From the Department of Medicine, University of
California, San Francisco and Veterans Affairs Medical Center, San
Francisco, California 94121, the ¶ Laboratory for Cell Analysis,
Cancer Center, University of California, San Francisco, California
94143, the
Department of Biophysics and Cell Biology, Medical
University School of Debrecen, Debrecen 4012, Hungary, and the
** Departments of Internal Medicine and Molecular and Human Genetics,
Baylor College of Medicine and Veterans Affairs Medical Center,
Houston, Texas 77030
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ABSTRACT |
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Although the glycoprotein (GP) Ib-IX-V complex
and FcRIIA are distinct platelet membrane receptors, previous
studies have suggested that these structures may be co-localized. To
determine more directly the proximity of GP Ib-IX-V and Fc
RIIA, we
assessed the effects of anti-GP Ib
monoclonal antibodies on
Fc
RIIA-mediated platelet aggregation and on the direct binding of
polymeric IgG to human platelets. In addition, we directly examined the
proximity of Fc
RII and GP Ib-IX-V using flow cytometric fluorescence
energy transfer and immunoprecipitation studies. Preincubation of
platelets with either of two monoclonal antibodies (AN51 or SZ2)
directed against GP Ib
completely blocked platelet aggregation by
polymeric IgG. Similarly, these antibodies totally inhibited platelet
aggregation by two strains of viridans group streptococci known to
induce aggregation via Fc
RIIA. In addition, AN51 and SZ2
significantly reduced the binding of polymeric IgG to washed fixed
platelets. When assessed by flow cytometry, significant levels of
bidirectional energy transfer were detected between Fc
RIIA and GP
Ib
, indicating a physical proximity of less than 10 nm between these
receptors. This energy transfer was not due to high receptor density,
because no homoassociative energy transfer was seen. Moreover,
immunoprecipitation of Fc
RIIA from platelet lysates also
co-precipitated GP Ib
. These results indicate that GP Ib
and
Fc
RIIA are co-localized on the platelet membrane and that this
association is not random.
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INTRODUCTION |
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The glycoprotein (GP)1
Ib-IX-V complex and FcRIIA are distinct platelet membrane receptors
for von Willebrand factor and polymeric IgG, respectively. The GP
Ib-IX-V complex contains four polypeptides (GP Ib
, GP Ib
, GP IX,
and GP V), which are present in a stoichiometry of 2:2:2:1 on the
platelet plasma membrane (1). The precise configuration by which these
peptides form a functional receptor is unknown, although more than one
copy of each polypeptide per receptor complex is probable (1). This
receptor mediates platelet adhesion to sites of blood vessel injury by
binding von Willebrand factor in the subendothelium and participates in
platelet activation by thrombin by providing a high affinity binding
site for this agonist. In contrast to the GP Ib-IX-V complex, the
platelet receptor for the Fc portion of IgG, Fc
RIIA, is much simpler
in structure, consisting of a single transmembrane polypeptide. This
receptor is responsible for the aggregation and activation of platelets induced by immune complexes, opsonized bacteria, and certain antibodies that bind other platelet surface proteins (2).
Although GP Ib-IX-V and FcRIIA are structurally unrelated, a
functional interaction between these receptors has been suspected for
some time. Moore et al. reported that both polymeric IgG and IgG Fc fragments could inhibit platelet aggregation induced by von
Willebrand factor and ristocetin (3). The reciprocal phenomenon was
also observed, i.e. prior incubation of platelets with von Willebrand factor and ristocetin inhibited platelet aggregation by
polymeric IgG. More recent studies examining heparin-induced platelet
aggregation, which is mediated by anti-heparin antibodies, further
suggest a proximity of GP Ib-IX-V and Fc
RIIA. In particular, platelet aggregation by heparin can be blocked by antibodies directed against either Fc
RIIA or GP Ib
(4, 5). Moreover, anti-GP Ib
antibodies inhibit heparin-induced aggregation of platelets obtained
from normal donors but not of platelets from individuals with the
Bernard-Soulier syndrome, which lack the GP Ib-IX-V complex (4).
Although these findings suggest a physical proximity or functional
interaction of FcRIIA and the GP Ib-IX-V complex, such an
association has never been demonstrated directly. In the current studies, we used three distinct approaches to determine whether these
receptors are physically associated. First, we assessed whether anti-GP
Ib
monoclonal antibodies would affect either Fc
RIIA-mediated
platelet aggregation (induced by polymeric IgG or antibody-coated
bacteria) or the direct binding of polymeric IgG to human platelets.
Second, we directly examined the proximity of Fc
RIIA and GP Ib-IX-V
on the platelet membrane as measured by flow cytometric fluorescence
energy transfer. Third, we assessed by co-immunoprecipitation
experiments whether these receptors were linked covalently. As
discussed below, these studies strongly indicate that GP Ib-IX-V and
Fc
RIIA are co-localized on the platelet membrane and that these
receptors may be functionally interactive.
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EXPERIMENTAL PROCEDURES |
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Polymeric IgG-- Polymeric IgG was produced either by heating or chemically cross-linking an IgG solution. For the former procedure, IgG was purified from human immunoglobulin (Miles Laboratory, Elkhart, IN) by protein A affinity chromatography. IgG aggregates were generated by heating a 2 mg/ml solution of the purified IgG in phosphate-buffered saline at 63 °C for 30 min (6). Macroscopic aggregates were removed by centrifugation (12,000 × g, 10 min), and the protein content of the supernatant was determined using a bicinchoninic acid assay (Pierce) (7). To produce chemically cross-linked IgG (8), a 100 mg/ml solution of purified human IgG1 (Sigma) in 0.1 M Tris buffer (pH 8.5) was treated with a 10-fold molar excess of dimethyl suberimidate for 2 h at 30 °C followed by dialysis, centrifugation, and determination of its protein content.
Monoclonal Antibodies--
The following monoclonal antibodies
(mAb), both unlabeled and conjugated with FITC, were obtained from
commercial sources: mAb AN51 (anti-GP Ib; Immunotech International,
Westbrook, ME); mAb SZ2 (anti-GP Ib
; DAKO, Carpinteria, CA); mAb
FA6.152 (anti-CD36, Immunotech). mAb WM23 (anti-GP Ib
) was a gift
from Dr. Michael Berndt of the Baker Research Institute, Prahran,
Victoria, Australia. mAb IV.3 (anti-Fc
RII) was purchased from
Mederex (West Lebanon, NH) or purified from the ascites of BALB/c
mice inoculated with hybridoma IV.3 (American Type Culture Collection,
Rockville, MD) using ammonium sulfate precipitation (9) and protein G
affinity chromatography as recommended by the manufacturer (Pharmacia
Biotech Inc.). Antibodies were conjugated with cyanine 3.18 (Cy3) using the Fluorolink-Ab Cy3 labeling kit as per the manufacturer's
instructions (Biological Detection Systems, Pittsburgh, PA). Labeling
of the mAbs with FITC or Cy3 had no effect on binding affinity as
measured by a previously described competitive assay (10).
Preparation of Human Platelets-- Platelet-rich plasma (PRP) and washed platelets were prepared from freshly obtained human blood as described previously (11). To produce PRP, blood was collected in tubes containing 3.8% buffered citrate solution (anticoagulant-to-blood ratio, 1:9) and centrifuged (100 × g, 15 min, 25 °C) to remove erythrocytes and leukocytes. PRP was recovered by collecting the uppermost two-thirds of the top layer.
Washed platelets were produced by collecting 5 volumes of blood into a syringe containing 1 volume of acid-citrate-dextrose (85 mM Na citrate, 111 mM glucose, 71 mM citric acid) anticoagulant and prostaglandin I2 (final concentration, 1 µg/ml; Sigma). The platelets were then washed three times in 140 mM NaCl, 20 mM HEPES, 6 mM glucose, 1 mM EDTA, pH 6.6, and then suspended in Tyrode's salt solution (TSS, Sigma) pH 7.2 (11). For flow cytometry, washed platelets were fixed in 0.8% paraformaldehyde (12), washed, and suspended in TSS. Platelets prepared by this technique were not activated, as shown by minimal expression of surface P-selectin (13), when measured by flow cytometry (data not shown).Preparation of Bacteria for
Aggregometry--
Streptococcus sanguis strain M99 and
S. salivarius strain D1 previously have been shown to induce
platelet aggregation via FcRIIA (14). To produce bacterial
suspensions for platelet aggregometry, each strain was grown for
18 h at 37 °C in Todd-Hewitt broth and then washed, sonicated,
and suspended in TSS as described (11). The concentrations of organisms
were determined by counting in a hemacytometer and adjusted to 3 × 109 bacteria/ml by the addition of TSS.
Platelet Aggregometry-- The ability of polymeric IgG or bacteria to aggregate platelets was tested by conventional light aggregometry using a single-channel aggregometer (Model 330, Chronolog, Haverton, PA). For polymeric IgG, 50 µl of a 10 mg/ml stock solution was added to 450 µl of washed platelets (3 × 108/ml) suspended in TSS. To assess platelet aggregation by streptococci, washed bacteria suspended in TSS were added to PRP at a final bacterium/platelet ratio of 1:1. Platelet aggregation was analyzed with regard to the time interval between the addition of an agonist to the platelet suspension and the onset of aggregation (lag phase), the rate of aggregation (slope at the midpoint of aggregation), and the maximum change in light transmission. All agonists were tested on multiple occasions using platelets from different donors. Human thrombin (1 unit/ml), ADP (10 µM), or ristocetin (1 mg/ml) served as controls for a positive aggregation response (all reagents from Sigma).
Binding of Polymeric IgG to Human Platelets-- The binding of polymeric IgG to human platelets was assessed by flow cytometry. Washed fixed platelets (1 × 108/ml) suspended in TSS containing 1% bovine serum albumin were incubated with 1 mg/ml polymeric IgG for 1 h at 4 °C. After three washings in TSS, the platelets were incubated with 1.5 µg/ml FITC-conjugated goat anti-human F(ab')2 IgG (Caltag, South San Francisco, CA). Platelets were diluted 500-fold, and 10,000 cells/sample were analyzed by flow cytometry using a Becton Dickinson FACScan (San Jose, CA). The threshold for forward scatter was set to eliminate small debris and gated to omit cell doublets and clumps. IgG binding was detected as the mean log FITC fluorescence emission in the Fl1 channel using a 530 ± 30 nm band-pass filter. Platelets incubated with secondary antibody alone served as controls for nonspecific binding. To determine the relative ability of the monoclonal antibodies to inhibit polymeric IgG binding, fixed platelets were incubated for 1 h with 10 µg/ml of mAb IV.3, mAb AN51, or mAb SZ2. After washing twice in TSS, the binding of polymeric IgG was assessed by flow cytometry as described above.
Fluorescence Resonance Energy Transfer--
To assess directly
the proximity of the FcRIIA receptor and the GP Ib-IX-V complex on
the cell surface, the efficiency of fluorescence energy transfer (FET)
between FITC-labeled monoclonal antibodies (donor) and CY3-conjugated
monoclonal antibodies (acceptor) was measured by flow cytometry as
described previously (10, 15, 16). A FACStar Plus flow cytometer
(Becton Dickinson) with dual-laser excitation (488 and 528 nm) was
used to determine platelet surface FET measurements. For each sample
analyzed, at least 20,000 events were collected. Using threshold
settings and light-scatter gating as described above, five intensities
were measured on a cell-by-cell basis: (i) forward scattered light at
488 nm; (ii) orthogonal scattered light at 488 nm; (iii) fluorescence emission at 510 ± 15 nm, when excited at 488 nm; (iv)
fluorescence emission at >580 nm, when excited at 528 nm; (v)
fluorescence emission at >580 nm, when excited at 488 nm (representing
energy transfer from FITC to Cy3). The contribution of autofluorescence was determined using unlabeled platelets. To calculate FET efficiency for dual-labeled cells on a cell-by-cell basis, correction factors for
spectral overlap were determined from single-labeled platelets.
Immunoprecipitation and Western Blotting-- Blood from healthy donors was drawn into acid-citrate-dextrose buffer containing prostaglandin I2, and PRP was prepared as described above. Immunoprecipitation studies were performed with and without the use of a chemical cross-linking reagent. For the former, 100 µl of PRP were incubated in 2 mmol of dithiobis(sulfosuccinimidylpropionate) (DTSSP, Pierce, Rockford, IL) for 30 min at room temperature, followed by lysis in buffer containing 1% digitonin and a mixture of protease inhibitors (20 mg/ml leupeptin, 1.6 µg/ml benzamidine, 1 mg/ml soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride). For the latter, identical aliquots of PRP were lysed directly without prior treatment with DTSSP.
FcStatistical Analysis-- Mean values were compared by the unpaired t test using analysis of variance and the Bonferroni correction for multiple comparisons.
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RESULTS |
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Effects of mAbs on Platelet Aggregation by Polymeric IgG-- Addition of 1 mg/ml of polymeric IgG to washed platelets resulted in a lag phase of 6.5 ± 5.3 min (mean ± S.D.) followed by irreversible platelet aggregation (n = 18) (Fig. 1A). As expected, this aggregation was blocked completely by 100 ng/ml mAb IV.3 (6). In addition, platelet aggregation by polymeric IgG was completely inhibited both by mAb AN51 and mAb SZ2, when either mAb was tested at 10 µg/ml (n = 5; Fig. 1B). At 1 µg/ml, the effects of these mAbs were variable, with aggregation being either partially inhibited (prolonged lag phase, decreased maximal change in optical transmission) or blocked completely. In control studies, washed platelets treated with SZ2, AN51, or IV.3 aggregated normally in response to thrombin (1 unit/ml), whereas FA6.152 (anti-CD36) had no effect on platelet aggregation induced by polymeric IgG, indicating that inhibition by the above mAbs was relatively specific.
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Effects of mAbs on Platelet Aggregation by Viridans Group
Streptococci--
Platelet aggregation by many strains of viridans
group streptococci requires opsonization of bacteria with specific IgG
followed by platelet activation via FcRIIA (11, 14, 21). To confirm that Fc
RIIA-mediated platelet aggregation could be inhibited by mAbs
to GP Ib
, we examined the effects of mAbs SZ2 and AN51 on platelet
aggregation by two strains of viridans group streptococci. Addition of
S. sanguis strain M99 or S. salivarius strain D1
to PRP resulted in mean lag phases of 9.5 ± 2.2 and 11.3 ± 5.1 min, respectively, followed by rapid and irreversible platelet
aggregation (n = 5; Fig. 1C). As with
aggregation induced by polymeric IgG, preincubation of the platelets
with mAb IV.3 (100 ng/ml) completely blocked aggregation by these
organisms. In addition, platelet aggregation by streptococci was also
blocked by 10 µg/ml mAb SZ2 or mAb AN51 (n = 4; Fig.
1D). Inhibition by these mAbs was selective; ADP normally
aggregated platelets pretreated with IV.3, AN51, or SZ2. Moreover,
FA6.152 failed to block platelet aggregation by either strain M99 or
D1, indicating that the inhibition by mAbs directed against platelet GP
Ib
was not merely due to a nonspecific or generalized effect of
antibody binding to the platelet surface.
Inhibition of Polymeric IgG Binding to Platelets--
We then
examined whether mAbs AN51 and SZ2 inhibited aggregation by reducing
binding of polymeric IgG to the platelets. As shown in Fig.
2, the binding of polymeric IgG to washed
fixed platelets could be readily detected by flow cytometry. When
probed with FITC-conjugated goat anti-human F(ab')2,
platelets incubated with polymeric IgG appeared as a discrete
population clearly separated from platelets treated with secondary
antibody alone. Preincubation of platelets with IV.3 reduced binding by
98.8 ± 21.1% (mean ± S.D., n = 8, p < 0.01), indicating that binding by polymeric IgG to
platelets was predominantly mediated by FcRIIA (Fig. 2C). Preincubation of platelets with 10 µg/ml of SZ2 or AN51 also reduced binding significantly, by 98.1 ± 19.8% (p < 0.01) and 73.0 ± 19.9% (p < 0.05), respectively
(Fig. 2D; n = 5). In control studies, platelets pretreated with anti-CD36 had levels of polymeric IgG binding
comparable to those seen with untreated platelets (data not shown).
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Analysis of Receptor Co-localization by Fluorescence Energy
Transfer--
The above results indicated that FcRIIA and GP
Ib-IX-V are in physical proximity on the platelet membrane. To examine
more definitively the potential co-localization of these receptors, we
measured FET between labeled mAbs directed against epitopes on each
receptor (Fig. 3). As expected, the two
epitopes on GP Ib-IX-V bound by AN51 and SZ2 showed significant levels
of FET (Table I). For example, percent
energy transfer from FITC-labeled AN51 to Cy3-labeled SZ2 was 16.3 ± 3.9%. Comparable levels of energy transfer were observed with
FITC-labeled SZ2 paired with Cy3-labeled AN51. FET measurements between
Fc
RIIA and GP Ib-IX-V also indicated significant proximity between
these receptors. For example, the mean energy transfer from
FITC-labeled AN51 to Cy3-labeled IV.3 was 9.2 ± 3.2%
(p < 0.001, compared with single-labeled controls).
Comparable levels of FET were observed between these epitopes when the
donor and acceptor dyes were reversed (FITC-labeled IV.3 and
Cy3-labeled AN51). Significant FET was also seen between FITC-labeled
IV.3 and Cy3-SZ2 (6.8 ± 3.0) as well as between these epitopes
when the fluorochrome labels were reversed (5.2 ± 1.4; p < 0.01).
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Physical Association between the GP Ib-IX-V Complex and
FcRIIA--
As another means of studying the association of the two
receptors, we attempted to co-immunoprecipitate GP Ib-IX-V complex with
Fc
RIIA using mAb IV.3 and digitonin lysates of platelets. These
experiments were performed with or without pretreatment of the
platelets with the bifunctional cross-linking agent DTSSP. When tested
with lysates of untreated platelets, mAb IV.3 co-precipitated GP Ib
in parallel with Fc
RIIA, but with considerable variation in the
amount of GP Ib
recovered on repeated testing. However, pretreatment
of platelets with DTSSP resulted in the consistent co-precipitation of
GP Ib
with Fc
RIIA in quantities comparable to those obtained with
antibodies against GP V or GP Ib
itself (Fig.
4).
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DISCUSSION |
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Although GP Ib-IX-V and FcRIIA are seemingly unrelated
receptors on the platelet membrane, previous reports had suggested a
functional cooperation or structural association of these surface structures. With this study, we provide strong evidence that the functional interaction between GP Ib-IX-V and Fc
RIIA is largely due
to a direct physical interaction. Several observations support this
conclusion. First, we found that mAbs to GP Ib
completely blocked
platelet aggregation induced by polymeric IgG, which is an
Fc
RIIA-mediated process. These antibodies apparently inhibited aggregation by blocking the binding of polymeric IgG to Fc
RIIA. Indeed, we found that mAbs directed against GP Ib
could
significantly reduce platelet binding by polymeric IgG and that these
mAbs inhibited binding by amounts comparable with those obtained with
the anti-Fc
RII mAb IV.3. Because the GP Ib
-specific mAbs do not
appear to bind Fc
RIIA directly (data not shown), these findings
indicate that the above inhibitory activity results from steric
hindrance, suggesting that Fc
RIIA and GP Ib
are in close
proximity on the platelet membrane.
We also demonstrated the proximity of these receptors more directly by
two methods, resonance energy transfer between antibodies bound to
FcRIIA and GP Ib
and co-immunoprecipitation experiments. Fluorophor-labeled antibodies bound to the respective antigens were
able to transfer emission energy from the FITC-labeled antibody to the
Cy3-labeled antibody, regardless of whether the donor dye was
associated with Fc
RIIA or GP Ib
. The detection of energy transfer
between epitopes on Fc
RIIA and GP Ib
indicates that these
receptors are within 10 nm of each other. This proximity cannot be
explained by the random co-localization of Fc
RIIA and GP Ib
.
Indeed, we observed no homoassociative energy transfer for epitopes on
either receptor, indicating that the densities of Fc
RIIA and GP
Ib
on the platelet membrane are too low to randomly produce FET. Our
calculations of receptor density also argue against the association
being due to random distribution. Based on previous estimates of
platelet surface area (20-80 µm2) (8) and receptor
number (25,000 and 500-8,000 copies/platelet for GP Ib
(1, 17) and
Fc
RIIA (2, 22), respectively), and assuming random receptor
distribution, the predicted distance between these receptors would
exceed the limits of detectable FET. Thus, the observed FET in our
studies indicates nonrandom association between Fc
RIIA and GP Ib
.
We obtained further evidence for a direct physical association between
the GP Ib-IX-V complex and FcRIIA in our platelet in
immunoprecipitation studies. Using mAb IV.3, GP Ib
was invariably coprecipitated with Fc
RIIA from platelet membranes that had been pretreated with DTSSP, a chemical cross-linking reagent that is membrane-impermeable (23). In the absence of this pretreatment, the GP
Ib
was still recovered, but the yield and frequency of recovery
varied considerably. These findings indicate that there is a direct
interaction between the GP Ib-IX complex and Fc
RIIA, although the
interaction is weak enough to be disrupted under certain conditions of
lysis. Moreover, our ability to chemically cross-link these receptors
on the surface of intact platelets provides additional evidence for
their proximity.
Of note, platelet aggregation by streptococci was also blocked by mAbs
to GP Ib. In addition to providing corroborative data for the
aggregation studies using polymeric IgG, these results may clarify some
inconsistent findings on the mechanisms of platelet aggregation by
these organisms. Our previous studies had demonstrated that platelet
aggregation by many strains was mediated by Fc
RIIA (11, 14). A
subsequent report, however, indicated that mAbs to GP Ib
could
inhibit platelet aggregation by viridans group streptococci, suggesting
a role for this receptor (24). Although these divergent observations
may reflect different mechanisms of platelet activation used by
different species, our current data suggest that the inhibitory
activity observed by Ford et al. (24) is due to steric
blockade of Fc
RIIA by the anti-GP Ib antibodies.
What are the potential consequences of the association between the GP
Ib-IX-V complex and FcRIIA? As yet no definitive information exists
to answer this question, but one possible reason for their association
may be for participation in signal transduction. The interaction
between the GP Ib-IX-V complex and von Willebrand factor leads to
signal transduction across the platelet membrane, resulting in tyrosine
phosphorylation of a number of proteins and an influx of calcium
(25-27). As yet, the mechanism for this signal transduction has not
been delineated, and in particular it is unclear as to how the GP
Ib-IX-V complex is coupled to the signal transduction machinery. Its
subunits do not have tyrosine kinase activity, are not known to bind to
G-proteins, and are not phosphorylated by tyrosine kinases. Recently,
one of the 14-3-3 proteins has been shown to associate with the GP
Ib-IX-V complex (28, 29), but as yet no clear mechanism has been
proposed for how this association is involved in signal transduction.
The association of Fc
RIIA with the complex may provide a mechanism for signal transduction by the complex. Fc
RIIA is able to transduce activation signals when cross-linked by aggregated IgG or IgG bound to
bacteria, as we have demonstrated here. This activation is partly
mediated by an interaction of an immune tyrosine-based activation motif
(ITAM) in the cytoplasmic domain of Fc
RIIA and the SH2 motif
containing kinase Syk, a member of the Src family of tyrosine kinases
(30, 31). Recent evidence indicates that the GP Ib-IX-V complex must be
cross-linked to transmit signals (32), a mechanism similar to that
employed by Fc
RIIA for signal transmission (31). Thus, if an agonist
is able to change the conformation of the GP Ib-IX-V complex such that
subcomponents of the complex are brought together, this action may also
bring together and cross-link associated Fc
RIIA polypeptides,
resulting in signal transduction.
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ACKNOWLEDGEMENTS |
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We thank Martin Bigos and Richard Stoval of Stanford University for valuable help with the FET studies and Margaret Chambers and David A. Smith for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI32506, AI41513, HL02463, and HL46416; by American Heart Association (Northern California) Grant-in-Aid 93220; by American Heart Association Grants 96012670 and 96002750; and by Grant OTKA T-019372 from the Hungarian Academy of Sciences.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.
§ To whom correspondence should be addressed: Veterans Affairs Medical Center, Division of Infectious Diseases (111W), 4150 Clement St., San Francisco, CA 94121. Tel.: 415-221-4810, ext. 2550; Fax: 415-750-0502; E-mail: sullam{at}sanfrancisco.va.gov.
Established Investigator of the American Heart
Association.
1 The abbreviations used are: GP, glycoprotein; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; PRP, platelet-rich plasma; TSS, Tyrode's salt solution; FET, fluorescence energy transfer; DTSSP, dithiobis(sulfosuccinimidyl propionate.
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REFERENCES |
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