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Prepublished online as a Blood First Edition Paper on May 8, 2003; DOI 10.1182/blood-2003-03-0717.
 Previous Article | Table of Contents | Next Article 
Blood, 1 September 2003, Vol. 102, No. 5, pp. 1701-1707
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion
Kazunobu Kato,
Taisuke Kanaji,
Susan Russell,
Thomas J. Kunicki,
Kenichi Furihata,
Sachiko Kanaji,
Patrizia Marchese,
Armin Reininger,
Zaverio M. Ruggeri, and
Jerry Ware
From the Roon Research Center for Arteriosclerosis and Thrombosis,
Division of Experimental Hemostasis and Thrombosis, Department of Molecular
and Experimental Medicine, The Scripps Research Institute, La Jolla, CA;
Department of Experimental Medicine Torrey Pines Institute for Molecular
Studies, San Diego, CA; and Department of Transfusion Medicine and
Hemostaseology, University of Munich, Munich, Germany.
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Abstract
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Platelet interaction with exposed adhesive ligands at sites of vascular
injury is required to initiate a normal hemostatic response and may become a
pathogenic factor in arterial diseases leading to thrombosis. We report a
targeted disruption in a key receptor for collagen-induced platelet
activation, glycoprotein (GP) VI. The breeding of mice with heterozygous GP VI
alleles produced the expected frequency of wild-type, heterozygous, and
homozygous genotypes, indicating that these animals had no reproductive
problems and normal viability. GP VInull platelets failed to
aggregate in response to type I fibrillar collagen or convulxin, a snake venom
protein and known platelet agonist of GP VI. Nevertheless, tail bleeding time
measurements revealed no severe bleeding tendency as a consequence of GP VI
deficiency. Ex vivo platelet thrombus formation on type I collagen fibrils was
abolished using blood from either GP VInull or
FcR- null animals. Reflection interference contrast
microscopy revealed that the lack of thrombus formation by GP
VInull platelets could be linked to a defective platelet activation
following normal initial tethering to the surface, visualized as lack of
spreading and less stable adhesion. These results illustrate the role of GP VI
in postadhesion events leading to the development of platelet thrombi on
collagen fibrils.
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Introduction
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Platelet membrane receptors interact with surface-bound adhesive ligands
and, as such, become essential for hemostasis and
thrombosis.1 There
are numerous unique receptors interacting with different adhesive ligands
suggesting that a large opportunity exists for functional redundancy in
platelet adhesion. However, an emerging theme of platelet biology is the
relevance of different membrane receptors in different areas of the
vasculature.2,3
A specific example is the exclusive role for the platelet glycoprotein (GP)
Ib-IX-V complex and von Willebrand factor in areas of the vascular system
where flow rates and high shear occur, such as in small arteries and
arterioles.4 Thus,
defining the physiologic relevance of an individual receptor and its ligand is
an important aspect for understanding participation of the platelet in
hemostasis and thrombosis.
Among adhesive ligands of the extravascular matrix, collagen is a
significant component with a number of known collagen receptors on the
platelet
surface.5,6
One of the more recently characterized collagen receptors is GP
VI.7 The molecular
cloning of GP VI revealed it to be a member of the immunoglobulin superfamily
of type I transmembrane
proteins.8-10
The surface expression of GP VI requires the concomitant expression of the
-subunit of the FcR receptor (FcR-) and their association is
functionally relevant as collagen binding to GP VI results in platelet
signaling via the immunoreceptor tyrosine–based activation motif (ITAM)
located in the FcR-
subunit.8,11-14
As with many of the platelet receptors, the in vivo relevance of GP VI was
established prior to its description and recognition as a protein membrane
receptor. In several clinical cases, patients lacking GP VI as a consequence
of autoantibody inhibition or possible gene deletion have been
described.15-19
Most commonly, these patients show a moderately prolonged bleeding time and
mild bleeding diatheses like subcutaneous bleeding. No severe bleeding
complication caused by the absent GP VI has been reported. Possible
explanations for the moderate bleeding phenotype include the presence of
multiple additional collagen receptors such as
 2 1 or GP
IV.14 It is also
not clear in the clinical cases of autoantibody inhibition of GP VI whether
such antibodies can produce a more global effect on platelet function. Indeed,
the possibility of an antiplatelet antibody stimulating platelet activation
pathways might mask the physiologic relevance of the receptor in these
autoimmune cases. The possible cases of genetic deletion of human GP VI have
yet to be defined at the molecular level, which makes the interpretation of
data from these individuals less than definitive as a case of GP VI
deficiency.
One experimental model to assess the physiologic relevance of individual
platelet receptors is the use of targeted gene deletions in mice. This
strategy generates an experimental model to directly assess the role of a
membrane receptor in hemostasis and thrombosis and the contribution of the
receptor to other biologic processes such as reproduction. Here, we report the
characterization of the murine GP VI gene and its targeted disruption for the
generation of a model of GP VI deficiency. Platelets from homozygous-deficient
mice completely lack the ability to respond in an aggregometer to stimulation
by type I fibrillar collagen. Yet, the in vivo tail bleeding times for GP
VInull animals are within the range obtained for wild-type animals.
In vitro models of thrombus formation demonstrated a direct role for GP VI in
the activation events that lead to platelet spreading. These results define
the in vivo relevance of an individual collagen receptor and its synergistic
contribution to the initiation and amplification of platelet thrombus
formation.
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Materials and methods
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Construction of mouse bone marrow cDNA library
Bone marrow was isolated from approximately 200 mouse femur bones and
immediately frozen in liquid nitrogen. The tissue was treated in 4 M
guanidinium isothiocyanate and total RNA purified by centrifugation through a
CsCl cushion as
described.20 Poly
A+–enriched RNA was prepared by standard affinity
purification using oligo-dT cellulose. The enriched poly A+ RNA was
used to construct a cDNA library in the  ZAP-CMV vector (Stratagene, La
Jolla, CA). The isolation of a mouse GP VI cDNA from the bone marrow cDNA
library was done using radiolabeled probes of a polymerase chain reaction
(PCR) fragment representing the full-length human GP VI
cDNA.8 Full-length
mouse GP VI cDNAs corresponded to the recently reported
sequence.9
Isolation of the mouse GP VI gene
A search of the high-throughput genomic sequence database using the mouse
GP VI cDNA sequence identified a mouse clone (GenBank Accession no.
AC087129
[GenBank]
.1) containing 8 GP VI exon sequences
(Figure 1) similar to the
exon/intron arrangement of the human GP VI gene. Oligonucleotides
from mouse GP VI exon 4 were supplied to Incyte Genomics (St Louis,
MO) for PCR screening to isolate a corresponding mouse bacterial artificial
chromosome (BAC) plasmid. Three positive clones were characterized by
restriction enzyme analysis and found to have a restriction fragment pattern
predicted from the GenBank sequence found in Accession no. AC087129
[GenBank]
.1. Limited
nucleotide sequence analysis was done on the BAC plasmid and no differences
were noted with the GenBank deposited sequence.
Generation of targeting vector for disruption of mouse GP VI
synthesis
As schematically illustrated in Figure
1, the mouse GP VI gene contains an initiating Met codon
within the putative exon 1 sequence. A 9-kb HindIII restriction
fragment spanning exons 1 to 3 was cloned into
pBS/KS-(Figure 1A). Within this
subcloned fragment a premature stop codon was created immediately 3' to
the initiation codon by double-stranded site-directed mutagenesis using
Platinum Pfx DNA polymerase (Stratagene). A unique restriction site was also
added 3' to the premature stop codon to allow insertion of a
phosphoglycerate-kinase neor cassette (kindly provided by Dr
Richard Hynes, Massachusetts Institute of Technology, Cambridge) within exon 1
(Figure 1). The final targeting
construct contained 4.5-kb arms, both 5' and 3', and the 1.7-kb
neor selectable marker immediately downstream to the initiating Met
codon (Figure 1C).
Generation of GP VInull mice
The targeting vector was linearized with NotI and electroporated
into DS2A embryonic stem (ES) cells at the Dartmouth Transgenic Facility
(Lebanon, NH). Transfected cells were selected for geneticin (G418) resistance
and approximately 180 clones were expanded for analysis by Southern blotting.
A HincII/HindIII restriction fragment outside the targeting
vector sequence was used to probe HincII digests of ES cell DNA.
Correct homologous recombination was further confirmed by Southern blot
analysis of an XbaI-digested ES cell DNA hybridized with a 1.1-kb
fragment containing exons 2 and 3. All probes were labeled by
[32P]dATP (deoxyadenosine triphosphate) using Prime-It II
Random Primer Labeling kit from Stratagene. Three clones with the predicted
homologous recombination event were confirmed with additional probes and PCR
analysis to validate the correct generation of a GP VI–targeted
allele (Figure 1D). The
positive ES cells were microinjected into C57BL/6J mouse blastocysts and were
implanted into pseudopregnant females. Chimeric mice were bred to Swiss Black
mice to test for germ line transmission. Two chimeric mice from different ES
cells produced germ line offspring as assessed by coat color transmission.
Heterozygous mice were crossed to produce the 3 genotypes: wild type (GP
VIWT), heterozygous (GP VIhet), and homozygous deficient
(GP VInull). Studies comparing the 3 genotypes, such as tail
bleeding times and aggregation assays, were performed using littermates from
GP VIhet x GP VIhet crosses prior to genotype
analysis. Mice deficient in the FcR- subunit were kindly provided by
Prof Toshiyuki Takai (Tohoku University, Sendai, Japan) and have been
previously
described.21
Chemicals, immunologic reagents, and flow cytometry
An anti–human GP VI monoclonal antibody was generated using
recombinant GP VI immunogen produced by expression using a baculovirus system
in Hi 5 cells. Primary hybridomas were screened by enzyme-linked immunosorbent
assay (ELISA) for reactivity of supernatants with recombinant human GP VI.
Upon characterization the monoclonal antibody was found to cross-react, albeit
less strongly, with mouse GP VI antigen. Convulxin was purified from
Crotalus durissus terrificus venom (Miami Serpentarium Laboratories,
Miami, FL) as
described.22 A
rabbit anti–FcR- chain polyclonal antibody was purchased from
Upstate Biotechnology (Lake Placid, NY). Horseradish peroxidase
(HRP)–conjugated rabbit antimouse IgG and HRP-conjugated goat antirabbit
IgG were obtained from Zymed Laboratories (South San Francisco, CA). A rabbit
anti–14-3-3 polyclonal antibody was purchased from Santa Cruz
Technologies (Santa Cruz, CA).
Mouse platelet preparation
Murine blood was withdrawn from the retro-orbital plexus using
heparin-coated micro-hematocrit capillaries (Fisher Scientific, Pittsburgh,
PA) and transferred to tubes with heparin at final concentration of 30
units/mL (Sigma, St Louis, MO). Platelet-rich plasma (PRP) was obtained by
centrifugation of whole blood at 500g for 6 minutes. To obtain
platelet lysates, platelets were washed one time in a modified Tyrode buffer
(5 mM HEPES
[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]
[pH 6.5]; 137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 2.8
mM Dextrose) with 5 U/mL apyrase and 10 µM prostaglandin E1 and
then centrifuged at 1500g for 5 minutes. The platelet pellet was
resuspended in 100 µL Tyrode buffer (pH 7.4) and lysed with an equal volume
of 50 mM Tris (tris(hydroxymethyl)aminomethane; pH 7.5) and 10% sodium dodecyl
sulfate (SDS) and boiled for 5 minutes. Platelet poor plasma (PPP) was
obtained by centrifuging at 2000g for 7 minutes.
Platelet aggregation
Following the preparation of PRP (see "Mouse platelet
preparation") the platelet counts were normalized to 240 x
109/L with PPP. Four hundred microliters of PRP was prewarmed at
37°C. Convulxin, bovine fibrillar collagen (type I), and 50 µM phorbol
12-myristate 13-acetate (PMA; Sigma) were added to PRP as agonists.
Aggregation profiles were generated in a Chrono-Log aggregometer (Havertown,
PA). PPP was used as a blank for aggregation study.
Immunoblotting
Proteins were separated by 4% to 20% SDS–polyacrylamide gel
electrophoresis (PAGE) and transferred to nitrocellulose membranes
(Invitrogen, Carlsbad, CA). Membranes were blocked with TBS-T (20 mM Tris [pH
7.5]; 150 mM NaCl, 0.05% Tween 20) containing 5% skim milk (30 minutes, room
temperature [RT]). Antibodies were incubated with the membranes for 1 hour at
RT. Afterward, the membranes were washed 3 times with TBS-T and the membranes
were incubated with either HRP rabbit antimouse IgG or HRP goat antirabbit IgG
(30 minutes, RT). Membranes were developed using an enhanced chemiluminescence
detection system (Amersham-Pharmacia Biotech UK Limited, Little Chalfont,
United Kingdom).
Bleeding time assays
Mouse tail bleeding times were determined as
described.23
Briefly, 1 to 3 mm of distal tail was removed and immediately immersed in
isotonic saline (37°C). A complete cession of bleeding was defined as the
bleeding time.
Analysis of platelet adhesion and thrombus formation by real-time
videomicroscopy
The blood of mice anesthetized by inhalation of methoxyflurane (Medical
Developments, Springvale, Australia) was drawn from the retro-orbital venous
plexus through a heparinized glass capillary tube and collected into plastic
tubes containing a solution of unfractionated heparin (sodium salt) from
porcine intestinal mucosa (grade III; Sigma) to give a final concentration of
40 U/mL. Apyrase (grade III; Sigma) was then added at a final concentration of
1.5 ATPase U/mL in order to prevent desensitization of platelet purinergic
receptors. Epifluorescence and confocal videomicroscopy were performed as
described in detail
elsewhere.3,24
In brief, purified fibrillar type I collagen from bovine tendon (acid
insoluble; Sigma) was coated onto a glass coverslip that was subsequently
assembled into a parallel plate rectangular flow chamber. Mouse platelets were
labeled in whole blood by addition of the fluorescent dye mepacrine
(quinacrine dihydrochloride; final concentration 10 µM) before perfusion
through the chamber by aspiration with a syringe pump (Harvard Apparatus,
Holliston, MA) at the desired flow rate. The perfusion chamber was mounted on
the stage of an inverted microscope (Axiovert 135M; Carl Zeiss, Thornwood, NY)
for real-time visualization of platelet adhesion and aggregation in flowing
blood, and the process was recorded continuously on videotape with a VCR
(SVO-9500MD; Sony, Inchinomiya, Japan) at the acquisition rate of 30
frames/second. Thrombus volume was measured in real time by confocal
videomicroscopy (LSM 410; Carl
Zeiss).3 We also
performed experiments to visualize in real time the interface between
platelets and immobilized collagen using reflection interference contrast
microscopy (RICM). In this technique, which does not require labeling of the
blood cells, interference colors indicate the distance between 2
surfaces.25,26
In our studies, the interference was caused by the glass surface onto which
the substrate was coated and by the membrane of cells flowing in its proximity
or interacting with it. Since we used a black-and-white video camera,
information on the separation between the 2 surfaces was obtained on a gray
scale, in which zero-order black corresponds to a distance of 4 to 12 nm and
white corresponds to a distance greater than 20 to 30
nm.25,26
Structures that are separated by a distance greater than 30 nm appear as out
of focus. All these experiments were recorded on S-VHS videotape. Image
analysis was performed off-line using the Metamorph software package
(Universal Imaging, West Chester, PA). The video clips available for viewing
on the Blood website (see the Supplemental Videos link at the top of
the online article) were prepared by digitizing and editing the recorded
analog tapes with Adobe Premiere (Adobe Systems, San Jose, CA).
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Results
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The mouse GP VI gene is composed of 8 exons schematically
represented in Figure 1A. The
intron/exon arrangement was deduced from a comparison of the mouse GP VI cDNA
sequence and a genomic clone present in GenBank, accession no. AC087129
[GenBank]
.1. A
targeting vector to promote homologous recombination in murine ES cells was
constructed in which a stop codon and a phosphoglycerate kinase
(PGK)–neomycin resistance (neor) cassette were
inserted immediately 3' to the putative Met codon present in exon 1
(Figure 1C). Accordingly,
successful homologous recombination after transfection of the targeting vector
would generate ES cellular DNA in which a 7-kb HincII DNA fragment is
replaced with an 8.7-kb HincII fragment
(Figures 1B,D).
A total of 184 G418-resistant ES colonies were screened for homologous
recombination and the recombination event depicted in
Figure 1D was confirmed in 2
different colonies using additional restriction enzymes, Southern blotting,
and PCR analysis. Both clones with an altered GP VI gene were chosen
for microinjection into mouse blastocysts and chimeric males derived from 2
different cell lines produced germ line offspring. Mice containing
heterozygous GP VI loci (GP VIhet) were bred (GP
VIhet x GP VIhet) and DNA analysis of the
resultant offspring revealed all 3 expected GP VI genotypes: wild-type
(GPVIWT), heterozygous (GP VIhet), and
homozygous-deficient (GP VInull) animals
(Figure 1E). To date, we have
observed no unusual ratios in the generation of expected genotypes. Thus, it
appears the absence of GP VI has no impact on the fertility of mice, nor do we
have any evidence the absence of GP VI impairs development or viability of the
animal. As a platelet-expressed receptor, hematologic parameters were also
determined and no striking differences were observed as a consequence of GP VI
deficiency (data not shown).
A murine monoclonal antibody was prepared against a recombinant fragment of
human GP VI and found to have cross-reactivity, albeit more weakly, with a
60-kDa protein found in mouse platelet lysates. A characterization of platelet
lysates from each of the GP VI genotypes revealed a complete absence of GP VI
polypeptide sequence in GP VInull platelets
(Figure 1F). Similarly, a gene
dosage effect was observed for the presence of GP VI alleles, as
evidenced by the GP VIhet genotype containing less GP VI antigen
compared with the GP VIWT
(Figure 1F). An internal
platelet antigen, 14-3-3, was expressed at similar levels in each
platelet sample and used to verify platelet proteins in each lane
(Figure 1G).
The platelet GP VI polypeptide requires the presence of FcR- for
efficient surface expression. Indeed, others have shown the inability of GP VI
to be membrane expressed without a concomitant expression of
FcR-.8,11,12
Figure 2 demonstrates that the
expressed levels of FcR- do not change in the absence of GP VI. Whether
the expressed FcR- reaches the membrane surface or is associated with
another receptor in the absence of GP VI was not determined because of the
lack of an immunologic reagent that specifically recognizes the short
extracytoplasmic domain of the FcR- subunit.
PRP was prepared from littermates produced from GP VIhet x
GP VIhet crosses. The ability of type I fibrillar collagen to
support aggregation was characterized using PRP from each of the genotypes. As
shown in Figure 3, there was no
aggregation using platelets from GP VInull animals. Platelets from
the GP VIhet animals failed to aggregate using 4 µg/mL collagen,
unlike their WT counterpart, yet a downward shift in the aggregation profile
suggested a platelet shape change. Higher concentrations of collagen, such as
8 µg/mL, did produce aggregation using GP VIhet platelets. At
the highest collagen concentration tested (100 µg/mL) no platelet
aggregation was observed using GP VInull platelets (data not
shown). Another property of GP VI is its ability to aggregate platelets in the
presence of the snake venom protein, convulxin. Convulxin was unable to
support platelet aggregation using GP VInull PRP
(Figure 3). Using a nonspecific
platelet agonist, PMA, platelet aggregation was indistinguishable among GP
VIWT, GP VIhet, and GP VInull samples
(Figure 3).
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Figure 3.. Aberrant collagen and convulxin-induced aggregation in GP
VInull mice. Blood was withdrawn from GP VIWT, GP
VIhet, and GP VInull mice. Mouse platelet-rich plasma
was obtained by pooling blood from animals with the same genotype. The
platelet number in each sample was normalized to 240 x 109/L
with platelet-poor plasma. Indicated concentrations of acid-insoluble
fibrillar collagen (type I), convulxin, and PMA were added to stirred
platelets and the aggregation profiles are presented.
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Although the platelet aggregometer identified an abnormality in GP
VI–deficient mice, the extent to which that abnormality would translate
to an effect on hemostasis and thrombosis is less clear. Shown in
Figure 4 are tail bleeding time
assays done on littermates from GP VIhet x GP
VIhet crosses prior to their genotyping. The absence of GP VI does
not have a major impact on the tail bleeding time.
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Figure 4.. Tail bleeding time assays. Littermates from GP VIhet
x GP VIhet crosses were subjected to tail bleeding assays. A
2- to 3-mm portion of distal tail was removed from 5-week-old animals and the
cessation of bleeding time was recorded. Following a determination of the
bleeding time, genotype analysis was performed and the data are presented
correlating the bleeding time with the genotype. Data obtained from individual
animals are shown.
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To evaluate directly the role of platelet GP VI in thrombus formation,
perfusion experiments were performed using whole blood obtained from GP
VIWT, GP VIhet, GP VInull, and
FcR-null animals. Platelets were labeled with fluorescent
mepacrine in whole blood containing heparin and apyrase. Type I collagen
(acid-insoluble) was coated on a glass surface and the blood was perfused over
the surface. Representative images are shown in
Figure 5 after 2.5 minutes of
blood flow (Video 1, available online, provides a more detailed representation
of these results). Blood from GP VIWT animals displayed large
thrombi throughout the perfusion chamber reflecting the generation of platelet
aggregates at all flow rates examined. Quantitation of the thrombus formed at
a shear rate of 1500 s–1 revealed a volume of 3.4
x 105 µm3. Platelet adhesion for both GP
VInull and FcR-null animals was significantly
reduced to values corresponding to a background monolayer of platelets visible
as individual fluorescent particles (Figure
5).
After observing a lack of thrombus formation in GP VInull
platelets, we investigated the dynamics of platelet adhesion to surface-bound
collagen using reflection interference contrast microscopy. This analysis
allowed us to visualize the surface membrane interactions occurring during
platelet adhesion. As shown in Figure
6, single platelets are visibly adhering to fibrillar collagen as
early as 5 seconds after the initiation of flow (Video 2, available online).
By 45 seconds, platelets from control animals have spread and several of them
in close proximity form the surface-contacting base of the thrombi illustrated
in Figure 5. In contrast,
platelets from the GP VInull animals interacting with the collagen
surface for the same duration remain round and isolated and never show the
spreading that is indicative of activation. Thus, the defect in a GP
VInull platelet is an inability to activate following the initial
adhesion event. The inability to activate produces the platelet monolayer seen
in Figure 5 and precludes
platelet accumulation and formation of a thrombus.
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Figure 6.. Time course analysis of the contact interface between platelets and
collagen fibrils. Blood was obtained as described in the legend to
Figure 5, with the exception
that no mepacrine was added. The technique of RICM, described in
"Materials and methods," allows visualization of the larger
collagen fibrils immobilized on the glass bottom of the flow chamber before
the beginning of blood perfusion (time 0 s). After 5 seconds of perfusion with
either normal or GP VInull blood, comparable numbers of single
platelets (arrows) are seen interacting with the surface. Their shape is round
indicating that spreading following activation has not yet taken place. After
45 seconds, in the case of normal blood perfusion the adherent platelets have
become spread and occupy a larger portion of the surface; in contrast, in the
case of GP VInull blood perfusion, platelets have the same
morphology as after 5 seconds, indicating that they have not become activated
and, thus, have not spread. The insets to the right present a larger
magnification of the surface after 45 seconds of perfusion. Note that the
individual boundaries of spread platelets tend to be lost. In this technique,
the darker color of spread platelets compared with those that have not spread
indicates a closer proximity to the collagen fibrils. Video 2 presents a more
detailed view of these events and demonstrates that the spread platelets
represent the base of large thrombi attached to the collagen fibrils and
protruding into the flow path.
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Discussion
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Platelet GP VI is a relatively recent addition to the repertoire of
receptors supporting adhesion and aggregation at a wound site. The recent
focus to GP VI was greatly aided by the molecular cloning of the human GP VI
cDNA and
gene.8-10
However, clinical cases with a presumed defect in GP VI or autoantibodies
directed against GP VI were described several years
earlier.15-19
To date, the molecular defects associated with these rare forms of GP
VI–deficient platelets have not been defined. Thus, interpreting the
mild bleeding phenotype typical of most of these patients or the potential
relevance of GP VI in thrombus formation has been difficult since the
molecular basis of the dysfunctional human GP VI remains unknown.
Our study was undertaken to make a predefined genetic lesion in the mouse
GP VI gene and abolish synthesis of the GP VI polypeptide. The chosen
strategy would place a stop codon immediately 3' to the initiating Met
codon. Upon generation of homozygous-deficient mice the absence of GP VI
polypeptide was confirmed by negative results by Western blot analysis of GP
VInull platelets (Figure
1F) and a complete absence of platelet aggregation induced by the
snake venom protein, convulxin (Figure
3). Our preliminary expansion of the GP VInull colony
has not identified any obvious phenotypic consequences of GP VI deficiency
other than its role as a platelet adhesion and activation receptor. The
absence of type I fibrillar collagen–induced platelet aggregation was
taken as further evidence that GP VI synthesis was abolished.
Previous studies have documented the requirement of the adapter protein,
FcR-, for efficient surface expression of GP
VI.11,12
Thus, the normal levels of FcR- that we observe in the absence of mouse
GP VI (Figure 2) might be
considered surprising. Indeed, studies of human GP VI–deficient
platelets have demonstrated reduced or undetectable levels of
FcR-.11 A
number of possibilities could explain the presence of FcR- in GP
VI–deficient platelet lysates. First, it should be recognized that the
GP VI–deficient platelets cited in the human study were from a patient
with a novel antiplatelet antibody, presumably directed against GP
VI.15 Thus, the
previous study on human GP VI–deficient platelets and our mouse model
represent 2 very different situations. In the GP VInull mouse
model, the FcR- never associates with a GP VI polypeptide, whereas in
the case of an immune-induced model of GP VI deficiency, an assembled GP
VI–FcR- complex is most likely removed in its entirety from the
platelet surface. The FcR- polypeptide can be expressed independently
as confirmed by different groups using heterologous cell transfection but even
this result might be misleading for conclusions on stability since the
FcR- is
overexpressed.27,28
It is interesting to see the expanding number of polypeptide chains in
addition to GP VI for which FcR- can associate, such as members of the
leukocyte inhibitory receptor (ILT) family, their murine homologues (PIRA),
and the high-affinity FcRI receptor for
IgE.29 These
results illustrate the utility of FcR- as an adapter protein for
intracellular signaling whose role goes beyond GP VI and even raises the
possibility that in the genetic absence of mouse GP VI, the FcR-
polypeptide might associate with another platelet protein.
The genetic removal of GP VI from the platelet surface directly confirms
the importance of GP VI in collagen-induced platelet aggregation
(Figure 3). Our data
demonstrate using fibrillar type I collagen that other collagen receptors do
not participate in this process or this type of aggregation requires an
initial interaction with GP VI that must precede the engagement of other
collagen receptors. Indeed, the ablation of the integrin receptor,
21, in mouse platelets did not prevent
aggregation in response to fibrillar collagen but did result in a lag time for
the aggregation
response.30,31
Perhaps the delay in aggregation seen with
21-deficient platelets reflects a more
global change in the platelet membrane that occurs and prevents an immediate
engagement of the GP VI–FcR- complex. This statement is supported
by our result demonstrating that the aggregation observed with type I
fibrillar collagen is completely dependent upon a functional GP VI receptor.
The issue is different with soluble collagen, as blockage of
21 function with a monoclonal antibody
prevents platelet thrombus formation in flowing blood exposed to immobilized
pepsin-digested type I collagen but not to the same collagen renatured to form
fibrils32; and
21-deficient platelets do not aggregate in
the presence of enzymatically digested
collagen.30
However, the physiologic relevance of soluble collagen to hemostasis and
thrombosis has yet to be defined.
Antibody inhibition of mouse GP VI has previously been shown to moderately
increase the tail bleeding
time,33 unlike our
data from the GP VInull platelet. However, this antibody was not
completely effective in inhibiting aggregation using higher concentrations of
fibrillar
collagen,34 leading
the investigators to conclude the presence of a second collagen receptor or
second collagen binding site on mouse GP VI important for aggregation. Our
data do not support this conclusion as we observed a complete absence of
aggregation at concentrations exceeding those used by investigators using
inhibitory
antibodies.34
Moreover, in more recent
work,35 the
attainment of an antibody-induced platelet GP VI deficiency produced a lack of
platelet adhesion and aggregation on a damaged vessel wall. Our results do not
support a direct participation of GP VI in the initial tethering of platelets
to a surface presenting collagen type I fibrils (Figures
5,
6; Videos 1-2), in agreement
with the concept that essential for this process under high flow conditions is
the interaction of GP Ib with collagen-bound von Willebrand
factor.3 The absence
of GP VI severely impaired the transition from a transient interaction to
irreversible adhesion. The latter is required for subsequent aggregation and
the absence of GP VI resulted in a complete blockade of platelet spreading
indicative of defective activation. Our results, therefore, are consistent
with the characterization of GP Ib-IX-V as a critical platelet adhesion
receptor and underlines how distinct adhesion and activation pathways provide
their unique synergistic contribution to achieve an efficient progression from
initial platelet tethering to stable adhesion and aggregation.
Perhaps a more intriguing problem posed by the present study is how to
reconcile the required function of GP VI in platelet aggregation
(Figure 3) and ex vivo thrombus
growth (Figure 5) with the
nonessential role for GP VI in supporting the hemostasis associated with a
tail bleeding assay (Figure 4).
Indeed, conclusions from each experimental assay must be carefully considered
when trying to understand the physiologic impact of GPVI on normal hemostasis
and thrombosis. The aggregometer can be appreciated as an experimental tool
that has provided much of the fundamental knowledge on platelet function.
However, the aggregometer's ability to mimic the complex processes occurring
at a wound site or the site of pathologic thrombosis is limited. In this
regard the aggregometer might be better viewed as a unique binding assay
between platelets and their ligand with the outcome or readout being the
ability of the platelets to agglutinate and aggregate. In contrast, the
mechanisms controlling tail bleeding time assays are more likely global with
contributions from blood coagulation, as hemophilic mice have a prolonged
bleeding
time,36,37
and platelet components, as absent platelet receptors GP Ib-IX or
IIb3 also produce prolonged bleeding
times23,38
coupled to a potentially heterogeneous subendothelium. Thus, the results and
conclusions from experiments performed in the aggregometer compared with the
conclusions derived from the tail bleeding assay may represent 2 extreme
experimental conditions examining platelet function.
Our results would suggest that GP VI is an essential receptor for thrombus
growth yet its absence does not prolong the bleeding time. As an adhesive
ligand of extravascular matrix, collagen has long been thought of as an
essential component of thrombus
formation.39
Indeed, our mouse in vivo bleeding times seem to be consistent with the
apparent minor bleeding tendencies associated with the few human patients that
have been characterized as GP VI deficient. Again, although the genetic
defects in this minor group of individuals remain to be defined, similar
results and conclusions were recently made by Goto et
al40 using human
blood from one of these individuals. However, we are faced with the intriguing
possibility that GP VI may be more relevant for pathologic thrombus formation
and less essential for normal hemostasis, an exciting possibility when
considering potential targets for antithrombotic therapies.
|
Acknowledgements
|
|---|
The authors acknowledge the Sam and Rose Stein Charitable Trust for the
establishment of the DNA Core Facility within the Department of Molecular and
Experimental Medicine at The Scripps Research Institute. The laboratory of Dr
Steven Fiering (Dartmouth Medical School, Lebanon, NH) is acknowledged for the
transfection and injection of mouse ES cells. The authors also acknowledge
help by Rolf Habermann in the editing and generation of videos.
|
Footnotes
|
|---|
Submitted March 6, 2003;
accepted April 27, 2003.
Prepublished online as Blood First Edition Paper, May 8, 2003; DOI
10.1182/blood-2003-03-0717.
Supported by grants from the Heart, Lung, and Blood Institute of the
National Institutes of Health, HL50545 (J.W.); HL46979 (T.J.K.); HL42846,
HL48728, HL31950 (Z.M.R.).
The online version of the article contains a data supplement.
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.
Reprints: Jerry Ware, MEM175, 10550 North Torrey Pines Rd, La Jolla, CA
92037; e-mail:
jware{at}scripps.edu.
|
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T. J. Kunicki, A. B. Federici, D. R. Salomon, J. A. Koziol, S. R. Head, T. S. Mondala, J. D. Chismar, L. Baronciani, M. T. Canciani, and I. R. Peake
An association of candidate gene haplotypes and bleeding severity in von Willebrand disease (VWD) type 1 pedigrees
Blood,
October 15, 2004;
104(8):
2359 - 2367.
[Abstract]
[Full Text]
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B. Boylan, H. Chen, V. Rathore, C. Paddock, M. Salacz, K. D. Friedman, B. R. Curtis, M. Stapleton, D. K. Newman, M. L. Kahn, et al.
Anti-GPVI-associated ITP: an acquired platelet disorder caused by autoantibody-mediated clearance of the GPVI/FcR{gamma}-chain complex from the human platelet surface
Blood,
September 1, 2004;
104(5):
1350 - 1355.
[Abstract]
[Full Text]
[PDF]
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J. M. Gibbins
Platelet adhesion signalling and the regulation of thrombus formation
J. Cell Sci.,
July 15, 2004;
117(16):
3415 - 3425.
[Abstract]
[Full Text]
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M. J. E. Kuijpers, V. Schulte, C. Oury, T. Lindhout, J. Broers, M. F. Hoylaerts, B. Nieswandt, and J. W. M. Heemskerk
Facilitating roles of murine platelet glycoprotein Ib and {alpha}IIb{beta}3 in phosphatidylserine exposure during vWF-collagen-induced thrombus formation
J. Physiol.,
July 15, 2004;
558(2):
403 - 415.
[Abstract]
[Full Text]
[PDF]
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P. Nurden, M. Jandrot-Perrus, R. Combrie, J. Winckler, V. Arocas, C. Lecut, J.-M. Pasquet, T. J. Kunicki, and A. T. Nurden
Severe deficiency of glycoprotein VI in a patient with gray platelet syndrome
Blood,
July 1, 2004;
104(1):
107 - 114.
[Abstract]
[Full Text]
[PDF]
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T. J. Kunicki
Of men; why not mice?
Blood,
May 1, 2004;
103(9):
3251 - 3252.
[Full Text]
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T.-T. Li, S. Larrucea, S. Souza, S. M. Leal, J. A. Lopez, E. M. Rubin, B. Nieswandt, and P. F. Bray
Genetic variation responsible for mouse strain differences in integrin {alpha}2 expression is associated with altered platelet responses to collagen
Blood,
May 1, 2004;
103(9):
3396 - 3402.
[Abstract]
[Full Text]
[PDF]
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A. Kasirer-Friede, M. R. Cozzi, M. Mazzucato, L. De Marco, Z. M. Ruggeri, and S. J. Shattil
Signaling through GP Ib-IX-V activates {alpha}IIb{beta}3 independently of other receptors
Blood,
May 1, 2004;
103(9):
3403 - 3411.
[Abstract]
[Full Text]
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P. R.-M. Siljander, I. C. A. Munnix, P. A. Smethurst, H. Deckmyn, T. Lindhout, W. H. Ouwehand, R. W. Farndale, and J. W. M. Heemskerk
Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood
Blood,
February 15, 2004;
103(4):
1333 - 1341.
[Abstract]
[Full Text]
[PDF]
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P. A. Smethurst, L. Joutsi-Korhonen, M. N. O'Connor, E. Wilson, N. S. Jennings, S. F. Garner, Y. Zhang, C. G. Knight, T. R. Dafforn, A. Buckle, et al.
Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody
Blood,
February 1, 2004;
103(3):
903 - 911.
[Abstract]
[Full Text]
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S. Gruner, M. Prostredna, V. Schulte, T. Krieg, B. Eckes, C. Brakebusch, and B. Nieswandt
Multiple integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo
Blood,
December 1, 2003;
102(12):
4021 - 4027.
[Abstract]
[Full Text]
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S. Kanaji, T. Kanaji, K. Furihata, K. Kato, J. L. Ware, and T. J. Kunicki
Convulxin Binds to Native, Human Glycoprotein Ib{alpha}
J. Biol. Chem.,
October 10, 2003;
278(41):
39452 - 39460.
[Abstract]
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Top
Abstract
Introduction
) and introns were identified. By sequence alignment
with the human GP VI cDNA, a putative initiating Met codon (ATG) is shown in
exon 1. (B) An expanded region of the wild-type mouse GP VI gene containing
exons 1 to 3 is shown. (C) A 9-kb HindIII restriction fragment
spanning exons 1 to 3 was subcloned into a pBS/KS-vector and altered via
site-directed mutagenesis to place a stop codon (TGA) immediately 3' to
the initiating Met codon followed by an XhoI restriction site.
Following successful mutagenesis a neocassette was cloned within the exon 1
sequence using the restriction site created during mutagenesis. (D) Successful
homologous recombination in mouse ES cells results in the replacement of a
7-kb HincII restriction fragment with an 8.7-kb HincII
fragment. (E) Germ line transmission of the ES cell genotype was obtained
producing GP VIhet mice. The breeding of GP VIhet mice
produced the expected 3 genotypes and shown is a representative Southern blot
of each of the genotypes revealing the wild-type GP VI gene (7 kb) and the
targeted allele (8.7 kb). (F) A mouse monoclonal antibody recognizing both
human and mouse GP VI proteins was used to verify the lack of the GP VI
polypeptide in platelet lysates from GP VInull mice. Shown is a
Western blot of platelet lysates from each of the 3 genotypes. A gene dosage
effect of GP VI levels is seen as a consequence of the altered GP VI allele.
(G) The same filter was reprobed with an anti–14-3-3
