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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Research Center and Department of
Clinical Pathology, University Hospital La Fe, Valencia, Spain;
Division of Hematology and Medical Oncology, Department of Medicine, VA
New York Harbor Healthcare System, New York, NY; and Division of
Hematology and Medical Oncology, Departments of Medicine and Pathology,
Weill Medical College of Cornell University, New York, NY.
Activated platelets release biologically active compounds, which
then recruit additional platelets into an evolving thrombus. We studied
activation of When platelets interact with the subendothelial
matrix of an injured vessel, they become activated, release components
of their intracellular granules, and generate metabolic products. These
products include adenine nucleotides, eicosanoids, and
serotonin.1 In turn, the platelet releasate functions as
agonist for recruitment of additional platelets into the evolving
thrombus.1-5
By using an experimental approach to evaluate platelet activation and
recruitment independently, we previously demonstrated that cell-cell
interactions between activated platelets and intact erythrocytes (RBCs)
amplify both platelet activation and the proaggregatory capacity of
cell-free releasates.2-4 In contrast, we found that platelet-neutrophil interactions down-regulate platelet reactivity in
our system.5
One of the earliest events in platelet reactivity is activation of
platelet P-selectin exposure is a measure of platelet-granule release on
activated platelets.9 P-selectin interactions between
platelets stabilize the initial
Our previous studies of cell-cell interactions between activated
platelets and erythrocytes indicated that biochemical communication between these cell types is initiated upon platelet activation, because, without a platelet agonist, erythrocytes cannot promote platelet activation or the recruiting activity of cell-free
releasates.3,4 We, therefore, hypothesize that components
of the platelet releasate act as extracellular mediators that bring
about biochemical modifications generating prothrombotic activity on erythrocytes.
Among components of platelet releasates, eicosanoids are
physiologically important for thrombus formation.1
Moreover, metabolic interactions of erythrocytes with activated
platelets modulate, within 1 minute of collagen addition, platelet
arachidonate release and eicosanoid formation.3 This
process promotes significant increases in platelet
cyclooxygenase (TXA2) and lipoxygenase (12-HETE) metabolites. In the same time frame, in the presence of erythrocytes, extracellular free arachidonate increases 3- to 10-fold upon platelet stimulation with collagen.3
The effects of prostanoids have been examined in several cell types by
studying agonist-induced changes in second messengers, including levels
of cytosolic calcium
([Ca++]i).16 A rise in
[Ca++]i is associated with loss of
phospholipid asymmetry and exposure of phosphatidylserine (PS) on the
erythrocyte membrane.17 This transforms the erythrocyte
membrane to a procoagulant surface on which a prothrombinase complex
can assemble, leading to thrombin generation.17-19
With the above in mind, we set the following aims: (1) to determine
whether cell-free releasates from collagen-stimulated platelets act as
agonists to induce Our present data indicate that the releasate of collagen-stimulated
platelets induces Materials
Blood cell collection
Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared by differential centrifugation (200g and 2500g, respectively, 15 minutes, 22°C). After removal of PRP, PPP, and buffy coat, 1 mL of erythrocytes was removed from the central area of the erythrocyte zone of each Vacutainer tube. The absence of platelets in the erythrocyte suspensions was verified by phase contact microscopy. Measurement of cytosolic calcium in erythrocytes Cytosolic Ca++ ([Ca++]i) in erythrocytes was measured by spectrofluorimetry. Fura-2 AM was dissolved in dimethyl sulfoxide at a concentration of 1 mM and stored in aliquots at 20°C. Isolated RBCs were washed
twice with HEPES (123 mM NaCl, 5 mM KCL, 1 mM MgCl2, 10 mM
glucose, 25 mM HEPES, 1 mM CaCl2, pH 7.4, 285 mOsm/L) (HEPES-A). Packed cells were diluted to 1% hematocrit with HEPES-A containing 0.05% bovine serum albumin (HEPES-B). RBCs were loaded with
fura-2 AM by incubation with 0.5 M fura-2 AM
for 45 minutes at 37°C in the dark.27 Final dimethyl
sulfoxide concentrations were always less than 0.1%. To remove
extracellular fura-2 AM, RBCs were diluted 1:10 with
HEPES-A and washed twice (10 minutes, 300g) with the same
buffer. The cells were suspended at 0.1% hematocrit and transferred to
a quartz cuvette for fluorescence measurements, performed at 37°C
under constant stirring (approximately 300 rpm), using excitation at
340 and 380 nm, and emission at 510 nm.28 [Ca++]i was calculated according to Poenie et
al.29 To verify that fluorescence increases were not due
to fura-2 AM de-esterification or leakage extracellularly,
control experiments were performed with 1 mM Mn2+ in the
cell suspension.30 We evaluated the effects of different concentrations of free AA, U46 619 (TXA2 analog), 12-HETE,
prostacyclin (PGI2), or appropriate solvents on the
erythrocyte [Ca++]i. Ionomycin (1 µM) was
used as positive control because A23 187 interferes with fura-2
AM fluorescence measurements.
Because TXA2 is very labile, we used the U46 619 analog, a thromboxane receptor ligand in platelets as well as other human cells.16 The concentrations of AA and eicosanoids used did not induce cell lysis; U46 619 (0.5-1 µM) and AA (5-10 µM) aggregated washed platelets (not shown). Measurement of platelet activation and recruitment Platelet activation and recruitment were independently evaluated by using the activation-recruitment system, an in vitro dual cell system previously described.3-5 In the generating system, platelets alone (PRP) (2 × 108platelets/mL), platelet-erythrocyte mixtures (PRP + RBCs), or whole blood (WB) were preincubated (10 minutes, 37°C). Collagen (1 µg/mL) was added as primary platelet agonist; the tube contents were mixed by inversion (10 seconds) and rapidly centrifuged (13 000g; 50 seconds). The centrifugation step promotes cell-cell contact between platelets and other cells in the generating system and yields a cell-free, collagen-free releasate within 1 minute of collagen addition.3 In this releasate, biochemical studies can be performed to assess platelet activation by collagen in the generating system (eg, platelet granule release, TXA2 synthesis). In addition, an aliquot of this releasate was used as an agonist for recruitment of other platelets in the second cell system (assay system). This system was monitored by optical aggregometry in PRP3 or by flow cytometry of recruited platelets in which IIb 3 activation and P-selectin exposure
were evaluated.
Flow cytometry On recruited platelets.
FITC-labeled PAC-1, an antibody to the active conformation of the
Annexin-V binding to erythrocytes. WB (5 µL) was diluted in 200 µL HEPES-C buffer in a polypropylene tube. Then, 13.4 µL of 25 mM CaCl2 in HEPES-C buffer was added, (final Ca++ concentration, 1.5 mM).32 Small volumes (0.5-1.5 µL) of different concentrations of AA, U46 619 (the TXA2 mimetic), 12-HETE, PGI2, as well as appropriate solvent controls, were added. Ca++-ionophore A23 187 and ionomycin were used as positive controls. Samples were kept without stirring (5 minutes, 22°C). To each sample 5 µL FITC-annexin-V and 5 µL PE-antiglycophorin-A antibody were added (10 minutes, 4°C, undisturbed, dark). Samples were quench-diluted with 600 µL ice-cold HEPES-C (with 1.5 mM Ca++), kept at 4°C, and examined within 60 minutes by flow cytometry.32 Erythrocytes were identified by gating of forward and side scatter and as PE-antiglycophorin-A positive. Erythrocytes that exposed PS on the extracellular membrane leaflet were identified as both FITC-annexin-V positive and PE-antiglycophorin-A positive. Data were collected by the System II software, and results were expressed as percentage of 250 000 events. To standardize, the flow cytometer was calibrated daily by using Immunocheck microbeads. Flow rate was adjusted to yield less than 3000 events/second by appropriately diluting samples with buffer. Integrity of the erythrocyte suspensions following treatment with the different agonists was established by lactate dehydrogenase (LDH) assays on cell supernatants.
Releasates from collagen-stimulated PRP induced P-selectin
exposure and
Releasates from platelet-erythrocyte mixtures enhanced P-selectin
exposure and Increased P-selectin exposure has additional implications for
thrombus evolution. We previously demonstrated inhibition of platelet
reactivity by cell-cell interactions with neutrophils.5 The inhibitory effect of neutrophils was amplified by P-selectin blockade with a specific antibody, suggesting a prothrombotic role for
P-selectin in this setting.5 Moreover, P-selectin binding
to monocytes can induce tissue factor activity34 and promote fibrin formation.15 This erythrocyte-promoted
increase of P-selectin on recruited platelets constitutes an additional prothrombotic parameter. P-selectin binding to neutrophils is known to
initiate activation and signal transduction by CD11b/CD18 (Mac-1),35 which promotes interactions with platelet
When the action of releasates was examined 2 hours after ingestion of
ASA, a significant reduction in Data from the present (Figures 1, 2A) and previous studies2-4 indicate an absolute requirement for platelet activation to elicit the enhancing effect of erythrocytes on platelet reactivity. This finding indicates that erythrocytes may respond to one or more components of the platelet releasate. Signaling by free AA and several of its eicosanoid metabolites was evaluated in this study. We measured cytosolic Ca++ levels and alterations in erythrocyte membrane lipid asymmetry (exposure of phosphatidylserine). This is a Ca++-dependent process in platelets and erythrocytes, with implications for hemostasis and thrombosis.17,18 Erythrocyte cytosolic Ca++ was measured at 1-second
intervals for 600 seconds, and maximal
[Ca++]i was determined. Mean basal
[Ca++]i in erythrocytes was 34 nM (Figure
3), in agreement with previous reports.30 Addition of the TXA2-analog
U46 619 (0.5-2 µM) or sodium arachidonate (5-20 µM) to
erythrocytes dose dependently increased maximal
[Ca++]i (Figure 3). In contrast, cytosolic
Ca++ was not modified by the endothelial COX-1 metabolite
PGI2 (0.5-2 µM), or the platelet lipoxygenase product
12-HETE (0.1-1 µM) (not shown). This finding indicates specificity of
the effects of different eicosanoids on erythrocyte
[Ca++]i. For these experiments, the positive
control (1 µM ionomycin) induced an increase in
[Ca++]i of 105 nM (Figure 3, legend). This
rise in [Ca++]i was similar to that observed
after treatment of erythrocytes with AA or U46 619 (Figure 3),
although the kinetics differed (Figure
4). Arachidonate induced a rapid initial
increase, followed by a slow rise during the 10-minute assay period
(Figure 4A). The maximum increase of [Ca++]i
with U46 619 occurred at a later time (Figure 4B). Ionomycin produced,
as expected, a virtually instantaneous rise in
[Ca++]i (Figure 4C). These differences in
kinetics of the [Ca++]i rise suggest that
different signal transduction mechanisms are involved.
U46 619 is a TXA2-analog and a specific ligand for TXA2 receptors on different human cells,16 whereas free AA (without enzymatic or nonenzymatic oxygenation) is a signaling molecule in different cell types.39 Because U46 619 and free AA increase cytosolic Ca++ in erythrocytes, our data demonstrate that TXA2 and free AA are involved in intercellular signaling between activated platelets and erythrocytes. This mechanism could contribute to the enhancing effect of erythrocytes on platelet reactivity3,4 (Figures 1, 2). Our data adds additional support to the concept that erythrocytes are signaling cells, as previously suggested by other investigators,40-42 and further exemplified by thrombospondin-mediated erythrocyte adhesion of sickle erythrocytes by integrin-associated signal transduction mechanisms.24 Interestingly, all substances tested thus far eliciting these effects (TXA2, free AA, PGE2, lysophosphatidic acid, and thrombospondin) are components of the releasate of activated platelets.24,40-42 This finding constitutes further support for an important role for regulated biochemical interactions between platelets and erythrocytes in (patho)physiology. The increase of cytosolic Ca++ in erythrocytes is associated with disruption of phospholipid asymmetry and exposure of PS on the outer leaflet of the erythrocyte membrane, a modification with important implications for hemostasis and thrombosis.17,18 We, therefore, examined the effect of different eicosanoids on PS exposure on erythrocytes in WB, using annexin-V binding measurements by flow cytometry. A small quantity (< 0.5%) of normal circulating erythrocytes exposes
PS on their membrane (Figure 5), as
reported previously.32,43-45 Addition of U46 619 (1-3 µM) dose dependently increased the number of erythrocytes binding
annexin-V over resting levels to 2.2% to 3.6% (Figure 5). Sodium
arachidonate (20-80 µM) also produced a significant increase (2- to
4-fold) over basal levels in the percentage of erythrocytes binding
annexin-V (Figure 5). Lower AA concentrations did not produce an
increase in annexin-V binding (not shown). The difference in AA
concentrations that induced a rise in [Ca++]i
(Figure 3) and annexin-V binding on erythrocytes (Figure 5) is likely
due to the presence of albumin in diluted WB and to a higher
extracellular Ca++ concentration in the annexin-V binding
experiments, factors known to reduce availability of free fatty acid to
act on the cells.39
PGI2 (0.5-2 µM) and 12-HETE (0.1-1 µM) did not increase annexin-V binding to erythrocytes (not shown). This paralleled the lack of effect on [Ca++]i. The positive control, Ca++-ionophore A23 187 (1 µM), is known to have a time-dependent effect.32 In the time frame of our study (5 minutes), A23 187 induced PS exposure in 8.4% of the erythrocytes in agreement with previous data.32 Ionomycin (1 µM, used as positive control for cytosolic Ca++ measurements because A23 187 interferes with fluorescence determinations) induced PS exposure in 1.91% ± 0.19% of erythrocytes (X ± SEM, n = 10). This ionomycin-induced PS exposure was similar in extent to that generated by1 µM U46 619 or 40 µM AA (Figure 5) as was the case for the increase in cytosolic Ca++ (Figure 3). However, the kinetics of the rise in cytosolic Ca++ differed among the 3 agonists (Figure 4), suggesting distinct biochemical mechanisms. A novel aspect of our data is the demonstration that erythrocytes
present PS on the outer leaflet of their membrane when exposed to
components of the activated platelet releasate. This may be mediated by
eicosanoid-induced increases in [Ca++]i,
because treatment of erythrocytes with Ca++-ionophores
induces PS exposure.20,32 The parallel effects on both
[Ca++]i increases and PS exposure by
ionomycin, U46 619, and AA support this mechanism. This finding is of
pathophysiologic significance because erythrocytes exposing PS
stimulate prothrombinase activity and enhance thrombin generation. This
action occurs in patients with sickle cell disease (in which 2% to 3%
of the erythrocytes expose PS)32,43 as well as
Another implication for thrombus formation on exposure of PS on erythrocytes is promotion of thrombospondin-mediated adhesive processes between PS-expressing erythrocytes and subendothelial matrix.20,21 The increase in PS-expressing erythrocytes facilitates close proximity of erythrocytes to platelets and endothelial cells, facilitating thrombus formation. These mechanisms are especially pertinent for the microcirculation, because the increase in [Ca++]i in erythrocytes by U46 619 and AA also reduces erythrocyte deformability,25 a phenomenon that promotes microvascular occlusion.26 Our results, therefore, suggest that a subpopulation of erythrocytes in proximity to activated platelets can acquire a prothrombotic phenotype that contributes to thrombin generation and microvascular occlusion. We conclude that erythrocytes up-regulate the agonistic effects
of platelet releasates on
We gratefully acknowledge the technical assistance of M. Carmen Insa, Amparo Garrido, and Pilar Ferriz.
Submitted January 29, 2001; accepted January 23, 2002.
Supported in part by grants FIS 98/0906 and FIS 01/1208 from the Fondo de Investigaciones Sanitarias (J.V., M.T.S., and J.A.), by a Merit Review grant from the Department of Veterans Affairs, and by grants HL-47073, HL-46403, and NS-41462 from the National Institutes of Health (M.J.B. and A.J.M.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Juana Vallés, Research Center, University Hospital La Fe, Avda Campanar, 21, 46009 Valencia, Spain; e-mail: valles_jua{at}gva.es.
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J. Valles, M. T. Santos, A. Moscardo, M. A. Dasi, and J. Aznar Role of Erythrocytes in Signal Transduction Mechanisms of Recruiting Platelets: Effects on Platelet Tyrosine Phosphorylation and Cytoskeletal Reorganization. Blood (ASH Annual Meeting Abstracts), November 16, 2004; 104(11): 3887 - 3887. [Abstract]
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D. J. Macdonald, R. M. Boyle, A. C. A. Glen, and D. F. Horrobin Cytosolic phospholipase A2 type IVA is present in human red cells Blood, May 1, 2004; 103(9): 3562 - 3564. [Abstract] [Full Text] [PDF]
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 A. J. Marcus, M. J. Broekman, J. H. F. Drosopoulos, N. Islam, D. J. Pinsky, C. Sesti, and R. Levi Metabolic Control of Excessive Extracellular Nucleotide Accumulation by CD39/Ecto-Nucleotidase-1: Implications for Ischemic Vascular Diseases J. Pharmacol. Exp. Ther., April 1, 2003; 305(1): 9 - 16. [Abstract] [Full Text]
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 P. Hermand, P. Gane, M. Huet, V. Jallu, C. Kaplan, H. H. Sonneborn, J.-P. Cartron, and P. Bailly Red Cell ICAM-4 Is a Novel Ligand for Platelet-activated alpha IIbbeta 3 Integrin J. Biol. Chem., February 7, 2003; 278(7): 4892 - 4898. [Abstract] [Full Text] [PDF]
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F. Krotz, H. Y. Sohn, M. Keller, T. Gloe, S. S. Bolz, B. F. Becker, and U. Pohl Depolarization of Endothelial Cells Enhances Platelet Aggregation Through Oxidative Inactivation of Endothelial NTPDase Arterioscler Thromb Vasc Biol, December 1, 2002; 22(12): 2003 - 2009. [Abstract] [Full Text] [PDF]
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IIb
3 integrin receptor activation and
P-selectin expression during platelet recruitment: down-regulation
by aspirin ex vivo
Top
Abstract
Introduction
ATP dependence and the effects of modifiers of stimulation and recovery.
J Biol Chem.
1995;270:10468-10474