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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4112-4121
Glycoprotein V-Deficient Platelets Have Undiminished Thrombin
Responsiveness and Do Not Exhibit a Bernard-Soulier Phenotype
By
Mark L. Kahn,
Thomas G. Diacovo,
Dorothy F. Bainton,
Francois Lanza,
JoAnn Trejo, and
Shaun R. Coughlin
From the Department of Medicine, University of Pennsylvania,
Philadelphia, PA; the Department of Pediatrics and Pathology,
Washington University, St Louis, MO; the Department of Pathology,
Cardiovascular Research Institute, and Departments of Medicine and
Cellular and Molecular Pharmacology, University of California, San
Francisco, CA; and INSERM U.311, Strasbourg, France.
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ABSTRACT |
Adhesion of platelets to extracellular matrix via von Willebrand
factor (vWF) and activation of platelets by thrombin are critical steps
in hemostasis. Glycoprotein (GP) V is a component of the GPIb-V-IX
complex, the platelet receptor for vWF. GPV is also cleaved by
thrombin. Deficiency of GPIb or GPIX results in Bernard-Soulier
syndrome (BSS), a bleeding disorder in which platelets are giant and
have multiple functional defects. Whether GPV-deficiency might also
cause BSS is unknown as are the roles of GPV in platelet-vWF interaction and thrombin signaling. We report that GPV-deficient mice
developed normally, had no evidence of spontaneous bleeding, and had
tail bleeding times that were not prolonged compared with wild-type
mice. GPV-deficient platelets were normal in size and structure as
assessed by flow cytometry and electron microscopy. GPV-deficient and
wild-type platelets were indistinguishable in botrocetin-mediated
platelet agglutination and in their ability to adhere to mouse vWF A1
domain. Platelet aggregation and ATP secretion in response to low and
high concentrations of thrombin were not decreased in GPV-deficient
platelets compared with wild-type. Our results show that (1) GPV is not
necessary for GPIb expression and function in platelets and that GPV
deficiency is not likely to be a cause of human BSS and (2) GPV is not
necessary for robust thrombin signaling. Whether redundancy accounts
for the lack of phenotype of GPV-deficiency or whether GPV serves
subtle or as yet unprobed functions in platelets or other cells remains
to be determined.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE ABILITY OF PLATELETS to adhere to
sites of vessel injury under conditions of flow is critical for
hemostasis.1 The molecular basis for initial platelet
adhesion after vessel disruption appears to involve the coordinate
actions of 2 sets of adhesive receptors: the glycoprotein (GP) Ib-V-IX
complex2,3 and the integrins  2 1 and IIb3.
The Ib-V-IX complex is composed of 4 related transmembrane GPs, ie,
GPIb, Ib, V, and IX, which are associated with each other in a
stoichiometry of 2:2:1:2.4,5 Within this complex, Ib and
Ib are disulfide linked and noncovalently associated with
IX.4,6 GPV is noncovalently associated with GPIb-IX,5 and surface expression of GPV is decreased in
GPIb and GPIX deficiency states.7 Approximately 11,000 copies of the GPIb-IX complex are found on the surface of human
platelets.4,5 This high density is consistent with its role
in cell adhesion under conditions of shear, the Ib-V-IX complex
mediates reversible platelet binding to the subendothelial matrix of
the vessel wall by binding to von Willebrand factor (vWF)8
via sites on the Ib chain.9 Subsequent platelet
activation triggers integrin activation and irreversible binding to vWF
by IIb3.
Loss of GPIb-V-IX function causes Bernard-Soulier syndrome (BSS), a
severe bleeding disorder.3,7,10 BSS is characterized by
abnormal, giant circulating platelets with defective adhesion to vWF
and reduced thrombin responsiveness.11 Classic BSS is associated with the loss of the entire Ib-V-IX complex.7
Nonclassic presentations have also been reported in which the complex
is present in normal amounts but defective in
function.12,13 Mutations in the genes encoding both
Ib12,13 and IX14 have been shown to cause BSS.
GPV has been reported to enhance surface expression of Ib-IX in some
heterologous expression systems,15,16 but not
others.17 It is not known whether mutations in the gene encoding GPV will affect platelet expression of GPIb-IX and/or cause BSS.
Interestingly, GPV is a substrate for thrombin,18-21 a
potent platelet activator. Thrombin binds to GPIb on human
platelets,22 perhaps positioning itself to cleave an
adjacent GPV molecule. These observations conjured the hypothesis that
GPV might contribute to thrombin signaling.23,24 The
observation that antibodies that inhibited thrombin cleavage of GPV
failed to inhibit platelet activation by thrombin25
suggested that GPV cleavage was not necessary for platelet activation
by thrombin, and the identification of 3 distinct G protein-coupled
receptors (GPCRs) for thrombin provided an alternative explanation of
how platelets respond to thrombin.26-29 Nonetheless, the
fact that thrombin cleavage site in GPV is conserved in the mouse, rat,
and human proteins suggests that this sequence may be important for the
structure or function of GPV,30 and available data do not
formally exclude a contribution by GPV to thrombin signaling.
To address the role of GPV in vivo, we have generated GPV-deficient
mice using gene targeting. GPV null mice developed normally and
exhibited no spontaneous bleeding. GPV null platelets were normal in
size and shape, responded normally to thrombin, and exhibited wild-type
adhesion to mouse vWF A1 domain under shear. Thus, in the mouse, GPV is
not necessary for surface expression or function of platelet Ib, for
normal platelet cytoskeletal structure, or for normal responsiveness to
thrombin. Our results suggest that loss of function mutations in the
GPV gene are unlikely to be a cause of human BSS and support recent
studies that suggest that platelet thrombin responses of shape change,
aggregation, and granule secretion are mediated by protease-activated
receptors (PARs).29,31
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MATERIALS AND METHODS |
Targeted inactivation of the GpV gene.
Two P1 bacteriophage clones that contained the GpV gene were
obtained by polymerase chain reaction (PCR) screen of a mouse genomic
library (Genome Systems, St Louis, MO). A 1.3-kb Sal
I/BamHI fragment 5' of exon 1 and a 7.0-kb
EcoRI/Xho I fragment 3' of exon 2 were cloned
into the pNTK vector32 to create the targeting vector
(Fig 1A). The 5' Sal I site
was contributed by the backbone vector (pAd10SacBII). A 0.6-kb
Bgl II/EcoRV fragment of the GpV gene 5' of the short arm of homology was used as a probe to
identify both the wild-type and targeted alleles (Fig 1A). RF8 ES
cells33 (129/SvJae) were electroporated with the targeting
construct, and clones resistant to G418 and FIAU were selected and
screened by Southern blot. A highly chimeric male mouse derived from
GpV+/ ES cells was bred to C57Bl/6 females to generate
approximately 30 F1 GpV+/ mice. All experiments reported
here were performed using the F2 offspring of these mice.
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| Fig 1.
Generation of GPV-deficient mice. (A) Gene-targeting
strategy. A replacement vector69 was used to substitute a
neomycin phosphotransferase expression cassette (Neo) for the entire
GpV gene. The wavy line represents plasmid backbone; TK, HSV
thymidine kinase expression cassette. X1 and X2 represent exons 1 and 2 of the GpV gene, with the coding region shown as a white box
and the 5' and 3' untranslated regions shown as shaded
boxes. (Sal1) indicates a Sal I restriction endonuclease site
found in the P1 bacteriophage vector containing the GpV gene
that was used to construct the targeting vector. (B) Southern blot
analysis of Bgl2-digested genomic DNA from the tails of pups derived
from GpV+/ matings using 5' flanking probe (A).
Targeting removed an endogenous Bgl2 site and introduced a new Bgl2
site. The 4.2- and 5.5-kb bands correspond to wild-type and targeted
alleles, respectively. (C) RT-PCR analysis of GpV+/+ and
GpV/ mouse spleen total RNA using GpV (right
panel) and Par3g (left panel) primers.
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Reverse transcription-PCR (RT-PCR) of mouse spleen.
Total RNA was obtained from individual mouse spleens using Trizol
(GIBCO, Grand Island, NY). Two micrograms of total RNA was used as template to create first-strand cDNA using random hexamer primers and a commercially available kit per the manufacturer's instructions (Superscript; GIBCO). One microliter of the 20 µL reaction was used for PCR amplification of the GpV and
Par3ggenes with 2 µmol/L primers as detailed below. For
GpV, the product size was 440 bp, the sense strand primer for
PCR was 5'-TGC CTA CGA ACC TCA CAC ACA TC-3', and the
antisense primer for PCR was 5'-GCT TAA CTT GAG CCC CAA GCA
G-3'. The conditions used were as follows: 94°C for 4 minutes
and 72°C for 1 minute with the addition of Taq; then 94°C for
45 seconds, 60°C for 1 minute, and 72°C for 1 minute for 40 cycles; and then 72°C for 8 minutes. For Par3g, the product
size was 511 bp, the sense strand primer for PCR was 5'-TCC TCA
CTT GCA TGG GCA TC-3', and the antisense primer for PCR was
5'-TCT AGG CAG CTA TTC AGG CTC CC-3'. The conditions used
were as follows: 94°C for 4 minutes and 72°C for 1 minute with
addition of Taq; then 94°C for 45 seconds, 58°C for 1 minute, and 72°C for 1 minute for 45 cycles; and then 72°C for 8 minutes.
Tail bleeding time.
The tail bleeding times of 45 progeny of GPV+/ matings 6 to 7 weeks of age were assayed using the technique of Dejana et al.34 The bleeding time was performed in a blinded fashion
before tail cutting for genotyping by Southern blot.
Electron microscopy of murine platelets.
Platelets were fixed in 1.5% glutaraldehyde for 2 hours at 22°C in
sodium cacodylate buffer, postfixed in 1% OsO4 in
veronal-acetate buffer, stained with aqueous 1% uranyl acetate,
dehydrated in ethyl alcohol, infiltrated with propylene oxide, and
embedded in Epon (Ted Pella Inc, Redding, CA).
Flow cytometry.
For size analysis, washed platelets were fixed in 1% paraformaldehyde
for 20 minutes at 4°C, washed 3 times with platelet buffer (20 mmol/L Tris-HCl, pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L KCl, 1 mmol/L
MgCl2, 1 mg/mL glucose, and 0.5% bovine serum albumin [BSA]) and analyzed by flow cytometry.
For measurement of GPIb-IX surface expression, washed platelets were
fixed in 0.5% paraformaldehyde for 15 minutes at room temperature and
then washed 3 times with phosphate-buffered saline (PBS). Platelets (5 × 107) were incubated in 0.25 mL Tyrode's buffer
with BSA plus 1:1,000 (vol/vol) nonimmune rabbit serum or GPIb-IX
immune serum (antiserum no. 3584; generously provided by Drs Sylvie
Meyer and Beat Steiner, Hoffmann-LaRoche, Basel,
Switzerland)35 for 1 hour at 4°C. Platelets were then
washed once with PBS, incubated with fluorescein isothiocyanate (FITC)-goat antirabbit monoclonal antibody (Molecular Probes, Sunnyvale, CA) diluted 1:500, and analyzed by flow
cytometry.36
Platelet aggregation and secretion.
Blood was collected into citrate buffer from the inferior vena cava of
pentobarbital-anesthetized mice. Blood from 3 to 4 GPV/ mice or their wild-type littermates was
pooled for each platelet study. Platelet-rich plasma (PRP) was prepared
by centrifugation of whole blood at 200g for 7 minutes. EDTA
(10 mmol/L) and prostaglandin E1
(PGE1; 1 µmol/L) were then added and PRP was
centrifuged at 500g for 10 minutes. Platelets were then washed
in platelet buffer containing 1 mmol/L EDTA and 1 µmol/L
PGE1, collected by centrifugation, resuspended to an
OD500 of 1.0 (~2.5 × 108 platelets/mL)
in platelet buffer lacking EDTA and PGE1, and incubated on
ice for 30 minutes before use. Aggregation and secretion were measured
in a Chrono-Log lumiaggregometer (Havertown, PA). Three hundred
microliters of platelet suspension was added to the aggregometer chamber. Luciferase (880 U/mL), luciferin (8 µg/mL), and
CaCl2 (1 mmol/L) were then added, and aggregation was
followed as change in light transmission with time after addition of
agonist. Results were expressed as  light transmission, defined as
the percentage increase in light transmission over that of the
unactivated platelet suspension, with 100% representing light
transmission of platelet buffer alone. Platelet ATP secretion was
measured as luminescence generated by platelet-released ATP compared
with that of an ATP standard. Studies using botrocetin
were performed in PRP diluted using platelet-poor plasma (PPP; obtained
by centrifugation of the remaining blood at 1,200g for 10 minutes) to an OD500 of 1.0, with 100% light transmission
representing light transmission of PPP alone. Botrocetin was obtained
as a kind gift from M. Berndt (Victoria, Australia).
Platelet adhesion to murine vWF: Construction of murine vWF-A1
domain expression vector.
A region corresponding to the human vWF-A1 (475-709) was cloned from
mouse genomic DNA and used to produce recombinant protein (T. Diacovo,
manuscript in preparation). The murine vWF-A1 domain was
subcloned into the expression vector pQE9 (Qiagen, Valencia, CA) and expressed in Escherichia coli, and the
protein was purified.37 Protein concentrations were
determined using the BCA method (Pierce Chemical Co, Rockford, IL).
Coomassie-blue staining of sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) gels showed that greater than 99% of the
protein in the prep was vWF-A1 domain.
Purified recombinant mouse vWF-A1 protein (diluted to 100 µg/mL with
10 mmol/L Tris, 150 mmol/L NaCl, pH 7.4) was loaded into microslides
(rectangular glass tubes with a cross-section of 300 µm × 30 mm; H.P. Scientific, Inc, Concord, CA) by capillary action and stored
overnight at 4°C. Coated microslides were subsequently rinsed and
incubated with PBS containing 1% human serum albumin for 30 minutes at
37°C to block nonspecific interactions. Protein-coated microslides
were secured to the stage of an inverted phase microscope (Diaphot-TMD; Nikon, Garden City, NJ), and plastic tubing was attached
to each end. A uniform wall shear rate was generated by aspirating
platelets through the microslide with a syringe pump (Harvard
Apparatus, Holliston, MA). For attachment assays, platelets were
purified by centrifugation of anticoagulated blood obtained from the
retroorbital venous plexus of anesthetized mice. Platelets were washed
twice in HEPES buffer (145 mmol/L NaCl, 10 mmol/L HEPES, 0.5 mmol/L
Na2HPO4, 5 mmol/L KCl, 2 mmol/L
MgCl2, 0.2% BSA, pH 7.4), resuspended at a concentration
of 2 × 108/mL, and used within 2 hours. Platelet
suspensions were drawn through microslides at shear rates of 50 or 600 s 1 for 5 minutes. The wall shear stress was
calculated assuming a Newtonian fluid and a viscosity of 1.0 cP.
Attached platelets were observed with phase contrast objectives and
quantitated by analysis of videotape images. The number of platelets
attached per unit area (0.67 mm2) was quantitated using 4 fields of view for each data point.
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RESULTS |
Generation of GPV-deficient mice.
The GpV gene was inactivated using a targeting vector that
eliminated exons 1 and 2 by homologous recombination (Fig 1A). The
targeted allele was detected by Southern blotting using the 5'
flanking probe shown and loss of GPV mRNA was confirmed by RT-PCR of
total mouse spleen RNA (Fig 1B and C). Spleen is a hematopoietic organ
in the mouse and contains megakaryocytes. GpV mRNA was detected in spleen RNA from wild-type mice but not knockout mice. Par3g mRNA, which is known to be expressed in mouse
megakaryocytes,38 was detected in samples from both
wild-type and knockout (Fig 1C).
GPV-deficient mice developed normally and exhibited normal
hemostasis.
GpV+/ × GpV+/ matings produced
GpV/ offspring at the expected rate (eg, 31 +/+, 77 +/, and 34 /); thus, there is no evidence for loss
of GPV-deficient embryos during development. GPV-deficient mice were
grossly normal at birth, grew like their wild-type littermates, and
were fertile. They showed no evidence of spontaneous bleeding and had
hematocrit levels and platelet counts indistinguishable from those of
their wild-type littermates (data not shown). To test platelet function
in vivo, tail bleeding times were determined on the progeny of
heterozygous matings before genotyping (Fig
2). GPV-deficient mice had bleeding times indistinguishable from those
of their wild-type littermates, consistent with their lack of any overt
bleeding.
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| Fig 2.
Tail bleeding times of wild-type and GPV-deficient mice.
The bleeding times of 6- to 7-week-old progeny of heterozygote matings
were obtained in a blinded manner before genotyping. The results for
mice subsequently identified as wild-type (+/+) and GPV-deficient
(/) are shown.
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GPV-deficient platelets were normal in size and structure.
The morphology of platelets from GPV-deficient and wild-type mice was
examined by transmission electron microscopy. Electron micrographs of
wild-type and GPV-deficient mouse platelets were indistinguishable. In
particular, GPV-deficient platelets showed no giantism or cytoskeletal
disruption typical of BSS (Fig 3). Granule
counts of 300 platelets derived from 3 distinct platelet preparations
of each genotype (100 each) were also equivalent: 300 wild-type
platelets contained 1,200 granules and 300 GPV-deficient platelets
contained 1,174 granules. On average, both wild-type and GPV-deficient
platelets had 4.0 -granules per thin section of platelet.
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| Fig 3.
Transmission electron micrograph of mouse platelets. Two
major granule types are present: the  -granules, which are the
predominant population (a), and the serotonin-containing dense granule
(d) population. Some -granules contain small tubules cut in
cross-section (A; arrow, and at higher magnification in [B], arrow)
that is probably vWF.67 Others contain a regular fibrillar
array, which is probably partially polymerized fibrinogen (C;
arrowhead). Although the majority of granules are round, some have
a tent-like shape (D) and a few are very large (E; denoted by
asterisk). The platelets shown are GPV-deficient, but indistinguishable
granules were also observed in the platelets of wild-type mice.
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An independent analysis of the size of wild-type and GPV-deficient
platelets was obtained by using flow cytometry to measure the forward
and side scatter of light (Fig 4).
GPV-deficient platelets exhibited the same range of forward and side
scatter as that observed for wild-type platelets, confirming the
microscopic observation that loss of GPV had no effect on platelet size
or shape.
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| Fig 4.
Assessment of platelet size and shape using flow
cytometric analysis of wild-type (+/+) and GPV-deficient (/)
platelets. Washed platelets were fixed and analyzed by flow cytometry
for light forward and side scatter. Shown is the analysis of 10,000 platelets for each group.
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Adhesion to vWF was not impaired in GPV-deficient platelets.
Platelet adhesion to vWF is dependent on GPIb function.8,39
GPIb's interaction with vWF is species specific, and mouse GPIb will
not bind human vWF.40 To measure the function of the mouse
GPIb in wild-type and GPV-deficient mouse platelets, we therefore
measured platelet adhesion to immobilized recombinant mouse vWF A1
domain. VWF-A1 domain contains unique sequences that provide a distinct
binding site for GPIb.41,42 The importance of this domain
with respect to vWF function is evident based on studies of type 2M vWD
mutations. Patients with this genotype have mutations within the vWF-A1
domain that result in the impairment of hemostasis, but retain normal
vWF multimer structure.43,44 Under conditions of both low
and moderate shear, no difference in the steady-state number of
adherent platelets was observed between GPV-null and wild-type
platelets (Fig 5). Similarly, no difference
was noted in the rate at which wild-type and GPV-deficient platelets
rolled on the vWF-coated surface (data not shown). As observed for
full-length vWF, the ability of recombinant vWF-A1 protein to support
platelet adhesion in flow is species specific; murine, but not human,
vWF-A1 bound murine platelets under flow conditions (T. Diacovo,
manuscript in preparation), and no platelet adherence was
noted when surfaces were not coated with vWF (not shown). Thus,
GPV-deficient mouse platelets demonstrated no evidence for defective
GPIb-vWF interaction in this functional assay.
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| Fig 5.
Accumulation of platelets on immobilized monomeric murine
vWF-A1 during flow. Washed murine platelets were infused through
recombinant vWF-A1-coated microslides at a shear rate of 50 or 600 s1. After 5 minutes of continuous flow, adherent
platelets were counted in 4 different fields of view. Data are averaged
from 2 experiments performed. Error bars represent the standard
deviation.
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Other measures suggest normal GPIb expression in GPV-deficient
platelets.
Surface expression of GPIb in wild-type and GPV-deficient platelets was
also assessed by 2 other measures. First, botrocetin-mediated platelet
agglutination was examined. Like ristocetin, the snake venom-derived
botrocetin induces platelet agglutination in PRP by facilitating
GPIb-vWF binding,45 but, unlike ristocetin, botrocetin is
active on rodent platelets.46,47 Platelets were incubated
with 1, 4, or 10 µg/mL botrocetin for 10 minutes with stirring, and
agglutination was observed as an increase in light transmission.
Representative tracings are shown in Fig 6.
The percentages of maximal change in light transmission (0% equals PRP
and 100% equals PPP) for wild-type versus GPV-deficient platelets were
5.7 ± 0.6 versus 6.0 ± 1.0 (mean ± SD; n = 3) at 1 µg/mL, 37 ± 6 versus 34 ± 2 at 4 µg/mL (n = 3), and 44 ± 4 versus 47 ± 4 at 10 µg/mL (n = 9), respectively. Thus,
botrocetin-induced agglutination of wild-type and GPV-deficient
platelets were indistinguishable, suggesting normal vWF binding of
platelet GPIb in the absence of GPV.
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| Fig 6.
Platelet agglutination by botrocetin. PRP were stirred
and botrocetin was added at a final concentration of 10 (A), 4 (B), or
1 µg/mL (C). Agglutination was measured as the change in light
transmission. +/+, wild-type platelets; /, GPV-deficient
platelets. This experiment was replicated 3 times.
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Surface expression of GPIb-IX was also measured using a polyclonal
antibody to Ib-IX and flow cytometry. No difference in antibody binding
was detected in wild-type versus GPV-deficient platelets. For wild-type
platelets, the values for nonimmune and immune antibody binding in
average fluorescence units (mean ± SD; n = 3) were 10 ± 2 and
88 ± 35, respectively. The cognate values for GPV-deficient
platelets were 8 ± 1 and 97 ± 5, respectively. These data
suggest normal GPIb-IX expression in the absence of GPV, consistent
with the functional data on platelet rolling on vWF-coated surfaces and
botrocetin-induced agglutination.
Activation of platelets by thrombin and other agonists.
To determine if thrombin cleavage of GPV contributes to activation of
platelets by thrombin, we measured shape change, aggregation, and ATP
secretion by wild-type and GPV null platelets in response to low (0.5 and 1 nmol/L) and high (30 nmol/L) concentrations of thrombin. Unlike
PAR3 null mouse platelets28 or PAR1-inhibited human
platelets,29 GPV null platelets responded to 1 nmol/L thrombin like wild-type platelets (Fig 7A).
The rate and extent of shape change and aggregation by wild-type and
GPV-deficient platelets in response to 0.5 nmol/L thrombin, a
concentration close to the threshold for aggregation under these
conditions, were also indistinguishable (n = 5 for wild-type and n = 6 for GPV-deficient platelet preparations, data not shown). In addition, the time to half-maximal secretion, a sensitive measure of thrombin signaling in platelets,29,31 did not differ between
wild-type and GPV-deficient platelets stimulated with 0.5, 1, or 30 nmol/L thrombin. The mean time to half-maximal secretion in seconds for wild-type and GPV-deficient platelets, respectively, was 53.33 (n = 3)
and 54.5 (n = 4) at 0.5 nmol/L thrombin, 47.25 (n = 4) and
50.67 (n = 4) at 1.0 nmol/L thrombin, and 4.8 (n = 6) and 5.0 (n = 6)
at 30 nmol/L thrombin. Even at 30 nmol/L thrombin, a concentration of
thrombin demonstrated to efficiently cleave GPV,48 there
was no detectable difference in the rate or extent of platelet
aggregation or ATP secretion (Fig 7 and data not shown). GPV-deficient
platelets also responded like wild-type to the thromboxane A2 analog
U46619 (10 µmol/L) and to collagen (20 µg/mL, data not shown).
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| Fig 7.
Platelet activation in response to thrombin. (A) Platelet
aggregation. Washed wild-type (+/+) and GPV-deficient (/)
platelets were stirred and exposed to 1 (top) or 30 nmol/L (bottom)
thrombin at 0 seconds. Results are representative of 3 experiments. (B)
Platelet secretion of ATP. Washed wild-type (+/+) and GPV-deficient
(/) platelets were exposed to 1 or 30 nmol/L thrombin and the
peak ATP secretion was measured by lumiaggregometry. Data represent the
mean ± SD of 4 to 6 experiments.
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DISCUSSION |
Since its identification as a platelet surface GP more than 20 years
ago,18 GPV has been hypothesized to play 2 potential roles
in hemostasis and thrombosis. It was proposed as a possible platelet
thrombin receptor based on its proximity to the GPIb thrombin binding
site and its susceptibility to thrombin cleavage.18,23 GPV
was also postulated to be necessary for normal expression and function
of GPIb based on GPV's association with Ib and IX on the platelet
surface49 and its ability to enhance Ib expression in some
studies.15,16 These ideas were concordant with the loss of
GPV expression and vWF binding and decreased thrombin responses
reported in BSS platelets7,50 and raised the important question of whether mutations in the GPV gene itself might cause BSS.
The cloning of GPV showed a type I membrane glycoprotein with 15 extracellular leucine-rich repeats (LRRs), a thrombin cleavage site
just amino terminal to the transmembrane domain, and a short intracellular carboxyl tail.20,21 LRRs are also present in the extracellular domains of Ib,51 Ib,52
and IX.53 The functions of these presumed protein-protein
interaction motifs in GPV and the other members of the Ib-V-IX complex
are unknown. They may mediate interactions among Ib, V, and IX or bind
to vWF or to as yet unidentified ligands. Especially intriguing is that the thrombin cleavage site of GPV is conserved in the mouse, rat, and
human proteins,30 suggesting possible functional importance for this sequence and again raising the possibility that GPV might contribute to thrombin signaling.
To address the importance of GPV for Ib expression and function and for
thrombin signaling, we generated GPV-deficient mice. These mice
developed normally, were fertile, and had no overt bleeding phenotype.
Their platelets showed normal secretion and aggregation responses to
thrombin and U46619. They were normal in number and size, exhibiting
none of the giant platelet morphology associated with human
BSS.11 By contrast, mice lacking Ib do have giant
platelets and spontaneous hemorrhage,54 and vWF-deficient mice have a bleeding diathesis.55 The abnormal size and
shape of platelets from patients with BSS were readily detected by flow cytometry56 and electron microscopy.57 These
observations suggest that our results with GPV-deficient mice are
unlikely to be due to insensitivity of the measurements used or to a
species difference in the roles of the Ib-V-IX complex and vWF.
Instead, our results show that loss of GPV is not sufficient to disrupt thrombin signaling or GPIb expression or function. The lack of a
hemostatic defect and apparently normal adherence to vWF suggest that
GPV is not necessary for vWF binding. This finding is consistent with
recent studies comparing the function of Ib-IX and Ib-V-IX expressed
heterologously.58 The normal number, size, shape, and
cytoskeletal morphology of GPV-deficient platelets suggest that GPV is
not required for normal thrombopoiesis or for formation of a normal
platelet membrane cytoskeleton.59
The observation that disruption of the GpV gene in mouse had no
deleterious effect on platelet thrombin responses is consistent with
the observation that blocking thrombin cleavage of GPV with antibodies
had no effect on the thrombin activation of human
platelets25 and clearly demonstrates that GPV is not
necessary for thrombin-triggered platelet secretion and aggregation.
Indeed, available data suggest that these responses are mediated by G
protein-coupled PARs.26,28,29,38,60,61 In human platelets,
PAR1 and PAR4 can mediate thrombin signaling, and inhibition of both
receptors virtually ablated responsiveness to thrombin.29
In mouse platelets, PAR3 and PAR4 mediate thrombin signaling.28,38 Knockout of the G-protein subunit Gq in
mice ablated platelet aggregation and secretion to
thrombin,62 which is also consistent with the notion that
thrombin signaling is mediated, directly or indirectly, by GPCRs.
Interestingly, thrombin-triggered shape change was intact in
Gq-deficient platelets. However, shape change was inhibited
by blockade of PAR1 and PAR4 in human platelets, and shape change was
normal in GPV-deficient platelets. Thus, available data suggest that
thrombin triggers platelet shape change through PARs via a G protein
other than Gq, probably G12/13.63 Indeed, when Gq-deficient platelets were exposed to the PAR4-activating peptide GYPGKF, they underwent rapid shape change but not aggregation or secretion (M.L.K. and S.R.C., unpublished
observations). Thus, the lack of an effect of
GPV-deficiency on thrombin signaling in platelets is consistent with
the model that PARs are the major mediators of this process. Whether
known PARs completely account for thrombin signaling in platelets will
ultimately be tested in knockout mouse models.
As noted above, the observation that the knockout of Ib results in a
BSS-like phenotype suggests that the Ib-V-IX complex plays similar
roles in mouse and human. Failure to observe thrombocytopenia, giant
platelets, platelet cytoskeletal abnormalities, or bleeding in
GPV-deficient mice suggests that loss of function mutations in the GPV
gene is unlikely to be uncovered as a cause for BSS in humans.
As an aside, it is interesting to note that, although our transmission
EM studies of mouse platelets did not show differences between
wild-type and GPV-deficient mouse platelets, they did show interesting
differences between mouse and human platelets. When compared with human
platelets,64-67 the -granules of mouse platelets
displayed more variations in shape and content (Fig 3). These
variations were present in both wild-type and GPV-deficient mouse platelets.
In summary, our results with GPV-deficient mice provide strong evidence
that GPV is not necessary for GPIb function or thrombin signaling and
is unlikely to be a cause of BSS. It is possible that GPV plays a
subtle or redundant role in the function of the Ib-V-IX complex or in
thrombin signaling. Alternatively, GPV may play a role in platelet
function not probed in the present study. For example, the LRRs of GPV
might bind an as yet unidentified ligand(s), or GPV shed from the cell
surface upon cleavage by thrombin or another protease might signal to
other cells. Lastly, GPV may play a role in cells other than platelets.
Indeed, GPV expression has been reported in human endothelial
cells.68 The GPV-deficient mouse provides a critical
reagent for probing the role of GPV in endothelial cells and for
testing other hypotheses regarding GPV's function as they are generated.
| |
ACKNOWLEDGMENT |
The authors thank Violetta Bigornia, Ivy Hsieh, and Martine Morales for
their technical assistance and Michael Berndt for his kind gift of botrocetin.
| |
FOOTNOTES |
Submitted May 28, 1999; accepted August 4, 1999.
Supported in part by National Institutes of Health (NIH) Grants No.
HL44907 and HL59202 and by the Daiichi Research Center, University of
California, San Francisco (S.R.C.). M.L.K. was supported by NIH Grant
No. HL03731-01.
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 Shaun R. Coughlin, MD, PhD, CVRI, UCSF, 505 Parnassus Ave, San Francisco, CA 94143-0130; e-mail:
shaun_coughlin{at}quickmail.ucsf.edu.
| |
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ABSTRACT
INTRODUCTION