(Circulation. 1999;99:2577-2582.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Platelet Microparticles Promote Platelet Interaction With Subendothelial Matrix in a Glycoprotein IIb/IIIa–Dependent Mechanism
From the Department of Internal Medicine, Divisions of Hematology (P.T.) and Cardiology, University of Texas Houston Medical School.
Correspondence to Claude R. Benedict, MD, Department of Internal Medicine, Division of Cardiology, University of Texas Houston Medical School, 6431 Fannin, MSB 6.039, Houston, TX 77030.
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Abstract |
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Background—Platelets, on activation, release vesicular particles called platelet microparticles. Despite their procoagulant activity, their functional role in platelet–vessel wall interactions is not known.
Methods and Results—We examined the binding of microparticles to
vessel wall components in vitro and in vivo. Microparticles bound to
fibrinogen-, fibronectin-, and collagen-coated surfaces. Compared with
activated platelets, we observed minimal binding of
microparticles to vitronectin and von Willebrand
factor. The glycoprotein IIb/IIIa (GP IIb/IIIa)
inhibitors abciximab and eptifibatide (Integrilin)
inhibited the binding to fibrinogen and fibronectin but had minimal
effect on binding to collagen. Furthermore, monoclonal antibodies to GP
Ib or anionic phospholipid-binding proteins
(ß2-glycoprotein I or annexin V) had no
effect in these interactions. Microparticles did not bind to monolayers
of resting or stimulated human umbilical vein
endothelial cells (HUVECs), even in the presence of
fibrinogen or von Willebrand factor. However, under similar
conditions, microparticles bound to extracellular matrix produced by
cultured HUVECs. Abciximab inhibited this interaction by 
50%. In a
rabbit model of arterial endothelial
injury, the infusion of 51Cr-labeled microparticles
resulted in a 3- to 5-fold increase of microparticle adhesion to the
injured site compared with the uninjured site
(P<0.05%). Furthermore, activated
platelets bound to surface-immobilized microparticles
in a GP IIb/IIIa–dependent mechanism. This binding increased in the
presence of fibrinogen by 30%.
Conclusions—Platelet microparticles bind to subendothelial matrix in vitro and in vivo and can act as a substrate for further platelet binding. This interaction may play a significant role in platelet adhesion to the site of endothelial injury.
Key Words: platelets • microparticles • endothelium • glycoproteins
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Introduction |
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After activation of platelets with certain stimuli, there is a release of vesicular particles called platelet microparticles. These microparticles have been shown to accelerate thrombin generation.1 2 3 4 5 The membranes of microparticles contain platelet GPs Ib, IIb, and IIIa as well as P-selectin and thrombospondin.6 7 Elevated levels of microparticles have been detected in patients with disseminated intravascular coagulation,8 unstable angina,9 myocardial infarction,10 coronary angiography,10 transient ischemic attacks,11 and diabetes mellitus12 and during cardiopulmonary bypass.13 Conversely, a deficiency of platelet microparticle generation leads to a bleeding disorder with isolated prolonged bleeding time.14
In addition to their procoagulant effect, microparticles may interact with components of the vessel wall, which contributes to their prothrombotic activities. Thus, microparticles were suggested to promote platelet adhesion to subendothelium, but the precise mechanism was not elucidated.15 Our findings demonstrate that platelet microparticles adhere to subendothelial matrix and provide a substrate for subsequent binding of platelets in a GP IIb/IIIa–dependent manner.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Materials
Anti-CD42b (IgG1 mouse, clone SZ2, Immunotech) binds to platelet GP Ib. Abciximab (Reopro) was purchased from Eli Lilly. CP8 is a murine monoclonal antibody (mAb) to GP IIb/IIIa and was a generous gift from Dr Zaverio Ruggeri (Scripps Clinic, La Jolla, Calif). Integrilin (a gift from Cor-therapeutics, San Francisco, Calif) is a cyclic heptapeptide that specifically blocks GP IIb/IIIa. Anti–GP IIIa (IgG1 mouse, clone B79.7), anti–GP IIb (IgG1 mouse, clone G1.9), and rabbit anti–polyclonal GP IIb/IIIa have been described previously.16 Control mAb K4.40 is a murine IgG1 directed against platelet GP IV. Peroxidase-conjugated goat anti-mouse IgG1 was purchased from Calbiochem. Peroxidase-conjugated protein A was obtained from Promega, and phycoerythrin-labeled anti–GP IIIa was obtained from Pharmingen. ß2-Glycoprotein (ß2GPI),17 annexin V,18 fibrinogen,19 fibronectin,20 vitronectin,21 and von Willebrand factor (vWF)22 were isolated as previously described. Thrombin receptor peptide (SFLLRNA) was synthesized by Dr T.C. Liang (University of Texas, Houston). Recombinant human interleukin-1ß was purchased from R & D Systems Inc. [51Cr]NaCrO4 was obtained from Amersham. Human collagen types I and III and all other reagents were obtained from Sigma Chemical Co.
Cells
Primary human umbilical vein endothelial cells
(HUVECs) purchased from American Type Culture Collection (CRL-1730)
were cultured in F12K medium containing 10% FBS as previously
described.16
Platelet/Microparticle Preparation
Platelet microparticles were isolated from blood obtained by
venipuncture from healthy adult volunteers in a
modification of previously described methods.2 For the
preparation of microparticles, washed platelets were resuspended in
medium M199 supplemented with 1 mmol/L CaCl2
and then activated with calcium ionophore A23187 (25
µmol/L) at 37°C for 10 minutes. After activation, the platelets
were stirred in siliconized cuvettes for 15 minutes and then sedimented
twice at 1000g for 5 minutes. The resulting supernatant,
containing microparticles, was sedimented and washed twice at
10 000g for 10 minutes. The protein content of
microparticles was determined by the protein assay of Bradford
according to the manufacturer's instructions (Bio-Rad). Flow
cytometric analysis of the microparticles was performed with a
FACScan flow cytometer (Becton Dickinson) and a phycoerythrin-labeled
anti–GP IIIa antibody as previously described.2 This
analysis revealed that the preparation of microparticles
contained >97% microparticles and the washed platelet preparation
contained >97% platelets.
Binding Assays
Microparticle/Platelet Binding to Extracellular Matrix
Proteins
Substrates for binding studies were prepared in 96-well
polystyrene microtiter plates (MaxiSorp F96, Nunc). A 50-µL solution
containing either fibrinogen (0.5 µg), fibronectin (0.5 µg),
vitronectin (2 µg), collagen (2 µg), or vWF (2 µg) in
Tris-buffered saline (TBS) (0.15 mol/L NaCl, 0.02 mol/L Tris, pH 7.5)
was added to each well and incubated at 4°C overnight. The optimal
concentration of each protein for microparticle binding was determined
in preliminary experiments. The plates were then blocked with 200 µL
TBS containing 3% BSA at 4°C for 1 hour. Microparticles were added
to each well in various amounts from 0.25 to 5 µg protein. After a
60-minute incubation at 37°C, nonbound microparticles were removed by
3 vigorous washes with TBS. Thereafter, the samples were fixed for 15
minutes at room temperature by the addition of 200 µL freshly
prepared 4% paraformaldehyde to each well. The plates
were then washed 3 times and blocked with TBS containing 3% BSA at
4°C for 1 hour. Rabbit polyclonal anti–GP IIb/IIIa (dilution 1:100)
in TBS containing 1% BSA was then added to each well, and the bound
microparticles were detected by peroxidase-conjugated protein A and
o-phenylenediamine as substrate in an ELISA
reader (MR 5000, Dynatech). To correct for nonspecific binding,
adsorbence due to microparticles bound to BSA was subtracted from total
adsorbence. The abciximab concentration for maximal inhibition of
microparticle binding to fibrinogen and especially fibronectin was
found to be 50 µg/mL in preliminary experiments. For inhibition
assays, microparticles were incubated with either abciximab (50
µg/mL), eptifibatide (Integrilin) (50 µg/mL), SZ2 (20 µg/mL),
ß2GPI (200 µg/mL), annexin V (20 µg/mL), or
control mAb K4.40 (50 µg/mL) at 37°C for 1 hour before their
addition to the wells.
Binding assays with platelets were performed as previously
described with the modification of ELISA for platelet
detection.23 Platelets (106
to 107) were activated with 10
µmol/L thrombin receptor peptide (SFLLRNA) and added to wells coated
with various proteins. For inhibition assays, activated
platelets were incubated with abciximab (50 µg/mL), Integrilin
(50 µg/mL), or CP8 (50 µg/mL) at room temperature 10 minutes before
their addition. The bound platelets were detected by ELISA as
described for microparticles. All the experiments were performed at
least 3 times.
Microparticle Binding to HUVECs and Subendothelial Matrix
Confluent monolayers of HUVECs (96-well plates, Falcon 3072)
were stimulated with either thrombin receptor peptide (SFLLRNA,
100 µmol/L) for 30 minutes or recombinant human interleukin-1ß
(100 pg/mL) for 4 hours at 37°C. Subendothelial
matrix was prepared by removing monolayers of HUVECs cultured for 5
days with 0.02 mol/L ammonium hydroxide for 5 minutes as described by
Gospodarowicz et al.24 The wells were blocked with 3% BSA
at 4°C for 1 hour, and microparticles were then added in various
amounts from 0.25 to 5 µg protein either to resting or stimulated
HUVECs or to subendothelial cell matrix. After a
60-minute incubation at 37°C, nonbound microparticles were removed by
5 washings and aspirations of the supernatant. After the integrity of
the cell monolayer had been confirmed by direct microscopy, the samples
were fixed with freshly prepared 4% paraformaldehyde
for 15 minutes. The bound microparticles were detected by ELISA with
mAbs to GP IIb/IIIa (CP8) or GP Ib (SZ2) as primary antibodies and goat
anti-mouse IgG peroxidase as secondary antibody. These experiments were
repeated at least 3 times.
Platelet Binding to Surface-Immobilized Microparticles
Wells of 96-well microtiter plates (MaxiSorp F96, Nunc) were
incubated with 0.2 µg protein of microparticles in 50 µL M199 per
well at 37°C for 1 hour. After 1 washing step, the plate was blocked
with TBS containing 3% BSA at 4°C for 1 hour. The binding of
platelets to the surface-bound microparticles was examined in 4
different experiments: (1) "resting" platelets added in the
presence of 1 µmol/L PGE1; (2)
"activated" platelets prepared by incubation with
10 µmol/L thrombin receptor peptide (SFLLRNA) at room
temperature 10 minutes before their addition; (3) addition of
fibrinogen (300 µg/mL) before the activation of platelets; and
(4) addition of activated platelets incubated at room
temperature with either abciximab (50 µg/mL), Integrilin (50
µg/mL), CP8 (50 µg/mL), SZ2 (20 µg/mL),
ß2GPI (200 µg/mL), annexin V (20 µg/mL), or
control mAb K4.40 (50 µg/mL) 10 minutes before their addition to the
wells. In these experiments, the platelets were added at
106 to 107 platelets
in 100 µL M199 to each well and incubated at 37°C for 1 hour.
In dose-response assays, microparticles were coated at concentrations
varying from 0.25 to 5 µg protein of microparticles per well. After 1
washing step, the plate was blocked with 3% BSA for 1 hour, and
1.25x106 activated platelets were
added to each well. The subsequent steps were the same as described for
microparticles. Because the polyclonal anti–GP IIb/IIIa antibodies
recognize both platelets and microparticles, the adsorbence due to
antibodies bound to surface-coated microparticles was subtracted from
the total adsorbence. Each of these experiments was performed at least
3 times.
SDS-PAGE and Immunoblotting
Substrate-bound microparticles were solubilized in boiling TBS
containing 2% SDS and 5% mercaptoethanol, subjected to SDS-PAGE, and
transferred electrophoretically to PVDF membranes, and the platelet
GP IIb was identified by immunoblotting with an mAb to
GP IIB (G1.9).
Radioactive Labeling of Microparticles
Microparticles isolated from 1 U blood were resuspended in 250
µL PBS containing 250 µCi 51Cr and incubated
for 60 minutes. The 51Cr-labeled microparticles
were then washed 3 times by centrifugation at
10 000g and resuspended in HEPES-buffered saline. The
radioactivity of the labeled microparticles was assessed by a
gamma-counter, LKB-Wallac Clini Gamma 1272 (Wallac Oy). The labeling
efficiency of microparticles with 51Cr was
7%.
Adhesion of Microparticles in a Rabbit Model of Endothelial
Injury
New Zealand White rabbits (3 kg) were anesthetized
with ketamine and xylazine percutaneously, and
the abdominal aorta and iliac arteries were surgically exposed in a
modification of previously described methods.18 A balloon
catheter with a balloon length of 20 mm was introduced into the
abdominal aorta 1 cm above the aortic bifurcation. The catheter was
forwarded to place the balloon into one of the iliac arteries just
below the aortic bifurcation. The balloon was inflated 5 times to a
pressure of 8 atm for 30 seconds with intervals of 30 seconds. After
immediate removal of the balloon, 51Cr-labeled
microparticles in 3 mL HEPES-buffered saline (1200 µg protein,
2.7x106 cpm) were injected via catheter into
the abdominal aorta 1 cm above the aortic bifurcation. After 3
minutes, the rabbits were killed, and 1-cm pieces of the injured and
the contralateral uninjured iliac arteries were removed. After 3
washings with physiological saline solution, the
radioactivity of each piece was assessed by gamma-counter. These data
were analyzed by ANOVA followed by the Bonferroni test.
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Results |
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Microparticle Binding to Extracellular Matrix Proteins
Microparticles bound to surface-bound fibrinogen, fibronectin, and collagen I and III (Figure 1
, Ia through
Ic). Compared with binding studies with activated platelets
under similar conditions (Figure 1, IIa through IIe), we found
that microparticles bound minimally to vitronectin and vWF
(Figure 1, Id and Ie). The binding of microparticles to
fibrinogen and fibronectin was inhibited by abciximab (50 µg/mL), the
Fab fragment of chimeric mouse/human mAb against GP IIb/IIIa, to
95% and 90%, respectively (Figure 1, Ia and Ib). The cyclic
heptapeptide Integrilin (50 µg/mL) inhibited this interaction by
95% and 90%, respectively (Figure 1, Ia and Ib). There was
only minimal inhibition of microparticle binding to collagen I and III
by abciximab (Figure 1, Ic). The mAb to GP Ib SZ2 (20 µg/mL)
or a control mAb (50 µg/mL) did not affect binding of microparticles
to all the examined extracellular matrix (ECM) proteins (Figure 1). Furthermore, the anionic phospholipid-binding proteins
annexin V (20 µg/mL) and ß2GPI (200 µg/mL)
had no effect, suggesting that anionic phospholipids do not play a role
in this interaction (Figure 1).
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, Medium 199 only; 
, abciximab (50 µg/L); 
,
Integrilin (50 µg/mL); 
, CP8 (50 µg/mL); x, SZ2 (20 µg/mL);
, ß2GPI (200 µg/mL); *, annexin V (20 µg/mL); +,
control mAb (50 µg/mL). C: 
, binding
to collagen III;
Microparticle Binding to Endothelium
We examined the binding of microparticles to intact monolayers of
HUVECs. The bound microparticles were detected with an mAb to GP
IIb/IIIa (CP8). No significant binding of microparticles to a monolayer
of resting HUVECs was detected, even in the presence of fibrinogen (300
µg/mL) or vWF (2 µg/mL) (Figure 2).
After pretreatment of the endothelial monolayer with
thrombin receptor peptide (SFLLRNA, 100 µmol/L), microparticles
did not bind to the endothelial cells in the presence
of fibrinogen (300 µg/mL) or vWF (2 µg/mL) (Figure 2).
Similar results were obtained after stimulation with recombinant human
interleukin-1ß (100 pg/mL). Also, using an mAb to GP Ib (SZ2) to
detect microparticle binding gave similar results. To further support
these findings, the binding of microparticles was also assessed by
means of SDS-PAGE and immunoblotting with a GP IIb mAb
(G1.9). In these immunoblot studies, we could not
demonstrate binding of microparticles to endothelial
cells, whereas binding of microparticles to fibrinogen or fibronectin
could be shown under similar conditions (Figure 3).
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Microparticle Binding to Subendothelial Matrix
To determine whether microparticles bind to
subendothelial matrix, we studied the binding of
microparticles to ECM produced by cultured HUVECs. As shown in Figure 2
, microparticles bound to subendothelial matrix
produced by HUVECs. This interaction was inhibited by abciximab to
50%, suggesting that microparticles bound to
subendothelial matrix at least partly in a GP
IIb/IIIa–dependent manner.
Adhesion of Microparticles to the Site of
Endothelial Injury in a Rabbit Model
To determine whether microparticles will interact with the
subendothelium in vivo, the endothelium
of one iliac artery of New Zealand White rabbits (n=3) was injured by
repetitive balloon inflation. 51Cr-labeled
microparticles were infused into the abdominal aorta above the aortic
bifurcation. There was a 3- to 5-fold increase in radioactivity
associated with the injured site compared with the contralateral
uninjured site (P<0.05%) (Figure 4).
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Platelet Binding to Surface-Immobilized Microparticles
A previous study suggested that microparticles promote subsequent
platelet binding to subendothelium.15
To determine whether surface-immobilized microparticles
mediate this interaction, we studied the binding of platelets to
surface-immobilized microparticles. Our results demonstrate
that resting platelets bound minimally to microparticles (Figure 5A). After activation, however, there was
significantly increased (4-fold) binding of platelets to
surface-immobilized microparticles in a dose-dependent
manner (Figure 5A and 5B). The binding of activated
platelets was further increased in the presence of fibrinogen (300
µg/mL) by 30% (Figure 5A). This binding was inhibited by
abciximab (70%), CP8 (75%), or Integrilin (25%) (Figure 6), suggesting that activated
platelets bind to surface-bound microparticles in a GP
IIb/IIIa–dependent manner. In contrast, mAb to GP Ib, control
antibody, or anionic phospholipid-binding proteins (annexin V or
ß2GPI) did not have an effect in this
interaction (Figure 6).
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Discussion |
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In this study, we showed that platelet-derived microparticles bound to adhesive glycoproteins, fibrinogen, fibronectin, and collagen I and III in a saturating manner, similar to platelets. Abciximab and Integrilin inhibited binding of microparticles to fibrinogen and fibronectin, suggesting a GP IIb/IIIa–dependent binding mechanism. However, there was only minimal inhibition of microparticle binding to collagen I and III by abciximab, suggesting that collagen is not a major ligand for platelet GP IIb/IIIa. This minimal inhibitory effect could have been due to transdominant inhibition of a collagen-binding integrin.25 Unlike platelets, microparticles showed only minimal binding to vitronectin and vWF. This functional difference could be the result of conformational changes of GP IIb/IIIa in microparticle membranes.26
Microparticles also bound to subendothelial matrix, but not to resting or stimulated intact monolayers of HUVECs, under similar experimental conditions. Using flow cytometric analysis, Gawaz et al27 showed that microparticles adhered to thrombin-activated endothelial cells, which involved the GP IIb/IIIa receptor. The difference in our study results could be because the ELISA and immunoblot techniques used may not be as sensitive as flow cytometric analysis. Alternatively, the removal of the endothelial monolayer and separation into a single-cell suspension by repetitive pipetting, as done by Gawaz et al,27 could have exposed the ECM, which could have resulted in the apparent binding of microparticles to endothelial cells. Furthermore, we could show that in a rabbit model of arterial endothelial injury, 51Cr-labeled microparticles localized to the site of endothelial injury, emphasizing that a similar interaction might even occur in vivo under flow conditions.
A previous study suggested that microparticles promote platelet adhesion to subendothelium.15 The precise mechanism involved in this process, however, is not clear. To test the hypothesis that ECM-bound microparticles may provide a substrate for further platelet binding, we used surface-immobilized microparticles to study platelet binding. Resting platelets bound only minimally to surface-immobilized microparticles, whereas activated platelets showed significant binding. This binding was further increased in the presence of fibrinogen and was inhibited by the GP IIb/IIIa inhibitors abciximab and Integrilin. The increased binding in the presence of fibrinogen may be partly due to the formation of platelet aggregates. These findings suggest that activation of platelets is a prerequisite for binding to microparticles, probably because platelet GP IIb/IIIa undergoes conformational changes on activation that provide binding sites for soluble fibrinogen. Fibrinogen may then act as a bridging molecule between platelet and microparticle GP IIb/IIIa. In support of this, recent investigations showed that microparticles bind fibrin28 and soluble fibrinogen and coaggregate with platelets.29 This interaction of microparticles with platelets may even lead to further platelet activation.30
Platelets activated by high shear stress in arteries with severe stenosis may lead to elevated levels of microparticles in circulation.31 Microparticles would be farther in the periphery of the blood stream than platelets because of their smaller size, according to the concept of size-dependent radial distribution of particles in flow.32 33 However, their membrane function seems to be comparable to that of activated platelets. Therefore, they would be more likely to bind initially to exposed subendothelial matrix, thus providing a substrate for further platelet adhesion via GP IIb/IIIa–fibrinogen bridging. This interaction may play a significant role in hemostasis and atherosclerosis.
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Acknowledgments |
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This work was supported by National Institutes of Health/National Heart, Lung, and Blood Institute grants HL-39916, HL-50653, HL-50100, and HL-40860 and a Grant-in-Aid from the American Heart Association.
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Footnotes |
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Guest Editor for this article was Joseph Loscalzo, MD, PhD, Boston University Medical Center, Boston, Mass.
Received October 23, 1998; revision received February 3, 1999; accepted February 16, 1999.
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  S. K. Dasgupta, H. Abdel-Monem, P. Niravath, A. Le, R. V. Bellera, K. Langlois, S. Nagata, R. E. Rumbaut, and P. Thiagarajan Lactadherin and clearance of platelet-derived microvesicles Blood, February 5, 2009; 113(6): 1332 - 1339. [Abstract] [Full Text] [PDF]
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 N. Li Platelet-lymphocyte cross-talk J. Leukoc. Biol., May 1, 2008; 83(5): 1069 - 1078. [Abstract] [Full Text] [PDF]
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 N. Pacienza, R. G. Pozner, G. A. Bianco, L. P. D'Atri, D. O. Croci, S. Negrotto, E. Malaver, R. M. Gomez, G. A. Rabinovich, and M. Schattner The immunoregulatory glycan-binding protein galectin-1 triggers human platelet activation FASEB J, April 1, 2008; 22(4): 1113 - 1123. [Abstract] [Full Text] [PDF]
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 B. Salanova, M. Choi, S. Rolle, M. Wellner, F. C. Luft, and R. Kettritz beta2-Integrins and Acquired Glycoprotein IIb/IIIa (GPIIb/IIIa) Receptors Cooperate in NF-{kappa}B Activation of Human Neutrophils J. Biol. Chem., September 21, 2007; 282(38): 27960 - 27969. [Abstract] [Full Text] [PDF]
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 R. A. Preston, W. Jy, J. J. Jimenez, L. M. Mauro, L. L. Horstman, M. Valle, G. Aime, and Y. S. Ahn Effects of Severe Hypertension on Endothelial and Platelet Microparticles Hypertension, February 1, 2003; 41(2): 211 - 217. [Abstract] [Full Text] [PDF]
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F. Sabatier, V. Roux, F. Anfosso, L. Camoin, J. Sampol, and F. Dignat-George Interaction of endothelial microparticles with monocytic cells in vitro induces tissue factor-dependent procoagulant activity Blood, May 13, 2002; 99(11): 3962 - 3970. [Abstract] [Full Text] [PDF]
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 H. Horigome, Y. Hiramatsu, O. Shigeta, T. Nagasawa, and A. Matsui Overproduction of platelet microparticles in cyanotic congenital heart disease with polycythemia J. Am. Coll. Cardiol., March 20, 2002; 39(6): 1072 - 1077. [Abstract] [Full Text] [PDF]
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 M. Merten and P. Thiagarajan Role for Sulfatides in Platelet Aggregation Circulation, December 11, 2001; 104(24): 2955 - 2960. [Abstract] [Full Text] [PDF]
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M. Merten, J. F. Dong, J. A. Lopez, and P. Thiagarajan Cholesterol Sulfate : A New Adhesive Molecule for Platelets Circulation, April 24, 2001; 103(16): 2032 - 2034. [Abstract] [Full Text] [PDF]
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M. Merten and P. Thiagarajan P-Selectin Expression on Platelets Determines Size and Stability of Platelet Aggregates Circulation, October 17, 2000; 102(16): 1931 - 1936. [Abstract] [Full Text] [PDF]
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