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Next Article 
Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1149-1155
Cytosolic Calcium Changes in a Process of Platelet Adhesion and
Cohesion on a von Willebrand Factor-Coated Surface Under Flow
Conditions
By
Mitsuhiro Kuwahara,
Mitsuhiko Sugimoto,
Shizuko Tsuji,
Shigeki Miyata, and
Akira Yoshioka
From the Department of Pediatrics, Nara Medical University,
Kashihara, Nara, Japan.
 |
ABSTRACT |
Recent flow studies indicated that platelets are transiently
captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to
be activated, thereafter becoming firmly adhered and cohered on the
surface. In exploring the mechanisms by which platelets become
activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca2+]i)
concomitantly with the real-time platelet adhesive and cohesive process
on a vWF-coated surface under flow conditions. Reconstituted blood
containing platelets loaded with the Ca2+ indicators Fura
Red and Calcium Green-1 was perfused over a vWF-coated glass surface in
a flow chamber, and changes in [Ca2+]i were evaluated
by fluorescence microscopy based on platelet color changes from red
(low [Ca2+]i) to green (high [Ca2+]i)
during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s 1, no change in
[Ca2+]i was observed during translocation of platelets,
but [Ca2+]i became elevated apparently after platelets
firmly adhered to the surface. Platelets preincubated with anti-GP
IIb-IIIa antibody c7E3 showed no firm adhesion and no
[Ca2+]i elevation. The intracellular Ca2+
chelator dimethyl BAPTA did not inhibit firm platelet adhesion but
completely abolished platelet cohesion. Although both firm adhesion and
cohesion of platelets have been thought to require activation of GP
IIb-IIIa, our results indicate that [Ca2+]i elevation
is a downstream phenomenon and not a prerequisite for firm platelet
adhesion to a vWF-coated surface. After platelets firmly adhere to the
surface, [Ca2+]i elevation might occur through the
outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand,
thereby leading to platelet cohesion on the surface.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
PLATELET THROMBUS formation represents an
essential physiologic defense mechanism in primary hemostasis to arrest
bleeding at sites of vessel disruption and can contribute to generation of pathologic intravascular thrombosis.1,2 von Willebrand factor (vWF) plays a central role in both physiologic and pathologic thrombus formation.3 Indeed, the critical contribution of
vWF to processes of platelet plug generation, especially under high shear rate conditions, has become clear through platelet functional studies that take physiologic blood flow situations into
consideration.4-6 Recent analysis of platelet function on a
vWF-coated glass surface in a real-time flow system under high shear
rates showed that the interaction between surface-immobilized vWF and
platelet glycoprotein (GP) Ib leads to the rapid but transient capture
of flowing platelets onto the surface.7 This initial
tethering of platelets results in platelet translocation along the
surface. During translocation, the GP IIb-IIIa complex is assumed to
become gradually activated, presumably through an intraplatelet signal
generated from the vWF-GP Ib interaction. This activation, in turn,
leads to firm platelet adhesion through binding of activated GP
IIb-IIIa to immobilized vWF. Cohesion of platelets firmly adhering to
the surface occurs thereafter through the binding of soluble adhesive proteins, such as vWF or fibrinogen, to activated GP IIb-IIIa, resulting in platelet aggregate accumulation on a vWF-coated
surface.7,8
Most studies to clarify the molecular mechanisms by which platelets
become activated during platelet aggregation processes have been
conducted in static or closed stirring experimental conditions.9,10 However, little is known about the precise mechanisms involved in the real-time process of platelet adhesion and
cohesion on a thrombogenic surface under physiologic blood flow
conditions, perhaps because of the technical difficulties in evaluating
the state of platelet activation under flow conditions.
To address this issue, we analyzed changes in platelet cytosolic
Ca2+ concentrations ([Ca2+]i), a well-known
indicator of platelet activation, in a colorimetric system that allows
visualization of [Ca2+]i changes during the platelet
adhesive process on a vWF-coated surface under flow conditions. We show
that the initial phase of GP IIb-IIIa activation, which may be
generated through the interaction between surface-immobilized vWF and
GP Ib during translocation and is necessary for firm platelet adhesion
to the vWF-coated surface, is independent of [Ca2+]i
elevation. However, platelet [Ca2+]i elevation,
presumably triggered by binding of activated GP IIb-IIIa to immobilized
vWF (firm adhesion), is a prerequisite for the second phase of GP
IIb-IIIa activation necessary for subsequent platelet cohesion.
| |
MATERIALS AND METHODS |
Materials.
Visible light-excitable Ca2+ indicator Fura Red
acetoxymethyl ester (AM), Calcium Green-1 AM, and intracellular
Ca2+ chelator 5,5'dimethyl 1,2-bis (2-aminophenoxy)
ethane-N,N,N',N'-tetraacetic acid (dimethyl BAPTA) AM were
purchased from Molecular Probes, Inc (Eugene, OR). The Fab fragment of
human/mouse chimeric anti-GP IIb-IIIa monoclonal antibody c7E3, which
totally inhibits the ligand-binding functions of GP IIb-IIIa at
concentrations up to 0.3 µmol/L,11,12 was purchased from
Eli Lilly and Co (Indianapolis, IN). AP1, an anti-GP Ib monoclonal
antibody that completely blocks the vWF-GP Ib interaction at
concentrations up to 0.1 µmol/L, was a kind gift from Dr Thomas
Kunicki (The Scripps Research Institute, La Jolla, CA).6,13
The F(ab')2 fragment of AP1 was prepared by pepsin
digestion of IgG at low pH and the collection of flow-through fractions
in protein A-Sepharose (Pharmacia-LKB Japan, Tokyo, Japan) column
chromatography as described.6,14 The antithrombin agent
argatroban was supplied by Mitsubishi Chemical Corp (Tokyo, Japan).
Apyrase (grade VIII) was from Sigma-Aldrich Japan Co (Tokyo, Japan),
and bovine serum albumin (fraction V) was from Calbiochem (La Jolla,
CA). Human native vWF containing the highest molecular weight
multimers, as judged by sodium dodecyl sulfate (SDS)-1.5% agarose gel
electrophoresis,15 was purified from cryoprecipitates, as
described.16-18
Ca2+ indicator-loading on platelets.
Blood was collected as described12,19 from nonsmoking
healthy donors who had not taken any medication for at least 2 weeks using argatroban at a final concentration of 125 µg/mL as
anticoagulant. Platelet-rich plasma (PRP) was obtained by
centrifugation at 180g for 10 minutes. Apyrase was added (final
concentration, 3 U/mL) to PRP and the mixture was centrifuged on a 40%
albumin cushion at 700g for 2 minutes.6,20 The
platelet fraction was resuspended in HEPES buffer (137 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl2, 3 mmol/L
NaH2PO4, 5.5 mmol/L glucose, 0.35% albumin,
3.5 mmol/L N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid,
pH 7.2) containing 3 U/mL apyrase, 30 µmol/L Fura Red AM, and 20 µmol/L Calcium Green-1 AM at 37°C for 30 minutes.
Ca2+ indicator-loaded platelets were centrifuged again and
resuspended in HEPES buffer (pH 7.4). Erythrocytes were washed 3 times
in HEPES buffer. Ca2+ indicator-loaded platelets and washed
erythrocytes were mixed together and adjusted to 2 to 3 × 107 platelets/mL and a 35% hematocrit.
Perfusion chamber and epifluorescence videomicroscopy.
The perfusion chamber used varies the shear rate in a linear manner as
originally described by Usami et al.21 In brief, this
chamber was designed to reproduce shear rates starting from a
predetermined maximum value at the entrance and decreasing to 0 at the
exit.21 Naflon tape (Iuchi Inc, Osaka, Japan) was used to
adjust flow path height to 0.1 mm. Glass coverslips (24 × 50 mm;
Matsunami Glass, Osaka, Japan) were coated with 200 µL of suspension
of purified vWF (100 µg/mL), placed in a humid environment at room
temperature for 60 minutes, and rinsed with 10 mL of HEPES buffer
before perfusion. The perfusion chamber was assembled and mounted on
BX60 Olympus epifluorescence microscope (Tokyo, Japan). The inner
surface of the vWF-coated area was focused with a 100× objective
lens (UPlanApo 100× oil Iris; Olympus), the focus depth of which
was 0.49 µm. Thus, the [Ca2+]i changes as well as
adhesive processes of platelets were observed preferentially in
platelets adjacent to the vWF surface. Microscopy was equipped with an
epifluorescent illumination apparatus (BX-FLA; Olympus; the excitation
filter/dichroic mirror/barrier filter combination was
MBP490/DM505/BA515IF, respectively) attached to a color charge-coupled
device (CCD) camera (U-VPT-N; Olympus).
Before perfusion, CaCl2 (final concentration, 1 mmol/L) was
added to cell suspensions containing erythrocytes and Ca2+
indicator-loaded platelets in HEPES buffer. Reconstituted blood containing platelets were aspirated through the chamber by a syringe pump (Model 935; Harvard Apparatus, South Natick, MA) at a constant flow rate of 0.285 mL/min and 37°C situations in a thermostatic air
bath (Model UI-50; Iuchi Inc). In the present study, platelets were
perfused through the chamber a single time and not recirculated. [Ca2+]i changes and the entire adhesive and cohesive
process were recorded with a Hi-8 video cassette recorder (VL-HL1;
Sharp Inc, Osaka, Japan), with a video rate of 30 frames per second,
ie, time resolution of 0.033 seconds for each frame. Only platelets
interacting with the vWF-coated surface were analyzed in this study.
Freely flowing platelets in perfused blood can be discriminated from
platelets interacting with the vWF surface by (1) the effective focus
depth directed to the vWF surface and (2) the limit of time resolution of videotapes. Therefore, the background signals (flowing platelets) that were usually less than 20 pixel values can be separated from platelets interacting with the vWF surface (>140 pixel values) and
subtracted as background by a computer-assisted image analysis using
Win ROOF software (Mitani Corp, Fukui, Japan), as
described.12,19
Evaluation of platelet [Ca2+]i changes during the
platelet adhesive and cohesive process under flow conditions.
The emission peaks of Calcium Green-1 and Fura Red were 531 nm and 637 to 657 nm, respectively.22 Elevated [Ca2+]i
results in increased fluorescence intensity of Calcium Green-1 and
decreased fluorescence intensity of Fura Red at an excitation wavelength of 490 nm. Thus, the combined use of these 2 Ca2+ indicators allows colorimetric analysis of cytosolic
calcium changes, with ordinal fluorescence in which platelets glow from red ( orangeyellow-green) to green with
increasing platelet [Ca2+]i.22 Analogue
records of video tape images were digitized by a frame grabber (DIG98;
Detect, Tokyo, Japan) and analyzed by an image processing application
(Win ROOF), as described.12,19 Changes in platelet
[Ca2+]i were evaluated based on a ratio of signal
intensity at G-channel (sensitive peak at 534 nm, green) relative to
R-channel (sensitive peak at 594 nm, red) from digitized images. In
this regard, we used in this study considerably higher concentrations
of Ca2+ indicators (30 µmol/L Fura red AM and 20 µmol/L
Calcium Green-1 AM) than usual to obtain sufficient signal intensity,
which is crucial for precise colorimetric evaluation of
[Ca2+]i changes during observation periods. These
concentrations of Ca2+ indicators were selected based on
our preliminary experiments, in which various concentrations of
Ca2+ indicators on [Ca2+]i changes under flow
on a collagen- or vWF-coated glass surface were tested. In addition, to
address the possibility that the combination loading of such high
concentrations of Ca2+ indicators seriously alters platelet
functions, we compared the entire adhesive and cohesive process of
Ca2+ indicator-loaded platelets with that of platelets
labeled with mepacrine at a concentration (10 µmol/L) assumed to
preserve normal platelet functions.5,7 No significant
differences between these 2 approaches were observed in the extent of
platelet translocation or final platelet surface coverage (see below).
Next, to minimize the effect of photo-activation of platelets or
photo-bleaching of fluorescence, our observation of platelet adhesive
processes was performed by limiting the continuous photo-exposure time. In fact, preliminary experiments confirmed that 3-second consecutive illumination did not induce significant photo-activation of platelets and that any photo-bleaching had no effect on evaluation of platelet [Ca2+]i.
Evaluation of the extent of firm platelet adhesion and cohesion.
During the platelet adhesive process on a vWF-coated surface under flow
conditions, platelets that initially translocate along the surface
gradually become firmly adhered to the surface. The extent of firm
platelet adhesion was determined by counting translocating platelets,
defined as those moving along the surface at a distance greater than
their diameters in 1 second as evaluated based on the logical OR
algorithm of superimposed images (10 frames in 1 second) and Win ROOF
computer software, within a defined area. Platelet adherence to the
surface was evaluated using Win ROOF software to determine the
percentage of the area covered by adhering platelets in a defined area
after background subtraction and binarization of each image.
| |
RESULTS |
Shear-dependency of [Ca2+]i elevation in a platelet
adhesive and cohesive process on a vWF-coated surface.
Platelet [Ca2+]i changes during the adhesive process on a
vWF-coated surface were evaluated at shear rates of 50 s1, 750 s1, or 1,500 s1, representing low, intermediate, or high shear
rate, respectively (Fig 1). After 8 minutes
of blood perfusion at 50 s1, platelet adhesion was
sparse and platelets appeared mostly reddish, indicating only slight
[Ca2+]i elevation. Even at a shear rate of 750 s1, most platelets remained red, although a larger
number of platelets, some of which certainly cohered, adhered to the
surface. At 1,500 s1, the surface area covered by
platelets was greatly increased, and green-colored platelets (high
[Ca2+]i), most of which cohered, were more abundant.
These observations indicate the shear-dependency of
[Ca2+]i elevation as well as platelet adhesion and
cohesion on a vWF-coated surface. Thus, all further experiments to
examine real-time changes in [Ca2+]i during the platelet
adhesive and cohesive process were performed at a wall shear rate of
1,500 s1.
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| Fig 1.
Platelet adhesion, cohesion, and [Ca2+]i
elevation on a vWF-coated surface under varying shear rates.
Reconstituted blood containing platelets loaded with the
Ca2+ indicators Fura Red and Calcium Green-1 was perfused
over a vWF-coated surface with various shear rates. (Upper panels)
Images obtained at 8 minutes of perfusion at a shear rate of 50 s1, 750 s1, or 1,500 s1,
representative of 5 independent perfusions using blood from 5 donors.
Note greenish cohered platelets (high [Ca2+]i) at 1,500 s1, but red, sparsely adhered platelets (low
[Ca2+]i) at 50 s1. Images represent an
area of 2,380 µm2 in a single frame with an original
magnification of 1,000×. (Lower panels) Extent of platelet
[Ca2+]i elevation and platelet adhesion/cohesion at
various shear rates (50, 250, 500, 750, 1,500, and 3,000 s1) evaluated as the signal ratio of green/red channels
(G/R ratio) and the percentage of surface area covered by adhering and
cohering platelets. Images obtained at 8 minutes of perfusion at the
various shear rates were analyzed. Data represent the mean and standard
deviation (SD) of results from 5 areas (4,760 µm2 each)
randomly selected from images derived from 5 independent perfusions.
Note that the G/R ratio (preperfusion value, 0.82 ± 0.06) and surface
coverage of platelets increased as a function of shear rate, reaching a
plateau at 1,500 s1 (P < .01; evaluated by
one-way repeated measures ANOVA with assist of Stat View computer
software; Abacus Concepts Co, Berkeley, CA).
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Time course of [Ca2+]i changes during a platelet
adhesive and cohesive process on a vWF-coated surface at a shear rate
of 1,500 s1.
The platelet adhesive and cohesive process on a vWF-coated surface
involves (1) an initial platelet attachment to the vWF-coated surface;
(2) translocation of platelets along the surface mediated by
interaction between immobilized vWF and GP Ib; (3) firm adhesion to the
surface mediated by the interaction between immobilized vWF and
activated GP IIb-IIIa; and (4) platelet cohesion mediated by binding of
soluble adhesive proteins to activated GP IIb-IIIa, as determined under
experimental conditions involving direct fluorescence-labeling of
platelets in whole blood.7,8 All of these events were recapitulated in the present system using reconstituted blood containing washed platelets labeled with fluorescent Ca2+
indicators (Fig 2). Our approach also
enabled evaluation of the platelet [Ca2+]i changes in the
real-time platelet adhesive and cohesive process. Under flow conditions
at a shear rate of 1,500 s1, no elevation in
[Ca2+]i was observed during translocation of platelets,
but [Ca2+]i increased apparently after platelets firmly
adhered to the surface (Fig 2). However, the time-lag from firm
adhesion to the [Ca2+]i elevation was highly variable
among adhering platelets, with some firmly adhering platelets turning
to green in a few seconds, whereas others remained red throughout the
observation period (results not shown). A similar heterogeneity was
also reported in the case of platelet adhesion on a fibrinogen-coated
surface under low shear rate conditions.23 Platelet
cohesion then occurred, using green platelets with fully elevated
[Ca2+]i as a base (Fig 2). In this regard, adhesive
proteins, vWF, or fibrinogen released from platelets were thought to
play a role, because reconstituted blood free of plasma components was
used in this study.
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| Fig 2.
Time-course of [Ca2+]i changes during a
platelet adhesive and cohesive process on a vWF-coated surface under a
shear rate of 1,500 s1. Reconstituted blood containing
platelets loaded with Ca2+ indicators was perfused over a
vWF-coated surface with a shear rate of 1,500 s1. (Upper
panels) Images obtained at 10 seconds, 1 minute, 4 minutes, and 8 minutes of perfusion (original magnification × 1,000). To distinguish
translocating platelets from those firmly adhering to the surface,
images were reconstructed by superimposition of 10 frames (2,380 µm2 each) obtained every 0.1 seconds (total, 1 seconds)
at each time point indicated. Thus, individual platelets translocating
along the surface are seen as multiple images in a line (arrow 1),
linked with a motion trace that appears as an artifact in a digitized
video image when platelets are moving in a manner beyond the limit of
time resolution of the videotape, whereas noncohered platelets not
moving for at least 1 second are seen as single entities (arrows 2 and
3). Translocating platelets are reddish, whereas cohering platelets
preferentially seen at late stages of platelet adhesive and cohesive
processes (4 and 8 minutes of perfusion) are green (arrow 4). Single
platelets firmly adhering to but not yet cohering on the surface are
heterogeneous in color, with some appearing reddish (arrow 2) and
others greenish (arrow 3). These time-course images were taken from the
different locations in the same perfusion to minimize a possible
photo-activation of platelets. (Lower panels) Time-course changes of
G/R ratio (left) and platelet surface coverage (right). Data represent
the mean and SD of results obtained from 5 independent perfusions at a
shear rate of 1,500 s1 (see Fig 1 legend). Note that the
G/R ratio and surface coverage of platelets increase as a function of
time.
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Effect of intracellular Ca2+ chelator dimethyl
BAPTA, anti-GP Ib, or anti-GP IIb-IIIa on [Ca2+]i
changes during the platelet adhesive and cohesive process on a
vWF-coated surface under a shear rate of 1,500 s1.
To confirm the time point and significance of the platelet
[Ca2+]i elevation in the platelet adhesive process on a
vWF-coated surface, we evaluated the effect of the intracellular
Ca2+ chelator dimethyl BAPTA, an inhibitory anti-GP Ib, or
anti-GP IIb-IIIa antibody on [Ca2+]i in our system. As
judged by the visual observation at 8 minutes after perfusion,
[Ca2+]i chelation did not block firm platelet adhesion
after platelet translocation, but it did abrogate [Ca2+]i
elevation and subsequent cohesion (Fig 3).
This phenomenon was also confirmed by the time point observation at 30 seconds, 1 minute, 2 minutes, 4 minutes, and 6 minutes after the
beginning of perfusion (results not shown). Neither
[Ca2+]i elevation nor firm platelet adhesion was observed
by the blockage of functional sites on GP IIb-IIIa (Fig 3). Indeed,
statistical image analysis (Fig 4)
supported the visual findings.
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| Fig 3.
Effect of intracellular Ca2+ chelator
dimethyl BAPTA, anti-GP Ib, or anti-GP IIb-IIIa on
[Ca2+]i changes in the platelet adhesive and cohesive
process on a vWF-coated surface under a shear rate of 1,500 s1. Experimental conditions were identical to those
described in the Fig 2 legend, except that platelets were preincubated
with 15 µmol/L of dimethyl BAPTA AM, 0.1 µmol/L of
F(ab')2 of AP1 (anti-GP Ib), 1 µmol/L of c7E3
(anti-GP IIb-IIIa) for 30 minutes at 37°C before perfusion.
Superimposed images (total 30 frames of 3 seconds) were obtained at 8 minutes of perfusion. Unlike the control without blockers, platelets
preincubated with dimethyl BAPTA evidenced no [Ca2+]i
elevation or no platelet cohesion. However, most platelets firmly
adhered to the vWF surface to an extent comparable to that in the
control experiment. Note the highly impaired firm adhesion (and no
[Ca2+]i elevation or cohesion) in the presence of
anti-GP IIb-IIIa antibody. In the presence of inhibitory anti-GP Ib,
no significant platelet-surface interaction was observed at this shear
rate.
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| Fig 4.
Statistical evaluation of effect of Ca2+
chelator dimethyl BAPTA, anti-GP Ib, or anti-GP IIb-IIIa on
[Ca2+]i changes in the platelet adhesive and cohesive
process on a vWF-coated surface under a shear rate of 1,500 s1. Experimental conditions were as described in the Fig
3 legend. To evaluate the state of platelet adhesion, the number of
translocating platelets, defined as those moving along the surface in a
greater distance than the corresponding platelet diameter in 1 second
(see Materials and Methods), within a defined area, was determined, in
addition to the G/R ratio and surface coverage. Data represent the mean
and SD obtained by analysis of 5 areas (4,760 µm2 each)
randomly selected from images derived from 5 independent perfusions (8 minutes at 1,500 s1). One-way factorial ANOVA and
Scheffe's method were used for analysis of variance and comparison of
each agent against control, respectively, with assist of Stat View
computer software. Asterisks (*) denote statistically significant
differences from a control (P < .01). These statistical
analyses support the findings obtained by visual recognition in Fig 3;
dimethyl BAPTA inhibited both [Ca2+]i elevation and
surface coverage without affecting firm adhesion of platelets, and
clear inhibition of all 3 parameters was observed in the presence of
anti-GP IIb-IIIa antibody.
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| |
DISCUSSION |
Both firm adhesion of individual platelets and subsequent platelet
cohesion on a vWF-coated surface have been thought to require activation of GP IIb-IIIa. Our results demonstrate that platelet [Ca2+]i elevation is a downstream phenomenon and not a
prerequisite for firm platelet adhesion to a vWF-coated surface.
Although intraplatelet signaling plays a role in firm platelet
adhesion, as evidenced by previous observations that prostaglandin
E1 (PGE1)-induced elevation of
intraplatelet cAMP levels blocks the firm adhesion,7,8 our
results show that the first step in GP IIb-IIIa activation, which
occurs during platelet translocation by the inside-out signaling from
interaction between immobilized vWF and GP Ib, is independent of
platelet [Ca2+]i elevation. In contrast, platelet
[Ca2+]i elevation appears to be necessary for subsequent
platelet cohesion on the surface, in which platelet-platelet engagement
is achieved by binding of soluble adhesive ligands to activated GP
IIb-IIIa.
Current studies on the activation of integrins have raised the
possibility that there are at least 2 distinct phases in modulation of
GP IIb-IIIa (also known as integrin  IIb 3), namely an increase in
affinity or an increase in avidity.24 An increase in
affinity is caused by conformational changes in the heterodimer itself, resulting in a greater strength of ligand binding, whereas an increase
in avidity is induced by clustering or multimerization of GP IIb-IIIa
on the plasma membrane.24 Although alternative interpretations may be possible, it is interesting to assume that firm
adhesion of individual platelets is a consequence of the affinity
modulation of GP IIb-IIIa, independent of [Ca2+]i
elevation, and that platelet cohesion requires the avidity modulation
of GP IIb-IIIa, a more drastic GP IIb-IIIa activation. Platelet
[Ca2+]i elevation may serve to trigger this avidity
modulation of GP IIb-IIIa. Indeed, recent studies using cell types
other than platelets suggested that [Ca2+]i elevation
triggers clustering of several membrane receptors, including other
members of integrin family.25-27 When [Ca2+]i
is elevated, the Ca2+-binding protein complex activates the
myosin light chain kinase, which is known to activate actin-activated
myosin ATPase.28-30 The contraction of actin fibers evoked
by the myosin ATPase may play a role in clustering of membrane
receptors on the cell surface.
With regard to mechanisms by which [Ca2+]i is elevated in
platelets during platelet aggregation processes in a closed suspension system, Chow et al,31 using a cone-and-plate type
viscometer, demonstrated that cooperative functions of GP Ib and GP
IIb-IIIa are essential for platelet [Ca2+]i elevation in
platelet aggregation mediated by high shear stress. On the other hand,
Ikeda et al,32 in a similar experimental system, reported
that platelet [Ca2+]i elevation is absolutely dependent
on the vWF-GP Ib interaction without GP IIb-IIIa functions. The basis
for these discrepant findings remains uncertain, but may rest in the
different blocking agents used in the respective inhibition studies.
Although our experimental system involving observation of platelet
adhesive and cohesive process on a surface is quite different from
systems that analyze platelet aggregation in the soluble phase, our
findings that platelet [Ca2+]i elevation occurs only
after firm adhesion indicates that the binding of GP IIb-IIIa to
immobilized vWF is indispensable in this regard. An outside-in
signaling from the GP IIb-IIIa complex, generated when occupied by
ligands, is likely the trigger for platelet [Ca2+]i
elevation. Indeed, earlier studies, albeit in static experimental conditions, indicated the critical involvement of GP IIb-IIIa functions
in activation of platelet membrane calcium channels.33,34
Although the vWF-GP Ib interaction is also involved in
[Ca2+]i elevation by capturing rapidly flowing platelets
onto a surface under high shear rate conditions, our results confirmed
that the contribution of this interaction is only indirect, indicating the lack of any substantial role for the inside-out signaling generated
from GP Ib in [Ca2+]i elevation during platelet translocation.
In conclusion, our approach involving observations of changes in
platelet [Ca2+]i during a real-time platelet adhesive and
cohesive process under flow conditions showed distinct phase-specific
mechanisms of platelet activation (adhesion and cohesion) that have not
been demonstrable in previous static or closed stirring experiments.
Studies under physiologic flow conditions to determine in detail the
process of platelet activation will further contribute to an
understanding of the complex mechanisms involved in mural platelet thrombogenesis.
| |
ACKNOWLEDGMENT |
The authors thank Marina Hoffman for editorial assistance.
| |
FOOTNOTES |
Submitted December 18, 1998; accepted April 19, 1999.
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.
Presented in part at the American Society of Hematology Meeting in
Miami Beach, FL, December 4-8, 1998 (abstr no. 1423).
Address reprint requests to Mitsuhiko Sugimoto, MD, Department of
Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara
634-8522, Japan; e-mail: sugi-ped{at}naramed-u.ac.jp.
| |
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