(Received for publication, November 14, 1996, and in revised form, January 16, 1997)
From the Neutrophil elastase (NE) and cathepsin G are two
serine proteinases released concomitantly by stimulated
polymorphonuclear neutrophils. We previously demonstrated that while NE
by itself does not activate human platelets, it strongly enhances the
weak aggregation induced by a threshold concentration of cathepsin G
(threshold of cathepsin G) (Renesto, P., and Chignard, M. (1993) Blood 82, 139-144). The aim of this study was to delineate
the molecular mechanisms involved in this potentiation process. Two main pieces of data prompted us to focus on the activation of the
platelet fibrinogen receptor, the Thrombosis and inflammation are processes which result from
complex relationships between various vascular cell types,
i.e. endothelial cells, leukocytes, and platelets (1, 2). As part of such a cell cooperation network, polymorphonuclear neutrophils contribute to vessel injury not only by their own, but also through interactions with platelets. Thus, neutrophils are found admixed with
platelets in the core of vascular occlusions in several experimental models (1, 3), and more importantly, a neutrophil-dependent platelet deposition has been described in arterial injuries (4-6). Neutrophil-mediated platelet activation can be demonstrated in vitro by adding specific neutrophil agonists such as the
formyl-Met-Leu-Phe (fMLP) peptide, tumor necrosis factor- Platelet aggregation is primarily mediated by the binding of the
bifunctional adhesive protein fibrinogen to the surface of adjacent
activated platelets, and considerable evidence has established the
integrin In view of these data, and considering the potential physiopathological
importance of the process, the aim of the present study was to
delineate the molecular mechanism(s) involved in the potentiation
exerted by NE on cathepsin G-induced platelet aggregation. For this
purpose, we considered both the possible involvement of intracellular
signaling pathways, and the changes in the structure and biological
activity of the Antibodies and Reagents
Except for the monoclonal antibody PAC-1, which was provided by
the University Cell Center of Pennsylvania (Philadelphia, PA), the
murine monoclonal and rabbit polyclonal domain-specific anti-
Domain-specific Unité de Pharmacologie Cellulaire,
Laboratoire de Spectrométrie de Masse
Bioorganique,
Laboratoire Franco-Luxembourgeois de Recherche
Biomédicale, Centre Universitaire, Grand-Duchy, Luxembourg
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
IIb
3
integrin. First, previous studies have shown this integrin to be
particularly prone to proteolytic regulation of its function. Second,
we found that the potentiating activity of NE on the threshold of
cathepsin G-induced platelet aggregation was strictly dependent on the
presence of exogenous fibrinogen. Using flow cytometry analysis, NE was shown to trigger a time-dependent binding of PAC-1 and
AP-5, two monoclonal antibodies specific for the activated and
ligand-occupied conformers of
IIb
3.
Furthermore, the potentiated aggregation was shown to result from an
increased capacity of platelets to bind fibrinogen. Indeed, the
combination of NE and threshold of cathepsin G increased the binding of
PAC-1
5.5-fold over basal values measured on nontreated platelets,
whereas this binding raised only by
3-fold in threshold of cathepsin
G-stimulated platelets (p < 0.05). By contrast,
phosphatidic acid accumulation, pleckstrin phosphorylation, and calcium
mobilization produced by the combination of NE and threshold of
cathepsin G were not significantly different from those measured with
threshold of cathepsin G alone (p > 0.05), indicating
that the phospholipase C/protein kinase C pathway is not involved in
the potentiation of aggregation. The foregoing data, as well as the
requirement of catalytically active NE to trigger
IIb
3 activation and potentiate threshold
of cathepsin G-initiated platelet aggregation, led us to examine
whether the structure of this integrin was affected by NE. Immunoblot
and flow cytometry analysis revealed a limited proteolysis of the
carboxyl terminus of the
IIb subunit heavy chain
(
IIbH), as judged by the disappearance of the epitope
for the monoclonal antibody PMI-1. Mass spectrometry studies performed on a synthetic peptide mapping over the cleavage domain of
IIbH predicted the site of proteolysis as located
between Val837 and Asp838. Treatment by NE of
ATP-depleted platelets or Chinese hamster ovary cells expressing human
recombinant
IIb
3 clearly established that
activation of the integrin was independent of signal transduction events and was concomitant with the proteolysis of
IIbH.
In support of this latter observation, a close correlation was observed
between the kinetics of proteolysis of
IIbH on platelets
and that of expression of the ligand binding activity of
IIb
3 (r2 = 0.902, p
0.005). However, only a subpopulation (
25%)
of the proteolyzed
IIb
3 appeared to fully
express the ligand binding capacity. Altogether, these results
demonstrate that NE up-regulates the fibrinogen binding activity of
IIb
3 through a restricted proteolysis of
the
IIb subunit, and that this process is relevant for
the potentiation of platelet aggregation.
, or
interleukin-8 to autologous neutrophil-platelet mixed suspensions
(7-10). Cathepsin G, a serine proteinase stored in the azurophilic
granules of neutrophils and released upon their stimulation, has been
established as the major mediator of this cell-to-cell interaction
(11-13). Acting similarly to
-thrombin, another serine proteinase
agonist of platelets, cathepsin G-induced platelet activation results
in massive exocytosis and aggregation reactions. The potent signal
transduction triggered by this neutrophil proteinase includes the
activation of an as yet unidentified proteinase-activated membrane
receptor, and the subsequent stimulation of the phospholipase C
(PLC)1/protein kinase C (PKC) and
Ca2+ pathways (14-16). Another important serine proteinase
released from the azurophilic granules concomitantly with cathepsin G
is neutrophil elastase (NE). While NE fails to trigger platelet
aggregation and exocytosis (17-19), it has been demonstrated that when
cathepsin G and NE are added together at concentrations comparable to
those released by fMLP-activated neutrophils, NE potentiates the
capacity of platelets to aggregate in response to cathepsin G (18, 19). However, the molecular mechanism underlying this synergism remains unknown.
IIb
3 (glycoprotein IIb-IIIa) as
the membrane receptor for fibrinogen, thus supporting platelet
aggregation (for review, see Refs. 20-22). As all other integrin
receptors (23),
IIb
3 is made of two
non-covalently associated subunits. The
IIb subunit is
made of two disulfide-linked glycosylated polypeptide chains originating from a single precursor. The heavy chain
(
IIbH, relative molecular mass,
Mr
126,000) is entirely extracellular,
whereas the light chain (
IIbL, Mr
23,000) contains a single transmembrane domain. The
3 subunit (Mr
110,000) is
made of one glycosylated polypeptide chain with a single transmembrane
domain, and presents a complex pattern of intramolecular disulfide
bonds within its large extracellular domain (20). Although
IIb
3 is constitutively expressed on the
platelet plasma membrane, the receptor normally acquires its capacity
to bind fibrinogen only upon platelet activation. An inside-out
signaling process is likely responsible for converting this integrin
from a low-affinity to a high-affinity membrane receptor for
fibrinogen, through conformational modifications of its extracellular
domains (21, 22). During platelet exocytosis, translocation to the
plasma membrane of the fraction of
IIb
3 complexes associated with the internal
-granules (24) is another mean for increasing the capacity of activated platelets to bind fibrinogen (25). Finally, an alternative pathway for activation of
IIb
3 at the surface of platelets could be
a proteolytic modification of the extracellular regions of this
receptor. Thus, exposure of platelets to pancreatic or leukocyte
elastases (26, 27) or to
-chymotrypsin (28-30) has been reported to
induce the irreversible expression of fibrinogen-binding sites in the
absence of intracellular activation.
IIb
3 integrin brought
about by NE alone or in combination with cathepsin G.
IIb
3 antibodies used in this study
and listed in Table I were obtained from the Scripps Research Institute
(La Jolla, CA): PMI-1 and the polyclonal antiserum raised against the
peptide V41 (designated anti-V41) were kindly supplied by Dr. M. H. Ginsberg, AP-2 and AP-5 were generous gifts from Dr. T. J. Kunicki, and the polyclonal antiserum IIb-10 was kindly provided by Dr. S. E. D'Souza. Rabbit polyclonal antisera against purified SDS-denaturated whole
IIb or
3 (designated as
anti-
IIb and anti-
3) have been previously
described and characterized (30). Negative control IgG or IgM isotype
antibodies were from Sigma and DAKO (Glostrup, Denmark), respectively.
Fluorescein isothiocyanate-conjugated anti-IgG or anti-IgM were
obtained from DAKO and Sigma, respectively. Reagents for SDS-PAGE were
from Bio-Rad. Nitrocellulose membranes (0.45 µm pores) were from
Schleicher and Schuell (Dassel, Germany). Affinity-purified
staphylococcal 125I-Protein A and carrier-free sodium
[125I]iodide were from Amersham International plc (Little
Chalfont, United Kingdom). The PKC inhibitor GF 109203X was a kind gift from Dr. J. Kirilovsky (Laboratoire Glaxo-Wellcome, Les Ulis, France).
This compound was dissolved in Me2SO (which final
concentration in platelets was less than 0.5%, v/v). Eglin C was
generously provided by Dr. H. P. Schnebli (Ciba-Geigy Research, Basel,
Switzerland). Blood was obtained from the Centre National de
Transfusion Sanguine (Paris, France). Fibrinogen (Grade L) was
purchased from Kabi (Stockolm, Sweden) and treated with diisopropyl
fluorophosphate to inactivate coagulant contaminants and subsequently
dialyzed to remove the free inhibitor.
N-Succinyl-(Ala)2-Pro-Phe-p-nitroanilide (a cathepsin G substrate),
N-succinyl-(Ala)3-p-nitroanilide (a NE substrate), 2-deoxy-D-glucose, sodium azide
(NaN3), glucono-
-lactone, the proteinase inhibitors
phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin, soybean
trypsin inhibitor and aprotinin were from Sigma. Iscove's buffer was
from BioWhittaker (Belgium). The peptide FPQPPVNPLKVDWGL (using the
single-letter code for amino acids), corresponding to the sequence
827-841 of the
IIb subunit heavy chain, was synthesized
by Neosystem Laboratoire (Strasbourg, France). All other reagents were
obtained as indicated in Si-Tahar et al. (16).
IIb
3 antibodies used in this
study
Name
Mono/polyclonal
Isotype
Specificity on
IIb
3
Refs.
PAC-1
Monoclonal
IgM
RGDa-binding
site
31
AP-2
Monoclonal
IgG1
Complexed form of
IIb and
3
32
AP-5
Monoclonal
IgG1
LIBSb on the amino terminus
of
3
33
PMI-1
Monoclonal
IgG1
LIBS on the
carboxyl terminus of
IIbHc
34
IIb-10
Polyclonal
Amino terminus of
IIbH
30
Anti-V41
Polyclonal
Amino terminus of
IIbLc
34
a
RGD, Arg-Gly-Asp adhesion motif present in fibrinogen
(20).
b
LIBS, ligand-induced binding site.
c
IIbH,
IIbL, extracellular heavy chain
and transmembrane light chain, respectively, of the
IIb
subunit.
Purification of Neutrophil Cathepsin G and NE
Cathepsin G and NE were purified as described previously (16),
using a two-step chromatographic procedure (aprotinin-Sepharose affinity and CM-Trisacryl ion-exchange). The purity of the neutrophil proteinases was assessed by SDS-PAGE. Moreover, it was verified that
cathepsin G and NE preparations were devoid of each others proteinase
by monitoring spectrophotometrically the hydrolysis of
N-succinyl-(Ala)2-Pro-Phe-p-nitroanilide
and N-succinyl-(Ala)3-p-nitroanilide induced by purified NE and cathepsin G, respectively. For the determination of their active site concentrations, a constant amount of
the enzymes were reacted in the presence of increasing amounts of
titrated 1-antitrypsin and extrapolation of the
concentrations were performed using linear regression analysis.
To block the catalytic site of NE, the purified proteinase (70 µM) was incubated for 60 min at 25 °C with PMSF (1.25 mM), and the mixture was subsequently dialyzed to remove the free inhibitor. PMSF-treated NE was shown to be proteolytically inactive by testing the lack of hydrolysis of its specific synthetic substrate.
Preparation and Labeling of Platelets
Blood was obtained from healthy adult volunteers without any medication. The platelet-rich plasma was isolated by centrifugation of blood at 180 × g for 20 min and incubated with 5-[14C]HT (0.05 mCi/ml) for 30 min at 37 °C. For protein phosphorylation and phospholipid metabolism studies, platelets were labeled with [32P]phosphoric acid as described previously (16). Then, labeled platelets were washed by two successive centrifugations (1,600 × g, 10 min) and resuspended in Tyrode's buffer (composition, mM: NaCl, 137.0; KCl, 2.68; NaHCO3, 11.9; NaH2PO4, 0.42; CaCl2, 2.0; MgCl2, 1.0; glucose, 5.5; Hepes, 5.0; and bovine serum albumin, 0.35%, pH 7.4) supplemented with PGI2 (0.25 µM) and heparin (50 units/ml). Before the last centrifugation, the platelet pellet was resuspended in the same buffer without heparin. The final pellet was resuspended in Tyrode's buffer such that the final platelet concentration was 4 × 108/ml. The entire procedure was performed at 37 °C and isolated platelets were maintained at this temperature until use.
Generation of Stable CHO Cells Expressing Human
IIb
3
(CHO/
IIb
3)
The full-length cDNA encoding wild type human
IIb (35) or human
3 (36) were inserted
into the pBJ1 expression vector and cotransfected into CHO dhfr(neg)
cells as described previously (37). Briefly, 20 µg of each
IIb and
3 cDNA and 2 µg of
dihydrofolate reductase plasmid (pMDR901) were mixed with 40 µg of
LipofectAMINE in a final volume of 200 µl and added to the cells.
After a 48-h incubation of the cells in Iscove's medium supplemented
with 10% heat-inactivated fetal calf serum, the cells were grown in
nucleoside-free
-minimal essential medium supplemented with 10%
dialyzed fetal calf serum, used as selective medium. Positive
transfectants were selected for cell surface expression of recombinant
IIb
3 using the complex specific
anti-
IIb
3 monoclonal antibody AP-2 (32) and goat anti-mouse IgG-coated immunomagnetic beads. Cells were further
grown to confluence in T75 or T25 flasks in Iscove's buffer supplemented with glutamine, penicillin, streptomycin, and 10% fetal
calf serum, and routinely passaged after detachment using EDTA
buffer (composition, mM: NaCl, 126; KCl, 5; EDTA, 10;
HEPES, 50, pH 7.4).
Aggregation and Secretion Measurements
Platelet aggregation in 0.5-ml aliquots (4 × 108 platelets/ml) was recorded at 37 °C using a Dual Aggro-Meter (Chrono-Log Corp., Havertown, PA) under constant stirring (1,100 rpm). Samples were preincubated with imipramine (1 µM) to prevent the re-uptake of released 5-[14C]HT and with fibrinogen (0.7 mg/ml) for 2 min before stimulation. Activation was initiated by addition of cathepsin G, NE, or a threshold concentration of cathepsin G (threshold of cathepsin G) preceded by NE for 10 s, and the variations in light transmission were continuously recorded. The 5-[14C]HT- or 32P-labeled platelets were transferred to tubes containing 125 µl of a stopping solution made of 77 mM EDTA, 155 mM NaCl, 33% formaldehyde (1:9:8, v/v), or 1.8 ml of ice-cold chloroform, methanol, 12 M HCl, 0.1 M EDTA (20:40:1:2, v/v), respectively, to terminate the reaction. Supernatants containing released 5-[14C]HT were mixed with scintillation fluid for measuring radioactivity. Aggregation was expressed as the percent of changes in light transmission and the 5-[14C]HT release was expressed as the percent of total 5-[14C]HT platelet content.
Protein Phosphorylation and Polyphosphoinositide Metabolism
Chloroform/distillated water (0.5 volume of each) was added to 32P-labeled platelets diluted in the stopping organic solution (see above). This suspension was vigorously shaken and centrifuged for 10 min at 10 °C. The upper aqueous phase was discarded. Proteins, concentrated at the interface, were solubilized according to the procedure of Laemmli (38). Radiolabeled proteins were then subjected to SDS-PAGE using a 12.5% resolving gel and a 5% stacking gel. After staining, dried gels were exposed to a Molecular Dynamics (MD; Evry, France) PhosphorImaging screen. Concurrently, the lower chloroformic phase was evaporated, washed according to Jolles et al. (39), and resuspended in chloroform. Phosphoinositides and phosphatidic acid (PtdOH) were separated by thin layer chromatography using chloroform/acetone/methanol/acetic acid/water (40:15:13:12:7, v/v) as the migration solvent. Upon drying, the chromatography plates were also exposed to a PhosphorImaging screen. PLC and PKC activities were evaluated by quantifying the radioactive signals associated to PtdOH and pleckstrin, respectively, using an MD PhosphorImager coupled to the ImageQuant software (version 3.3).
Calcium Flux Measurements
Platelets were prepared as described above with slight modifications. Following the resuspension in Tyrode's buffer supplemented with prostacyclin and heparin, platelets were incubated for 30 min at 37 °C with 3 µM Fura 2-acetoxymethylester, washed, and the final platelet concentration was adjusted to 4 × 108/ml in Tyrode's buffer. The basal fluorescence of a 1-ml aliquot of cell suspensions was monitored under stirring with a spectrofluorimeter Jobin Yvon JY 3D (Paris, France) thermostatted at 37 °C. Fluorescence excitation and emission wavelengths were 340 and 510 nm, respectively. Platelets preincubated for 2 min were challenged with cathepsin G, NE, or combinations of both proteinases, and the changes in fluorescence were recorded for 2 min.
Flow Cytometry Analysis
Analysis of Washed PlateletsCell samples were treated as
for platelet aggregation analysis, except for those used to analyze the
binding of PAC-1, for which exogenous fibrinogen was omitted as this
natural ligand of the activated IIb
3 may
competitively inhibit the binding of PAC-1 (31). In any case, once the
agonist has been added, the stirring was allowed for only 5 s to
homogenize the milieu, then samples were incubated undisturbed for
different periods of time at 37 °C to prevent platelet aggregate
formation, which would interfere with the flow cytometry analysis. The
reaction was stopped by the addition of 5 µM eglin C, an
inhibitor of cathepsin G and NE (40) and 2 mM PMSF.
Platelets were then immediately fixed with 1% (v/v) formaldehyde for
30 min at room temperature. Following the fixation, all samples were
diluted 10-fold in Tyrode's buffer and then incubated for 30 min at
4 °C with saturating concentrations of purified PAC-1 (2 µg/ml),
AP-2 (1 µg/ml), PMI-1 (5 µg/ml), AP-5 ascitic fluid (diluted
1/1000), or with nonimmune IgG or IgM as control isotypes. Incubations
were done in conical bottom 96-well plastic plates with 4 × 106 cells/well. After centrifugation of the plates at
80 × g for 10 min at 4 °C, platelets were washed
twice in Tyrode's buffer and incubated for 30 min at 4 °C with the
corresponding second fluorescein isothiocyanate-labeled antibody at
optimal concentration. Finally, platelets were centrifuged as above,
resuspended in the same buffer, and stored at 4 °C in the dark until
flow cytometric assays were performed within the next 24 h. It is
of note that when platelets were to be tested for the expression of the
PMI-1 epitope, they were first incubated with the proteinase, then with 5 mM EDTA for 15 min at room temperature to maximally
expose the PMI-1 epitope (41), before to be fixed and processed as
described above.
Cells were harvested from culture flasks using EDTA buffer
and washed once in Iscove's buffer without fetal calf serum and then
in Tyrode's buffer. The final pellet was resuspended in this latter
buffer such that the final concentration was 107 cells/ml.
Then, cells were incubated for 3 min at 37 °C with 400 nM NE or 550 nM cathepsin G under gentle
shaking. The reaction was stopped and part of the cells further fixed
with 1% (v/v) formaldehyde, whereas nonfixed cells were used for
immunoblot analysis (see below). Next, 106 fixed
CHO/IIb
3 cells were processed for PAC-1
or AP-2 or control isotype antibodies binding as described for
platelets.
In all cases, samples were analyzed using a FACScan flow cytometer
(Becton Dickinson Immunocytometry System, Mountain View, CA). Binding
of the different domain-specific
anti-IIb
3 antibodies to their epitopes is
expressed as the fold increase in median fluorescence intensity over
basal values measured on nontreated cells, following subtraction of the
background binding measured with the control isotypes.
Binding of 125I-Labeled Monoclonal Antibodies
The monoclonal antibodies PMI-1 and AP-5 were purified to
homogeneity from ascitic fluids by conventional Protein A- or Protein G-Sepharose chromatography, and labeled with 125I to a
specific activity of 4.5 × 104 becquerels/µg of
IgG using the chloramine T procedure (32, 41). Platelets were exposed
to NE (400 nM) in an aggregometer cuvette for increasing
periods of time (up to 3 min), and the proteinase was blocked by
addition of eglin C and PMSF as for flow cytometry analysis. Control
platelet suspensions were treated similarly except that NE was absent.
Platelets were immediately distributed (final concentration, 2 × 108/ml) in a Tyrode's medium containing either divalent
cations or EDTA (final concentration, 3 mM), and either one
of the 125I-labeled antibodies. Incubations were performed
at room temperature for 45 min, and platelet-bound antibodies were
separated from unbound by layering triplicate 50-µl aliquots of the
cell suspensions on 0.5 ml of 20% sucrose made in Tyrode's medium,
and centrifugation for 5 min at 14,000 × g. The
supernatant and sucrose were aspirated, and the platelet pellets at the
bottom of the tube cut and counted for 125I in a 1282 Compugamma CS counter (LKB Wallac, Turku, Finland). Binding of
125I-AP-5 on NE-treated platelets, which reflected
fibrinogen binding to the activated
IIb
3
integrin (33), was performed in the presence of divalent cations. Under
similar conditions, binding to control nontreated platelets was
negligible, as previously reported (33), and increased linearly as a
constant fraction (0.7%) of the antibody input. This was taken as
nonspecific binding. Maximal binding of 125I-AP-5 was
measured in the presence of EDTA (33) and found to saturate at 25 µg/ml IgG; this concentration was used throughout all subsequent
experiments. Binding of 125I-PMI-1 on NE-treated platelets,
which measured the proteolysis of the
IIb
3 integrin (see "Results"), was
performed in the presence of EDTA, to maximize the exposure of the
PMI-1 epitope on the platelet surface (41). In preliminary experiments,
the nonspecific binding was measured in the presence of a 50-fold
excess of unlabeled antibody, and found to represent a constant
fraction (0.25%) of the antibody input. Maximal binding of
125I-PMI-1 was measured on control platelets in the
presence of EDTA and found to saturate at 250 µg/ml IgG; this
concentration was used throughout all subsequent experiments. Isotherm
binding of increasing amounts of 125I-labeled antibodies to
nontreated or NE-treated platelets for 1 min showed that the
KD of each antibody for its epitope was unchanged
following proteolysis of
IIb
3 (data not
shown). All data are reported as specific binding, i.e.
total binding corrected for the nonspecific as defined above.
SDS-PAGE and Immunoblot Analysis
At the end of the exposure of platelets to proteinases in the
aggregometer cuvettes, and after addition of eglin C and PMSF to block
the enzymatic activity of cathepsin G and/or NE, 200 µl of platelet
suspensions were rapidly centrifuged at 12,000 × g for
4 min. The supernatant was carefully removed and the platelet pellets
were resuspended in the initial volume with 10 mM Tris/HCl, 150 mM NaCl, 3 mM EDTA, pH 6.8, and cells were
solubilized by addition of a one-fifth volume of 12% (w/v) SDS and 30 mM N-ethylmaleimide in 10 mM
Tris/HCl, pH 6.8, and heated at 100 °C for 5 min. SDS-PAGE was
performed according to the procedure of Laemmli (38). When needed,
disulfide bonds were reduced prior to electrophoresis by adding 5%
(v/v) 2-mercaptoethanol to the samples. For
CHO/IIb
3 cells solubilization, 1-3 × 106 nonfixed cells pretreated or not with NE or
cathepsin G as described above were centrifuged for 4 min at
12,000 × g. After discarding the supernatant, the cell
pellets were resuspended at 107 cells/ml in a lysis medium
(final concentrations, mM: Tris, 10; NaCl, 150; EDTA, 3;
N-ethylmaleimide, 5; PMSF, 1; benzamidine, 5; leupeptin,
0.1; soybean trypsin inhibitor, 0.0014, pH 7.4) to which 10% (v/v)
Triton X-100 was added for a final concentration of 1%. Protein
extraction was performed at 4 °C for 30 min with occasional
vortexing, then the extract cleared from cells debris and nucleus by a
15-min centrifugation at 12,000 × g at 4 °C. The
supernatant was then solubilized with SDS-N-ethylmaleimide, and the extract processed for SDS-PAGE.
Electrophoresis was performed after loading each well on gels with 10 or 20 µg of total platelet or CHO/IIb
3
cell proteins, respectively; then proteins were transferred to
nitrocellulose membranes and probed by immunoblotting using specific
antibodies exactly as described previously (30), and as specified in
the figure legends. Bound antibodies were detected following incubation of the membranes with 125I-Protein A (diluted 1/1000) and
autoradiography on Kodak X-Omat MA or AR films (Kodak-Pathé,
Paris, France) for various periods of time (0.25 to 4 days). For
Mr determinations, polyacrylamide gels were
calibrated using standard proteins with Mr in
the range 200,000 to 14,400.
Matrix-assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Analysis
The site of cleavage by NE was searched by MALDI-TOF mass
spectrometry on a 15-mer peptide, designated peptide 827-841,
corresponding to the amino acid sequence
Phe827-Leu841 of the IIbH
subunit (35). Enzymatic digestion assay was performed at 37 °C with
NE at a final concentration of 400 nM and the peptide 827-841 at 525 µM in 200 mM Tris acetate, pH
7.4. Following different incubation periods, a 1-µl aliquot was
withdrawn from the reaction medium and diluted in 0.1% aqueous
trifluoroacetic acid, a solution that quenches the enzyme reaction by
lowering the pH to
3. The stability of the substrate in the absence
of NE was assessed under the same conditions. The diluted medium was
then submitted to MALDI-TOF measurement. Samples were prepared as
follows: 1 µl of a 4-
-cyano-4-hydroxy-trans-cinnamic
acid was deposited on a stainless steel probe and allowed to evaporate
quickly. About 0.5 µl of the dilute digest solution was then
deposited on the matrix surface and allowed to air dry. At last, the
sample was washed according to Vorm et al. (42) with 0.5%
aqueous trifluoroacetic acid. Mass spectra were obtained using a Bruker
Biflex MALDI-TOF mass spectrometer (Bremen, Germany). The average error
on the MALDI-TOF derived mass is theoretically 0.1%, i.e.
1.7 Da in our mass range. However, the difference between the
experimental mass and the calculated average isotopic mass of our
peptide was usually less than 0.5 Da. Hence, the sequence of the
peptide(s) resulting from cleavage by NE could be unambiguously derived
from their masses. The instrument was calibrated prior to each
measurement with the monoprotonated molecular ions from a standard
mixture of angiotensin II, ACTH 18-39, and bovine insulin. The
sequences of the proteolytic fragments were predicted using the
MacProMass 1.2 software (Beckman Research Institute, Duarte, CA) on the
basis of the known sequence of the initial peptide and the determined molecular masses of the fragment(s).
Statistics
Results are expressed as mean ± S.E. for the indicated
number of independently performed experiments. Statistical significance between the different values was analyzed by Student's
t-test for unpaired data with a threshold of
p 0.05. The standard linear regression analysis was
applied to correlate the different parameters using the Statview
512+ software (BrainPower Inc., Calabasas, CA).
As previously established (11, 16), cathepsin
G alone added to platelet suspensions at the optimal concentration of
550 nM acts as a strong platelet agonist inducing extensive
platelet aggregation (83.5 ± 5.6%, n = 5),
accompanied by a marked exocytosis of intracellular granules as judged
by the release of 5-[14C]HT from dense granules
(76.2 ± 1.6%, n = 5; Fig. 1,
panels A and B). For each tested platelet
suspension, we determined the threshold of cathepsin G as that resulted
in platelet shape change followed by 5-10% of increase in light
transmission within 3 min of stirring (Fig. 1, panels A
and B). This concentration was always within the range
150 to 180 nM. Under these conditions, exocytosis remained
minimal at 3 min, with 3.1 ± 0.8% of 5-[14C]HT
release (n = 5; Fig. 1, panel B).
Platelet suspensions stirred for 3 min with 400 nM NE (and up to 800 nM) showed no evidence for aggregate formation (Fig. 1, panel A) and exocytosis of internal granules was barely detectable (0.8 ± 0.3% release of 5-[14C]HT, n = 5), in agreement with previous reports (17-19). By contrast, addition of 400 nM NE 10 s before stimulation of platelets with threshold of cathepsin G resulted in an extensive aggregation, similar to that induced by the optimal concentration of cathepsin G (Fig. 1, panels A and B). The potentiation exerted by NE on threshold of cathepsin G-induced platelet aggregation was already detectable with 100 nM NE, and was maximal in the range 200-800 nM NE (Fig. 1, panel B). Increasing the period of exposure of platelets to NE to 180 s before threshold of cathepsin G had no further effect on the extent of potentiation (not shown and Ref. 18).
Major observations in these experiments were that (i) whereas aggregation induced by 550 nM cathepsin G was associated with an extensive granule exocytosis, the aggregation induced by the combination of 100-800 nM NE with threshold of cathepsin G was accompanied by a limited release of granule contents, varying from 4.7 ± 1.4% to a maximum of 25.9 ± 0.9% secretion of 5-[14C]HT (n = 5; Fig. 1, panel B); and (ii) the potentiating activity of NE on threshold of cathepsin G-induced platelet aggregation was strictly dependent on the presence of exogenous fibrinogen (Fig. 1, panels A and C).
Activation of the Platelet Fibrinogen Receptor by NE and Cathepsin GPrevious reports have shown that platelet exposure to various
serine proteinases, including elastases, induces expression of
fibrinogen binding sites (26, 27). This, together with the requirement
for exogenous fibrinogen for the potentiation of platelet aggregation
(the present work) led us to consider that the synergism resulting from
the combination of NE and threshold of cathepsin G could be exerted at
the level of the IIb
3 integrin, the
platelet fibrinogen receptor. We first evaluated whether NE could
modify by itself the surface expression and the biological activity of
IIb
3 on platelets by using flow cytometry
analysis with a panel of monoclonal antibodies specific for distinct
conformations of this integrin (see Table I). These
included AP-2, an
IIb
3 complex-specific
antibody reacting with both the resting and active forms of the
receptor (32), PAC-1, which binds at one fibrinogen-binding site on
IIb
3 and only recognizes the active
conformation of the receptor (31), and AP-5, an anti-ligand-induced
binding site (LIBS) which specifically reveals the active and
fibrinogen-occupied integrin (33). As mentioned under "Materials and
Methods," when platelets were to be tested with PAC-1, prior
incubation with NE was without exogenous fibrinogen, while analysis of
the binding of AP-5 was necessarily performed on platelets incubated in
the presence of fibrinogen.
As illustrated in Fig. 2, exposure of platelet
suspensions to 400 nM NE at 37 °C, a concentration which
triggers a maximal potentiation of threshold of cathepsin G-induced
platelet aggregation, resulted in a rapid transition of the
IIb
3 conformation reflecting activation
of the receptor and binding of its ligand. Indeed, a
time-dependent increase in binding of PAC-1 occurred within 10 s of exposure to NE, and reached a plateau after 1 min
(panel A). A similar increase in the binding of AP-5 was
observed, with a plateau after 2 min of exposure to NE (panel
B). At 3 min, binding of PAC-1 and AP-5 on NE-treated platelets
was increased about 2.3-fold (n = 8, p < 0.001) and 3.4-fold (n = 4, p < 0.05), respectively, compared with nontreated platelets. However,
binding of these antibodies on NE-treated platelets was approximately
3.5-fold lower than that measured on platelets optimally stimulated
with 550 nM cathepsin G (compare panels A and
C, and B and D). Such a difference can
be largely explained by the ability of a high concentration of
cathepsin G to induce platelet shape change and extensive exocytosis of
-granules (43), thus allowing the translocation of the internal
fraction of
IIb
3 complexes to the plasma
membrane (25). Indeed, binding of AP-2 to platelets activated with 550 nM cathepsin G for 3 min was increased by 92.7 ± 16.8% when compared with nontreated platelets (n = 7, p < 0.001; see Fig. 3, panel B), an increase similar to that measured on platelets activated with 0.5 IU/ml thrombin (98.9 ± 9.2%, n = 8, p < 0.001). By contrast, the increase in AP-2 binding
to platelets exposed to 400 nM NE was only 9.6 ± 5.0% and remained nonsignificant (n = 8, p > 0.05; see Fig. 3, panel B).
Involvement of the Activation of
Considering that NE is able to up-regulate the
biological activity of the plasma membrane
IIb
3 integrin, we assumed that the
potentiation by NE of platelet aggregation induced by threshold of
cathepsin G resulted from an increased capacity of the platelet surface
to bind fibrinogen. To examine this hypothesis, platelet suspensions
were challenged for 3 min with 400 nM NE, threshold of
cathepsin G, or a combination of both proteinases, before being processed for analysis of AP-2 and PAC-1 binding by flow cytometry, the
latter antibody being taken as a fibrinogen-like probe. Thus, combination of the two proteinases increased the binding of PAC-1
5.5-fold, while activation of platelets with 550 nM
cathepsin G increased PAC-1 binding
7.5-fold, these values being not
statistically different (p > 0.05, n = 3). By contrast, the increase in PAC-1 binding induced at 3 min by
threshold of cathepsin G alone was
3-fold above the background
binding measured for nontreated platelets, a value similar to that
measured for NE-treated platelets in this series of experiments
(p > 0.05; Fig. 3, panel A). Of note is that PAC-1 binding to platelets activated with either threshold of
cathepsin G alone or with the combination of NE and threshold of
cathepsin G was significantly different (p < 0.05, n = 3). When platelet suspensions were similarly
treated in the presence of exogenous fibrinogen, then evaluated for the
binding of AP-5 as a marker of ligand-occupied
IIb
3, similar profiles were obtained (not
illustrated). Strikingly, the strong potentiating effect of NE on
threshold of cathepsin G-induced platelet aggregation occurred despite
a limited expression of the internal
IIb
3
fraction at the plasma membrane, as measured by the binding of AP-2
which was identical to that initiated by threshold of cathepsin G alone (
1.3-fold increase over basal value under both conditions,
p > 0.05; Fig. 3, panel B).
The enhanced
exposure of fibrinogen-binding sites induced by NE could be potentially
explained by the ability of this proteinase to enhance intracellular
signals by which cathepsin G normally initiates platelet activation and
thus up-regulates the activity of IIb
3.
It has been demonstrated (14-16) that the interrelated elements of the
PLC-Ca2+-PKC pathway act in concert to mediate such a cell
response. Thus, as indicated in Fig. 4, an optimal
concentration of cathepsin G (550 nM) triggered extensive
activation of the PLC and PKC pathways, as measured through PtdOH
accumulation (panel A) and phosphorylation of pleckstrin, a
47-kDa protein (P47) which is the main substrate for PKC in platelets
(44) (panel B). As expected, activation of PLC and PKC was
accompanied by a massive increase in cytosolic Ca2+
(panel C). By contrast, activation of platelets with
threshold of cathepsin G resulted in a detectable but limited metabolic activation compared with 550 nM cathepsin G. On the other
hand, exposure of platelets to NE alone at concentrations up to 800 nM failed to initiate PLC or PKC activities or
intracellular Ca2+ movements. Finally, and more
importantly, stimulation of platelets with the combination of 400 nM NE and threshold of cathepsin G resulted in an
activation which was not different with that produced by threshold of
cathepsin G alone (values of fold-increase over basal signals were
2.25 ± 0.25 and 2.63 ± 0.14 (n = 4) for
PtdOH accumulation, respectively, and 1.26 ± 0.05 and 1.24 ± 0.02 (n = 4) for P47 phosphorylation, respectively).
Furthermore, we determined that the activation of
IIb
3 by NE was not blocked by substances known to prevent platelet activation. Table II shows
that pretreatment of platelets with GF 109203X, a specific PKC
inhibitor (45), or with PGI2, a potent activator of
platelet adenylate cyclase and inhibitor of
IIb
3 metabolic activation (46), had no
inhibitory effect on the capacity of 400 nM NE to activate
IIb
3, as evaluated by the binding of
PAC-1. By contrast, and as expected, both GF 109203X and
PGI2 were potent inhibitors of the expression of activated
IIb
3 on the surface of platelets
stimulated with 0.5 IU/ml thrombin (45, 46). Altogether, these data
clearly indicate that the potentiating effect of NE on threshold of
cathepsin G-induced platelet aggregation is not related to an increased
intracellular signaling involving the PLC, PKC, and Ca2+
components.
|
A major feature in our study
was that PMSF-inactivated NE was totally unable to either up-regulate
the activity of IIb
3 as measured by the
binding of PAC-1 (2.3 ± 0.2- versus 1.1 ± 0.1-fold increase (n = 3) over basal values with intact
and PMSF-treated NE, respectively), or to potentiate cathepsin
G-induced platelet aggregation (not shown and Ref. 19), indicating that
the proteolytic activity of NE is required for both processes. This,
together with the known susceptibility of
IIb
3 to proteolysis by serine proteinases
(26-30) and the absence of intracellular metabolic activation by NE
(see above), prompted us to examine whether the integrin structure was
affected by this proteinase under our experimental conditions.
Panel A in Fig. 5 illustrates the analysis of
the 3 and
IIb subunits separated on
7-12% gradient acrylamide gels following reduction of intra- or
interchain disulfide bonds, and probed with polyclonal rabbit antisera
raised against each of the whole subunit. When compared with nontreated
control platelets (lane 1), platelets exposed to 400 nM NE for 3 min (lane 2) showed no proteolytic
modification of the
3 subunit (Mr
118,000), whose mobility and intensity remained unchanged. Only
longer exposure (15 min) to 400 nM NE, or to higher
concentrations of proteinase (1.2 µM) resulted in the
appearance of a minor membrane-associated fragment of
3
with Mr
66,000 (not shown and Ref. 27).
Under the conditions of electrophoresis used in this experiment, the
IIb subunit could be clearly resolved into its two heavy
and light polypeptide chains (
IIbH,
Mr
126,000, and
IIbL,
Mr
25,000).
IIbL appeared to
be unchanged for both its mobility and intensity in NE-treated
platelets. By contrast, the
IIbH subunit migrated as a
broader band in platelets exposed to NE (lane 2), as
compared with nontreated platelets (lane 1), with a
component (indicated by the open arrowhead in Fig. 5,
panel A) running slightly ahead of the intact
IIbH.
More detailed immunoblot analysis of the IIbH subunit
was performed following reduced SDS-PAGE on highly resolutive 5%
acrylamide gels, using a panel of domain-specific
IIb
antibodies (see Table I). As illustrated in panel B of Fig.
5, the anti-
IIb polyclonal antiserum clearly identified
two molecular species in NE-treated platelets (lane 2),
showing approximately equal intensity, one corresponding to the intact
IIbH (Mr
128,000) as seen in
control untreated platelets (lane 1), and the second to a
membrane-bound proteolytic fragment with Mr
123,000, designated
IIbHf. On several platelet samples
exposed to NE under identical conditions, the mean
Mr difference between
IIbH and
IIbHf was 6,470 ± 290 (n = 15).
Considering that
IIbH is entirely extracellular, such a
limited proteolysis must have occurred at one or both extremities of
the polypeptide chain. The polyclonal rabbit antiserum IIb-10, recognizing the
IIbH amino-terminal
Leu1-Pro14 sequence (30), reacted equally with
the intact
IIbH and the
IIbHf fragment.
By contrast, the murine monoclonal antibody PMI-1, which recognizes the
IIbH carboxyl-terminal
Pro844-Arg856 sequence (34), was reactive with
the residual intact
IIbH in NE-treated platelets, but
totally unreactive with
IIbHf (Fig. 5, panel
B). To ascertain that proteolysis was limited to
IIbH, similar experiments were performed on unreduced
samples (Fig. 5, panel C). Here, the anti-
IIb
antiserum identified the native
IIb subunit
(i.e. disulfide-linked
IIbH and
IIbL) in nontreated samples with
Mr
143,000 (lane 1). With
NE-treated platelets (lane 2), a second component could be
distinguished slightly ahead of intact
IIb. This
component, designated
IIbf, had
Mr
137,000. The
IIb light
chain within the
IIbf membrane-bound proteolytic species
was shown to have an intact amino terminus by the normal reactivity of
IIbf with the anti-V41 polyclonal antiserum, which recognizes the amino-terminal Gln860-Arg871
sequence of
IIbL (34).
Similar immunoblot analysis was further performed on SDS lysates of
platelets treated for 3 min with the combination of 400 nM
NE and threshold of cathepsin G (lanes 3 in Fig. 5). Results were strictly identical to those obtained with platelets treated with
400 nM NE alone. In addition, we previously demonstrated that high concentrations of cathepsin G alone have no proteolytic effect on the IIb
3 integrin (47). Taken
together, these data indicate that under optimal conditions of
potentiation by NE of threshold of cathepsin G-initiated aggregation,
NE specifically proteolyzes a short domain located at the carboxyl
terminus of the
IIbH polypeptide chain.
Since the above data pointed to the existence of a specific and
previously unreported modification of
IIb
3 by NE, we sought to confirm that it
occurred to a significant extent at the surface of platelets. Platelet
suspensions were thus exposed to 400 nM NE for increasing
periods of time (up to 3 min) and analyzed by flow cytometry to
quantitate the binding of the monoclonal antibody PMI-1 (this being
taken as a marker of proteolysis at the
IIbH carboxyl
terminus) since the PMI-1 epitope should be lost upon exposure to NE.
Results indicated a near complete disappearance of the PMI-1 epitope on
NE-treated platelets, with a binding after 3 min of proteolysis
decreased by 93 ± 4% (n = 5) compared with the
initial value measured on untreated platelets (data not
illustrated).
Considering the domain of
IIb proteolyzed by NE (see above), the relative
Mr difference measured between
IIbH and
IIbHf (
6,500), and the fact
that in resting platelets, the epitope recognized by PMI-1 is largely
cryptic (41), the site(s) of cleavage by NE were searched by MALDI-TOF
mass spectrometry on a peptide corresponding to the sequence
Phe827-Leu841 of
IIbH. Indeed,
this sequence maps from the carboxyl-terminal side of
Cys826, which is involved in the linkage of
IIbH to
IIbL (20), to the amino-terminal
side of the PMI-1 epitope (34) (Fig. 6, panel B). The lower tracing in panel A of Fig. 6
shows a mass spectrum which corresponds to the initial undigested
peptide 827-841, with a mass of 1708.0 Da. Upon a 5-min incubation of
this peptide with 400 nM NE (upper tracing in
panel A of Fig. 6), a single new peptide was generated with
a mass of 1236.8 Da, corresponding to the sequence Phe827-Val837. This indicates a proteolysis of
the initial peptide at a unique bond, between Val837 and
Asp838. Of note is that the cleavage was detectable as soon
as 30 s and was complete at 10 min, without detection of other
proteolytic products (not shown). The shorter tetrapeptide fragment
Asp838-Leu841 could not be detected under our
experimental conditions.
Relationship between Cleavage of the
When bindings of both PMI-1
and AP-5 were examined by flow cytometry on the same platelet samples
treated by NE, a relationship was observed between the time course of
proteolysis of the IIbH subunit (i.e. the
decreasing binding of PMI-1) and that of the ligand binding capacity of
IIb
3 (i.e. the increasing
binding of AP-5) (data not shown). To further support this inference
and to exclude an activation of the integrin resulting from NE acting on another membrane structure inducing signaling which may feed-back to
IIb
3, two series of experiments were
carried out. First, platelets were depleted in cytosolic ATP by
incubation for 15 min with 50 mM
2-deoxy-D-glucose, 0.05% NaN3, and 10 mM glucono-
-lactone to inhibit glycolysis, oxidative
phosphorylations, and glycogen phosphorylase, respectively (48). These
platelets were treated or not with 400 nM NE or 550 nM cathepsin G for 3 min, and PAC-1 binding was examined by
flow cytometry as an index of
IIb
3
activation. Results showed that while NE-induced activation of the
fibrinogen receptor remained unchanged (p > 0.05, n = 3), that produced through intracellular pathways by
cathepsin G (see Fig. 4) was inhibited by 73.8 ± 1.0%
(p < 0.001, n = 3). Second, CHO cells
expressing human
IIb
3 were used as it is
known that these cells do not support agonist-induced metabolic
activation of this integrin (49). Fig. 7 indicates that
treatment of CHO/
IIb
3 cells with NE
resulted in a 4.2 ± 0.7-fold increase over basal value for PAC-1
binding (n = 2). This was specific to NE since
incubation of these cells with 550 nM cathepsin G did not
trigger any increase in PAC-1 binding. Moreover, this process did not
result from an increase of
IIb
3 molecules
expression at the surface of CHO/
IIb
3 cells as values for AP-2 binding were unchanged compared with nontreated cells. Importantly, in both cases (i.e. platelets
pretreated with metabolic inhibitors and
CHO/
IIb
3 cells), activation of
IIb
3 by NE was accompanied by an
extensive proteolysis of
IIbH (Fig. 7).
We thus further examined whether cleavage of the IIbH
subunit by NE quantitatively correlates with the activation of
IIb
3. Direct binding of
125I-AP-5 and 125I-PMI-1 antibodies allowed
quantitative measurements of the number of NE-activated and occupied
IIb
3 molecules in relation to the number
of proteolyzed molecules, respectively. Panel A in Fig. 8 shows that the maximal number of binding sites for
125I-PMI-1 on control nontreated platelets (27,270 ± 5,360 molecules/platelet, n = 3) progressively
decreased upon exposure of cells to NE, to be reduced at 3 min by
96 ± 3%. Panel B demonstrates that, on a time course
basis, the proteolysis of the carboxyl-terminal domain of
IIbH correlates linearly with the increased capacity of
IIb
3 to bind exogenous fibrinogen
(r2 = 0.902, p
0.005). In
this series of experiments, the maximal binding of
125I-AP-5 (measured in the presence of EDTA) on nontreated
as well as NE-treated platelets amounted to 18,250 ± 770 molecules/platelet (n = 3), whereas the specific
binding measured in the presence of divalent cations and fibrinogen on
platelets treated with NE for 3 min amounted to 4,270 ± 760 molecules/platelet, i.e. 23 ± 3.6% of the maximal
AP-5 binding capacity of platelets. These data suggest that not all
proteolyzed
IIb
3 molecules acquire the
capacity to bind a ligand, and that the stoichiometry is about one
active integrin over four proteolyzed.
The aim of the present investigation was to characterize the
molecular mechanism underlying the synergistic activation of platelets
by the neutrophil-derived proteinases elastase and cathepsin G. The
major findings are as follows: (i) NE does not activate the platelet
intracellular signaling but specifically cleaves the IIb
subunit heavy chain of the
IIb
3 integrin,
likely between Val837 and Asp838; (ii) this
proteolysis correlates with an up-regulation of the fibrinogen receptor
function of this integrin; and (iii) this particular activation of
IIb
3 by NE is relevant for the
potentiation of platelet aggregation initiated by low concentrations of
cathepsin G.
The IIb
3 integrin has been shown to
undergo variations in affinity for fibrinogen that reflect
conformational changes within the
IIb and
3 subunits (20-22). Since cathepsin G stimulates platelets as potently as does thrombin and through a common signaling pathway (14-16, 50), it may be speculated that the mechanism which
regulates the fibrinogen-binding function is similar for both
proteinases. In thrombin-activated platelets, an intracellular signal
transduction pathway, including heterotrimeric GTP-binding proteins,
PLC, PKC, PI 3-kinase, low molecular weight GTP-binding proteins and
the cytoskeleton, affects the cytoplasmic domains of
IIb
3 and influence the conformation of
the extracellular domains (inside-out signaling) (22, 51). Hence,
cathepsin G-induced activation of the
IIb
3 integrin (the present study and Ref.
43 and 50) is likely controlled by this complex network of
intracellular signaling reactions. In addition to that exposed on the
cell surface,
IIb
3 has been identified in
an internal membrane compartment made of the surface-connected
canalicular system and the
-granules (24). Compared with resting
platelets, the density of
IIb
3 on the
platelet plasma membrane increases after strong stimulation, due to the
mobilization of these intracellular stores (25). This is confirmed in
the present work for a maximal concentration of cathepsin G (550 nM) using AP-2, a monoclonal antibody that recognizes a
complex-dependent determinant on
IIb
3, whose binding doubles under these
conditions, in agreement with previous findings (50).
Other serine proteinases have been shown to induce the activation of
IIb
3, but through a nonmetabolic pathway.
Thus, treatment of platelets with chymotrypsin (28-30) or elastases
(26, 27) results in a proteolytic-dependent exposure of
fibrinogen-binding sites at the platelet surface. In accordance with
these studies, measurements of the binding of PAC-1 and AP-5 antibodies
allowed us to demonstrate that NE rapidly induces the high affinity and ligand-occupied conformers of native
IIb
3. Unlike cathepsin G or thrombin,
this was not accompanied by an increase of the number of
IIb
3 expressed on the platelet surface.
Another difference with cathepsin G or thrombin is that the
up-regulation of
IIb
3 by NE occurs even
in the presence of the potent inhibitor of platelet activation
PGI2 or after the metabolic pool of ATP has been depleted. A similar stimulation of fibrinogen binding at the platelet surface, independent of PGI2-inhibitable pathways, has been observed
following incubation of platelets with Fab fragments of certain
anti-LIBS antibodies (52). It was hypothesized that these activating
antibodies shift a conformational balance to favor or stabililize the
high affinity of
IIb
3 for fibrinogen.
With regard to NE, its potential binding to the integrin might also
displace a structural equilibrium as do anti-LIBS antibodies. However,
our data rather indicate that the conformational shift which uncovers
binding site(s) for fibrinogen is associated with a proteolytic
processing. Indeed, the blockade of NE catalytic site by PMSF
suppressed the activation of
IIb
3.
Actually, immunoblot analysis as well as the measurement of the binding
capacity of platelets for PMI-1 antibody, revealed a limited
proteolysis by NE of the
IIb subunit heavy chain
carboxyl terminus. Furthermore, kinetics of this cleavage closely
correlated with that of the expression of the ligand binding activity.
It is of note, however, that these two events occur in a stoichiometry of about four
IIb
3 molecules proteolyzed
for one molecule activated. Taken together, these data mean that NE
cleaves almost all the
IIb
3 complexes
expressed at the platelet plasma membrane, but only a fraction
(
25%) of them shift from an inactive to an active conformer. This
observation is actually consistent with studies which have brought
evidence for the existence of distinct subpopulations of
IIb
3 at the platelet surface, showing
distinct conformations and/or susceptibility to acquire an activated
state (52-54). Interestingly, this functional heterogeneity appears
intrinsic to the
IIb
3 molecules. Thus,
after purification of the total platelet integrin, at least two
conformers can be identified, one inactive but still activable, and one
"naturally" active following solubilization (
20% of the total,
based on data reported in Ref. 54). The mechanism through which a
fraction of the platelet
IIb
3 molecules resists the activation shift remains to be determined. Finally, that NE
activates
IIb
3 directly through
proteolysis was confirmed using CHO cells transfected with human
IIb
3, knowing that the integrin expressed
in these cells is unable to respond to metabolic activation (49). All
these data concur to exlude that NE acts on another membrane protein
which may secondary stimulate
IIb
3.
MALDI-TOF mass spectrometry performed on the synthetic peptide
corresponding to the sequence Phe827-Leu841 of
IIbH gave an insight into the site of NE-induced
proteolysis by indicating a cleavage located between Val837
and Asp838. This finding is in agreement with the primary
specificity of NE for valine residues (55). As a result of this
cleavage by NE within the
IIbH carboxyl terminus, a
peptide of 19 amino acids, i.e.
Asp838-Arg856, including an
O-glycosylated serine residue at position 847 (20) (see Fig.
6), is expected to be released. The difference in
Mr found in SDS-PAGE between
IIbH
and its membrane-bound proteolytic derivative (
6,500) is in fair
agreement with the presumed mass of such a glycopeptide. A mechanism by
which this restricted cleavage can modulate global conformational
changes within the whole
IIb
3 integrin
can be hypothesized on the basis of different structural models of this
integrin (see Fig. 9). From the model proposed by Honda
et al. (33), it can be suggested that the
IIb
domain proteolyzed by NE is located in a structurally constrained
cluster of cryptic LIBS epitopes including PMI-1 (
IIbH
842-856), AP-5 (
3 1-6), LIBS-2 (
3
602-690), and an undefined region in
3 interacting with
the PMI-2 antibody. On the other hand, studies aimed to localize the
IIb sequences involved in the intrasubunit contacts have suggested that
IIbH would be folded on itself, notably
through interactions between its amino-terminal and carboxyl-terminal domains (56). Therefore, it may be speculated that through limited proteolysis of the carboxyl-terminal end of the
IIbH
subunit, NE removes a constraint in a confined but particularly
sensitive region. The generated conformational change may then
propagate over the whole
IIb
3 complex
(57), converting a subpopulation of the integrin molecules from a
resting to an active fibrinogen receptor.
While the PLC/PKC pathway controls the synergism resulting from
combination of low concentrations of platelet agonists such as thrombin
and epinephrine (58), it does not account for the enhanced activation
of IIb
3 and platelet aggregation induced by the combination of NE and cathepsin G (see Fig. 4). As one consequence, it can be ruled out that this process occurs through an
increase by NE of the catalytic activity of cathepsin G, or an increase
of cathepsin G receptor expression and/or affinity, which would have
resulted in an increase of intracellular signaling messengers. In fact,
on the basis of the foregoing data, and notably those showing the
activation by NE of surface-expressed
IIb
3, a hypothetical mechanism may be
proposed to construe how NE enhances the platelet aggregation initiated
by low concentrations of cathepsin G. During this process, cathepsin G
would initiate different intracellular transduction signals, including
the PLC-Ca2+-PKC pathway, with as one result, a basal
metabolic activation of
IIb
3, however,
insufficient to promote stable aggregate formation. In parallel, NE
would trigger a proteolysis of the carboxyl terminus of the
IIb subunit heavy chain with the subsequent spatial
reorientation of the extracellular domains within the
IIb and
3 subunits allowing increased
binding of fibrinogen. As more fibrinogen binds and cross-links
adjacent platelets, post-occupancy outside-in signaling events through
IIb
3 may amplify cell activation (51),
and finally maximize the recruitment of platelets into aggregates similar to those produced by high concentrations of strong platelet agonists acting through a solely intracellular signaling pathway. Consistent with this proposal, it is of interest to note that the
potentiating effect of NE is not restricted to cathepsin G, as NE can
also enhance platelet aggregation initiated by low concentrations of
collagen or the thromboxane A2 stable analog U46619 (not
shown and Ref. 19).
Numerous studies suggest that platelet activation in vivo
could be attributed to the combined action of pairs (or more) of agonists (58, 59). The potentiation by NE of cathepsin G-induced platelet activation reported in the present work might be relevant in
physiopathological situations in which both platelets and neutrophils can locally accumulate and physically interact such at sites of inflammatory or thrombotic lesions (4-6). In connection with these
studies, it is tempting to speculate that proteinase-induced enhancement of adhesive receptor function could be a wide and physiologically relevant process. Indeed, like the
proteolysis-dependent activation of
IIb
3 by NE described here, a recent study
provided evidence that serine proteinases proteolytically enhance cell adhesion mediated by the integrin
V
3
(60).
We thank Dr. Mark H. Ginsberg, Dr. Thomas J. Kunicki, and Dr. Stanley E. D'Souza, from the Scripps Research Institute (La Jolla, CA), for kindly providing antibodies essential for this project. We are indebted to Jean-Charles Théodet for his participation in the early steps of this study. We also thank Dr. Juan J. Calvete (Institut für Reproduktionsmedizin, Hannover, Germany) for helpful comments during these investigations.