|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4186-4194
Modulation by Heparin of the Interaction of the A1 Domain of von
Willebrand Factor With Glycoprotein Ib
By
Christelle Perrault,
Nadine Ajzenberg,
Paulette Legendre,
Ghassem Rastegar-Lari,
Dominique Meyer,
Jose A. Lopez, and
Dominique Baruch
From INSERM U143, Hopital de Bicetre, Bicetre, France; and the
Departments of Internal Medicine and Molecular and Human Genetics,
Baylor College of Medicine, Houston, TX.
 |
ABSTRACT |
The conformation of the A1 domain of von Willebrand factor (vWF) is
a critical determinant of its interaction with the glycoprotein (GP)
Ib/V/IX complex. To better define the regulatory mechanisms of vWF A1
domain binding to the GPIb/V/IX complex, we studied vWF-dependent
aggregation properties of a cell line overexpressing the GPIb ,
GPIb , and GPIX subunits (CHO-GPIb/IX cells). We found that
CHO-GPIb/IX cell aggregation required the presence of both
soluble vWF and ristocetin. Ristocetin-induced CHO-GPIb/IX cell
aggregation was completely inhibited by the recombinant VCL fragment of vWF that contains the A1 domain. Surprisingly, the substitution of heparin for ristocetin resulted in the formation of
CHO-GPIb/IX cell aggregates. Using monoclonal antibodies blocking
vWF interaction with GPIb/V/IX or mocarhagin, a venom metalloproteinase
that removes the amino-terminal fragment of GPIb extending from aa 1 to 282, we demonstrated that both ristocetin- and heparin-induced
aggregations involved an interaction between the A1 domain of vWF and
the GPIb subunit of the GPIb/V/IX complex. The involvement of
heparin in cell aggregation was also demonstrated after treatment of
heparin with heparinase that abolished CHO-GPIb/IX cell
aggregation. These results indicated that heparin was able to induce
vWF-dependent CHO-GPIb/IX cell aggregation. In conclusion, we
demonstrated that heparin is capable of positively modulating the vWF
interaction with the GPIb/V/IX complex.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
AFTER BLOOD VESSEL injury, von
Willebrand factor (vWF) is the only multimeric protein involved in
platelet adhesion to the extracellular matrix (ECM) under conditions of
high shear stress. vWF is synthesized by platelets and endothelial
cells and is found in their storage vesicles, in plasma, and in
vascular ECM. Mature subunits of vWF containing 2050 amino acids (aa)
are assembled in multimers ranging in molecular weight from 500 kD to
more than 10,000 kD. Extremely large multimers of vWF are the most
effective ones for promoting platelet adhesion. To initiate platelet
adhesion, vWF binds to the glycoprotein (GP) Ib subunit, which is
disulfide-linked to GPIb and noncovalently associated with GPIX. The
platelet GPIb/V/IX complex also includes GPV noncovalently bound to
GPIb-IX.1 Circulating plasma vWF does not interact spontaneously with platelets. Binding to the GPIb/V/IX complex requires
conformational changes of the A1 domain of vWF, which can be achieved
in vivo by immobilization of vWF onto the endothelial ECM or by high
shear stress conditions and in vitro by nonphysiological substances
such as ristocetin or botrocetin. Two different types of vWF sequences,
the regulatory sites on one hand and the direct binding sites on the
other, are involved in binding to GPIb and are located in the A1
domain. The asymmetric charge distribution of the A1 domain as well as
the high level of negative charges on GPIb itself suggests that
exposure of the GPIb-binding site on vWF is dependent on
electrostatic interactions. Ristocetin binds to 2 discontinuous,
proline-rich, negatively charged regions (aa 474-488 and 695-708)
flanking the disulfide bridge of the A1 domain. Botrocetin, a protein
isolated from the venom of the snake Bothrops jararaca, binds
to predominantly positively charged sequences in the A1 loop (aa
514-542, 539-553, 569-583, and 629-643).2,3 Interestingly,
both inducers may act through different mechanisms.4 Binding of ristocetin to vWF may relieve the effect of inhibitory sites
responsible for maintaining vWF in an inactive conformation, thus
indirectly inducing vWF binding to GPIb.4,5 In contrast, botrocetin favors direct binding of vWF to GPIb without a need for
relieving inhibitory sites. Additional binding sequences of vWF to
GPIb, involved independently of the inducer, are located in a
sequence between aa 596-616.5
Heparin is a glycosaminoglycan with anticoagulant properties related to
its ability to accelerate the inhibition of thrombin by
antithrombin-III.6 Besides its therapeutic use, heparin and
related sulfated glycosaminoglycans may be important in vivo, because
they are present in ECM and on cell surfaces. The significance of vWF
binding to heparin in physio-pathological conditions remains to be
established. Heparin has been reported to inhibit ristocetin-induced platelet agglutination or vWF binding to platelets and to reduce platelet deposition on injured arteries.7-9 However, the
major heparin binding sequence on vWF is located within the A1 loop and
overlaps a vWF binding site to botrocetin.10-12 Moreover,
we have previously reported that a monoclonal antibody (MoAb) 724 to
vWF is able to inhibit its binding to heparin and to platelet GPIb/V/IX
in the presence of botrocetin.13 Interestingly, binding of
MoAb 724 to vWF increased the extent of shear-induced platelet aggregation and of platelet aggregation induced by low ristocetin concentrations, an effect potentially related to a shift of soluble vWF
from an inactive conformation to an active one.14
The aim of the present work is to define the regulatory mechanisms
controlling the binding of the vWF-A1 domain to the GPIb/V/IX complex
and to determine the importance of the A1 domain conformation. To this
end, we compared the effect of ristocetin and heparin on the
aggregation properties of a stable CHO cell line overexpressing the
GPIb/V/IX receptor (CHO-GPIb/IX cells) in the presence of fluid-phase vWF. Our work indicates that heparin binding to vWF may
positively modulate the interaction of vWF with GPIb. This indicates
that heparin may participate in conformational changes of the A1 domain
that allow optimal vWF binding to the GPIb/V/IX complex.
| |
MATERIALS AND METHODS |
Plasma vWF and VCL recombinant fragment.
Human vWF was isolated from plasma high-purity concentrates (provided
by the Laboratoire Français du Fractionnement et des Biotechnologies, Les Ulis, France).15 The amount of vWF:Ag
was measured by enzyme-linked immunosorbent assay.13
Analysis of vWF subunit and its multimeric pattern was performed as
described,16 showing a single band of molecular weight (MW)
250 kD by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis under reducing conditions and a whole range of
multimers by agarose gel electrophoresis. Molar concentration of vWF
was calculated with an MW of the vWF subunit of 250 kD. VCL, a
monomeric recombinant vWF fragment extending between aa 504-728, was
expressed in Escherichia coli and purified on
carboxymethyl-Sepharose17 (kind gift of Dr L. Garfinkel,
Biotechnology General, Rehovot, Israel). VCL was reconstituted from its
lyophilized form in phosphate-buffered saline (PBS), pH 7.4, with
ice-cold sterile water to a concentration of 1.5 mg/mL (60 µmol/L).
Analysis of VCL by 3.5% to 20% gradient SDS-polyacrylamide gel
electrophoresis showed a single band of 25 kD. Molar concentration of
VCL was calculated with an MW of 25 kD.
Radiolabeling of proteins.
VCL or vWF was labeled with Na125I (Amersham, Les Ulis,
France) using Iodo-Gen (Pierce Chemical Co, Rockford, IL) as
described.18 Specific radioactivity was 0.04 µCi/µg for
125I-VCL and between 3 and 5.2 µCi/µg for
125I-vWF. Labeled proteins were stored at 4°C and used
within 1 week.
Binding of radiolabeled vWF or VCL to platelet GPIb/V/IX or heparin.
Binding of vWF to the GPIb/V/IX receptor was performed using final
concentrations of 2 nmol/L (0.5 µg/mL) 125I-vWF,
108 paraformaldehyde-fixed platelets/mL, 1 mg/mL ristocetin
(abp, New York, NY), or 0.5 µg/mL purified botrocetin,
as previously described.19 Binding of vWF or VCL to
unfractionated heparin (from porcine intestinal mucosa; Sigma, La
Verpillière, France) immobilized on aminoethyl-agarose beads
(Sigma) was performed as reported, using final concentrations of 3%
heparin-agarose beads (vol:vol) and 2 nmol/L (0.5 µg/mL)
125I-vWF, as reported.20 For competition with
unlabeled ligand, vWF or VCL was added to these mixtures. Lower
heparin-agarose concentration (0.6%, vol:vol) was used for
125I-VCL (1 µg/mL, 40 nmol/L) binding studies.
After 1 hour of incubation, duplicate aliquots of the mixtures were
centrifuged on 25% sucrose in 20 mmol/L MES-Tris, pH 6, 100 mmol/L
NaCl containing 0.5% bovine serum albumin (BSA; Calbiochem, La Jolla,
CA) to separate bound from free ligand. Radioactivity was counted in a
 -counter (MultiGamma; Pharmacia, LKB Biotechnology, St
Quentin-en-Yvelines, France). The percentage of bound radioactivity was
calculated as (bound/[free + bound]) × 100 radioactivity.
Antibodies.
We used MoAbs reported as blocking the interaction of vWF with platelet
GPIb/V/IX. We selected 2 MoAbs to vWF: MoAb 328 inhibiting vWF binding
to GPIb/V/IX in the presence of ristocetin and shear-induced platelet
adhesion or aggregation14,21,22 or MoAb 724, which blocks
vWF binding to GPIb/V/IX in the presence of botrocetin, as well as
heparin or botrocetin binding to vWF.13,21 We also used
MoAbs to GPIb/V/IX, SZ2 (Immunotech, Marseille, France), and 6D1
(kindly provided by Dr B. Coller, Mount Sinai School of Medicine, New
York, NY) directed against the amino-terminal 45-kD domain of
GPIb,23,24 as well as MoAb SZ1 to the GPIb/V/IX complex,
which binds to a conformation-sensitive epitope on GPIX (provided by Dr
C. Ruan, Suzhou Medical College, Suzhou, People's Republic of
China).25 These MoAbs were used as purified IgGs at 20 µg/mL. As control, nonimmune purified IgG was tested in comparison
with specific antibody.
Cell culture.
Chinese hamster ovary (CHO) cells defective in the dihydrofolate
reductase gene (CHOdhfr ; American Type Culture
Collection, Rockville, MD) were grown in Iscove's medium (Eurobio, Les
Ulis, France) supplemented with 10% fetal calf serum (FCS; Boehringer
Mannheim, Meylan, France), 2 mmol/L glutamine, 100 µmol/L
hypoxanthine, and 10 µmol/L thymidine (Eurobio). After cell
transfection with full-length cDNAs encoding Ib, Ib, and GPIX or
Ib and GPIX, 2 stable cell lines were selected expressing either the
CHO-GPIb/IX (in this study referred to as CHO-GPIb/IX
cells) or the GPIb/IX complex (CHO-GPIb/IX cells).25
Both cell lines were cultured in minimum essential medium (MEM;
Eurobio) containing 10% FCS, 2 mmol/L glutamine, and 400 µg/mL G418
(GIBCO, Cergy Pontoise, France). CHO-GPIb/IX cells were cultured in
the presence of 100 µmol/L methotrexate (Sigma). Cells were detached
with 0.5 mmol/L EDTA for 10 minutes at 37°C. Flow cytometry
experiments were performed in a FACScan flow cytometer (Becton
Dickinson, Le-Pont-de-Claix, France) as previously
described26 and confirmed the expected surface expression of GPIb on CHO-GPIb/IX cell line using MoAb 6D1 and of GPIb and GPIX on CHO-GPIb/IX and CHO-GPIb/IX cell lines using MoAb SZ1. Binding properties to vWF of CHO-GPIb/IX cells were similar to those of CHO-GPIb/V/IX, another cell line expressing the GPIb/V/IX complex, thus indicating little difference according to
the presence of GPV.
Enzymatic treatment.
To confirm the involvement of the GPIb subunit, enzymatic treatment
of CHO-GPIb/IX cells was performed using enzymes with known
cleavage sites within the extracellular domain of GPIb. Mocarhagin,
a cobra venom metalloproteinase generating the amino-terminal fragment
of GPIb (aa 1 to 282), was a kind gift of Dr M. C. Berndt (Baker
Medical Research Institute, Prahran Victoria, Australia) and was
incubated with cells at 10 µg/mL.27 The neutrophil
proteinase elastase, generating an amino-terminal fragment of GPIb
extending from aa 1 to 296, was a kind gift of Dr M. Chignard (INSERM
U485, Institut Pasteur, Paris, France) and was added to the cells at a
concentration of 200 nmol/L, followed by neutralization by eglin C (10 µg/mL).28 Trypsin (50 µg/mL; GIBCO) was added to the
cell suspension, followed by a 12.5-fold excess (wt/wt) of soybean trypsin inhibitor (Sigma). Confluent cells were washed with PBS and
incubated with the enzymes for 10 minutes at 37°C in PBS, except
for mocarhagin, which was incubated in PBS containing 1 mmol/L
CaCl2. Cells were washed with PBS before conducting the functional assays.
Aggregation assay.
CHO-GPIb/IX cell aggregation was performed on a table-top rotary
shaker according to the method of Dong et al,29 with some
modifications. Two hundred microliters of CHO-GPIb/IX cells (106 cells/mL) in PBS containing 1.2 mmol/L
CaCl2 was added to 48-well plastic plates (ATGC,
Noisy-le-Grand, France) in the presence of purified vWF (0.5 to 40 nmol/L) and either ristocetin (0.2 to 2 mg/mL) or heparin (10 to 250 µg/mL). In some cases, cells were preincubated with inhibitors for 10 minutes before the aggregation assay. In some experiments, heparin was
degraded by treatment with 2 U/mL heparinase I (EC 4.2.2.7;
Sigma) in 5 mmol/L NaCOOCH3, 1 mmol/L
CaCl2, 50 mmol/L Tris, pH 7, for 18 hours at 37°C. Cell aggregation was performed for 20 minutes at room temperature by subjecting the plates to a constant rotary shaking of 6 cycles/sec on a
table-top shaker (Rotatest 95220; Bioblock, Illkirch, France). This
orbital motion induced mechanical forces resulting in increased bond
formation between cells, without exerting any measurable shear stress.
Cell aggregation was observed by light microscopy in an inverted
microscope (Axiovert 135; Carl Zeiss, Göttingen, Germany) and
microphotographs were taken using a Yashica 108 camera (Kyocera, Tokyo,
Japan) at a 10-fold magnification. To quantitate cell aggregation, we
counted nonaggregated cells defined as singlets, doublets, and
triplets. Between 1,000 and 3,500 cells were counted on 5 microphotographs taken on contiguous fields. Results were expressed
relative to the number of nonaggregated cells obtained in the absence
of vWF (n0). We calculated the percentage of aggregated cells as ([n0  n]/n0) × 100, where n represents the number of nonaggregated cells in the presence of vWF.
Statistical analysis.
Mean values of percentages of cell aggregation and their standard
errors (SEM) were calculated from 3 to 4 experiments performed in
duplicate. The statistical significance of differences between means
was evaluated using the Student's t-test for paired samples; P values less than .05 were considered significant.
| |
RESULTS |
GPIb/V/IX and heparin binding properties of vWF and VCL.
The isolated A1 domain of vWF has been reported to bind GPIb/V/IX both
in the absence and presence of modulators. As a model of the vWF-A1
domain, we used the recombinant VCL fragment of vWF that contains the
A1 domain. By analyzing its inhibitory effect on 125I-vWF
binding to platelet GPIb/V/IX, we confirmed that VCL was a potent
inhibitor of ristocetin-induced binding of 125I-vWF to
platelet GPIb/V/IX with an IC50 (concentration able to inhibit 50% of
binding) of 0.34 µmol/L, in agreement with a reported value of 0.26 µmol/L.17 Interestingly, a 3-fold lower concentration of
unlabeled vWF (0.1 µmol/L) was required to inhibit 50% of
125I-vWF binding to platelet GPIb/V/IX. Heparin inhibited
the ristocetin-induced 125I-vWF binding to platelet
GPIb/V/IX with an IC50 of 6 µg/mL, a value in concordance
with published data.9 Although the VCL fragment overlaps a
heparin-binding domain, few data are available on its heparin binding
properties. Figure 1 shows some
heparin-binding properties of vWF and VCL. We found that unlabeled vWF
inhibited 125I-vWF binding to heparin-agarose beads with an
IC50 of 0.6 µmol/L. VCL was an efficient competitor of
125I-vWF binding to heparin-agarose with an IC50 of 1.8 µmol/L (Fig 1A). These data showed the need for a 3-fold higher
concentration of VCL compared with vWF to inhibit 125I-vWF
binding to heparin. Interestingly, inhibition of 125I-VCL
binding to heparin-agarose beads by unlabeled vWF or VCL yield IC50
values of 0.8 and 2 µmol/L, respectively, similar to those obtained
for inhibition of 125I-vWF binding to heparin (Fig 1B).
View larger version (11K):
[in this window]
[in a new window]
| Fig 1.
Binding of 125I-vWF and 125I-VCL
to heparin. Heparin-agarose beads were incubated for 1 hour at 20°C
with 125I-vWF (A) or 125I-VCL (B) in the
presence of varying concentrations of unlabeled vWF ( ) or VCL ( ).
The mean ± SEM of percentage of bound radioactivity was calculated as
described in Materials and Methods for 3 separate experiments.
|
|
vWF-dependent aggregation of CHO-GPIb/IX cells.
To further characterize the regulatory mechanism of soluble vWF binding
to the GPIb/V/IX complex, we studied the effect of vWF on the
aggregation of CHO-GPIb/IX cells expressing the GPIb/IX complex. CHO-GPIb/IX cell aggregation was performed essentially as described by Dong et al29 using ristocetin or heparin
and a rotary shaker. When shaken at 6 cycles per second,
CHO-GPIb/IX cells in suspension were unable to aggregate in the
absence of vWF (Fig 2A). Most cells
remained nonaggregated as single cells, and only a few doublets or
triplets were visible. The addition of vWF was not sufficient to induce
CHO-GPIb/IX cell aggregation, as shown by the fact that only
isolated cells were present (Fig 2B). In addition, we found that
ristocetin by itself was not responsible for cell aggregation in the
absence of vWF (Fig 2C). In contrast, the addition of both vWF and
ristocetin induced the formation of large cell aggregates, with very
few isolated cells remaining (Fig 2E). These cell aggregates had
different sizes, including a majority of large aggregates containing 35 to 45 CHO-GPIb/IX cells. Surprisingly, the substitution of
heparin for ristocetin also resulted in the formation of large
aggregates (Fig 2F). Heparin-induced cell aggregation was characterized
by the presence of large aggregates of a similar size as
ristocetin-induced aggregates. The aggregatory effect of heparin
required the presence of vWF associated with shaking forces, because
heparin alone was not able to induce cell aggregation in the absence of
vWF (Fig 2D). In addition, in the absence of rotary shaking, neither
heparin nor ristocetin was able to induce aggregation of cells
incubated with vWF. Thus, both ristocetin and heparin were able to
induce vWF-dependent CHO-GPIb/IX cell aggregation.
View larger version (121K):
[in this window]
[in a new window]
| Fig 2.
CHO-GPIb/IX cell aggregation. CHO-GPIb/IX
cells (106 cells/mL) were exposed to rotary shaking at 6 cycles per second for 20 minutes either in the absence of vWF (A, C,
and D) or in the presence of vWF (B, E, and F) to which either buffer
(A and B), ristocetin (C and E), or heparin (D and F) was added.
Microphotographs of cell aggregation were taken at a 10-fold
magnification. vWF-dependent cell aggregation was seen in the presence
of ristocetin or heparin.
|
|
Ristocetin-induced aggregation of CHO-GPIb/IX cells.
To better understand the similarities between both agonists on
vWF-mediated aggregation of CHO-GPIb/IX cells, we first
characterized the ristocetin-induced cell aggregation. To this end, we
determined dose-response curves of cell aggregation as a function of
vWF or ristocetin concentrations. The influence of vWF concentration was studied in the presence of 1.4 mg/mL of ristocetin
(Fig 3A). We found that CHO-GPIb/IX
cell aggregation increased with the vWF concentration, reaching a
plateau at 10 nmol/L of vWF, corresponding to 48.5% ± 0.5%
aggregated cells. In contrast, CHO cells expressing the GPIb/IX
complex and nontransfected cells (CHO dhfr) did not
aggregate in the presence of ristocetin, even at vWF concentrations as
high as 40 nmol/L. To determine whether ristocetin had a dose-dependent
effect on cell aggregation, CHO-GPIb/IX cells were shaken in the
presence of 10 nmol/L vWF and different ristocetin concentrations (Fig
3B). Cell aggregation increased with the concentration of ristocetin,
tending to a plateau of 52.0% ± 0.6% aggregated cells for 1.4 mg/mL ristocetin. In contrast, in the absence of vWF, hardly any cell
aggregation occurred, because it did not exceed 8.4% ± 2.8%
aggregated cells at the highest ristocetin concentration (2 mg/mL). These results indicate that cell aggregation depends on vWF
and ristocetin and requires the GPIb subunit.
View larger version (11K):
[in this window]
[in a new window]
| Fig 3.
vWF-dependent ristocetin-induced CHO-GPIb/IX cell
aggregation. (A) Aggregation of CHO-GPIb/IX (), CHO-GPIb/IX
(), or CHOdhfr ( ) cells was performed in the
presence of various concentrations of vWF (0 to 40 nmol/L) by adding
1.4 mg/mL ristocetin. (B) CHO-GPIb/IX cell aggregation was
performed in the absence () or in the presence () of 10 nmol/L
vWF and various concentrations of ristocetin (0 to 2 mg/mL). After 20 minutes of shaking at 6 cycles per second, microphotographs were taken
to measure the percentages of aggregated cells relative to the total
number of cells. The mean ± SEM of 4 separate experiments was
reported. Cell aggregation was dependent on the concentration of vWF
and ristocetin and required the GPIb subunit.
|
|
Heparin-induced aggregation of CHO-GPIb/IX cells.
To characterize this new role of heparin as an inducer of vWF binding
to GPIb, we analyzed the influence of heparin concentrations on
CHO-GPIb/IX cell aggregation. In the presence of vWF, cell aggregation increased as a function of heparin concentrations, reaching
a plateau of 57.0% ± 5.1% aggregated cells at 100 µg/mL heparin
(Fig 4). In the absence of vWF and in the
presence of increasing doses of heparin, cell aggregation did not
exceed 9.1% ± 3.0% at 250 µg/mL heparin. CHO-GPIb/IX cell
aggregation specifically involved the GPIb subunit, because the
association of vWF with heparin did not allow the aggregation of
CHO-GPIb/IX cells or CHO dhfr-cells (data not shown). To confirm the
involvement of GPIb, we performed enzymatic degradation of GPIb
by treatment of CHO-GPIb/IX cells
(Table 1). We found that trypsin or
elastase treatment of CHO-GPIb/IX cells completely abolished both
ristocetin- and heparin-induced cell aggregations (Table 1). The
involvement of the vWF-binding domain of GPIb was further
demonstrated by the potent inhibitory effect on cell aggregation of
mocarhagin, a cobra venom metalloproteinase that removes an
amino-terminal fragment of GPIb extending from aa 1 to 282 (Table
1). Enzymatic treatment of GPIb/IX cells by trypsin, elastase, or
mocarhagin was as effective in removing the vWF-binding domain of
GPIb, as shown by the absence of fluorescence staining with MoAb 6D1 or SZ2, whereas untreated CHO-GPIb/IX cells were positively labeled with these MoAbs (data not shown).
View larger version (15K):
[in this window]
[in a new window]
| Fig 4.
vWF-dependent heparin-induced CHO-GPIb/IX cell
aggregation. CHO-GPIb/IX cell aggregation was performed in the
presence () or in the absence () of 10 nmol/L vWF by adding
varying concentrations of heparin (0 to 250 µg/mL) at a rotary
shaking of 6 cycles per second. The mean ± SEM of 4 separate
experiments is shown. Heparin-induced dose-dependent CHO-GPIb/IX
cell aggregation in the presence of vWF.
|
|
The involvement of heparin in cell aggregation was also demonstrated
after treatment of heparin with heparinase. Degradation of heparin
significantly abolished vWF-dependent cell aggregation, resulting in
8.0% ± 5.7% aggregated cells, compared with 30.2% ± 1.3%
aggregated cells induced by 50 µg/mL heparin (P < .0025). Because vWF-dependent CHO-GPIb/IX cell aggregation has been reported in the absence of ristocetin at a higher rotary shaking frequency of 10 cycles per second,29 we investigated the
heparin effect in this condition. We found a 14.4% aggregation in the presence of vWF that was increased 3-fold in the presence of 50 µg/mL
heparin, reaching 47.2% aggregated cells. These results indicate the
ability of heparin to act as a positive modulator of vWF binding to
CHO-GPIb/IX cells.
The involvement of the A1 domain of vWF was further established by
showing the inhibitory effect of the VCL fragment on ristocetin- or
heparin-induced CHO-GPIb/IX cell aggregation
(Fig 5). We found that VCL fragment
completely blocked ristocetin-induced cell aggregation at 10 µmol/L
and that its IC50 was 0.3 µmol/L (Fig 5). As expected, in the
presence of ristocetin and in the absence of vWF, VCL was unable to
aggregate CHO-GPIb/IX cells due to its monomeric structure. Our
results indicate that VCL competes with vWF for binding to GPIb.
Interestingly, significant inhibition of heparin-induced aggregation
was observed when VCL was added as a soluble compound, leading to 50%
inhibition in the presence of 0.4 µmol/L (Fig 5).
View larger version (15K):
[in this window]
[in a new window]
| Fig 5.
Effect of VCL on vWF-dependent ristocetin- or
heparin-induced CHO-GPIb/IX cell aggregation. After preincubation
of CHO-GPIb/IX cells with increasing concentrations of VCL, cell
aggregation was performed at 6 cycles per second in the presence of 1.4 mg/mL ristocetin (, ) or in the presence of 50 µg/mL heparin
( ,  ). Solid symbols are samples incubated in the presence of 10 nmol/L vWF; open symbols are samples incubated without vWF. The mean ± SEM was obtained from 3 experiments. The VCL fragment competed with
vWF for binding to GPIb.
|
|
To determine the involvement of the vWF-A1 domain and the GPIb
subunit in both ristocetin- and heparin-induced cell aggregations, we
analyzed the effect of MoAbs that block the interaction of vWF with the
GPIb subunit (Table 2). Cell
aggregations were performed in the presence of 10 nmol/L vWF and
induced by either 1.4 mg/mL ristocetin or 100 µg/mL heparin. In the
presence of a control antibody, ristocetin- and heparin-induced cell
aggregations reached 50.7% ± 0.9% and 47.8% ± 1.5%
aggregated cells, respectively. Compared with control antibody, MoAbs
328 (anti-vWF) and 6D1 (anti-GPIb) completely inhibited
ristocetin-induced cell aggregation, which reached 5% aggregated
cells. Furthermore, MoAb 328 or 6D1 inhibited by 80% heparin-induced
cell aggregation, as shown by values of 8.5% ± 2.5% and 10.6% ± 2.7% aggregated cells, respectively. Interestingly, whereas MoAb
724 had no inhibitory effect on ristocetin-induced aggregation, it
resulted in a 63% reduction of heparin-induced aggregation that
reached 17.8% ± 1.6% aggregated cells (Table 2). Our results
indicate that both ristocetin- and heparin-induced aggregations involve
an interaction between the A1 domain of vWF and the GPIb subunit of
the GPIb/V/IX complex. These results suggest that heparin may act as a
positive modulator of the interaction of vWF with the GPIb subunit.
Interactions between heparin and ristocetin: Effect on cell
aggregation.
Because ristocetin and heparin are known to bind different domains of
vWF,4,10 we investigated whether these compounds could act
independently to enhance cell aggregation. To this end, we studied the
effect of heparin on ristocetin-induced cell aggregation and conversely
of ristocetin on heparin-induced cell aggregation (Fig 6). Our results showed that addition
of heparin resulted in a complete inhibition of ristocetin-induced cell
aggregation at 250 µg/mL, with a corresponding IC50 value at 50 µg/mL heparin (Fig 6A). Conversely, ristocetin dose-dependently
inhibited heparin-induced cell aggregation, with an IC50 of 0.84 mg/mL
(Fig 6B). In contrast, in separate assays of 125I-vWF
binding to heparin-agarose beads, we found that ristocetin neither
inhibited vWF binding to solid-phase heparin nor abrogated the
inhibitory effect of soluble heparin as a competitor of vWF binding to
solid-phase heparin (Table 3). These
results suggest that ristocetin does not prevent heparin binding to vWF
and that its inhibitory effect on heparin-induced cell aggregation is
not due to a direct neutralization between the positive charges of ristocetin and the negative charges of heparin. Taken together, our
results suggest that heparin and ristocetin each induce different active conformations of vWF. However, their combination may result in a
conformation that does not favor cell aggregation.
View larger version (10K):
[in this window]
[in a new window]
| Fig 6.
Effect of heparin and ristocetin on vWF-dependent
CHO-GPIb/IX cell aggregation. After incubation of
CHO-GPIb/IX cells with increasing concentrations of heparin or
ristocetin, cell aggregation was performed at 6 cycles per second in
the presence of 10 nmol/L vWF and was induced by 1.4 mg/mL ristocetin
(A) or 50 µg/mL heparin (B). The mean ± SEM was performed on 4 separate experiments. Heparin inhibited the ristocetin-induced
CHO-GPIb/IX cell aggregation and ristocetin inhibited the
heparin-induced CHO-GPIb/IX cell aggregation.
|
|
| |
DISCUSSION |
In the present study, we demonstrated a new role of heparin as a
promoter of vWF-GPIb interaction. Using ristocetin as a reference
activator of CHO-GPIb/IX cell aggregation in the presence of
mechanical forces, we demonstrated that heparin induced an interaction
of the A1 domain of vWF with the GPIb subunit.
Although binding of vWF to heparin is well established, its
physiological significance remains completely unknown.8,9 The proximity of the heparin- and GPIb-binding sites within the vWF
A1 domain prompted us to investigate the role of heparin in an
aggregation assay. To rule out an effect of vWF binding to the
activated IIb3 platelet receptor, which can be expressed when
platelets undergo even minimal activation during isolation procedures,
we performed our study using a stable CHO cell line expressing the
GPIb subunit.25 This CHO-GPIb/IX cell line was
previously reported to aggregate in response to mechanical forces and
ristocetin or at high shaking frequencies in the absence of
ristocetin.29 We have confirmed these findings in both
conditions, and, to improve the sensitivity for quantitating a positive
modulation of the GPIb-vWF interaction, we have selected an
intermediate shaking frequency of 6 cycles per second. We demonstrated
that aggregation was induced by ristocetin in a dose-dependent manner. This interaction required the GPIb subunit, as indicated by the abrogating effect of MoAb 6D1 or trypsin, thus confirming previous findings.29 We also used GPIb-specific proteinases such
as elastase and mocarhagin to show the involvement of the GPIb
subunit.27,28 Furthermore, we have extended these data by
demonstrating the involvement of the vWF A1 domain in
ristocetin-induced cell aggregation, because MoAb 328, an anti-vWF that
inhibits binding to GPIb in both static and high shear
conditions,14,21 was able to completely inhibit cell
aggregation. In addition, a recombinant vWF fragment containing the A1
domain (VCL fragment) blocked ristocetin-induced CHO-GPIb/IX cell
aggregation with an IC50 of 0.3 µmol/L. These results are in good
agreement with the reported 50% inhibition of ristocetin-induced
platelet aggregation by 0.24 µmol/L VCL.17
Ristocetin is a modulator of vWF A1 domain binding to platelet
GPIb/V/IX and acts by modifying the equilibrium between inhibitory sites and direct GPIb binding sites.4,5 The inhibitory
sites cooperate to maintain an inactive conformation that prevents
GPIb binding to vWF and include 2 acidic regions (497-511 and
687-698) and a basic region (540-578) corresponding to type 2B von
Willebrand disease (vWD) mutations that lead to an increased affinity
for GPIb. When bound to vWF, ristocetin may relieve the inhibitory sites and, thus, may be comparable to an endogenous activator that
stimulates binding of vWF to GPIb.
We have recently demonstrated that, upon binding to MoAb 724, soluble
vWF undergoes a conformational transition from an inactive state to an
active state that becomes sensitive to intermediate shear rates or to
low ristocetin concentrations, thus inducing GPIb-dependent platelet
aggregation.14 This MoAb has also been described as an
inhibitor of vWF binding to heparin.13 Therefore, we
investigated whether heparin may act as a positive modulator of vWF
interaction with GPIb/V/IX. Our results showed that, in the presence of
vWF, heparin can substitute for ristocetin and promote
CHO-GPIb/IX cell aggregation. The arguments for a potentiating effect of heparin on the interaction of vWF with GPIb were the following. Cell aggregation was induced by heparin in a dose-dependent manner. In addition, MoAb 6D1, as well as 3 enzymes involved in the
cleavage of the extracellular amino-terminal part of GPIb (trypsin,
mocarhagin, and elastase), completely blocked heparin-induced cell
aggregation, clearly establishing the involvement of vWF binding to
GPIb. This was confirmed by the absence of aggregation of control
cells lacking the GPIb subunit. Furthermore, the involvement of
heparin on vWF interaction with GPIb was shown by the abrogation of
CHO-GPIb/IX cell aggregation after enzymatic degradation of
heparin by heparinase. Altogether, these results demonstrated that
heparin reproduced the activating effect of ristocetin on vWF
interaction with GPIb subunit.
Interestingly, we found that anti-vWF MoAbs 328 and 724 directed to the
A1 domain were able to inhibit heparin-induced aggregation to a
significant extent (by 80% and 60%, respectively). However, this
inhibition of heparin-induced aggregation contrasted with the opposite
effect of both MoAbs on ristocetin-induced aggregation, which was
completely suppressed by MoAb 328, whereas it remained unaffected by
MoAb 724. It is likely that the absence of effect of MoAb 724 is
related to its inability to prevent the interaction with ristocetin,
whereas its effect on heparin-induced aggregation may be simply
explained by an inhibition of vWF binding to heparin.13 However, the inhibitory effect of MoAb 328 on heparin-induced aggregation was intriguing, because this MoAb is known as an inhibitor of vWF binding to GPIb/V/IX in the presence of ristocetin and of
shear-induced platelet adhesion or aggregation, but has no effect on
heparin binding to vWF.14,21,22 Our data thus suggested that, in the presence of mechanical forces, heparin could induce CHO-GPIb/IX cell aggregation by increasing the interaction
between vWF and GPIb. This putative activating effect of heparin is
further supported by recent data on the crystal structure of the vWF A1 domain, indicating that 2 heparin binding sites are found at the lower
surface of a GPIb-binding groove consisting of 2 adjacent -helices and a -sheet.30,31 Interestingly, the type
2B vWD mutations leading to increased GPIb binding are clustered on
this lower surface. This suggests that heparin-binding sites are
important for the stability of the GPIb-binding sites.
Our data are in favor of a complex model through which heparin and
ristocetin promote vWF-GPIb interaction by mutually exclusive mechanisms and by binding to distinct sites on vWF. Ristocetin and
heparin bind to different domains of the vWF A1 domain, because heparin
interacts with aa 565-587 and 642-645.12,32 To investigate whether their simultaneous addition would favor a shift toward a highly
active conformation, we studied the effect of heparin on
ristocetin-induced cell aggregation and conversely of ristocetin on
heparin-induced cell aggregation. Surprisingly, we found that heparin
inhibited ristocetin-induced cell aggregation with an IC50 value of 50 µg/mL heparin. This result was previously observed by Sobel et
al,8 who showed an IC50 of 24 µg/mL heparin on ristocetin-induced vWF binding to fixed platelets. Interestingly, we
found that ristocetin also inhibited heparin-induced cell aggregation. Because both compounds have opposite charges, it was of importance to
rule out a direct neutralization of their charges through electrostatic interactions. In the absence of GPIb, no effect of ristocetin was
observed on direct vWF binding to heparin-agarose beads. These results
suggested that, when added simultaneously, heparin and ristocetin were
unable to act in concert so as to induce cell aggregation. Whether
ristocetin could sterically hinder the accessibility of heparin and
impair its binding to the vWF-GPIb complex remains to be elucidated.
Most in vitro studies have considered heparin as an inhibitor of
platelet aggregation.8,9 Our results provide an explanation for the inhibitory effect by heparin on ristocetin-induced platelet aggregation. However, because in vivo no ristocetin is available, one
could question the contribution of heparin as an inhibitor of
vWF-GPIb interaction. Because we used heparin concentrations ranging
from 10 to 100 µg/mL (0.1 to 1 U/mL) within the plasma concentration
range obtained in vivo after therapeutic doses of heparin, we could
hypothesize that, in vivo, heparin might have an activating effect on
platelet aggregation. Heparin-induced thrombocytopenia (HIT) is one of
the most severe side effects of heparin therapy, often associated with
thrombocytopenia and thrombosis, and has been attributed to the
pathogenic effect of an IgG that activates platelets in the presence of
heparin via the complex platelet factor 4-heparin or to the role of
some chemokines-binding auto-antibodies.33 Beside HIT,
evidence for in vivo platelet activation in the presence of heparin has
been recently reported.34 It is far beyond the scope of
this in vitro study to determine whether the reported activating effect
of heparin on vWF-GPIb interaction may be involved in the
pathogenesis of HIT. In the cellular model that we have used, we have
avoided the presence of the IIb3 platelet receptor, which is
responsible for stable aggregation, either through binding to
fibrinogen in the absence of elevated shear stress or through an
interaction with vWF that can be demonstrated under high shear
conditions. Therefore, future studies will have to address whether
heparin may lead to platelet activation through a vWF-dependent mechanism.
| |
ACKNOWLEDGMENT |
The authors thank Dr B. Coller and Dr C. Ruan for providing antibodies,
Dr L. Garfinkel for providing the recombinant VCL fragment, Dr M.C.
Berndt for the gift of mocarhagin, and Dr M. Chignard for
elastase. Dr D. Pidard is thanked for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted October 26, 1998; accepted August 23, 1999.
Supported by EC Biomed2 BMH4-CT-98-3517 (D.B.), SANOFI and Fondation
pour la Recherche Médicale fellowship (C.P.), an INSERM grant
(N.A.), and a Ministere de la Recherche et de la Technologie grant
(G.R.L.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Dominique Baruch, MD, PhD, INSERM U143,
Hopital de Bicetre, 84 rue du Général Leclerc, 94276 Bicetre, Cedex, France; e-mail: baruch{at}infobiogen.fr.
| |
REFERENCES |
1.
Lopez JA:
The platelet glycoprotein Ib-IX complex.
Blood Coagul Fibrinol
5:97, 1994
[Medline]
[Order article via Infotrieve]
2.
Sugimoto M, Ricca G, Hrinda ME, Schreiber AB, Searfoss GH, Bottini E, Ruggeri ZM:
Functional modulation of the isolated glycoprotein Ib binding domain of von Willebrand factor expressed in Escherichia coli.
Biochemistry
30:5202, 1991
[Medline]
[Order article via Infotrieve]
3.
Berndt MC, Ward CM, Booth WJ, Castaldi PA, Mazurov AV, Andrews RK:
Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin.
Biochemistry
31:11144, 1992
[Medline]
[Order article via Infotrieve]
4.
Matsuhita T, Sadler JE:
Identification of amino acid residues essential for von Willebrand factor binding to platelet glycoprotein Ib.
J Biol Chem
270:13406, 1995
[Abstract/Free Full Text]
5.
Matsuhita T, Sadler JE:
Characterization of ligand binding to human von Willebrand factor domain A1 by single residue charged-to-alanine scanning mutagenesis.
Thromb Haemost
78:1489, 1997
(abstr, suppl)
6.
Choay J, Petitou M, Lormeau JC, Synaï P, Casu B, Gatti G:
Structure activity relationship in heparin: A synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity.
Biochem Biophys Res Commun
116:492, 1983
[Medline]
[Order article via Infotrieve]
7.
Badimon L, Badimon J, Rand J, Turitto V, Fuster V:
Platelet deposition on von Willebrand factor-deficient vessels: Extracorporeal perfusion studies in swine with von Willebrand disease using native and heparinized blood.
J Lab Clin Med
110:634, 1987
[Medline]
[Order article via Infotrieve]
8.
Sobel M, McNeill PM, Carlson P, Kermode JC, Adelman B, Conroy R, Marques D:
Heparin inhibition of von Willebrand factor-dependent platelet function in vitro and in vivo.
J Clin Invest
87:1787, 1991
9.
Sobel M, Bird K, Tyler-Cross R, Marques D, Toma N, Conrad E, Harris R:
Heparins designed to specifically inhibit platelet interactions with von Willebrand factor.
Circulation
93:992, 1996
[Abstract/Free Full Text]
10.
Fujimura Y, Titani K, Holland LZ, Roberts JR, Kostel P, Ruggeri Z, Zimmerman T:
A heparin-binding domain of human von Willebrand factor. Characterization and localization to a tryptic fragment extending from aminoacid residue Val-449 to Lys-728.
J Biol Chem
262:1734, 1987
[Abstract/Free Full Text]
11.
Sugimoto M, Mohri H, McClintock R, Ruggeri Z:
Identification of discontinuous von Willebrand factor sequences involved in complex formation with botrocetin.
J Biol Chem
266:18172, 1991
[Abstract/Free Full Text]
12.
Sobel M, Soler D, Kermode J, Harris R:
Localization and characterization of a heparin binding domain peptide of human von Willebrand factor.
J Biol Chem
267:8857, 1992
[Abstract/Free Full Text]
13.
Christophe O, Rouault C, Obert B, Pietu G, Meyer D, Girma JP:
A monoclonal antibody (B724) to von Willebrand factor recognizing an epitope within the A1 disulfide loop (Cys509-Cys 695) discriminates between type 2A and type 2B von Willebrand disease.
Br J Haematol
90:195, 1995
[Medline]
[Order article via Infotrieve]
14.
Depraetere H, Ajzenberg N, Girma JP, Lacombe C, Meyer D, Deckmyn H, Baruch D:
Platelet aggregation induced by a monoclonal antibody to the A1 domain of von Willebrand factor.
Blood
91:3792, 1998
[Abstract/Free Full Text]
15.
Nishino M, Girma JP, Rothschild C, Fressinaud E, Meyer D:
New variant of von Willebrand disease with defective binding to factor VIII.
Blood
74:1591, 1989
[Abstract/Free Full Text]
16.
Christophe O, Ribba AS, Baruch D, Obert B, Rouault C, Niinomi K, Pietu G, Meyer D, Girma JP:
Influence of mutations and size of multimers in type II von Willebrand disease upon the function of von Willebrand factor.
Blood
83:3553, 1994
[Abstract/Free Full Text]
17.
Gralnick H, Williams S, McKeown L, Kramer W, Krutzsch H, Gorecki M, Pinet A, Garfinkel L:
A monomeric von Willebrand factor fragment, Leu504-Ser728, inhibits von Willebrand factor interaction with glycoprotein Ib-IX.
Proc Natl Acad Sci USA
89:7880, 1992
[Abstract/Free Full Text]
18.
Fraker PJ, Speck JC:
Protein and cell membrane iodination with a sparingly soluble chloroamide, 1, 3, 4, 6-tetrachlor-3-6-diphenyl glycoluril.
Biochem Biophys Res Commun
80:849, 1987
19.
Girma JP, Kalafatis M, Pietu G, Lavergne JM, Chopek MW, Edgington TS, Meyer D:
Mapping of distinct von Willebrand factor domains interacting with platelet GPIb and GPIIb/IIIa and with collagen using monoclonal antibodies.
Blood
67:1356, 1987
[Abstract/Free Full Text]
20.
Baruch D, Ajzenberg N, Denis C, Legendre P, Lormeau JC, Meyer D:
Binding of heparin fractions to von Willebrand factorEffect of molecular weight and affinity for antithrombin-III.
Thromb Haemost
71:141, 1994
[Medline]
[Order article via Infotrieve]
21.
Girma JP, Takahashi Y, Yoshioka A, Diaz J, Meyer D:
Ristocetin and botrocetin involve two distinct domains of von Willebrand factor for binding to platelet membrane glycoprotein Ib.
Thromb Haemost
64:326, 1990
[Medline]
[Order article via Infotrieve]
22.
Baruch D, Denis C, Marteaux C, Schoevaert D, Coulombel L, Meyer D:
Role of von Willebrand factor associated to extra-cellular matrices in platelet adhesion.
Blood
77:519, 1991
[Abstract/Free Full Text]
23.
Ruan CG, Du XP, Xi XD, Castaldi PA, Berndt MC:
A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen.
Blood
69:570, 1987
[Abstract/Free Full Text]
24.
Coller BS, Peerschke EI, Scudder LE, Sullivan CA:
Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: Additional evidence in support of GPIb as a platelet receptor for von Willebrand factor.
Blood
61:99, 1983
[Abstract/Free Full Text]
25.
Lopez JA, Li CQ, Weisman S, Chambers M:
The GPIb-IX "complex specific" monoclonal antibody SZ1 binds to a conformation-sensitive epitope on GPIX: Implications for the target antigen of quinine/quinidine-dependent autoantibodies.
Blood
85:1254, 1995
[Abstract/Free Full Text]
26.
Perrault C, Lankhof H, Pidard D, Kerbiriou-Nabias D, Sixma JJ, Meyer D, Baruch D:
Relative importance of the glycoprotein Ib-binding domain and the RGD sequence of von Willebrand factor for its interaction with endothelial cells.
Blood
90:2335, 1997
[Abstract/Free Full Text]
27.
Ward CM, Andrews RK, Smith AI, Berndt MC:
Mocarhagin, a novel cobra venom metalloproteinase, cleaves the platelet von Willebrand factor receptor glycoprotein Ib. Identification of the sulfated tyrosine/anionic sequences Tyr-276-Glu-282 of glycoprotein Ib as a binding site for von Willebrand factor and -thrombin.
Biochemistry
35:4929, 1996
[Medline]
[Order article via Infotrieve]
28.
Pidard D, Renesto P, Berndt MC, Rabhi S, Clemetson KJ, Chignard M:
Neutrophil proteinase cathepsin G is proteolytically active on the human platelet glycoprotein Ib-IX receptor: Characterization of the cleavage sites within the glycoprotein Ib subunit.
Biochem J
303:489, 1994
29.
Dong JF, Hyun W, Lopez JA:
Aggregation of mammalian cells expressing the platelet glycoprotein (GP) Ib-IX complex and the requirement for tyrosine sulfation of GPIb.
Blood
86:4175, 1995
[Abstract/Free Full Text]
30.
Emsley J, Cruz M, Handin R, Liddington R:
Crystal structure of the von Willebrand factor A1 domain and implications for the binding of platelet glycoprotein Ib.
J Biol Chem
273:10396, 1998
[Abstract/Free Full Text]
31.
Celikel R, Varughese K, Madhusudan, Yoshioka A, Ware J, Ruggeri Z:
Crystal structure of the von Willebrand factor A1 domain in complex with the function blocking NMC-4 Fab.
Nat Struct Biol
5:189, 1998
[Medline]
[Order article via Infotrieve]
32.
Kroner P, Frey A:
Analysis of the structure and function of the von Willebrand A1 domain using targeted deletions and alanine-scanning mutagenesis.
Biochemistry
35:13460, 1996
[Medline]
[Order article via Infotrieve]
33.
Amiral J, Marfaing-Koka A, Poncz M, Meyer D:
The biological basis of immune heparin-induced thrombocytopenia.
Platelets
9:77, 1998
[Medline]
[Order article via Infotrieve]
34.
Xiao Z, Theroux P:
Platelet activation with unfractionated heparin at therapeutic concentrations and comparisons with a low-molecular-weight heparin and with a direct thrombin inhibitor.
Circulation
97:251, 1998
[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
L. Wang, P. Menendez, F. Shojaei, L. Li, F. Mazurier, J. E. Dick, C. Cerdan, K. Levac, and M. Bhatia
Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression
J. Exp. Med.,
May 16, 2005;
201(10):
1603 - 1614.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Uff, J. M. Clemetson, T. Harrison, K. J. Clemetson, and J. Emsley
Crystal Structure of the Platelet Glycoprotein Ibalpha N-terminal Domain Reveals an Unmasking Mechanism for Receptor Activation
J. Biol. Chem.,
September 13, 2002;
277(38):
35657 - 35663.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Griffin, H. M. Rinder, B. R. Smith, J. B. Tracey, N. S. Kriz, C. K. Li, and C. S. Rinder
The Effects of Heparin, Protamine, and Heparin/Protamine Reversal on Platelet Function Under Conditions of Arterial Shear Stress
Anesth. Analg.,
July 1, 2001;
93(1):
20 - 27.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION