platelet aggregation is, however, inhibted by fibrinolytic agents5’This effect is caused by plasminnediated proteolysis of vWF rather than proteolysis of memxane glycoproteins.
SHEAR STRESS-INDUCED PLATELET SIGNALING
The mechanisms by which shear stress induces platelet
Iggregation are not yet precisely defined, but specific intra:ellular activation signals are involved in the process. When
uspensions of washed platelets are subjected to different
evels of uniform shear stress in an optically modified cone
md plate viscometer capable of simultaneously monitoring
:ytoplasmic ionized calcium ([Ca”Ii) and aggregation of
Ilatelets, a IO-fold increase of [Ca”], develops. This elevaion of [Caz+liis accompanied by synchronous aggregation,
md both responses are dependent on the shear force and
T W F . EGTA
~~
chelation of extracellular Ca2+ completely
nhibits vWF-mediated increases in [Ca”], and aggregation
‘esponsesto shear stress. Blockade of vWF binding to GpIba
ilso completely inhibits both of these platelet responses to
;hear stress. The tetrapeptide RGDS and monoclonal anti)odies that inhibit the binding of cohesive ligands to GpIIb:IIa are less effective inhibitors of shear stress-induced in:reases in [Ca’+li and platelet aggregati~n.’~.~’
Blocking the
:ffectofADP
released from stimulated platelets inhibits
;hear stress-induced platelet aggregation without affecting
he shear-induced increase in [Ca2+]i.25
Neither the [Ca2+Ij
lor aggregation response to shear stress is inhibited by
)locking platelet cyclooxygenase with acetylsalicylic acid.25
These results indicate that GpIba and extracellular
-a 2 t are absolutely required for vWF-mediated [Ca2+Iiand
iggregation responses to shear stress. Shear stress-induced
:levations of platelet [Ca2+Ii,but not aggregation, are inde)endent of the effects of released ADP, and both responses
xcur independently of platelet cyclooxygenase. These red t s are consistent with the model that shear stress induces
he initial binding of vWF multimers to platelet GpIba. The
IWF-GPIba interaction then causes an increase oflCa2+],
1
1529
and platelet aggregation, both of which are potentiated by
vWF binding to the activated platelet GpIIb-IIIa complex in
the presence of released ADP.
To validate the hypothesis that vWF binding to platelets
causes activation resulting in secretion and aggregation,
shear stress-induced vWF binding to platelets was investigated. Using complementary approaches (radioligand binding and immunologic tags identified byflow cytometry),
direct measurements of shear-induced vWF binding have
been ~ b t a i n e d . ~ .vWF
~ ’ . ~binding
~
to platelets under shear
has been difficult to quantify because of the variables of
vWF multimeric composition and platelet secretion of stored
vWF from a-granules. Nonetheless, certain findings have
been consistent: shear stress induces less total vWF binding
than does ristocetin, only a minority of platelets bind vWF,
the binding is reversible and not saturable, and both GpIba
and GpIIb-IIIa contain the sites of shear-induced vWF binding. Binding studies suggest that vWF attachment to GpIba
precedes vWF binding to GpIIb-IIIa, a sequence that is consistent with the hypothesis that the initial vWF-GpIblIXN
interaction activates the GpIIb-IIIa c ~ m p l e x . ~ .Shear
~’
stress-induced activation of GpIIb-IIIa, reported as an increase in monoclonal antibody PAC- 1 fluorescence, has been
shown.54
Efforts have been made to determine how shear stressinduced vWF binding to GpIba initiates signals for activating the GpIIb-IIIa complex. Althoughthe specific second
messengers involved in switching GpIIb-IIIa into a ligandreceptive conformation are not yet identified, the switch involves a network of signals including [Ca“], , protein kinase
C (PKC), and other serine-threonine kinases, tyrosine kinases, and phosphatidylinositol 3-kinase~.”-~~
Within this
context, it has been observed that platelet aggregation induced by a pathologic level of arterial wall shear stress (90
dynes/cm2) is associated with the phosphorylation of pleckstrin, an M, 47,000 PKC substrate ( ~ 4 7 ) . ’Shear-induced
~
p47 phosphorylation depends entirely on vWF binding to
platelet receptors, and the inhibition of PKC activity suppresses the aggregation response to shear. Interestingly, in
contrast to activation by chemical agonists, shear stressinduced platelet PKC activation occurs independently of any
measurable change in diacylglycerol or hydrolysis of phosphatidylinositol4,5-bisphosphate.These results indicate that
when mechanical shear stress induces vWF to bind to platelet
GpIba, a diacylglycerol-independent pathway of PKC is activated that contributes to platelet aggregation. The molecules responsible for activating PKC under these conditions
are not yet known.Platelet tyrosine kinases are also activated
in response to pathologic shear stress (90 dynes/cm2), although the functional importance of this is uncertain.60As
with shear stress-induced elevations of [Ca2+Iiand PKC,
tyrosine kinase activation in response to shear in most cases
also depends on vWF binding to platelet GpIba and GpIIbIIIa. Cytochalasin D, which inhibits GpIbalcytoskeletal interactions, inhibits the tyrosine phosphorylation of an M, =
76,000 substrate. This M, 76,000 protein, along with p r ,
has been observed to translocate to the platelet cytoskeleton
during the course of shear-induced platelet activation, and
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1530
this is associated with functionally significant phosphatidylinositol 3-kinase (PI 3-kinase) activity.m”These results suggest that tyrosine phosphorylation and PI 3-kinase activity
may be involved in shear stress-induced aggregation.
Although the molecular mechanisms of shear stress-induced stimulus-response coupling are presently unknown,
most signaling is likely to be initiated by vWF binding to
platelet GpIba. GpIb is a transmembranous heterodimeric
leucine-rich glycoprotein having M, 165,000 (composed of
GpIba M, 143,000 disulfide-linked to GpIb@M, 22,000) that
forms a noncovalent complex with GpIX and GpV; there
are approximately 25,000 GpIbAXN complexes per platelet.
The intracytoplasmic domain of GpIba interacts extensively
with the platelet cytoskeleton,6@This interaction between
the primary receptor for vWF and the submembranous cytoskeleton appears to form a component of the transmembranous structural apparatus linking extracellular shear forces
with intracellular signaling.m The GpIba chaincanbe
lengthened by a genetic polymorphism that adds 13 amino
acid tandem repeats to the mucin-like macroglycopeptide
region,and this had led to speculation that this region may
be susceptible to shear-induced conformational alterations.“
Once bound to vWF, platelet GpIba appears to function
as a signal molecule, although the mechanism of GpIbainitiated signaling is not characterized. One hypothesis proposed to explain the mechanism of shear-induced platelet
activation states that platelet GpIba, following shear-induced
binding of VWF?’,~’undergoes a conformational change that
directly, or indirectly through one or more coupling proteins,
triggers signals for cellular activation. Studies of thrombinplatelet interactions have shown that a-thrombin bindsto
platelet GpIba and that this may affect platelet signal transduction. There is also indirect evidence in support of the
hypothesis that the GpIbAXN complex is involved in signal
transduction: CAMP, which inhibits platelet activation, promotes the phosphorylation of the @-chainof GpIb6’ and decreases thrombin binding to platelets.”
Shear stress-induced vWF-mediated platelet signaling
develops generally under conditions associated with aggregation, suggesting that vWF bridging platelets is required
for shear stress-induced activation, and that vWF-mediated
platelet signaling is inseparable from vWF-mediated aggregation.25,52.59,h0This phenomenon is different from what happens to platelets activated by strong chemical agonists such
as a-thrombin, which stimulate phospholipase C-mediated
second messenger formation even in unstirred and unaggregated suspensions.
Studies by Huang and HellumsMshow that both collision
frequency (the number of platelet-platelet contacts per unit
time) and collision efficiency (platelet-platelet collisions that
result in cohesion, or aggregation) are directly related to
platelet concentration and shear ~ t r e s s . ~Therefore,
’
itis
likely that shear stress promotes platelet aggregation, at least
in part, by increasing both the numberandefficiencyof
platelet-platelet collisions. Increased collision efficiency at
higher shear stresses may result from the direct effects of
shear stress on platelets (perhaps specifically on GpIblIW
V and GpIIb-IIIa) to induce shear-dependent vWF binding
KROLL ET AL
resulting in platelet a g g r e g a t i ~ n . ” ~ ~It~ ,is~ ’also
~ ~ ’possible
that an ephemeral alteration and “activation” of vWF occurs
under theinfluence of pathologic shear stress, although direct
evidence in support of this is currently l a ~ k i n g . ~A~primary
,~’
goal in this field of research isto untangle andrankthe
various factors in Virchow’s triad thatcontribute to the initiation of shear stress-induced vWF-mediated platelet aggregation. It is becoming increasingly clear that signal transduction mechanisms that mediate platelet aggregation by
chemical stimuli (agonist-receptor interactions) and shear
stress may be distinct.
SHEAR STRESS-INDUCEDPLATELETADHESION
In humans, physiologic time-averaged mean shear stress
levels in the arterial circuit reach 20 to 30 dynes/cm2 (shear
rates of whole blood equal to 500 to 750 S - ’ ) , and pathologic
levels (ie, as in a stenosed coronary artery) may reach >350
dynes/cm2 (shear rate of 8,750 s ” ) . ’ ~ . ~ ’The
. ’ ~ latter clinical
situation is often associated with platelet thrombus formation. As described above, pathologic stenosis can directly
lead toshear-induced aggregation of platelets from the blood
in constricted arteries. Elevated liquid or wall shear stresses
also lower the threshold concentration atwhich platelets
become activated in response to chemical agonists, such as
e~inephrine.~’,~?
Of equal or greater importance, however,
is adhesion of blood platelets onto exposed atherosclerotic
subendothelium in a region of endothelial cell desquamation
(for example, after atherosclerotic plaque rupture), with extensive subsequent platelet aggregation.
There are many established systems for the in vitro investigation of platelet thrombus formation after adhesion, some
of which are described briefly in Table 2.66”4These experimental systems have yielded extensive data that form the
foundation for many of the conclusions presented inthis
review. It is interesting to note that, despite the heterogeneity
of design in constructing in vitro models of arterial thrombosis, identical bioengineering principles define the majority
of the phenomena observed in different flow chambers. Consequently, some general conclusions can be made regarding
mechanisms of arterial thrombosis initiated by platelets adhering to damaged blood vessels in vivo.
To begin, one should first describe the phenomena observed in any flow chamber into which “subendothelium”
is placed. However, the interpretation of such phenomena
must account for differences in the composition of the subendothelial tissue depending upon the site and species from
which it is derived. Nonetheless, it is clear that in all cases
platelets adhere, change shape (“spread”), and aggregate
(“thrombus formation”) when whole bloodis perfused over
de-endothelialized mammalian vessel segments. These responses depend on mechanical shear forces. The range of
shear stresses over which platelet adhesion and subsequent
aggregation are observed is approximately 1 to 200 dynes/
cm’. Within the range of elevated shear stresses (>30 dynes/
cm’), platelet thrombus production depends on vWF binding
to platelet GpIb/IX/V and GpIIb-IIIa.’5~16~1y~~1~~4~4”~hy
The
source of vWF can be plasma,”.“ platelet a-gran~les.’~~’~
or
the subendothelial extracellular matrix.” Larger vWF
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PLATELETS AND SHEAR
1531
Table 2. Different Systems for Studying Shear Stress Effects on Blood Cells and Blood Vessels
Type
Cone plate isc comet er^^^^'^^^^^^
Annular
Tubular chambeP7*"
Parallel plate ~ h a m b e r ~ ' . ~ '
Flow through viscometer"
Filter aggregation
Continuous flow
microaffinity co~umn"
Exposure Time
Seconds to
Seconds to
Seconds to
Seconds to
<l
S
Unknown
<l S
hours
minutes
minutes
minutes
Shear Stress
(dyneslcm')
< l to >200
<l0 to 800
<IO to >250
< l to >500
>1,000
Unknown
15-52
Surface
Measurement
None, cells, protein, ECM
Everted aorta
ad, sec, agg, biochemistry
ad, agg
ad, sec, agg
ad. agg
Protein, ECM, prosthetic
None, cells, protein,
ECM, prosthetic
None
Glass fibers
Protein
sec
Platelet retention
ad, morphology
Abbreviations: ad, adhesion; sec, secretion; agg, aggregation; ECM, extracellular matrix; prosthetic, prosthetic surfaces.
multimers are more effective than smaller vWF multimers
at promoting platelet thrombus formation in perfusion experiment~.~
Platelet
'
GpIb/IXN is involved in the initiation of
adherent platelet-subendothelial interactions under arterial
level shear stress, and v W F attachment to GpIIb-IIIa is required for subsequent aggregation and thrombus propagat i ~ n Acetylsalicylic
.~~
acid treatment of blood does not inhibit shear-dependent platelet thrombus formation in parallel
plate flow chamber~,4~,~'
but it has been reported to have
a moderate inhibitory effect in other flow systems.79PG12
(prostacyclin) derived from endothelial cells inhibits shearinduced platelet thrombus formation on subendothelium.80
Shear-induced platelet thrombus formation appears to promote the generation of fibrin through the activity of soluble
coagulation factors.8' This is very important because, in the
arterial circulation, the higher shear stresses associated with
increased flow tend to dilute out certain procoagulant molecules, such as fibrinogen and perhaps prothrombin and
thrombin, thereby preventing the formation of insoluble fibrin. The platelet-dependent arterial thrombus represents a
dynamic rheological process, however, and as luminal diameter decreases, so does blood flow and flow velocity, such
that fibrin generation ultimately develops. Based on in vitro
studies of whole blood perfused over vessel wall constituents
(eg, collagen") it is clear that the activation of the soluble
coagulation systems is a secondary, but significant, event in
shear-induced thrombus formation that occurs in vivo after
platelet adhesion and aggregati~n.'~
Platelets subjected to elevated shear stresses do not adhere
to an intact endothelial cell m~nolayer.~'
Platelets do, however, bind tightly to exposed subendothelium, become activated to express a functional GpIIb-IIIa receptor complex,
and subsequently aggregate.76Many candidate proteins have
been studied in perfusion systems in attempts to define the
critical vessel wall factors operating in Virchow's triad of
arterial thrombosis. Collagen, insoluble vWF, fibrin(ogen),
thrombospondin, laminin, and fibronectin have been investigated as potential mediators of shear-induced platelet-surface
interactions. In addition, platelets interact with prosthetic
surfaces under shear conditions, and these interactions mediate vascular graft occlusions.
Collagen. In constructing a model of subendothelium,
the following observations are relevant: (1) fibrillar collagen
types I and 111 are present in highest concentrations, along
with collagen types N ,V, and VI, in human adult arteriesws8;
(2) most of the collagen in the relatively pristine subendothelium of arterial vessels during childhood is type IIIS9;
( 3 ) both collagen types I and I11 increase in the thickened
subendothelium of atherosclerotic arteries; however, most of
the accumulating collagen is type IN; and (4) collagen types
I, 111, and VI bind to V W F . ~ ' - % Monomeric vWF contains
two binding sites for collagen types I and 111, only one of
which may be physiologically relevant.94The vWF binding
site for type VI collagen is uncertain.y3
Most studies of shear-induced platelet-collagen interactions in the three types of perfusion chambers have used
morphometric or radioactive techniques to evaluate platelet
accumulation at the end of an experiment. Recently, the
combination of parallel plate perfusion (Fig 3 ) with computerized epifluorescence videomicroscopy has allowed the observation, videotaping, and three-dimensional representation
of platelet adhesion and aggregation in real-time. Because
results have been generally consistent using different systems,48.Y7.Y8
a description of the dynamics of platelet-collagen
interactions in the parallel plate flow chamber in real-timeg7
willbeusedas
a paradigm of the basic mechanisms of
platelet thrombus formation over bovine type I collagen
monolayers in the presence of shear stress. It should be noted
that that there is experimental evidence using an annular
perfusion chamber that collagen is nottheprimary
vWF
binding site within a complex subendothelial milieu.92 However, more recent studies of transverse sections of human
coronary artery atheromatous plaques show that the "thrombogenicity" of excised endothelialized plaques subjected to
64 dyneskm' shear stress in a parallel plate perfusion chamber results only from plaque collagens type I and 111 binding
to platelets through a process that depends, at least in part,
on plasma vWF."
In the parallel plate flow chamber, the number of individual platelets and platelet monolayers per unit area, a measure
of platelet adhesion, increase during the initial 15-second
exposure to 60 dyneskm' wall shear stress. As individual
platelet thrombi develop and merge into larger aggregates
at 30 and 60 seconds of flow, the number of individual
adhesive events decrease concomitantly. In this system, the
total number of platelets deposited from normal blood onto
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KROLL ET AL
1532
5% CO2 in Air
2
I
I
1
f
I
I
Fig 3. A schematic of the parallel plate flow chamber. The fluid
pumped through the flow chamber can be collected or recirculated.
The interaction of blood cells with thevessel wall can be monitored
in real-time by epifluorescent videomicroscopy. This type of flow
chamber was used to generate the computer analysis of shear-dependent adhesion and aggregation shown in Fig 4.
collagen-coated slides using different anticoagulants (0.38%
citrate, 10 U/mL unfractionated porcine heparin, 1 U/mL
LMWH, or 200 U/mL recombinant hirudin) is not significantly different.
Three-dimensional reconstructions of the thrombi formed
using blood from a patient with severe vWD in the presence
or absence of purified vWF are shown in Fig 4. In severe
vWD blood (with < 1.5 U/dL plasma vWF and undetectable
platelet vWF forms) without vWF, platelet adhesion after
60 seconds' exposure to 60 dynes/cm*is much less than that
observed using severe vWD blood in the presence of 100
U/dL of purified vWF. Because of the decreased adhesion
in severe vWD blood, subsequent platelet aggregation is also
considerably diminished. This is further evidence in support
of the conclusion that there is direct adhesion-aggregation
coupling. Figure 4 also shows that the release of stored vWF
from shear-activated platelets has a significant effect on
platelet thrombus formation. Comparative studies of severe
vWD platelets in normal plasma, versus normal platelets in
severe vWD plasma, confirmthatbothplasma
vWF and
vWF released from platelets contribute to platelet thrombus
formation. Platelet adhesion onto collagen-coated surfaces
is inhibited by a monoclonal antibody to GpIba. Under identical shear conditions, there is greater platelet adhesion onto
collagen using severe vWD blood than in the presence of
an anti-GpIba antibody (-30% v -5% of controls, respectively), suggesting that only a small quantity of vWF immobilized with collagen is sufficient to initiate platelet adhesion
via GpIba. Either an anti-GPIIb-IIIa antibody, or an antibody
against the RGD sequence in vWF that mediates vWF binding to GpIIb-IIIa, is less effective as an inhibitor of platelet
surface accumulation than the anti-GpIba antibody. In platelets deficient in GpIIb-IIIa, or in the presence of inhibitors
of GpIIb-IIIa, platelet adhesion is minimally inhibited?X,7s.7h
In contrast, subsequent platelet aggregation is almost eliminated. ADP promotes platelet aggregation subsequent to initial adhesion, but blockade of platelet cyclooxygenase has
no significant effect on either platelet adhesion or aggregation under the conditions of these perfusion experiments.
It is uncertain if direct collagen-platelet interactions potentiate vWF-dependent platelet adhesion onto insolubilized
collagen fibrils. Under nonshear conditions platelets directly
bind collagen throughat least three receptors (GpIdIIa,
CDIV. and CDVI"). GpIdIIa-initiated platelet-collagen interactions underlow shear conditions operate throughthe
activation of the GpIIb-IIIa complex.""
vWF. Compared with vWF multimers from
normal
plasma, insoluble subendothelial vWF derived from endothelial cells may be more active in initiating platelet adhesion
under flow.'" Insoluble vWF alone does not support rapid
platelet attachment in the absence of shear forces. However,
the threshold wall shear stress for platelet adhesion to vWFcoated surfaces appears to be somewhat lower thanthe
threshold liquid shear stress for direct platelet aggregation
mediated by soluble vWF."'* Both GpIba and GpIIb-IIIa are
involved in shear-dependent platelet adhesion onto vWFcoated perfusion chambers.'".""
It is known that vWF becomes rapidly insolubilized onto
the exposed subendothelium of human arteries, as well as
onto collagenous and noncollagenous components of the vesselwall,andthat
these events may precede and augment
platelet adhesion.'"l"04As previously described, in vitro experiments indicate that vWF multimers immobilizedwith
human collagen type I attach predominantly to platelet GpIb/
I X N receptors, suggesting that a single platelet site for vWF
binding is exposed under these conditions to effect adhesion.
The relative importance of direct platelet-collagen binding
compared to direct platelet-vWF binding within a complex
subendothelial milieu isnot established, although studies
from excised atheromatous human coronary arteries provide
data that both are involved in platelet adhesion under elevated shear stress conditions.'"
Fihrin(c1gen). Plasma fibrinogen is cleaved by thrombin
to fibrin monomers that insolubilize by polymerization and
are subsequently cross-linked by a transglutaminase (factor
XIII). Fibrinogen can be found on the surface of vascular
endothelium,"15 and fibrinogen and fibrinare present in atherosclerotic plaques."'6 Fibrinogen is also deposited on vascular prostheses, where itis the major ligand mediating platelet thrombus growth on artificial
Platelets
deposit onto fibrin-coated coverslips in a perfusion chamber
at12, 18, and 52 dynes/cm*.'"'~''' At the highest of these
shear stresses platelet thrombus formation on fibrin is relatively more dependent on vWF and Gplba, whereas at the
lower shear stresses platelet thrombus formation on fibrin
is relatively more dependent on GpIIb-IIIa.'"' It has been
suggested thatunder pathologically elevated shear stress
conditions, GpIIb-IIIa binding directly to fibrin"slows
down" the passing platelets, but that vWF bridging fibrin
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PLATELETS AND SHEAR
1533
m
4
I-.
'
m
u
b
Severe V W D Blood + 100% Purified vWF
60 sec of Flow
Severe vWDBlood, 60 sec of Flow
I
P-
-
---
v
b
,
i
Healthy Donor, 60sec of Flow
Fig 4. These are computer-generated three-dimensional topographic representations platelet
of
thrombi formed on Bovine type I collagen.
The results presented show adhesion (x andy planar surface) and aggregation (z-axis height) of platelets from citrate-anticoagulated whole
blood subjected to 60 dyneslcm' for 60 seconds. Note that replacing plasma vWF levelsto normal only partially restores the adhesion and
aggregation of platelets on type I collagen. Thisis so because platelet secretion of stored vWF
is required foroptimal adhesion and aggregation
in response to elevated shear stress. (Reprinted with permission.-)
to GpIbrw "locks" the platelets into a stable thrombus capable of remaining attachedunder high shear stresses.'w.''' The
vWF binding site(s) on fibrin(ogen)have not been identified.
Other subendothelialmatrix proteins. Platelet adhesion
to fibronectin occurs only at low shear stresses (eg, 0 to 12
dynedcm'). Evidence has been presented that this adhesion
is partly dependent on the platelet integrins GpIIb-IIIa and
GpIcDIa (VLAS), which directly interact with fibronectin."'
Adhesion is also completely dependent on vWF and
GpIb~x."~
Because there is no evidence that either vWF or GpIblIXN
binds directly to fibronectin, these results suggest that adhesion is maintained only whenvWF forms an adluminal interplatelet scaffolding through GpIb/IX/V come~tions."~Fibronectin
may
also enhance platelet
accumulation
on
collagens type I and In under low shear conditions.'I4Laminin also supports platelet adhesion under low shear conditions (4dynes/cm2).'I5This depends on platelet VLA-6, but
is independent of vWF, GpIbIIXN, and GpIIb-IIIa. Thrombospondin supports platelet adhesion in a shear stress-dependent manner,withmaximal adhesion at 64 dynes/cm2.''6
This adhesion is also apparently independent of vWF, GpIb/
I X N ,and GpIIb-IIIa.The platelet receptor that binds thrombospondm under shear conditions in these studies has not
beenidentified,but is not GpIIb-IIIa,GpIa/IIa, GpIV, or
Cu,/3,.''6
EFFECTSOFSHEARSTRESS
ON VESSEL WALL
REGULATIONOFPLATELETTHROMBUS
FORMATION
Situated at the interface betweenbloodand the vessel
wall throughout the circulatory system, endothelial cells are
strategically positioned to respond to hemodynamic forces.
These forces include shear stress and strain. Strain is the
abluminal force that results from wall shear (the force which
would"tear"a
cell from its adherent state), resulting in
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1534
vascular distention that affects endothelial cells and subendothelial components (eg, smooth muscle cells and extracellular matrix). Flow-induced hemodynamic forces signal endothelial cells to modulate the synthesis and release of a variety
of vasoactive and antithrombotic substances, the general net
effect of which is to promote blood fluidity. The phenomenon of flow-induced adaptive
is mediated
by the release of vasoactive products, such as PGIz (prostacyclin) and endothelium-derived relaxing factor or nitric oxide (NO), from shear-stimulated endothelial cells.’2o”” Endothelial cells mayact as shear sensors in their role of
regulating flow-dependent arterial diameter under normal
and pathologic condition^.'^^ In addition, shear stress induces
endothelial cells to release platelet inhibitory and fibrinolytic
products, which, in the presence of intact endothelium, tend
to counterbalance the proaggregatory effects of pathologically elevated shear stress in the arterial circulation.’26
PG12 acts as both a potent vasodilator and inhibitor of
platelet aggregation by raising intracellular levels of CAMP
in vascular smooth muscle cells and platelets, respectively.
Shear stress stimulates PG12 synthesis by endothelial
~ e l l s . ~Using
~ ’ ~ ‘a ~parallel-plate
~
flow chamber with an immobilized monolayer of endothelial cells, it has been observed that the sudden onset of flow abruptly increases PGIz
production, followed by a decrease in production to a steady
state within several minutes. The steady-state release of
prostacyclin by endothelial cells subjected to pulsatile shear
stress is more than double that released by cells exposed to
constant shear stress.lZ8These findings indicate that endothelial cells release bursts of PGIz in response to step increases
in shear stress.129PG12 is likewise produced by endothelial
cells subjected to the cyclic strain or stretch which occurs
with each pulse wave.13”,’3’
NO is also both a potent vasodilator and an inhibitor of
platelet adhesion and aggregation. These effects are mediated by elevations of intracellular levels of cyclic GMP in
smooth muscle cells and platelets. NO is the by-product of
the conversion of L-arginine to L-citrulline by a family of
NO synthases (NOS).’32One of the isoforms of this enzyme
is a membrane-associated, Ca2+/calmodulin-dependent,
constitutive NOS (cNOS), which is found in endothelial cells
(hence termed “eNOS”)’33”35and can bedistinguished from
the constitutive NOS present in neuronal tissue.’36The release ofNO from endothelial cells is stimulated by both
shear stress137-145
and cyclic train."^.'^' The stimulation of
endothelial cell NO production in response to both shear
stress’42and cyclic strain’43is at least partly regulated by
upregulation of cNOS mRNA. The response to shear stress
is greater when flow is pu1~atile.l~~
Similar to the observations with shear-induced PG12production, exposure of endothelial cells to the sudden onset of laminar flow results in a
rapid burst of NO release that increases further with the
superimposition of increases in flow above the preexisting
Because eNOS is Ca2+/calmodulin-dependent,
the
initial rapid endothelial cell synthesis of NO in response to
shear stress (as well as chemical agonists) has an obligatory
requirement for an increase in the concentration of cytosolic
free Ca2’ 140.144.14s
KROLL ET AL
Tissue plasminogen activator (tPA)-generated plasmin is
notonly fibrinolytic butcan also proteolyze large vWF
multimers and, under certain conditions, inhibit platelet function.147.148 Asnoted previously, tPA and plasmin inhibit
shear-induced platelet aggregation. Arterial levels of shear
stress (>15 dynes/cm2) stimulate secretion of tPA by endothelial cells, whereas the secretion of plasminogen activator
inhibitor type 1 (PAI-1) remains unaffected by shear stress
over the physiologic range.149This enhancement of the fibrinolytic potential of endothelial cells in response to hemodynamic forces is at least partly transcriptionally regulated,
as tPA mFWA is increased by laminar shear
The mechanisms by whichthe principal hemodynamic
forces of shear stress and cyclic strain transduce signals to
alter the structural and functional properties of endothelial
(and other) cells are poorly understood. In addition, fluid
shear stress and cyclic strain mayuse different signaling
pathways, because downstream gene regulation and protein
secretion are quite different in response to these two mechanical stimuli.’” “Mechanotransduction” may be initiated directly, by shear-induced changes in the state of flow sensors
at the luminal surface of endothelium’s2or elsewhere in the
cell after transmission of the force via the cytoskeleton.’”
Alternatively, mechanotransduction may occur indirectly by
changes in local endothelial cell surface concentrations of
chemical agonists created by the convective transport of
these mediators from the bulkfluid to the cell boundary
layer.’54~‘ss
As reviewed by Davies and Tripathi,’“ hemodynamic forces generate a diverse set of intracellular signals
in endothelial cells: some signals develop extremely rapidly,
and these maybe followed after several hours by altered
gene expression and by cytoskeletal reorganization if the
forces are sustained. Rapid responses to flowmay involve
second messengers that are indistinguishable from those
that result from chemical agonist-receptor coupling in vascular cells, including activation of different ion channe~s,llS.lS00,1S7.1S8 increases
’
in cytosolic free Ca2+,1s9”h3
and
membrane phosphoinositide turnover with the formation of
inositol 1,4,5-trisphosphate (IP3)1a~’h6
and activation of
PKC.Ih7It has been proposed that rapid responses to flow
may activate two signal transduction pathways in endothelial
cells. One pathway is Ca2+-dependentand involves activation of phospholipase C and increases of [Ca”], , whereas a
second pathway is Ca2’-independent and involves a guanine
nucleotide regulatory (G) protein-mediated activation of
PKCand mitogen activated protein (MAP) kinases.lh8In
contrast to these second messenger-mediated rapid responses, a delayed response of endothelial cells to flow may
reflect the upregulation of expression of certain genes. The
pathways through which externally applied hemodynamic
forces signal the endothelial cell nucleus to activate gene
transcription may involve second messengers or cytoskeletal
reorganization. In addition, a cis-acting shear stress-responsive element has been identified inthe promoter ofthe
PDGF-b gene.” This sequence is also present in the promoters of other endothelial cell genes upregulated by shear
stress, including thetPAgene.The
relationship between
shear-stimulated transcription factor^'^^.^'^ and their binding
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PLATELETS AND SHEAR
1535
sites in the promoter regions of shear-responsive genes is an
area of considerable interest. The transcriptional factor NFKB, which accumulates in the nuclei of endothelial cells
exposed to fluid shear stress, binds to the shear stress response element and has been implicated in the transactivation of endothelial cell genes responding to rheologic stimu1i.171
Genes that do not contain the shear stress-responsive element in their 5' promoter regions, such as the tissue factor
gene, also respond to shear stress. More recently, it has been
found that the phorbol ester TPA-responsive element (TRE)
may be a functional shear responsive element, leading to
shear-induced immediate (or early) gene expression in endothelial and other cells.172Further characterization of these
and other positive (and negative) shear-responsive regulatory
elements, and the proteins that regulate them, will facilitate
our understanding of biomechanical stimulation of endothelial cell function.173Thus, observations to date indicate that
shear stress stimulates the production of certain endotheliumderived mediators (eg, prostacyclin) by activating constitutive stimulus-response effector mechanisms, and the production of other mediators (eg, tPA) by activating gene transcription.
The modulation of platelet activation by shear-activated
vascular cells is more complex than the above-described
shear effects on the release of individual endothelium-derived vasoactive and platelet inhibitory mediators. Shearinduced release of endothelium-derived prostaglandins, NO
and tPA, may act synergistically to inhibit platelet activation.174-176 On the other hand, the platelet inhibitory actions
of NO are quenched by hemoglobin in whole bl00d.I~~
Nevertheless, NO may still be able to inhibit platelet thrombus
formation over areas of intact endothelium because, in flowing blood, red blood cells tend to stream to the center of
the vessel lumen whereas platelets distribute to the lumen
periphery where they could be exposed to inhibitory concentrations of NO. Furthermore, endothelial cells interact, directly or via humoral mediators, with other constituents of
the vessel wall (eg, vascular smooth muscle cells and extracellular matrix), which may further modify platelet responses
to shear stress.'25
In summary, shear stress can directly activate platelets.
Under normal circumstances, however, intact endothelial
cells exposed to the same rheologic forces produce a potent
array of vasodilatory and platelet inhibitory mediators that
mitigate the activation of platelets by shear stress to preserve
blood fluidity. However, when the vessel wall component
of Virchow's triad is perturbed these endothelium-derived
factors cannot oppose direct shear stress-induced platelet
aggregation and the shear-induced platelet activation and
aggregation that occur subsequent to initial platelet-subendothelia1 adhesion. The result is platelet thrombus formation
associated with downstream tissue ischemia and infarction.
HEMOSTASIS
Experimental data from in vitro flow systems and in vivo
models of arterial thrombosis provide a solid foundation
for the concept that mechanical shear forces affect platelet
thrombus formation under conditions of physiologic and
pathologic arterial flow. There are also data that provide a
reasonable hypothesis to explain why proaggregatory shear
stresses under physiological arterial flow (inthe vast "microvasculature") don't result in spontaneous thrombosis: shear
in the arteriolar circuit simultaneously stimulates endothelial
cell release of antiaggregatory factors17' (see above). However, these facts do not necessarily explain primary hemostasis. It is established that platelet-vessel wall interactions are
required for hemostasis, and that hemostasis depends on
vWF, platelet GpIbnXN, and platelet GpIIb-IIIa. Primary
hemostasis must also occur in the low shear vasculature:
venules and capillaries. Is it possible, therefore, to relate
shear-dependent mechanisms of thrombosis to mechanisms
of hemostasis? Once again, clues that provide a solution to
this puzzle are gathered if one places its pieces within the
context of Virchow's triad.
If the blood factors responsible for primary hemostasis and
thrombosis are heldconstant,buttherheologicfactorsthat
affect hemostasis and thrombosis are different, one might look
closely at vessel wall factors that direct a hemostatic response
as possibly being different from those that effect thrombosis.
As described above, both fibronectin and laminin support low
shear-induced platelet adhesion, but the adhesioni s vWF-independent.Therefore,itbecomesnecessarytoconsiderother
components of the vessel wall that could be involved in vWFdependent hemostasis at low shear stresses.
Type VI collagen has been identified as a component of
subendothelial microfibrils capable of binding VWF.~'.''~
Type VI collagen is codistributed with vWF in the subendothelium of human umbilical vein endothelial cells in the
apparent absence of collagen types I and 111. The pattern of
distribution of collagen VI in the subendothelium of normal
arteries and veins, or atherosclerotic arteries, is notyet
known. It has recently been reported that platelet adhesion
and subsequent aggregation on purified type VI collagen in
a parallel plate flow chamber are extensive at low shear rates
(4 dyneskm'). In contrast, little adhesion occurs at high
shear rates (40 dyneslcm2). These shear-related effects are
exactly opposite to those observed with collagen type I.y7The
low-shear platelet deposition on type VI collagen depends on
vWF, GpIba, and GpIIb-IIIa. Experiments using whole
blood from a patient with severe vWD suggest that there
may also be some vWF-independent interactions between
platelets and type VI collagen.'*' These interactions may be
mediated by platelet GpIa-IIa.18' It is also possible that platelet adhesion to fibronectin and/or laminin could contribute
to vWF-independent adhesion atlow shear
Therefore, it is possible that a multistep process develops
rapidly in the low-shear environment of cutaneous and mucosal terminal venules and capillaries (eg, in bleeding time
wounds) that involves direct platelet interactions with fibronectin, laminin, and/or type VI collagen, and indirect interaction between platelets and type VI collagen through vWF
bridges to GpIbIIXN or GpIIb-IIla.
CLINICAL CONDITIONS
The importance of shear stress-induced platelet aggregation in human pathology is mostly inferred. The most rele-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
KROLL ET AL
1536
vant clinical condition epidemiologically is arterial stenosis
caused by chronic atherothrombotic occlusion of the coronary, carotid, and peripheral arteries. There is evidence that
agents that interfere with shear stress-induced platelet responses may beneficially affect these diseases and improve
clinical outcome.
The in vivo administration of the mouse-human chimeric
monoclonal Fab fragmentagainst GpIIb-IIIa (c7E3; 0.25 mg/
kg; Centocor, Malvern, PA) to patients in association with
coronary angioplasty results in almost completeinhibition of
shear-dependent aggregation subsequent to vWF-mediated
adhesion onto type collagenI for at least 24 hours after 7E3
infusion.lRZ Thesein vitro observations are associated with
improved reperfusion and diminished reocclusion of the obstructed coronary artery following angioplasty.IR3 Integrelin
(CorTherapeutics,SouthSanFrancisco,CA),
a cyclic
KGD-containing heptapeptide that antagonizes the binding
of fibrinogen andvWF toplatelet GpIIb-IIIa, has been investigated in angioplasty patients alsoreceiving aspirin and heparin. Blood was collected before and during Integrelin infusion (bolus of 135 mg/kg, followed by 0.5 or 0.75 mg/kg
min). As with c7E3, vWF-mediated platelet adhesion to collagen I was not inhibited at any time-point by in vivo Integrelin. In contrast, using blood collected after 45 minutes of
Integrelin infusion, there was a significant decrease in
the
size of theplateletaggregatesthat
formedsubsequentto
vWF-mediated adhesion at high shearstress. There was also
an associated -50% inhibition by the infused Integrelin of
ex vivoplatelet aggregation induced by > 100 dynes/cm2
shear stress in the cone-plate viscometer. No studies to date
elucidate the clinical importance of inhibiting vWF-platelet
GpIb/IX/V interactions.
It has been observed that larger vWF forms are considerably more effective, compared with smaller vWF multimers
purified from normalcryoprecipitate,
in mediating fluid
shear stress-induced platelet aggregation (see above). This
observation was recently extended to a clinical situation. It
has been found that children with chronic relapsing thrombotic thrombocytopenicpurpurahave
“unusually large”
(UL) vWF forms in their plasma in association with excessive shear stress-induced platelet aggregation in vitro at 90
to 180 d y n e s / ~ m ~ .ULvWF
’*~
forms (normally secreted abluminally by endothelial cells) may be especially effective in
promoting the adhesion of platelet G p I M X N to subendothelium containing collagens I, 111, or VI. Theexcessive
release of ULvWFmultimersinto thebloodstream in response to endothelial cell toxins may be fundamental to the
pathophysiology of TTPand thehemolytic-uremic
syndrome.’XS
FUTURE DIRECTIONS
It will be necessary to refine further in vitro model systems
of shear stress that more closely simulate normal and pathological conditions of flow invivo. Thismustbedoneto
elucidate fully the complex
interactions between cellular and
extracellular matrix components of the vessel wall that are
likely to modulate both vascular responses to shear stress,
as well as the intravascular responses of platelets
to shear
stress. The contributions of other bloodconstituents, particularlyred bloodcellsand
leukocytes,havelikewisebeen
incompletely defined in the process of platelet-vessel wall
interactions under various conditions of shear stress. The
observation that elevated shear stress activates platelets via
Gplba pathways that are different from chemical agonists
suggests the possibility of “lesion-specific” anti-thrombotics that affect platelets under conditionsof high shearwithout
perturbing the molecular interactions that result in hemostasis. Further investigations are required to determine if this
hypothesis is valid.
ACKNOWLEDGMENT
Wethank Bill Durante for critically reviewing the manuscript.
We also thank Dianna Wilson, Andrea Bethel, Patty Ryan and Rita
Laddimore for assistance with manuscript preparation, and Nancy
Turner for providing Fig 4.
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1996 88: 1525-1541
Platelets and shear stress
MH Kroll, JD Hellums, LV McIntire, AI Schafer and JL Moake
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