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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 886-893
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Structural and functional characterization of the mouse von
Willebrand factor receptor GPIb-IX with novel monoclonal antibodies
Wolfgang Bergmeier,
Kirsten Rackebrandt,
Werner Schröder,
Hubert Zirngibl, and
Bernhard Nieswandt
From the Department of Molecular Oncology, General Surgery,
University of Witten-Herdecke, and the BAYER Pharma Research Center,
Wuppertal, Germany.
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Abstract |
Five novel monoclonal antibodies (mAbs; p0p 1-5) were used to
characterize the structural and functional properties and the in vivo
expression of the murine GPIb-IX complex (von Willebrand factor
receptor). The molecular weights of the subunits are similar to
the human homologs: GPIb (150 kd), GPIb (25 kd), and GPIX (25 kd). Activation of platelets with thrombin or PMA predominantly induced
shedding of glycocalicin (GC; 130 kd) but only low levels of receptor
internalization. The GC concentration in normal mouse plasma was found
to be at least 10 times higher than that described for human plasma
(approximately 25 µg/mL versus 1-2 µg/mL). Two additional cleavage
sites for unidentified platelet-derived proteases were found on
GPIb, as demonstrated by the generation of 3 N-terminal fragments
during in vitro incubation of washed platelets (GC, 60 kd, 45 kd).
Occupancy of GPIb with p0p mAbs or F(ab)2-fragments resulted in aggregate formation in vitro and rapid irreversible thrombocytopenia in vivo, irrespective of the exact binding
epitopes of the individual antibodies. GPIb-IX was not detectable
immunohistochemically on endothelial cells in the major organs under
normal or inflammatory conditions. The authors conclude that the mouse
system might become an interesting model for studies on GPIb-IX
function and regulation.
(Blood. 2000;95:886-893)
© 2000 by The American Society of Hematology.
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Introduction |
Platelet adhesion to sites of vascular injuries is
mediated by the interactions of glycoprotein (GP) membrane receptors on circulating platelets with their distinct adhesive
ligands.1 In particular, under high shear stress, the
platelet GPIb-IX-V receptor complex contributes to this process
initiating hemostasis through interactions with the adhesive ligand von
Willebrand factor (vWF).2-4 The human GPIb-IX-V
complex consists of 4 distinct gene products: GPIb , Ib, IX, and
V.5,6 Unstimulated platelets express approximately 25 000
copies of GPIb-IX and 12 000 copies of GPV on their
surfaces.6 Binding sites for vWF and -thrombin have been
identified on the N-terminal 45-kd region of GPIb.7-9 Interactions with vWF molecules only occur when the latter are bound to subendothelium,10 fibrin,11 or
collagen,12 and they require high shear
forces.13 Interactions are also detectable during
ristocetin- or botrocetin-induced platelet agglutination, probably
because of neutralization of the repulsive negative charges by the
positively charged ristocetin or botrocetin molecules.14-16 Subsequent activation of the fibrinogen receptor GPIIbIIIa leads to the
formation of stronger bonds and therefore platelet adhesion and
aggregation.4,17 In contrast, other platelet agonists such
as adenosine diphosphate, collagen, or thrombin induce the binding of
vWF to GPIIbIIIa.18,19 The effect of platelet
activation on the surface expression of GPIb-IX in vitro has been a
matter of controversy. Although most investigators demonstrate the
translocation of GPIb-IX-complexes from the plasma membrane to the
surface-connected canalicular system in response to thrombin (without
receptor shedding),20-22 significant shedding has been
observed by others.23 Furthermore, White et
al24 found no effect of thrombin activation on
GPIb-IX surface expression. In contrast, it is commonly accepted
that GPIb can be proteolyzed by neutrophil cathepsin
G,25 neutrophil elastase,26 or platelet
calpain.27
The platelets of patients with Bernard-Soulier syndrome, a congenital
bleeding disorder, show diminished agglutination to vWF in the presence
of ristocetin and a reduced response to low doses of
thrombin. This results from a reduced expression or a malfunction
of GPIb-IX28 that coincides with the release of "giant" platelets,1,2 indicating a role of the
GPIb-IX-V complex in maintaining circulating platelet
morphology. Reports describing the expression of GPIb or even
all subunits of the receptor complex on cells of
nonhematopoietic origin, particularly endothelial cells (EC), raised
speculations about unrecognized functions of
GPIb-IX-V.29-31 However, these data are not
without controversy because others could not reproduce
the findings.32 No systematic investigations of
GPIb-IX expression on a cellular level in situ have been
performed to date.
As proposed recently,33 the vWF-receptor complex may become
an interesting pharmacologic target for the prevention of thrombotic and inflammatory complications. Because in vivo investigations are
obviously limited in humans and nonhuman primates, there is a need for
small animal models allowing for in vivo studies on GPIb-IX-V functions
under normal and inflammatory conditions. In the mouse system, adequate
animal models exist, but limited information about structure, function,
and regulation of the murine receptor complex has been available. In
the current study, we investigated the structural and functional
properties of mouse GPIb-IX and examined the in vivo expression of the
complex with novel monoclonal antibodies.
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Materials and methods |
Animals
Specific-pathogen-free mice (NMRI, BALB/c) 6 to 10 weeks of age were
obtained from Charles River (Sulzfeld, Germany) and kept in our animal facilities.
Reagents
EZ-Link sulfo-NHS-LC-biotin (Pierce, Rockford, IL),
immobilized pepsin (Pierce), ristocetin (EUROPA, Cambridge, UK),
phorbol 12-myristate 13-acetate (PMA; Sigma, Deisenhofen, Germany),
high molecular weight heparin (Sigma), thrombin (Boehringer Mannheim, Mannheim, Germany), Collagen A1 (Biochrom, Berlin, Germany), and streptavidin-horseradish peroxidase (HRP; DAKO, Glostrup, Denmark) were
purchased. Lipopolysaccharide (LPS from Salmonella minnesota 9700) was obtained from Difco Laboratories (Detroit, Michigan).
Antibodies
Rat antimouse P-selectin mAb RB40.34 was kindly provided by D. Vestweber (Münster, Germany). Polyclonal rabbit antibodies to
human fibrinogen and vWF were purchased from DAKO and were modified in
our laboratories. Rabbit anti-fluorescein isothiocyanate (FITC)-HRP and
rabbit anti-rat Ig-FITC were purchased from DAKO. All other
antibodies were generated, produced, and modified in our laboratories:
MWReg30 (anti-GPIIbIIIa, IgG1), JON1 (anti-GPIIbIIIa, IgG2b), EDL1
(anti-GPIIIa, IgG2a).
Platelet preparation and counting
Mice were bled under ether anesthesia from the retro-orbital plexus.
Blood was collected in a tube containing 10% (vol/vol) 0.1 mol/L
sodium citrate or 7.5 U/mL heparin, and platelet-rich plasma was
obtained by centrifugation at 300g for 10 minutes at room
temperature (RT). The platelets were washed twice with
phosphate-buffered saline (PBS) by centrifugation at 1300g for
10 minutes and were used immediately. Isolated platelets did not show
any signs of activation as shown by flow cytometry (staining for
P-selectin and surface-expressed fibrinogen). For determination of
platelet counts, blood (20 µL) was obtained from the retro-orbital
plexus of anesthetized mice using siliconized microcapillaries and
immediately diluted 1:100 in Unopette kits (Becton Dickinson,
Heidelberg, Germany). The diluted blood sample was allowed to settle
for 20 minutes in an Improved Neubauer Hemocytometer (Superior, Bad
Mergentheim, Germany), and platelets were counted under a
phase-contrast microscope at ×400 magnification.
Production of monoclonal antibodies
Female Wistar rats, 6 to 8 weeks of age, were immunized repeatedly
with mouse platelets or with purified antigens. The rat spleen cells
were then fused with mouse myeloma cells (Ag8.653), and hybridomas were
selected in HAT medium. Hybridomas secreting mAbs directed against
platelet receptors were identified by flow cytometry. Briefly, a 1:1
mixture of resting and thrombin-activated platelets (106)
was incubated with 100 µL supernatant for 30 minutes at RT, washed
with PBS (1300g, 10 minutes) and stained with FITC-labeled rabbit anti-rat Ig (DAKO) for 15 minutes. Samples were analyzed on a
FACScan (Becton Dickinson) in the set-up mode. Platelets were gated by
FSC/SSC-characteristics. Positive hybridomas were subcloned twice
before large-scale production. Monoclonal antibodies were produced and
purified according to standard methods. Isotype subclasses were
determined by enzyme-linked immunosorbent assay (ELISA) with alkaline
phosphatase (AP)-conjugated isotype-specific antibodies (Pharmingen):
p0p 1, IgG2a; p0p 2, IgG1; p0p 3, IgG2a; p0p 4, IgG2b; p0p 5, IgG1.
Modification of antibodies
Affinity-purified antibodies were fluoresceinated to a
fluorescein-protein ratio of approximately 3:1 by standard methods with FITC (Sigma) and separated from free FITC by gel filtration on a
PD-10 column (Pharmacia, Uppsala, Sweden). HRP conjugation of mAbs was
performed with a labeling kit (Boehringer Mannheim, Mannheim, Germany).
F(ab)2-fragments of p0p 3 and p0p 4 were generated by
24-hour incubation of 10 mg/mL mAb with immobilized pepsin (Pierce).
Purity of the F(ab)2-fragments was checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
Immunoprecipitation and immunoblotting
Immunoprecipitation was performed as described
previously.34 Briefly, 108 washed platelets
were surface labeled with EZ-Link sulfo-NHS-LC-biotin (Pierce; 100 µg/mL in PBS) and subsequently solubilized in 1 mL lysis buffer
(Tris-buffered saline containing 20 mmol/L Tris/HCl, pH 8, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride,
2 µg/mL aprotinin, 0.5 µg/mL leupeptin, and 0.5% Nonidet P-40, all
from Boehringer Mannheim). Cell debris was removed by centrifugation
(15 000g, 10 minutes). After preclearing (8 hours), 10 µg
mAb was added together with 25 µL protein G-Sepharose (Pharmacia), and precipitation took place overnight at 4°C. Samples were
separated on 9% to 15% gradient SDS-PAGE along with a molecular
weight marker and were transferred to a polyvinylidene difluoride
(PVDF) membrane. The membrane was incubated with streptavidin-HRP (1 µg/mL) for 1 hour after blocking. After extensive washing,
biotinylated proteins were visualized by echochemiluminescence (ECL; Amersham).
For immunoblotting, platelets were not surface labeled. After lysis,
whole-cell extract was run on an SDS-PAGE gel and transferred to a PVDF
membrane. The membrane was first incubated with 5 µg/mL FITC-labeled
p0p 5 followed by rabbit anti-FITC-horseradish peroxidase (1 µg/mL).
Proteins were visualized by ECL. Immunoprecipitation of GC from 1 mL of
1:10 diluted (PBS) mouse plasma was performed overnight at 4°C.
Flow cytometry
Freshly isolated platelets were washed twice with PBS and then
resuspended in platelet buffer (20 mmol/L Tris HCl, pH 7, 0.9% NaCl, 1 mmol/L CaCl2, and 1 mmol/L MgCl2) at a
concentration of 4 × 104/µL. Samples containing
25 µL of this dilution were stimulated with agonists for 10 minutes
at RT, followed by the addition of saturating amounts of
fluorophore-labeled antibodies or vice versa. After 15 minutes of
incubation at RT, the samples were analyzed on a FACScan. PMA (50 ng/mL; Sigma) or thrombin (0.2 U/mL; Boehringer Mannheim) was used as
agonists. For preincubation experiments, platelets were incubated with
unlabeled mAbs for 15 minutes followed by the addition of saturating
amounts of fluorophore-labeled antibodies. For analysis of
ristocetin-induced platelet activation, platelets were incubated with
1.5 mg/mL ristocetin in the presence of 1 U/mL apyrase (grade III;
Sigma) for 10 minutes at RT.
Immunohistochemistry
Acetone-fixed cryosections (6 µm) were blocked (5% normal goat
serum, 5 mg/mL bovine serum albumin in PBS) for 30 minutes at RT.
Primary mAbs were added at a final concentration of 2 µg/mL. After 90 minutes, the sections were washed 3 times with PBS and subsequently
incubated with the adequate HRP-labeled secondary antibodies at a final
concentration of 2 µg/mL for 60 minutes at RT. The AEC substrate was
added after 3 washing steps, and the sections were then counterstained
with hematoxylin.
Lipopolysaccharide treatment
Mice were injected with the indicated amounts of LPS (in 0.5 mL
sterile PBS) intraperitoneally.
Sequencing
The antigen of 5 × 1010
unbiotinylated platelets was immunoprecipitated with p0p
3. After electrophoresis on SDS-PAGE and transfer to PVDF membrane, the
150-kd band was cut out and enzymatically deblocked with pyroglutamate
aminopeptidase (sequencing grade; Boehringer Mannheim) as
described.35 The deblocked protein was subjected to an
Applied Biosystems (Foster City, CA) protein sequencer (model 494) with
an online PTH-analyzer.
Glycocalicin ELISA
Microtiter plates were coated with 15 µg/mL p0p 3 in coating
buffer (50 mmol/L NaHCO3, pH 9) overnight at 4°C. After
blocking, serial dilutions of plasma or platelet supernatants were
added to duplicate wells (1 hour, 37°C). Plates were washed and
subsequently incubated with HRP-conjugated p0p 4 (5 µg/mL, 1 hour,
37°C). After extensive washing, TMB was added to each well, and the
reaction was stopped by the addition of 2 N
H2SO4 after 10 to 15 minutes. Absorbance at 450 nm was recorded on a Multiskan MCC/340 (Labsystems, Lugano, Switzerland).
Glycocalicin standard
The copy number of GPIb on mouse platelets was estimated by flow
cytometry by comparing Fl2 signals obtained with R-phycoerythrin (PE)
conjugated p0p 3-5 on mouse platelets and a PE-conjugated antihuman
GPIb mAb on human platelets at identical instrument settings. The
signal intensities obtained were in a similar range. Thus, the number
of GPIb molecules on human and mouse platelets was assumed to be
similar (approximately 25 000). Washed platelets (5 × 108/mouse) from 10 healthy mice (5 NMRI, 5 Balb/c) were suspended in each 500 µL PBS (109
platelets/mL) and activated with PMA (50 ng/mL) for 20 minutes at RT.
Efficiency of GC shedding in each sample was determined by flow
cytometry (75.6% ± 3.4%). Platelets and debris were removed by
centrifugation (15 000g, 15 minutes). The supernatants were tested in the GC-ELISA and were found to contain virtually identical amounts of GC. Based on the estimated copy number of 25 000/platelet GPIb, it was calculated that approximately 18 750 GC molecules had
been shed from each platelet. Therefore, the supernatants were assumed
to contain approximately 1.875 × 1013 GC molecules per milliliter (18 750 × 109). Based on a
molecular weight of 130 kd of GC, a GC concentration of 23.8 µg/mL in
the pooled supernatants was calculated. The supernatant was diluted
1:100 in PBS, and aliquots were snap frozen in liquid nitrogen and
stored at  70° C. Serial dilutions of this standard were used
as a control of known GC concentration in each experiment. The GC
concentration of the standard was defined as 1 arbitrary unit.
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Results |
p0p mAbs are directed against mouse GPIb-IX
A series of novel mAbs recognizing a highly expressed membrane
glycoprotein complex on mouse platelets was generated. Five of these
mAbs (p0p 1-5) were used to characterize the recognized antigen. As
shown in Figure 1a, p0p 1-5 precipitated
proteins of identical molecular weights (150 and 25 kd under reducing
conditions) from resting surface-biotinylated mouse platelets. Although
the 150-kd band was precipitated in comparable amounts by all mAbs, different quantities of the 25-kd chain were observed. We used p0p 5 for Western blot analysis of the immunoprecipitates, which demonstrated
that the 150-kd proteins precipitated by the p0p mAbs were identical
(Figure 1b). N-terminal amino acid sequencing of the enzymatically
deblocked protein (see "Materials and Methods") identified the
150-kd protein as mouse GPIb36 [H(T)(X)(S)ISKVTSLLEV]. Flow cytometric experiments demonstrated that p0p 1-5 recognized nonoverlapping epitopes (not shown). GPIb and GPIIbIIIa are expressed in comparable amounts on the surface of resting mouse platelets as
determined flow cytometrically by comparing fluorescence intensities obtained with the p0p mAbs and anti-GPIIbIIIa mAbs (JON1, MWReg30).
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| Fig 1.
p0p mAbs are directed against mouse GPIb-IX.
(A) Immunoprecipitation from surface-biotinylated resting platelets by
p0p 1-5. NP-40 lysates were incubated with nonimmune rat IgG1 (control)
or p0p 1-5, followed by protein G-Sepharose. Proteins were separated by
9% to 15% gradient SDS-PAGE under reducing conditions, transferred to
a PVDF membrane, and detected by streptavidin-HRP and ECL. (B)
Unlabeled platelet proteins were immunoprecipitated with nonimmune rat
IgG1 (control) or p0p 1-5, followed by SDS-PAGE and immunoblotting with
FITC-labeled p0p 5. Bound p0p 5 was detected by HRP-labeled rabbit
anti-FITC.
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Shedding of GPIb is the dominant mechanism of GPIb-IX
down-regulation on activation with thrombin or PMA
As shown in Figure 2a,
activation of platelets with thrombin or PMA resulted in decreased
binding of the p0p mAbs to the platelet surface, whereas signals for
GPIIbIIIa (JON1) and P-selectin (not shown) significantly increased.
Although staining with p0p 3-5 was almost completely abolished on
activation, p0p 1,2 signals were just slightly decreased. To
discriminate between receptor internalization and proteolytic cleavage,
platelets were first incubated with fluorophore-labeled mAbs and
activated with thrombin or PMA after 15 minutes (Figure 2b). Again, the
results obtained with p0p 1,2 differed significantly from those
obtained with p0p 3-5. Fl1 intensities of p0p 1,2 were virtually
unaffected by both forms of activation under these conditions,
indicating that the respective epitopes had been internalized. In
contrast, fluorescence signals of p0p 3-5 decreased significantly on
activation with thrombin or PMA, suggesting that the epitopes
recognized by these mAbs had been cleaved from the platelet surface to
approximately 47% and 75%, respectively. Immunoprecipitation studies
demonstrated that p0p 3-5 recognized a soluble 130-kd fragment of the
receptor (GC) in the supernatant of platelets activated with PMA
(Figure 2c) or thrombin (not shown), whereas p0p 1,2 and control IgG1 did not. We concluded that p0p 1,2 either bound to GPIX or epitopes on
GPIb/ distinct from the GC portion. We were unable to localize the binding epitopes of p0p 1,2 on either GPIX, GPIb, or GPIb because separation of the peptide chains by different approaches always
abrogated binding of the mAbs. However, because binding of p0p 1,2 was
unaffected by proteolytic cleavage of GPIb, we expected these 2 mAbs
to precipitate the truncated rest of this polypeptide chain (t-GPIb)
retained in the membrane after GC shedding (approximately 20 kd).
Because this fragment is disulfide linked to GPIb (25 kd), an
approximately 45-kd band had to be detectable under nonreducing
conditions. Both the 20-kd band (red.) and the 45-kd band (nonred) were
identified in the immunoprecipitates of p0p 1,2 but not of p0p 3 (Figure 2d) or p0p 4,5 (not shown).
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| Fig 2.
Effect of -thrombin and PMA on p0p 1-5 binding to
mouse platelets.
Platelets were first incubated with 0.2 U/mL -thrombin or 50 ng/mL
PMA for 10 minutes at RT and subsequently stained with FITC-labeled
mAbs for 15 minutes at RT (A) or vice versa (B). Samples were analyzed
on a FACScan. The results shown are representative of 6 individual
experiments. (C) Immunoprecipitation with p0p 1-5 and control IgG1 from
NP-40 lysates of surface-biotinylated resting platelets (lanes 1, 3, 5, 7, 9, 11) or supernatant of PMA-activated surface-biotinylated
platelets (lanes 2, 4, 6, 8, 10, 12; 10 minutes, at 15 000g).
Proteins were separated by 9% to 15% SDS-PAGE under reducing
conditions, transferred to a PVDF-membrane, and detected by
streptavidin-HRP/ECL. (D) Detection of GPIb and the
membrane-anchored truncated 20-kd remainder of GPIb (t-GPIb).
Surface-biotinylated platelets were stimulated with PMA (50 ng/mL) for
10 minutes at RT, washed twice, and lysed with NP-40.
Immunoprecipitates of p0p 1, p0p 2, and p0p 3 (control) were separated
under reducing and nonreducing conditions and blotted onto a
PVDF-membrane, and biotinylated proteins were detected by
streptavidin-HRP/ECL.
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Mouse plasma contains approximately 25 µg/mL glycocalicin
We performed immunoprecipitation with p0p 1-5 and JON1 (control)
from normal mouse plasma and tested the immunoprecipitates for the
presence of GC by Western blot analysis with p0p 5. As shown in Figure
3a, p0p 3-5, but not p0p 1,2 or JON1, had
precipitated significant amounts of GC. For better quantification of GC
in mouse plasma, we established an ELISA system using p0p 3 as capture and HRP-conjugated p0p 4 as detection antibody. Serial dilutions of
supernatants from activated (PMA or thrombin) or resting platelets, normal mouse plasma, and a standard of known GC concentration ("Material and methods") were tested (Figure 3b). Results showed that normal mouse plasma contains approximately 25 µg/mL GC and that
a similar amount was shed from 109/mL PMA-activated
platelets (physiological count). Therefore, the platelet-GPIb:plasma
GC ratio is approximately 1:1 in mouse blood, which was confirmed by a
2-fold increase of GC in platelet-rich plasma on PMA-induced
activation. Plasma GC concentrations did not differ significantly
between individual mice tested (± 5.8%; n = 30; different mouse
strains).
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| Fig 3.
Detection and quantification of glycocalicin in mouse
plasma.
Blood (50 µL) was collected in 200 µL heparinized PBS and diluted
1:10 with PBS. Cells and microparticles were removed by centrifugation.
(A) Immunoprecipitation from normal mouse plasma with p0p 1-5 and a
nonimmune IgG1 (control), followed by immunoblotting with p0p 5-FITC.
Bound p0p 5 was detected by HRP-labeled rabbit anti-FITC/ECL. (B)
Detection and quantification of glycocalicin in mouse plasma and the
supernatants of mouse platelets using a sandwich-type ELISA (p0p
3,4-HRP). Washed platelets (109/mL) or platelet-rich plasma
were activated with either PMA (50 ng/mL) or thrombin (thr; 0.2 U/mL)
or were incubated without agonist (control) for 15 minutes at RT.
Supernatants were prepared by centrifugation at 15,000g for 10 minutes and were tested along with normal mouse plasma and a
standard of known GC concentration (23.8 µg/mL) in serial dilutions.
See "Materials and methods" for details.
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Mouse GPIb contains 3 different cleavage sites for
platelet-derived proteases
Flow cytometric studies demonstrated that incubation of washed mouse
platelets at RT resulted in time-dependent proteolytic degradation of
GPIb. Binding of p0p 1,2 remained virtually unaffected for 6 hours,
whereas the epitopes recognized by p0p 5 and, to a greater extent p0p
3,4, were down-regulated, indicating progressive shedding of GPIb
rather than internalization of the receptor complex (not shown). We
took advantage of this observation, incubated freshly isolated
surface-biotinylated platelets for 6 hours at RT, and subsequently
generated an NP-40 lysate containing the solubilized membrane proteins
and all proteolytically cleaved fragments and release products. This
lysate was used for immunoprecipitation with the p0p mAbs. Three
different band patterns were found on the blot (Figure
4). p0p 1 (lane 2) and p0p 2 (not shown)
had precipitated identical bands. Based on these results, we were able
to identify 3 different cleavage sites on GPIb, defining fragment
pairs of 20 + 130 kd, 90 + 60 kd, and 105 + 45 kd, respectively. p0p 3,4 precipitated the 45-kd band (fragment 1a), whereas p0p 1, 5 precipitated the 105-kd band (fragment 1b). Based on the finding that
p0p 1 precipitates the membrane-anchored fragment (see Figure 2d), we
concluded that fragment 1a represents the cleaved N-terminal 45-kd
fragment of GPIb, whereas fragment 1b is the truncated 105-kd
remainder of GPIb retained in the membrane. Therefore, the binding
epitopes of p0p 3,4 are located on the N-terminal 45-kd portion of the
receptor known to contain the binding sites for vWF and
thrombin.7-9 Furthermore, p0p 3-5 precipitated the 60-kd
band (fragment 2a), whereas the 90-kd band (fragment 2b) was
exclusively recognized by p0p 1 (and p0p 2). The latter therefore
represented the membrane-anchored truncated remainder of GPIb. We
concluded that p0p 5 binds to an epitope on the N-terminal 60-kd
portion of GPIb located between cleavage sites 1 and 2. The 130-kd
band (fragment 3a), recognized by p0p 3-5, was identified as GC,
whereas the 20-kd band (fragment 3b) exclusively recognized by p0p 1,2 represented the corresponding membrane-anchored truncated form of
GPIb (t-GPIb).
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| Fig 4.
Immunoprecipitation of 6 different proteolytic fragments
of GPIb.
Surface-biotinylated mouse platelets were incubated for 6 hours at RT
and lysed directly. Immunoprecipitation was performed with p0p 1,3,4,5 and control IgG1. Proteins were separated by 9% to 15% SDS-PAGE under
reducing conditions and were detected by streptavidin-HRP/ECL. (right)
Schematic drawing of the murine GPIb-IX complex. (arrows ) Proposed
cleavage sites (1, 2, 3). (left, arrows) Assumed binding sites of p0p
1-5. (middle) Schematic drawing of fragment pairs shown on the
blot.
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Occupancy of GPIb induces irreversible aggregate formation in
vitro and rapid thrombocytopenia in vivo
Standard aggregometry demonstrated that none of the p0p mAbs (at
concentrations of 10, 30, or 100 µg/mL) interfered with aggregation induced by adenosine diphosphate, collagen, PMA, or ristocetin (not
shown). We observed, however, that ristocetin (at concentrations greater than 1 mg/mL) did not induce passive agglutination (as described for human platelets) but did induce active aggregation of
mouse platelets as evidenced by rapid surface expression of P-selectin,
fibrinogen, and vWF in the presence of this antibiotic (Figure
5).
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| Fig 5.
Ristocetin induces activation of mouse platelets.
Platelets (106) were incubated with 1.5 mg/mL ristocetin in
the presence of 1 U/mL apyrase at RT for 10 minutes. Subsequently,
FITC-labeled antibodies were added in saturating amounts, and the
samples were analyzed on a FACScan after 15 minutes. (shaded area)
Staining of resting platelets. (solid lines) Staining of
ristocetin-activated platelets.
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Although no inhibitory effect of the p0p mAbs on platelet aggregation
could be detected, direct platelet activation by all mAbs directed
against the GC portion of GPIb was obvious. Addition of (10-100 µg/mL) p0p 3-5 or F(ab)2-fragments of p0p 3,4, but not
p0p 1,2 or control IgG, to platelet-rich plasma under stirring conditions (1000 rpm, 37°C) induced the formation of
microaggregates of 3 to 5 platelets (not shown). Platelet activation
induced by p0p 3-5 became more evident after mild centrifugation of the
samples (1300g, 5 minutes, RT).
After resuspension, large aggregates were found, whereas the single
platelet count was drastically reduced (Figure
6a). Flow cytometric analysis of the
aggregates demonstrated no increased surface expression of P-selectin,
vWF, or fibrinogen (not shown). In correlation with platelet
aggregating effects in vitro, we found rapid and irreversible platelet
depletion on the injection of 25 µg per mouse p0p 3-5 or
F(ab)2-fragments of p0p 3,4, whereas thrombocytopenia
induced by p0p 1,2 or EDL1 (anti-gpIIIa) was less effective (Figure
6b).
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| Fig 6.
p0p 3-5 induce aggregate formation in vitro and rapid
thrombocytopenia in vivo.
(A) Washed platelets (2 × 106) were incubated with
10 mg/mL of the indicated mAbs or without antibody (control) for 15 minutes at RT, followed by centrifugation at 1300g for 5 minutes. After resuspension of the pellets by vortexing for 5 seconds,
the single platelet count in the samples was determined by flow
cytometry. Results are shown as the mean ± SD for 3 independent
experiments. (B) Normal female NMRI mice received 25 µg purified p0p
1,2,5 or F(ab)2-fragments of p0p 3,4 intravenously in 200 µL sterile PBS. The anti-GPIIIa mAb EDL1 was used as a control.
Platelet counts were determined at the indicated times using an
Improved Neubauer hemocytometer. Injection of a nonimmune IgG1 had no
significant effect on the platelet count (not shown). Intact p0p 3,4 induced comparable thrombocytopenia as the F(ab)2-fragments
(not shown). Results of platelet count are shown as the
mean ± SD for groups of 3 mice. The experiment was
repeated twice with comparable results.
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GPIb-IX is not detectable on endothelial cells under normal or
inflammatory conditions
To examine the in vivo protein expression of GPIb-IX, cryosections
from the major organs (spleen, lung, liver, kidney, heart, brain,
intestine, skin, and thymus) from normal mice (n = 6) were stained
for GPIb-IX with a 1:1 mixture of p0p 1 and p0p 4 (each at 2 µg/mL).
Antibodies against vWf were used as a control for staining the
platelets, megakaryocytes, and endothelial cells. p0p1,4, and anti-vWF
specifically stained megakaryocytes and platelets in the red pulp of
the spleen (Figure 7). In the lungs, only
platelets but not endothelial or other cells were stained with p0p 1 and p0p 4, whereas anti-vWF stained platelets and endothelial cells. In
the liver, kidney, and heart, few platelets were detectable with p0p 1, p0p 4, and anti-vWF. As in the lungs, specific staining of endothelial
cells was only detectable with anti-vWF. The same result was obtained
with brain, intestine, skin, and thymus (not shown), in which virtually
no platelets were detected. The staining pattern obtained with p0p 1, p0p 4, and JON1 (anti-GPIIbIIIa, not shown) was identical in all organs
examined. Thus, no expression of GPIb-X on cells other than
platelets/megakaryocytes was detectable in any organ of normal healthy
mice. To test the hypothesis that GPIb-IX might be expressed on
endothelial cells in response to inflammatory
stimuli,10,29,30 we treated mice with bacterial LPS (20 mg/kg, n = 20). It is well documented that such treatment induces the
production of a variety of cytokines, resulting in systemic
inflammatory responses in mice.37 Significant
thrombocytopenia (platelet count, 61.4% ± 4.9% of normal) and
hypothermia (3.3°C ± 0.6°C) developed in all mice
within 12 hours. Organs from 5 mice were sampled after 6, 12, 24, or 48 hours, and cryosections were stained with p0p 1, p0p 4, anti-vWF, or
JON1. Although all antibodies stained platelets and megakaryocytes,
specific staining of endothelial cells in these organs was only
observed with anti-vWF. A significantly increased number of platelets
in the livers of LPS-treated mice was detected with p0p 1 and p0p 4 (and JON1), whereas staining of platelets in the lungs and
platelets/megakaryocytes in the spleens was apparently unchanged
compared with that in the control mice. As in the control mice, kidney,
heart, skin, intestine, brain, and thymus appeared virtually free of
platelets (the latter 4 organs are not shown).
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| Fig 7.
Immunohistochemical detection of GPIb-IX.
Acetone-fixed frozen sections from normal (untreated) mice and mice 24 hours after injection of 20 mg/kg LPS were stained for GPIb-IX with p0p
1,4. As a positive control for platelet/megakaryocytic and endothelial
expression, sections from normal mice were stained for vWF (left).
Representative sections of (A) spleen, (B) liver, (C) lung, (D) kidney,
and (E) heart are shown.
|
|
| |
Discussion |
In the current study we investigated the structural and functional
properties of mouse GPIb-IX and systematically examined the in vivo
expression of the receptor on a cellular level for the first time. For
our studies we used 5 newly developed monoclonal antibodies that
recognized different, nonoverlapping epitopes on the complex.
Immunoprecipitation and Western blot analysis showed that the
individual components of the complex had apparent molecular weights
similar to those of human homologs: GPIb (approximately 150 kd),
GPIb (approximately 25 kd), and GPIX (approximately 25 kd) (Figures
1, 2). Using p0p 1-5 for flow cytometric and biochemical analyses, we
were able to examine and quantify the regulation of different epitopes
on mouse GPIb-IX under various experimental conditions. In our
experiments, we found that thrombin-induced activation of mouse
platelets resulted in approximately 47% shedding of GPIb (GC) and
only approximately 31% internalization of the GPIb-IX complex. In
contrast, thrombin has been reported to induce internalization of
GPIb-IX complexes from the platelet surface to the surface-connected
canalicular system without evidence for receptor shedding on human
platelets,20-22 though this finding is not commonly
accepted.23,24 Therefore, it seems likely that mouse
GPIb is more susceptible to proteolytic cleavage during platelet
activation. This hypothesis may be supported by the detection of at
least 10-fold higher GC concentrations in mouse plasma than in human
plasma (approximately 25 µg/mL versus approximately 2 µg/mL38). Certainly, the higher platelet counts in mice
(1.0-1.2 × 106/µL versus
0.2-0.4 × 106/µL) also contribute to the marked
difference in plasma GC concentrations between the 2 species.
Although GC is the only known fragment of human GPIb generated by
platelet-derived proteases, an additional N-terminal 45-kd fragment can
be generated experimentally by trypsin digestion.8 This
45-kd fragment, isolated by tryptic digestion or expressed by
recombinant DNA methods, contains the binding sites for vWF and
thrombin and essentially mimics the functional properties of GPIb-IX-V
as a soluble receptor.7-9 In our immunoprecipitation experiments with washed platelets, we identified 3 different soluble fragments (GC, 60 kd and 45 kd) and the 3 corresponding
membrane-anchored truncated forms of GPIb (20 kd, 90 kd, and 105 kd), demonstrating that these fragments must have been generated by
platelet-derived proteases (Figure 4). To our knowledge, this is the
first report describing the proteolytic generation of both the 45-kd
and the 60-kd N-terminal fragment of GPIb by platelet-derived
proteases. Based on our data, we propose 3 cleavage sites on murine
GPIb (Figure 4), suggesting complex regulation mechanisms of GPIb-IX function in vivo. In contrast, in vitro activation of platelets with
thrombin or PMA only resulted in proteolytic generation of GC but not
of the 45-kd and 60-kd fragments of GPIb (not shown). Most
information on GPIb-IX-V regulation in humans is derived from studies
using exogenous platelet-activating stimuli. This may explain why GC is
the only known proteolytic fragment of GPIb cleaved from activated
human platelets.
The GPIb-vWF interaction has become a potentially interesting
target for antithrombotic therapies,39 leading to the
development of strategies for receptor or ligand blockage in vivo.
Although mAbs against the A1-domain of vWF efficiently blocked
GPIb-vWF interaction in vivo,39 there are conflicting
reports about the in vivo effects of anti-GPIb mAbs. Becker et
al40 reported that F(ab)2-fragments of an
anti-GPIb mAb inhibited GPIb-vWF interactions in vivo and ex vivo
without significant effects on platelet counts in guinea pigs. In
contrast, a more recent study performed in baboons showed that the
injection of anti-GPIb-F(ab)2-fragments immediately
resulted in profound irreversible thrombocytopenia.41 We
observed similar effects in the mouse. All mAbs or
F(ab)2-fragments against the GC portion of GPIb,
irrespective of their exact binding epitope, induced aggregate
formation in vitro and a greater than 95% drop of platelet count
within 2 minutes in vivo (Figure 6). Although the mechanisms underlying
this cytotoxic effect could not be identified in the current study, it
seems possible that attempts to block certain epitopes on GPIb with
modified antibodies in vivo may generally result in thrombocytopenia.
In contrast to the inhibition of GPIIbIIIa
function,42 in vivo blockage of certain epitopes on GPIb
may, therefore, not be a promising antithrombotic strategy.
Monoclonal antibodies directed against human GPIb have been reported
to inhibit ristocetin-induced platelet agglutination and to interfere
with collagen-induced platelet aggregation.43 Our
aggregometric studies with p0p 1-5, however, showed that none of the
mAbs had significant influence on aggregation induced by adenosine
diphosphate, collagen, and PMA (not shown). Furthermore, p0p 1-5 had no
effect on ristocetin-induced platelet aggregation, which was always
associated with classical platelet activation. Concentrations greater
than 1 mg/mL ristocetin induced surface expression of P-selectin,
fibrinogen, and endogenous vWF, as determined by flow cytometry (Figure
5). The direct activation of mouse platelets by ristocetin contrasts
its passive agglutination of human platelets.14 Thus,
ristocetin may not be suited for studies on GPIb-vWF interactions in the mouse system.
The expression of GPIb-IX on cells of nonmegakaryocytic origin
(particularly endothelial cells) has been controversial. Although the
importance of the complex in normal megakaryocyte and platelet physiology is clear and well documented, some authors have speculated on a second, unrelated role of GPIb or the GPIb-IX-V
complex.29-31 This hypothesis is based on the observation
that cultured human endothelial and smooth muscle cells express the
individual subunits of the complex.31,44-46 Furthermore,
endothelial GPIb has been proposed to be involved in
platelet-endothelial cell interactions under inflammatory
conditions.29,30,47 On the other hand, these data are not
without debate because the findings could not be reproduced by
others.32 Recently, Fujita et al48 made the first attempt to examine the in vivo expression of GPIb in the mouse. The authors reported consistent and reproducible GPIb gene
activity in nonhematopoietic organs, including lung and heart, and they
speculated on GPIb expression on cells other than
platelets/megakaryocytes. Although GPIb-IX-V can be detected
immunohistochemically on cultured human endothelial
cells,29-31 no systematic studies on the in vivo expression
of the receptor on a cellular level have been performed to date. We
examined GPIb-IX expression under normal and inflammatory conditions
immunohistochemically for the first time and found specific staining in
the major organs of the mouse only on platelets and megakaryocytes, but
not on endothelial cells (Figure 7). Comparison between JON1
(anti-GPIIbIIIa) and p0p 1,4 (anti-GPIb-IX)-stained sections
demonstrated identical staining patterns in all organs examined. In
contrast, antibodies against vWF clearly stained platelets/megakaryocytes and endothelial cells. Although the
sensitivity of immunohistochemical techniques is limited, our studies
did not support the hypothesis that GPIb-IX is expressed on endothelial cells in vivo.
In conclusion, the results presented in this article indicate that
mouse GPIb-IX was exclusively expressed on platelets and megakaryocytes
and, despite some differences, shared many structural and functional
properties with the human receptor. The p0p mAbs proved powerful tools,
and they may be helpful for further studies on the function and
regulation of the GPIb-IX complex. The availability of transgenic and
knockout strains predestinates the mouse system for such investigations.
| |
Acknowledgments |
We thank N. Huss for critically reading the manuscript, E. Rieke for assisting with photography and computer data, and W. Heil for
permission to use his aggregometer and chemicals. We also thank R. Müller-Peddinghaus, P. G. Höher, and J. Werner for their
support throughout the study.
| |
Footnotes |
Submitted March 16, 1999; accepted September 20, 1999.
Supported by BAYER AG, Germany.
Reprints: Bernhard Nieswandt, IMMI, Klinikum Wuppertal,
Universität Witten-Herdecke, Heusnerstrasse 40, D-42283
Wuppertal, Germany; e-mail: nieswand{at}klinikum-wuppertal.de.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction