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J Biol Chem, Vol. 275, Issue 13, 9758-9766, March 31, 2000
From the Platelet function is influenced by the platelet
thiol-disulfide balance. Platelet activation resulted in 440% increase
in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1b The platelet thiol-disulfide balance is important for platelet
function. Perturbation of platelet thiol status effects platelet aggregation and release. The low Mr thiol
compounds, reduced glutathione (GSH), cysteine, and 6-mercaptopurine,
inhibit platelet aggregation induced by several agonists, while the
disulfide-bond reducing agents dithiothreitol and Protein-disulfide isomerase
(PDI)1 is a noncovalent
homodimer with a subunit molecular mass of 57 kDa that catalyzes
thiol-disulfide interchanges that can result in formation, reduction,
or rearrangement of protein disulfide bonds. It is generally considered
that PDI is important for proper folding and disulfide bonding of
nascent proteins in the endoplasmic reticulum (8-10). PDI also
functions as the Increase or decrease in PDI on the surface of HT1080 human fibrosarcoma
cells is associated with increase or decrease in cell surface protein
thiols (28) and cell surface PDI has been implicated in the increase in
surface protein thiol content of human lymphocytes following mitogen
activation (19, 29). These observations indicated that secreted PDI can
control the redox state of existing exofacial protein thiols or
reactive disulfide bonds. We have examined the redox properties of PDI
on the platelet plasma membrane.
Platelet activation and aggregation resulted in 440% increase in
surface protein thiol groups. Both PDI and the von Willebrand factor
receptor, glycoprotein 1b Reagents and Chemicals--
HEPES, apyrase (grade VII),
leupeptin, phenylmethylsulfonyl fluoride, streptavidin-agarose,
iodoacetamide, GSH, and dithiothreitol were purchased from Sigma, and
Trasylol (aprotinin) from Bayer Australia, Sydney, New South Wales,
Australia. D-Phe-Pro-Arg-chloromethyl ketone was obtained
from Calbiochem, San Diego, CA.
3-(N-Maleimidylpropionyl)biocytin (MPB) was purchased from
Molecular Probes, Eugene, OR, and sulfosuccinimidobiotin (SSB) from
Pierce. PolyScreen polyvinyldiethylene fluoride (PVDF) transfer
membrane, Western blot chemiluminescence reagent, and reflection
autoradiography film were purchased from DuPont, Boston, MA. All other
chemicals were of reagent grade.
Proteins--
PDI was purified from human placenta according to
the method of Lambert and Freedman (30) with modifications described by Hotchkiss et al. (31). Human Platelet Preparation--
Platelets were isolated from whole
blood as described previously (34), with the following modifications.
The platelets were washed and incubated at 37 °C for 15 min in
Ca2+- and bovine serum albumin (BSA)-free Tyrode buffer
containing 2 units/ml apyrase to minimize platelet activation.
Following two further washes in apyrase-free Tyrode buffer the
platelets were resuspended in 20 mM HEPES, 137 mM NaCl, 4 mM KCl, 0.5 mM Na2HPO4, 0.1 mM CaCl2,
pH 7.4, buffer. Activation of platelets was with 30 nM
human Labeling of Platelets with SSB or MPB--
Labeling with SSB was
a modification of the method of Ingalls et al. (35), while
labeling with MPB was a modification of the method of Roffman et
al. (36). Resting or activated platelets (1 ml of ~7 × 108 per ml for SSB and ~0.4-2 × 109
per ml for MPB) were incubated with SSB (1 mM) or MPB (100 µM) for 30 min at room temperature on a rotating wheel.
On some occasions, resting platelets (1 ml of 1.3 × 109 per ml) were incubated with 50 µM
dithiothreitol for 30 min at room temperature prior to labeling with
MPB. On another occasion, resting platelets (1 ml of 0.4 × 109 per ml) were incubated with 20 µg/ml ReoPro (Centocor
B.V., Leiden, The Netherlands) and 5 mM EDTA for 30 min at
room temperature, prior to activation with thrombin and labeling with
MPB. Unreacted SSB was quenched with glycine (2 mM), and
unreacted MPB with GSH (200 µM), for 30 min at room
temperature. The unreacted GSH was quenched with iodoacetamide (400 µM) for 10 min at room temperature. The labeled platelets
were washed twice with PBS and sonicated as described above. An aliquot
of labeled platelet sonicate was set aside for Western blotting with
streptavidin-HRP. Streptavidin-agarose beads (100 µl of packed beads
for SSB or 25 µl of packed beads for MPB) were incubated with the
platelet sonicates for 1 h at 4 °C on a rotating wheel to
isolate the biotin-labeled proteins. The streptavidin-agarose beads
were washed 5 times with 50 mM Tris-HCl, 0.15 M
NaCl, 0.05% Triton X-100, pH 8.0, buffer. The beads were suspended in
200 µl (SSB) or 50 µl (MPB) of SDS-Laemmli buffer and boiled for 2 min.
MPB was also used to label purified human placenta PDI. PDI (0.5 µM), or PDI preincubated with dithiothreitol (5 µM) for 30 min, was labeled with MPB (100 µM) for 30 min. Unreacted MPB was quenched with GSH (200 µM) for 10 min and iodoacetamide (400 µM) added for 10 min to quench unreacted GSH. All incubations were performed at room temperature in 20 mM HEPES, 0.14 M NaCl, pH 7.4, buffer.
Immunoprecipitation of Platelet Surface
Glycoproteins--
Resting or activated platelets were labeled with
either SSB or MPB as described above and resuspended in PBS containing
1% BSA. The labeled platelets were incubated for 30 min at 4 °C on a rotating wheel with 10 µg/ml of either control murine monoclonal antibody (MOPC21) or murine monoclonal antibodies that recognize either
GP1b SDS-PAGE and Western Blotting--
Samples were resolved
on 10, 12, or 5-15% SDS-PAGE under nonreducing conditions according
to Laemmli (37), transferred to PVDF membrane, developed
according to the manufacturers instructions (DuPont), and visualized
using chemiluminescence. Affinity-purified rabbit anti-PDI polyclonal
antibodies were used at a final dilution of 1:5000, swine anti-rabbit
HRP-conjugated antibodies at 1:1000 dilution, and streptavidin-HRP at
1:2000 dilution.
Quantitation of SSB- and MPB-labeled Platelet Surface
Proteins--
SSB- or MPB-labeled platelet surface proteins were
quantitated by densitometry using a Model GS-300 Hoeffer Scientific
Instruments scanning densitometer. SSB- or MPB-labeled PDI was
quantitated by relating band intensity to a standard curve constructed
using purified placenta PDI. Total protein estimations (BCA Protein Assay, Pierce, Rockford, IL) were performed on all platelet samples prior to SSB or MPB labeling and after platelet sonication to correct
for platelet loss during the labeling and washing procedures. This loss
was always <20%.
Quantitation of Platelet Low Mr Thiol
Compounds--
Platelet releasate was separated from 1.5 × 109 thrombin-activated platelets in 1 ml by centrifugation
at 2,000 × g for 20 min at 4 °C. The total protein
in releasate and platelet sonicate was 0.21 and 1.5 mg/ml,
respectively, using the BCA protein assay (Pierce, Rockford, IL). The
platelet low Mr thiol compounds were derivatized
with the fluorescent compound, 7-benzo-2-oxa-1,3-diazole-4-sulfonic acid, and resolved by reverse-phase HPLC as described previously (38).
The starting sample was diluted 9.45-fold during derivatization and 20 µl was injected onto HPLC.
Flow Cytometry--
Flow cytometry was performed using a FACStar
Plus cytometer (Becton Dickinson, San Jose, CA) with argon ion laser
exitation at 488 nm. Emission spectra were collected using a 530 ± 30 nm band pass filter for fluorescein isothiocyanate and Alexa-488, or a 585 ± 45 nm band pass filter for phycoerythrin (PE). Ten thousand platelets were acquired at a flow rate of 500-1000 particles per second.
Binding of vWF to Platelets--
Purified human plasma vWF was a
gift from Dr. M. Berndt (39). The vWF was labeled with the
fluorochrome, Alexa-488, according to the manufacturer's instructions
(Molecular Probes, Eugene, OR). Briefly, vWF was mixed with Alexa-488
at pH 8.3 with constant stirring at room temperature for 60 min. The
reaction was quenched with hydroxylamine and the unconjugated dye
removed by dialysis against PBS. The concentration of the vWf-Alexa
conjugate was determined by protein assay (BCA Protein Assay, Pierce).
vWF and Alexa-488-labeled vWF were resolved on 1% agarose gel
electrophoresis according to Ruggeri and Zimmerman (40). There was no
apparent difference in the multimer distribution of unlabeled
versus Alexa-488-labeled vWF.
Washed platelets were prepared as described above and resuspended at
2 × 106 platelets/ml in PBS containing 1% BSA and 10 mM EDTA. Platelets were incubated with 20 µg/ml of the
anti-platelet Fc Fluorescence Resonance Energy Transfer (FRET)--
To assess the
proximity of PDI and GP1b Effect of Anti-PDI Polyclonal Antibodies on MPB Labeling of
GP1b Binding of Anti-GP1b-IX Monoclonal Antibodies to
Platelets--
Resting platelets (2 × 106 in 100 µl of BSA-free Tyrodes buffer) were incubated with 200 µg/ml of
either preimmune rabbit IgG F(ab)2 fragments or rabbit
anti-PDI F(ab)2 fragments for 30 min at room temperature.
The F(ab)2 fragments were made using the Pierce
F(ab)2 Preparation Kit (Pierce). The platelets were
activated with thrombin as described above and incubated with 10 µg/ml of the following fluorescein isothiocyanate-conjugated
monoclonal antibodies: MOPC21 (irrelevant control, Becton Dickinson,
San Jose, CA), SZ1 (anti-GPIX, Immunotech, Marseille,
France), AN51 (anti-GPIb Platelet Aggregation Triggered an Increase in Platelet Surface
Protein Thiol Groups--
The membrane impermeable biotinylated
thiol-specific reagent, MPB, was used to label sulfhydryl groups on
resting or thrombin-activated platelets allowed to aggregate or
prevented from aggregating with ReoPro and EDTA (Fig.
1A). Platelets were labeled
with MPB, sonicated, resolved on 12% SDS-PAGE, transferred to PVDF
membrane, and blotted with streptavidin peroxidase.
The amount of platelet surface proteins labeled with MPB increased
markedly upon platelet activation/aggregation. Densitometric analysis
of the profiles indicated that 460% more protein was labeled with MPB
on activated/aggregated platelets compared with resting platelets. In
contrast, far less protein was labeled with MPB on activated platelets
prevented from aggregating with ReoPro/EDTA, 60% increased labeling
compared with resting platelets. The same result was observed if EDTA
alone was used to minimize platelet aggregation (not shown). This
result implied that platelet activation and aggregation, not activation
alone, triggered reduction of platelet surface protein disulfides or
presentation of previously cryptic protein thiols. The intensity of
labeling of individual proteins with MPB increased with platelet
activation/aggregation. In addition, activation/aggregation-specific
MPB-labeled proteins were also observed. This feature of activated
platelets was examined in more detail by measuring the changes in total
protein on the resting versus activated platelet surface and
that fraction of protein that presented free sulfhydryl group(s).
Quantitation of Thiol- versus Amine-containing Platelet Surface
Proteins--
Platelet surfaces were labeled with either MPB or SSB.
SSB reacts with primary amines and, therefore, is a measure of total surface protein. The ratio of thiol- to amine-labeled surface protein
was used to estimate the relative proportion of total platelet surface
protein that presented free sulfhydryl group(s). MPB or SSB was
incubated with resting or thrombin-activated/aggregated platelets. The
labeled platelets were sonicated, resolved on 10% SDS-PAGE,
transferred to PVDF membrane, and blotted with streptavidin-peroxidase to detect the SSB or MPB label. The profiles of the labeled proteins are shown in Fig. 1B. The greater prevalence of primary
amines compared with sulfhydryl groups on the platelet surface
necessitated labeling of 22 times more platelets with MPB than with
SSB.
The amount of platelet surface proteins labeled with SSB increased
significantly upon platelet activation/aggregation. Densitometric analysis of the profiles indicated that 130% more protein was labeled
with SSB on activated/aggregated platelets compared with resting
platelets. In contrast, 440% more protein was labeled with MPB on
activated/aggregated platelets compared with resting platelets (see
also Fig. 1A). The ratio of thiol- to amine-labeled protein
on resting versus activated/aggregated platelets was a measure of the relative increase in surface protein thiols with respect
to total surface protein. Platelet activation/aggregation resulted in a
~190% increase in surface protein thiol groups. At least 11 proteins
presented free thiol(s) that were labeled with MPB on the
activated/aggregated platelet surface but were poorly or not labeled on
the resting platelet surface.
The finding that platelet activation/aggregation resulted in
significantly more labeling of certain platelet surface proteins with
MPB, implied that activation influenced the redox state of the
thiol/disulfide groups of these proteins. PDI is a platelet surface
protein (26, 27) whose activity is regulated by the redox state of its
active site dithiols/disulfides (31). Moreover, PDI has been shown to
regulate the redox state of proteins on the surface of cultured human
fibroblasts (28) and lymphocytes (29). These findings suggested that
PDI might similarly regulate the redox state of platelet surface
proteins. We investigated the consequence of platelet
activation/aggregation for the redox state of platelet surface PDI.
Platelet Activation Resulted in Reduction of Surface-bound
PDI--
The specificity of labeling of oxidized versus
reduced PDI with MPB is shown in Fig.
2A. The active site dithiols
of PDI purified from liver (44) or placenta (31) are oxidized. MPB did
not label purified placenta PDI, as expected, but did label PDI
activated by a low concentration of dithiothreitol (5 µM).
The ratio of thiol- to amine-labeled PDI was used to estimate the
relative proportion of total platelet surface PDI that contained active
site dithiols (Fig. 2B). MPB or SSB was incubated with resting or thrombin-activated/aggregated platelets and the
biotin-labeled proteins purified by affinity chromatography on
streptavidin-agarose beads. The labeled proteins were resolved on 10%
SDS-PAGE, blotted with anti-PDI polyclonal antibodies, and quantitated
using densitometry. Control reactions omitting the label were performed
to ensure that no unlabeled PDI was being nonspecifically carried
through the procedure (not shown).
Platelet PDI had the same apparent mass of ~57 kDa as placenta PDI on
SDS-PAGE (Fig. 2B), and they share the same amino-terminal sequence (26, 45). Amine-labeled platelet surface PDI increased 170%
upon platelet activation/aggregation (2,430 ± 120 to 6,480 ± 320 molecules of PDI per platelet). In contrast, thiol-labeled PDI
increased 750% upon platelet activation/aggregation (620 ± 50 to
5,250 ± 240 molecules of PDI per platelet). Therefore, 26% of
the total PDI on resting platelets was in a reduced conformation compared with 81% on activated/aggregated platelets (Table
I). It should be noted that PDI contains
two active site dithiols/disulfides and that this technique does not
distinguish between reduction of one or both of these disulfides.
Therefore, the reduced PDI measured on the platelet surface is the sum
of PDI molecules containing either one or two active site dithiols.
We hypothesized that the active site disulfides of platelet surface PDI
might be reduced by low Mr thiol compounds
secreted by activated platelets. To test this theory, whole platelet
and secreted low Mr thiol compounds were measured.
Characterization of Platelet Low Mr Thiol
Compounds--
Platelets contain low Mr thiol
compounds, in particular GSH and cysteinylglycine (CysGly) (for
example, see Ref. 46). We compared the low Mr
thiol content of thrombin-activated/aggregated platelets and activated
platelet releasate from two healthy individuals. The releasate from
1.5 × 109 thrombin-activated platelets/ml contained
<0.2 µM low Mr thiol compounds
(Fig. 3A). The average
concentrations of cysteine, cysteinylglycine, and GSH in normal plasma
are 9, 3, and 5 µM, respectively (47). This result
indicates that platelets are not a significant source of plasma low
Mr thiols and implied that platelet surface PDI was not reduced by secreted low Mr thiol
compounds. These observations suggested that
activation/aggregation-dependent reduction of platelet surface
PDI was mediated by protein-catalyzed events.
To further test this hypothesis, the susceptibility of the active site
disulfides of platelet surface PDI to reduction by dithiothreitol was
examined. Incubation of resting platelets with 50 µM
dithiothreitol for 30 min did not result in any further reduction of
platelet surface PDI (Fig. 3B). In contrast, incubation of
purified PDI with 5 µM dithiothreitol for 30 min was
sufficient to reduce the active site disulfides (Fig. 2A).
This result implied that the redox state of platelet surface PDI was
refractory to extracellular reducing agents.
The results shown in Figs. 1-3 demonstrated that the active site
dithiols/disulfides of PDI were in the dithiol state on the activated/aggregated platelet surface. At least 11 proteins contained free thiol(s) and were labeled with MPB on the activated/aggregated platelet surface but were poorly or not labeled on the resting platelet
surface (see Fig. 1B). These proteins were potential substrates for PDI. One of these proteins had a
Mr of ~120, which is the approximate
Mr of the platelet receptor for von Willebrand factor (vWF), GP1b Labeling of Thiol(s) in GP1b
GP1b
The results shown in Figs. 1-4 suggested that PDI and GP1b Inhibition of Binding of vWF to GP1b
Washed platelets were incubated with 20 µg/ml IV.3 and 0 to 200 µg/ml control or anti-PDI IgG and the binding of Alexa-labeled vWF
was measured by flow cytometry (Fig. 5).
The anti-PDI IgG reduced binding of vWF to platelets. The effects were
on the apparent dissociation constant for vWF binding with no
discernible effect on the apparent stoichiometry. The apparent
dissociation constant and maximal binding of vWF was 1.0 ± 0.2 µg/ml and 98 ± 6% in the presence of control IgG, and 1.5 ± 0.2 µg/ml and 97 ± 5% in the presence of 50 µg/ml
anti-PDI IgG. This corresponded to molar dissociation constants of
0.5 ± 0.1 and 0.8 ± 0.1 nM, respectively, assuming a weight average Mr for vWF of 2 × 106. This affinity is in good agreement with other
estimates of vWF binding (Ref. 49 and references therein).
These findings supported an association between PDI and GP1b Physical Proximity of PDI and GP1b
A FRET efficiency of 14.8 ± 4.9% (1 S.D.) was calculated from
three experiments. A 14.8% FRET efficiency means that on average the
PE fluorescence intensity of the FRET sample (labeled with both donor
and acceptor molecules) was 14.8% less than that of the donor sample
(labeled with donor molecule only). A FRET efficiency above 5% is
considered significant (41-43), therefore, our value of 14.8 ± 4.9% should be considered as significant. There was no FRET between
PE-labeled preimmune IgG and Cy5-labeled GP1b
It is unlikely that the FRET between PE-labeled PDI and
Cy5-labeled GP1b
The results shown in Fig. 4 implied that GP1b Enhancement of Labeling of GP1b
Incubation of resting platelets (1 × 109
platelets/ml) with dithiothreitol-activated PDI (0.5 µM)
for 10 min, or 200 µg/ml rabbit anti-PDI IgG for 30 min, at 37 °C
did not cause exposure of the free thiol in GP1b Enhancement of Binding of Anti-GP1b
The anti-PDI F(ab)2 fragments did not effect binding of the
irrelevant control antibody, MOPC21. The binding of SZ1, AN51, AK2, and
HIP1 to activated platelets significantly increased upon incubation
with anti-PDI F(ab)2 fragments, while the binding of SZ2
decreased (Fig. 8A).
Quantitation of the effects of anti-PDI polyclonal F(ab)2
fragments on the binding of the anti-GP1b-IX monoclonal antibodies is
shown in Fig. 8B. The anti-PDI F(ab)2 fragments did not
effect binding of any of the antibodies to resting platelets (not
shown).
Platelet activation/aggregation resulted in 440% increase in
exofacial protein thiol groups. This increase was dependent on platelet
aggregation, as negligible increase in surface protein thiol groups was
observed if activated platelets were prevented from aggregating. The
increase in surface thiols was due to expression of new
thiol-containing proteins on the platelet surface, generation of thiols
by reduction of disulfide bonds in existing proteins, or presentation
of previously cryptic protein thiols. At least 11 proteins presented
free thiol(s) that were labeled on the activated/aggregated platelet
surface but were poorly or not labeled on the resting platelet surface.
One of these proteins was PDI.
The ability of PDI to catalyze disulfide interchange in proteins
resides in two very reactive surface exposed dithiols/disulfides which
share the common sequence WCGPCK and have a redox potential of There were ~2,400 molecules of amine-labeled PDI on the resting
platelet surface versus ~6,500 molecules on the
activated/aggregated platelet surface, an increase of 170%. This
result implied that platelet activation/aggregation resulted in
recruitment of PDI to the platelet surface and/or that a fraction of
the PDI on the resting platelet surface was refractive to labeling with
SSB. Chen et al. (26) reported no clear difference in the
amount of PDI on resting versus activated platelets by
immunogold labeling and electron microscopy, which suggested that the
170% increase we observed was perhaps due to labeling differences
between resting and thrombin-activated/aggregated platelets. There is
evidence that resting platelets contain a surface connected
compartment, perhaps located in the platelet surface-connected
canalicular system, which can be freely entered by some compounds but
not others (51). It may be that the structure of SSB confers limited access to a pool of PDI on the resting platelet surface that becomes accessible following thrombin activation, which could account for the
observed increase in total PDI on the surface of activated/aggregated platelets.
PDI containing free sulfhydryl groups increased 750% upon platelet
activation/aggregation, and by comparison with amine-labeled PDI, 81%
of the PDI on activated platelets was in a reduced conformation compared with 26% on resting platelets. This result indicated that
platelet activation/aggregation triggered reduction of the active site
disulfides of surface PDI. Although every effort was made to minimize
activation/aggregation of the resting platelets, activation/aggregation
of a fraction of the platelets during the washing procedure was almost
unavoidable. The small amount of reduced PDI on the surface of resting
platelets may have represented the fraction of the total platelets that
is activated/aggregated. Therefore, the difference in amount of reduced
PDI on the surface of resting versus activated/aggregated
platelets may have in fact been greater then we have reported.
No detectable low Mr thiol compounds were
secreted upon platelet activation/aggregation and PDI on the surface of
resting platelets was refractory to reduction by 50 µM
dithiothreitol. These observations suggested that reduction of platelet
surface PDI was triggered by a secreted or plasma membrane protein or proteins. It was possible that platelet surface PDI was intrinsically reduced but that the active site dithiols were masked by a protein at
the platelet surface that was displaced upon platelet
activation/aggregation. However, the activated/aggregated platelet
surface contained 440% more protein thiol groups other than those of
PDI, which implied a more general reduction event. The report that
existing lymphocyte surface thiols are involved in the generation of
additional surface thiols (29) supports the notion that the active site
disulfides of PDI may have been reduced by an existing platelet surface
protein whose activity was controlled by platelet
activation/aggregation. One possibility is the plasma membrane
NADH-oxidoreductase system (52) which has been implicated in reduction
of extracellular protein disulfide bonds.
Another protein labeled with MPB on the activated/aggregated platelet
surface but not labeled on the resting platelet surface was GP1b PDI and GP1b Incubation of resting platelets with anti-PDI antibodies followed by
activation with thrombin enhanced labeling and binding of three
monoclonal antibodies to the N-terminal region (residues 1-74) of
GP1b These studies point to a control mechanism that is analogous to
activation of platelet We thank Wei Yu Fu and Xue Wei Guo for
assistance with determination of platelet low Mr
thiols and Dr. M. Berndt for purified vWF.
*
This work was supported by grants from the National Health
and Medical Research Council of Australia, the National Heart
Foundation of Australia, and an Infrastructure Grant from the New South
Wales Health Department.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Contributed equally to the results of this report.
The abbreviations used are:
PDI, protein-disulfide isomerase;
BSA, bovine serum albumin;
CysGly, cysteinylglycine;
GP, glycoprotein;
FRET, fluorescence resonance energy
transfer;
GSH, reduced glutathione;
HRP, horseradish peroxidase;
MPB, 3-(N-maleimidylpropionyl)biocytin;
PBS, phosphate-buffered
saline;
PVDF, polyvinyldiethylene fluoride;
PAGE, polyacrylamide gel
electrophoresis;
SSB, sulfosuccinimidobiotin;
vWF, von Willebrand
factor;
PE, phycoerythrin;
HPLC, high performance liquid
chromatography.
Physical Proximity and Functional Association of Glycoprotein
1b
and Protein-disulfide Isomerase on the Platelet Plasma
Membrane*
§,§,
,,, and
Centre for Thrombosis and Vascular Research,
School of Pathology, University of New South Wales and the Department
of Haematology, Prince of Wales Hospital, Sydney NSW 2052, Australia,
the ¶ Department of Molecular and Cellular Oncology, St.
Vincent's Hospital, Garvan Institute of Medical Research, Darlinghurst
NSW 2010, Australia, the Department of Cardiovascular Medicine,
Prince Henry Hospital, Little Bay NSW 2036, Australia, and the
** Department of Biophysics and Cell Biology, Medical University School
of Debrecen, Debrecen 4012, Hungary
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(GP1b). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on
resting platelets was in the dithiol form, compared with 81% in the
dithiol form on activated platelets. Similarly, GP1b presented one
or more free thiols on the activated platelet surface but not on
resting platelets. Anti-PDI antibodies increased the dissociation
constant for binding of vWF to platelets by ~50% and PDI and GP1b
were sufficiently close on the platelet surface to allow fluorescence
resonance energy transfer between chromophores attached to PDI and
GP1b. Incubation of resting platelets with anti-PDI antibodies
followed by activation with thrombin enhanced labeling and binding of
monoclonal antibodies to the N-terminal region of GP1b on the
activated platelet surface. These observations indicated that platelet
activation triggered reduction of the active site disulfides of PDI and
a conformational change in GP1b that resulted in exposure of a free thiol(s).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol
promote aggregation (1). In addition, reaction of platelet sulfhydryl
groups with the thiol specific compounds, diamide and
N-ethylmaleimide, inhibits in vitro aggregation
and clot retraction (2-5). These results imply that certain platelet
thiol groups are critical for platelet aggregation. Furthermore, the
observation that specific depletion of platelet GSH by
1-chloro-2,4-dinitrobenzene only marginally effects platelet
aggregability implies that the critical thiol groups are associated
with protein (6). In support of this notion, Yamada et al.
(7) have shown that the anti-platelet aggregation actions of
2,2'-dithiobis(N-2-hydroxypropylbenzamide) are mediated
through interaction of the compound with platelet protein thiol groups.
subunits of prolyl-4-hyroxylase (11, 12) and the subunit of triglyceride transfer protein complex (13, 14). Bovine
aortic endothelial cells (15), rat hepatocytes (16), rat pancreatic
cells (17), and human B cells (18, 19) secrete PDI which associates
with the cell surface, and murine fibroblasts secrete PDI in response
to treatment with calcium ionophore (20). Cell surface PDI has been
implicated in reduction of the disulfide-linked diptheria toxin
heterodimer (21, 22), cell surface events which trigger entry of the
human immunodeficiency virus into lymphoid cells (23), shedding of the
human thyrotropin receptor ectodomain (24), and as a cell surface
recognition/adhesion molecule during neuronal differentiation of the
retina (25). PDI is also on the external surface of the platelet plasma
membrane and can catalyze rearrangement of disulfide bonds in scrambled
ribonuclease (26, 27).
(GP1b), expressed free thiols on the
activated platelet surface. Moreover, PDI and GP1b were in close
proximity on the activated platelet surface. These findings demonstrated that platelet activation/aggregation triggered reduction of the active site disulfides of PDI and a conformational change in
GP1b that resulted in exposure of a free thiol(s). The close proximity of PDI and GP1b on the activated platelet surface
suggested that PDI may have participated in the conformational change
in GP1b.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thrombin was prepared as
described previously (32), and the active enzyme concentration
determined by active site titration (33). Rabbit polyclonal antibodies were developed against purified human placenta PDI in New Zealand White
rabbits and affinity purified on a PDI-Affi-Gel 15 matrix (Bio-Rad).
Streptavidin-horseradish peroxidase (HRP) was from Amersham Australia,
Sydney, NSW, and swine anti-rabbit IgG HRP conjugated antibodies were
from Dako Corporation, Carpinteria, CA. All proteins were aliquoted and
stored at 
80 °C until use.
-thrombin for 2 min at 37 °C on a rotating wheel. Thrombin
and platelet enzymes were quenched with 5 µM
D-Phe-Pro-Arg-chloromethyl ketone and 10 µM
leupeptin. Platelet releasate was separated from activated platelets by
centrifugation at 2,000 × g for 20 min at 4 °C. The
total protein in releasate from 1 × 109 activated
platelets per ml was 0.14 mg/ml using the BCA protein assay (Pierce).
Platelet lysates were prepared by washing platelets twice with
phosphate-buffered saline (PBS) by centrifugation at 2,000 × g for 20 min at 4 °C, resuspension in 50 mM
Tris-HCl, 0.5 M NaCl, pH 8.0, buffer containing 1% Triton
X-100, 10 µM leupeptin, 10 µM aprotinin, 2 mM phenylmethylsulfonyl fluoride, and 5 mM EDTA, and sonication at 4 °C. The total protein in 1 × 109 sonicated platelets per ml was 1.0 mg/ml using the BCA
protein assay (Pierce).
(AK3) or IIb3 (AP2). The platelets were washed three
times with PBS, lysed in 10 mM Tris-HCl, 0.15 M
NaCl, pH 8.0, buffer containing 0.5% Triton X-100, 0.05% Tween 20, 10 µM leupeptin, 10 µM aprotinin, 2 mM phenylmethylsulfonyl fluoride, and 5 mM
EDTA, sonicated as described above, clarified by centrifugation at
12,000 × g for 30 min at 4 °C, and incubated with
sheep anti-mouse coated Dynabeads (Dynal, Victoria, Australia) for
2 h at 4 °C on a rotating wheel. The Dynabeads were washed five
times with PBS, resuspended in SDS-Laemmli buffer, and boiled for 2 min.
RIIA receptor monoclonal antibody, IV.3, for 10 min
at room temperature and then with 0 to 200 µg/ml of either preimmune
rabbit IgG or anti-PDI IgG for a further 30 min at room temperature.
The blocked platelets were incubated with 0.1 to 10 µg/ml vWF-Alexa
and 1 mg/ml ristocetin (Sigma) for 10 min at room temperature. Unbound
vWF-Alexa was removed by washing the platelets three times with PBS
containing 1% BSA and the bound vWF-Alexa measured by flow cytometry.
on the platelet surface, the efficiency of
FRET between PE-labeled PDI and Cy5-labeled GP1b was measured by
flow cytometry as described previously (41-43). Platelets (2 × 107 in 100 µl of PBS containing 1% BSA) were incubated
with rabbit anti-PDI polyclonal antibodies (20 µg/ml) and/or the
anti-GPIb monoclonal antibody AK2 (20 µg/ml) for 20 min and washed
once with PBS containing 1% BSA. The primary antibodies were labeled by incubating for 20 min in the dark with PE-conjugated donkey anti-rabbit IgG (Becton Dickinson, San Jose, CA) or Cy5-conjugated donkey anti-mouse IgG (Jackson Immunoresearch, West Grove, PA). The
samples were diluted 4-fold with BSA-free Tyrodes and kept in the dark
until analysis. The FRET efficiency was expressed as the percentage of
the emission energy from donor (PE) taken up by acceptor (Cy5). This
method can detect proximity in the 2-10 nm range (41-43).
--
Resting platelets (1 ml of ~7 × 108
per ml for SSB and ~0.4-2 × 109 per ml for MPB)
were incubated with 200 µg/ml of either preimmune rabbit IgG or
rabbit anti-PDI IgG for 30 min at room temperature. The platelets were
activated with thrombin, labeled with either SSB or MPB, and the
GP1b-IX-V immunoprecipitated using AK3 monoclonal antibodies as
described above. The labeled glycoproteins were resolved on 10%
SDS-PAGE, transferred to PVDF membrane, and blotted with streptavidin
peroxidase to detect the SSB or MPB label.
, Dako Pharmaceuticals, Carpinteria, CA),
AK2 (anti-GPIb, Serotec, Oxford, United Kingdom), HIP1
(anti-GPIb, Becton Dickinson, San Jose, CA), and SZ2 (anti-GPIb,
Immunotech, Marseille, France). The samples were diluted 4-fold with
BSA-free Tyrodes and kept in the dark until analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
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Fig. 1.
Platelet aggregation triggered an increase in
platelet surface protein thiol groups. A, the membrane
impermeable thiol-specific reagent, MPB, was used to label sulfhydryl
groups on resting or thrombin-activated platelets allowed to aggregate
or prevented from aggregating with ReoPro (20 µg/ml) and EDTA (5 mM). Platelets were labeled with MPB, sonicated, resolved
on 12% SDS-PAGE, transferred to PVDF membrane, and blotted with
streptavidin peroxidase to detect the MPB label. The results represent
labeling of 5 × 106 platelets (5 µg). The positions
of Mr markers are shown at the left.
B, comparison of primary amines and thiols on the surface of
resting versus thrombin-activated platelets. Resting and
thrombin-activated/aggregated platelet surfaces were labeled with
either SSB or MPB as indicated, sonicated, resolved on 10% SDS-PAGE,
transferred to PVDF membrane, and blotted with streptavidin peroxidase
to detect the SSB or MPB label. The results represent labeling of
1 × 106 platelets (1 µg) with SSB and 3 × 107 platelets (30 µg) with MPB. The arrows at
right indicate proteins that were labeled with MPB on the
activated/aggregated platelet surface but were poorly or not labeled on
the resting platelet surface. The asterisks indicate
proteins with apparent Mr values of ~60,000
and ~120,000 which may be PDI and GP1b , respectively (see below).
The positions of Mr markers are shown on the
left. C, densitometric analysis of the
SSB-labeled (top) and MPB-labeled (bottom)
platelet surface proteins shown in B. The extent of labeling
with either SSB or MPB was calculated from the areas under the curves.
Platelet activation resulted in a 130% increase in labeling with SSB
and 440% increase in labeling with MPB.
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Fig. 2.
Platelet activation/aggregation resulted in
an increase in both total and reduced platelet surface PDI.
A, labeling of PDI with MPB. Purified placenta PDI (oxidized
PDI) or placenta PDI incubated with 5 µM dithiothreitol
(reduced PDI) was labeled with MPB, 100 ng of the PDI resolved on 10%
SDS-PAGE, transferred to PVDF membrane, and blotted with streptavidin
peroxidase to detect the MPB label. Placenta PDI (500 ng) blotted with
anti-PDI polyclonal antibodies is shown in lane 1 and
migrated at the expected Mr of 57,000. Oxidized
PDI was not labeled with MPB (lane 2), whereas PDI activated
with dithiothreitol incorporated MPB (lane 3). The positions
of Mr markers are shown at the left.
B, resting platelets and thrombin-activated/aggregated
platelets were labeled with either SSB or MPB as indicated, sonicated,
and incubated with streptavidin-agarose beads to collect the
biotin-labeled proteins. The labeled proteins were resolved on 10%
SDS-PAGE, transferred to PVDF membrane, and blotted with anti-PDI
polyclonal antibodies. The results represent labeling of 1.6 × 108 platelets (160 µg) with SSB (lanes 3 and
4) and 6.8 × 108 platelets (680 µg) with
MPB (lanes 5 and 6). Control reactions omitting
the SSB and MPB labeling were performed to confirm that no unlabeled
platelet PDI was being nonspecifically carried through the procedure
(data not shown). Controls are purified placenta PDI (100 ng,
lane 1) and sonicated whole platelets (8.3 × 106 platelets, 8 µg, lane 2). The positions of
Mr markers are shown at left.
Quantitation of amine- and thiol-labeled platelet surface PDI on
resting and thrombin-activated/aggregated platelets
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Fig. 3.
Platelet surface PDI is refractive to
reduction by low Mr thiols.
A, HPLC profiles of platelet low Mr
thiol compounds. Platelet low Mr thiol compounds
were derivatized with 7-benzo-2-oxa-1,3-diazole-4-sulfonic acid and
separated by reverse-phase HPLC (38). The left hand trace
show the standards, CysGly and GSH, followed by
thrombin-activated/aggregated platelet sonicate, the same platelet
sonicate spiked with either 2 µM CysGly or GSH to confirm
the identity of the platelet thiols, and finally the releasate from the
activated/aggregated platelets. The chromatograms represent thiols from
3 × 106 thrombin-activated/aggregated platelets (3 µg), or the releasate from the same platelets (0.42 µg). The
starting sample contained 1.5 × 109
thrombin-activated/aggregated platelets/ml (1, 500 µg), or the
releasate from the same platelets (210 µg). The HPLC procedure will
detect a lower limit of ~0.2 µM GSH or CysGly in the
starting sample. This pattern was repeated on three separate occasions.
B, platelet surface PDI was not reduced by dithiothreitol.
Resting platelets (lane 2), resting platelets incubated with
50 µM dithiothreitol for 30 min at room temperature
(lane 3), and thrombin-activated/aggregated platelets
(lane 4) were labeled with MPB, sonicated, and incubated
with streptavidin-agarose beads to collect the biotin-labeled proteins.
The labeled proteins were resolved on 10% SDS-PAGE, transferred to
PVDF membrane, and blotted with anti-PDI polyclonal antibodies. The
results represent labeling of 5.2 × 108 platelets
(520 µg). The control for the Western blot is sonicated whole
platelets (8.3 × 106 platelets, 8 µg, lane
1). The positions of Mr markers are shown
at left.
.
on the Activated Platelet
Surface--
Resting platelets and thrombin-activated/aggregated
platelets were labeled with either SSB or MPB and the GP1b or
IIb3 immunoprecipitated using either AK3 or AP2 monoclonal
antibodies, respectively. The labeled glycoproteins were resolved on
10% SDS-PAGE, transferred to PVDF membrane, and blotted with
streptavidin peroxidase to detect the SSB or MPB label (Fig.
4). Immunoprecipitation of GP1b
resulted in co-immunoprecipitation of the GP1b and GPIX components
of the GP1b-IX·GP-V complex.
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Fig. 4.
Labeling of GP1b
with MPB on the activated platelet surface. Resting
platelets and thrombin-activated/aggregated platelets were labeled with
either SSB or MPB as indicated, sonicated, and the GP1b or
IIb3 immunoprecipitated using either AK3 or AP2 monoclonal
antibodies, respectively. The labeled glycoproteins were resolved on
10% SDS-PAGE, transferred to PVDF membrane, and blotted with
streptavidin peroxidase to detect the SSB or MPB label. The results of
labeling of GP1b are shown in A, while the results of
IIb3 labeling are shown in B. The figure represents
labeling of 4 × 108 platelets (400 µg). GP1b,
GP1b, and GP-IX were labeled with SSB on resting and activated
platelets. Platelet activation resulted in a 180% increase in labeling
of GP1b with SSB. GP1b was labeled with MPB on activated
platelets, but was not labeled on resting platelets. Neither GP1b
nor GP-IX were labeled by MPB on resting or activated platelets.
IIb3 was labeled with SSB but not with MPB on resting and
activated platelets.
, GP1b, and GP-IX were labeled with SSB on resting and
activated platelets. GP1b was labeled with MPB on activated platelets, but was not labeled on resting platelets (Fig.
4A). Neither GP1b nor GP-IX were labeled by MPB on
resting or activated platelets. IIb3 is not known to contain
unpaired cysteines, therefore it was an appropriate control for MPB
labeling. Accordingly, IIb3 was labeled with SSB but not with MPB
on resting and activated platelets (Fig. 4B).
may have
been physically and/or functionally associated on the platelet surface.
This hypothesis was tested by measuring the effect of anti-PDI
antibodies on binding of vWF to platelet GP1b.
on the Platelet Surface by
Anti-PDI Polyclonal Antibodies--
In initial experiments we observed
that incubation of platelets with anti-PDI antibodies at concentrations
>200 µg/ml resulted in platelet activation measured by presentation
of P-selectin (48). Platelet activation by 300 µg/ml anti-PDI
antibody was blocked completely by the murine anti-FcRIIA antibody,
IV.3, at concentrations of either 50 or 20 µg/ml. This result
indicated that a small fraction of aggregated IgG in the anti-PDI
antibody preparation was binding to the platelet FcRIIA receptor and
triggering activation.
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Fig. 5.
Inhibition of binding of vWF to
GP1b on the platelet surface by anti-PDI
polyclonal antibodies. Washed platelets were incubated with 20 µg/ml of the anti-platelet FcRIIA receptor monoclonal antibody,
IV.3, for 10 min at room temperature and then with 0 to 200 µg/ml of
either preimmune rabbit IgG or anti-PDI IgG for a further 30 min at
room temperature. The blocked platelets were incubated with 0.1-10
µg/ml vWF-Alexa and 1 mg/ml ristocetin for 10 min at room temperature
and the bound vWF-Alexa measured by flow cytometry. The effect of
increasing control IgG (
) or anti-PDI IgG (
) on binding of 5 µg/ml vWF-Alexa to platelets is shown in A. The effect of
50 µg/ml of either control IgG or anti-PDI IgG on the binding of
0.1-10 µg/ml of vWF-Alexa to platelets is shown in B. The
lines in B represent the best fit of the data to
the rectangular hyperbolic binding equation by nonlinear least squares
regression. The apparent dissociation constant and maximal binding of
vWF was 1.0 ± 0.2 µg/ml and 98 ± 6% in the presence of
control IgG (
) and 1.5 ± 0.2 µg/ml and 97 ± 5% in the
presence of 50 µg/ml anti-PDI IgG (···). In another experiment
the apparent dissociation constant and maximal binding of vWF was
0.9 ± 0.4 µg/ml and 83 ± 6% in the presence of control
IgG and 1.5 ± 0.4 µg/ml and 84 ± 10% in the presence of
50 µg/ml anti-PDI IgG (data not shown).
on the
platelet surface. To examine directly the proximity of these proteins,
FRET between PE-labeled PDI and Cy5-labeled GP1b on the platelet
surface was measured by flow cytometry (41-43).
on the Platelet Plasma
Membrane Measured by FRET--
FRET efficiency is the probability that
an excited donor molecule will transfer its energy to an acceptor
molecule in a non-radiative process and can be determined either on the
donor side (quenching) or the acceptor side (enhancement). In our
experiments we monitored the fluorescence intensity on the donor side.
First, all fluorescence intensities were corrected for autofluorescence
(Fig. 6A, unlabeled), that is
the mean fluorescence intensity of unlabeled cells was subtracted from
the mean fluorescence intensity of labeled cells. Second, the mean
fluorescence intensity of sample labeled only with the donor secondary
antibody was subtracted from the mean fluorescence intensity of sample
labeled with donor primary and secondary antibodies and acceptor
secondary antibody (Fig. 6, anti-PDI/PE, AK2-PE). This value was
Fdonor,corrected and is corrected for
nonspecific binding of the donor PE-conjugated secondary antibody, which was negligible. Third, the mean fluorescence intensity of sample
labeled only with the donor secondary antibody and the sample labeled
only with the acceptor primary and secondary antibodies was subtracted
from the mean fluorescence intensity of sample labeled with both donor
and acceptor primary and secondary antibodies (Fig. 6, anti-PDI/PE,
AK2/Cy5-PE-AK2/Cy5). This value was
FFRET,corrected and is corrected for the
spectral contribution of acceptor Cy5 in the donor PE detection
channel, which was negligible. FRET efficiency was calculated
from the relationship; FRET efficiency = 1 (FFRET,corrected/Fdonor,corrected).
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Fig. 6.
Physical proximity of PDI and
GP1b on the platelet plasma membrane measured
by FRET. To assess the proximity of PDI and GP1b on the
platelet surface, the efficiency of FRET between PE-labeled PDI and
Cy5-labeled GP1b was measured by flow cytometry. The contribution of
autofluorescence was determined using unlabeled cells (a).
The binding of the PE-conjugated secondary antibody in the absence of
primary donor antibody is shown in b, while the overlap of
acceptor Cy5 in the donor PE channel is shown in c. FRET
between PE-labeled PDI and Cy5-labeled GP1b is shown in
d. A FRET efficiency of 14.8 ± 4.9% (1 S.D.) was
calculated from three experiments.
.
occurred by chance. The surface area of a platelet is 22 µm2 and there are 25,000 molecules of GP1b on
the platelet surface (49). Therefore, one molecule of GP1b will be
found in an area of 890 nm2. Assuming an even lattice-like
arrangement, the receptors will be 30 nm apart. This is well in excess
of the upper limit of the Förster energy transfer (8-10 nm).
underwent a
conformational change upon platelet activation resulting in the exposure of a free thiol(s). To test whether PDI might be involved in
this conformational change, resting platelets were incubated with
anti-PDI antibodies, activated with thrombin, and labeled with either
SSB or MPB or incubated with anti-GP1b-IX monoclonal antibodies.
on the Activated Platelet
Surface by Anti-PDI Polyclonal Antibodies--
Resting platelets were
incubated with 200 µg/ml of either preimmune rabbit IgG or rabbit
anti-PDI IgG for 30 min, activated with thrombin, labeled with either
SSB or MPB and the GP1b-IX·GP-V immunoprecipitated using AK3
monoclonal antibodies. The labeled glycoproteins were resolved on 10%
SDS-PAGE, transferred to PVDF membrane, and blotted with streptavidin
peroxidase to detect the SSB or MPB label (Fig.
7). The presence of anti-PDI polyclonal antibodies resulted in a 80% increase in labeling of GP1b with SSB
and a 130% increase in labeling with MPB.
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Fig. 7.
Enhancement of labeling of
GP1b on the activated platelet surface by
anti-PDI polyclonal antibodies. Resting platelets were incubated
with 200 µg/ml of either preimmune rabbit IgG or rabbit anti-PDI IgG
for 30 min, activated with thrombin, labeled with either SSB or MPB,
and the GP1b-IX·GP-V immunoprecipitated using AK3 monoclonal
antibodies. The labeled glycoproteins were resolved on 10% SDS-PAGE,
transferred to PVDF membrane, and blotted with streptavidin peroxidase
to detect the SSB or MPB label. The figure represents labeling of
4 × 108 platelets (0.4 mg). The presence of anti-PDI
polyclonal antibodies resulted in a 80% increase in labeling of
GP1b with SSB and a 130% increase in labeling with MPB. The
experiment was performed on two separate occasions with the same
qualitative result.
(not shown). These
findings suggested that the conformational change in GP1b upon
platelet activation was the result of specific events on the activated
platelet surface.
Monoclonal Antibodies to the
Activated Platelet Surface by Anti-PDI Polyclonal Fab
Fragments--
Resting platelets were incubated with 200 µg/ml of
either preimmune rabbit IgG F(ab)2 fragments or rabbit
anti-PDI F(ab)2 fragments for 30 min, activated with
thrombin, incubated with 10 µg/ml fluorescein
isothiocyanate-conjugated anti-GP1b-IX monoclonal antibodies and the
bound antibody measured by flow cytometry. The GP1b-IX·GP-V complex
is in close proximity to the FcRIIA receptor on the platelet surface
(48). To eliminate potential complications of anti-PDI polyclonal
antibody binding to the FcRIIA platelet receptor, F(ab)2
fragments of the anti-PDI antibodies were made and used. The binding of
one anti-GP-IX monoclonal antibody and four anti-GP1b monoclonal
antibodies was measured. SZ1 binds to GPIX in complex with GP1b.
AN51 binds to the N-terminal flank of GP1b (residues 1-35), while
AK2 binds to the first leucine-rich repeat, HIP1 to the second
leucine-rich repeat, and SZ2 to the anionic sulfated tyrosine sequence
(residues 269-282) (50).
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Fig. 8.
Enhancement of binding of
anti-GP1b monoclonal antibodies to the
activated platelet surface by anti-PDI polyclonal F(ab)2
fragments. A, resting platelets
were incubated with 200 µg/ml of either preimmune rabbit IgG
F(ab)2 fragments (filled histograms) or rabbit anti-PDI
F(ab)2 fragments (lined histograms) for 30 min, activated
with thrombin, incubated with 10 µg/ml fluorescein
isothiocyanate-conjugated anti-GP1b-IX monoclonal antibodies and the
bound antibodies measured by flow cytometry. MOPC21 (part i)
is an irrelevant control antibody, SZ1 (part ii) binds to
GPIX in complex with GP1b, while AN51 (part iii), AK2
(part iv), HIP1 (part v), and SZ2 (part
vi) bind to epitopes in GP1b (see text for antibody epitopes).
B, quantitation of the effects of anti-PDI polyclonal
F(ab)2 fragments on the binding of the anti-GP1b-IX
monoclonal antibodies shown in A. Monoclonal antibody
binding in the presence of control IgG F(ab)2 fragments was
gated at 50%, and the binding in the presence of anti-PDI
F(ab)2 fragments was measured. The experiment was performed
on three separate occasions with the same qualitative result.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
110 mV
(8-10). Secreted PDI controls the redox state of existing exofacial
protein thiols or reactive disulfide bonds on the surface of human
fibroblasts (28) and lymphocytes (29). The redox status of PDI on
resting and thrombin-activated/aggregated platelets was investigated by
labeling platelets with a thiol- or amine-reactive membrane impermeable probe.
.
GP1b is part of the GPIb-IX·GP-V complex which mediates adhesion
of platelets to vessel wall von Willebrand factor at high wall shear
(53). The thiol labeled in GP1b on the activated platelet surface
was perhaps the unpaired cysteine at position 65 in the second
leucine-rich repeat near the N terminus (54). This result indicated
that Cys65, or another cysteine, was not exposed on the
resting platelet surface and implied that platelet activation was
associated with a conformational change in GP1b which exposed
Cys65. It is noteworthy that substitution of
Cys65 for Arg in the N-terminal region of GP1b impairs
binding of vWF to GP1b (55). It should be noted, however, that there
may have been other free thiols in GP1b or other components of the GPIb-IX·GP-V complex that were inaccessible or refractive to labeling by MPB on the platelet surface.
were in close proximity on the activated platelet
surface. Anti-PDI antibodies increased the dissociation constant for
binding of vWF to platelets by ~50% and PDI and GP1b were sufficiently close on the platelet surface to allow FRET between chromophores attached to PDI and GP1b. There are approximately 6,500 molecules of PDI (Table I) and 25,000 molecules of GP1b (49) on the
activated platelet surface, therefore the PDI:GP1b molar ratio was
~1:4. There are two molecules of GP1b, GP1b, and GP-IX and one
molecule of GP-V in a GP1b-IX·GP-V complex on the platelet surface
(53). This translates to an average of one molecule of PDI for every
two (GP1b-IX)2·GP-V complexes.
on the activated platelet surface. In contrast, binding of a
monoclonal antibody to the anionic sulfated tyrosine sequence (residues
269-282) was inhibited by anti-PDI antibodies. The enhanced labeling
and antibody binding to GP1b in the presence of anti-PDI antibodies
may have been due to simple displacement of PDI from GP1b by the
anti-PDI antibodies, however, it was possible that the anti-PDI
antibodies were perturbing a PDI-catalyzed conformational change in
GP1b. The observation that anti-PDI antibodies caused enhanced
binding of three monoclonal antibodies but decreased binding of another
to GP1b supported a PDI-facilitated conformational change. The
nature of this putative conformational change is unknown, but it is
noteworthy that PDI can catalyze net formation, net rearrangement, or
net reduction of protein disulfide bonds depending on the nature of the
protein substrate, the redox conditions, and the presence of other
thiols and disulfides (10).
IIb3 (56). On the resting platelet surface
IIb3 is in a conformation that does not bind its ligands. Platelet activation triggers a conformational change in IIb3 which enables ligand binding (57). Similarly, platelet
activation/aggregation triggered reduction of the active site
disulfides of PDI and a conformational change in GP1b that resulted
in exposure of a free thiol(s). The fact that PDI and GP1b were in
close proximity on the activated platelet surface suggested that PDI
may have influenced the conformational change in GP1b. An important
question is what consequence the conformational change in GP1b has
for its interaction with vWF. It is possible that the change causes displacement of vWF from GP1b. This might facilitate platelet spreading.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Centre for
Thrombosis and Vascular Research, School of Pathology, University of New South Wales, Sydney NSW, 2052 Australia. Tel.: 61-2-9385-1004; Fax:
61-2-9385-1389; E-mail: p.hogg@unsw.edu.au.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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