Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Tria-toma infestans, which inhibited platelet aggregation induced by collagen but not by other a
Trang 1platelet aggregation inhibitor, triplatin, from salivary
glands of the assassin bug, Triatoma infestans
Akihiro Morita1, Haruhiko Isawa2, Yuki Orito1, Shiroh Iwanaga3, Yasuo Chinzei1and Masao Yuda1
1 Department of Medical Zoology, School of Medicine, Mie University, Edobashi, Tsu, Japan
2 Department of Medical Entomology, National Institute of Infectious Diseases, Toyama, Sinjyuku-ku, Tokyo, Japan
3 Laboratory of Chemistry and Utilization of Animal Resources, Faculty of Agriculture, Kobe University, Hyogo, Japan
Injury to the blood vessel wall exposes the
subendo-thelial extracellular matrix, which is rich in collagens,
providing a substrate for platelet adhesion and
aggre-gation This is the first step of hemostasis ending in
formation of the thrombus Several proteins on the
platelet membrane participate in collagen–platelet
interactions in a direct or indirect manner [1]
Glyco-protein (GP)VI [2–5] and a2b1 integrin [6] bind to
col-lagen directly, and GPIb-V-IX [7] and aIIbb3 integrin
[6,8] bind via von Willebrand factor (vWF) In the
cur-rent ‘two-site, two-step’ model of platelet–collagen
interaction [9–12], platelet aggregation proceeds as
fol-lows: first, GPIb-IX-V rapidly binds to vWF
immobi-lized on collagen so that passing platelets are tethered
to the latter Following this event, GPVI, a surface
signaling receptor, binds to collagen with low affinity,
which triggers the signaling cascade for platelet activa-tion This leads to ‘inside-out’ activation of a2b1 and
aIIbb3 integrins and secretion of platelet agonists such
as ADP and thromboxane A2, accelerating platelet aggregation and thrombus formation Therefore, GPVI has a central role in the initial phase of thrombus for-mation as the major signaling receptor for collagen GPVI is composed of two extracellular immunoglob-ulin domains, a mucin-rich stalk, a single transmem-brane domain, and a short cytoplasmic tail [2–4] GPVI is coupled to the Fc receptor (FcR) c-chain homodimer in the transmembrane domain via a salt bridge [5,9,10,13,14] The binding of collagen to GPVI leads to cross-linking of GPVI molecules [15], inducing tyrosine phosphorylation of the cytoplasmic tail of FcR c-chains This phosphorylation leads to binding
Keywords
collagen-induced platelet aggregation
inhibitor; triplatin; Triatoma infestans
Correspondence
M Yuda, Department of Medical Zoology,
School of Medicine, Mie University,
Edobashi, Tsu, Mie 514–8507, Japan
Fax: +81 59231 5215
Tel: +81 59231 5013
E-mail: m-yuda@doc.medic.mie-u.ac.jp
(Received 1 March 2006, revised 19 April
2006, accepted 4 May 2006)
doi:10.1111/j.1742-4658.2006.05306.x
To facilitate feeding, certain hematophagous invertebrates possess inhibi-tors of collagen-induced platelet aggregation in their saliva However, their mechanisms of action have not been fully elucidated Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Tria-toma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin Furthermore, these triplatins also inhibited platelet aggregation induced by collagen-related peptide, a specific agonist of the major colla-gen-signaling receptor glycoprotein (GP)VI Moreover, triplatin-1 inhibited
Fc receptor c-chain phosphorylation induced by collagen, which is the first step of GPVI-mediated signaling These results strongly suggest that trip-latins target GPVI and inhibit signal transduction necessary for platelet activation by collagen This is the first report on the mechanism of action
of collagen-induced platelet aggregation inhibitors from hematophagus invertebrates
Abbreviations
CRP, collagen-related peptide; FcR, Fc receptor; GP, glycoprotein; MBP, maltose-binding protein; PGE1, prostaglandin E1; PRP, platelet-rich plasma; vWF, von Willebrand factor.
Trang 2and subsequent activation of the tyrosine kinase, Syk,
which initiates downstream signaling events [16]
Various platelet inhibitors are found in
hematopha-gus invertebrates [17,18] These inhibitors interfere
with platelet aggregation in host animals and facilitate
blood feeding They include inhibitors of
collagen-sti-mulated platelet aggregation, but only a small number
of inhibitors of this type have been characterized
LAPP [19–21] and calin [22] were identified from
Haementeria officinalisand Hirudo medicinalis,
respect-ively They prevent both the binding to collagen of
a2b1and the binding of GPIb-IX-V to vWF, inhibiting
platelet aggregation and platelet adhesion to collagen
Moubatin [23,24] and pallidipin [25] were identified
from Ornitohdoros moubata and Triatoma pallidipennis,
respectively These proteins inhibit platelet aggregation
induced by collagen, but not platelet adhesion to
colla-gen under static conditions However their inhibitory
mechanisms and target molecules remain unknown
Here, we have identified the novel platelet
aggrega-tion inhibitors triplatin-1 and -2, from T infestans
They share sequence similarity with pallidipin and
spe-cifically inhibit platelet aggregation induced by
colla-gen We suggest that triplatins target GPVI and block
platelet activation induced by collagen
Results
Cloning and production of triplatin-1 and -2
recombinant proteins
A cDNA library was constructed from the salivary
glands of unfed T infestans Five hundred and fifty
clones were randomly picked from this library and
sequenced Among them, the most abundant species
(25 clones) corresponded to a cDNA encoding a 178
amino acid protein with a molecular mass of
19.5 kDa In addition, an isoprotein of this abundant
clone (four additional clones) was also found This
iso-form contains 182 amino acids with a molecular mass
of 19.8 kDa Both molecules were predicted to be
secretory proteins by the signal p program, and both
shared sequence similarities with pallidipin, a platelet
inhibitor found in T pallidipenis (Fig 1) Identities of
the abundant clone and its isoprotein with pallidipin
are 63 and 49%, respectively We named this salivary
gland protein and its isoprotein ‘triplatin-2’ and
‘trip-latin-1’, respectively
To investigate the function of these triplatins,
recombinant proteins were produced in a
baculovirus-insect cell system Secreted recombinant proteins
formed a major fraction of all the proteins in the
cell culture medium They were purified by
cation-exchange and gel filtration chromatography Purity was confirmed by SDS⁄ PAGE (Fig 2) Apparent molecular masses of purified recombinant triplatin-1 and -2 were approximately 17 kDa on SDS⁄ PAGE, which agreed with their predicted molecular masses of 17.9 and 17.1 kDa in secreted form
To identify native proteins in the saliva, antisera were raised against recombinant triplatin-1 In western blot analysis, the sera reacted with both triplatin-1 and -2, although relatively weakly with the latter (Fig 2) The sera also reacted with native proteins in salivary glands, showing that both triplatin-1 and -2 are indeed expressed there SDS⁄ PAGE and western blot analysis indicated that triplatin-1 and -2 are major proteins of
T infestanssaliva
Fig 1 Alignment of triplatin-1 and -2 sequences Multiple align-ment of triplatin-1 and -2 sequences with pallidipin from T pallidi-pennis by CLUSTAL W Underlining, asterisks, colons and periods indicate signal sequences, single fully conserved, strongly con-served and weakly concon-served residues, respectively.
Fig 2 Western blot analysis of triplatin-1 and -2 from the salivary gland of T infestans Extracts from a pair of salivary glands, recom-binant triplatin-1 and recomrecom-binant triplatin-2 were separated by 15% SDS ⁄ PAGE under reducing conditions and stained by Coo-massie brilliant blue and immunoblotted with anti-triplatin-1 serum (a-triplatin-1).
Trang 3Inhibition of platelet aggregation
by triplatin-1 and -2
To investigate the function of triplatin-1 and -2, their
effects on platelet aggregation were tested using
plate-let-rich plasma (PRP) and platelet agonists, because
they shared similarities with the known platelet
aggrega-tion inhibitor, pallidipin, as described above As shown
in Fig 3, triplatin-1 and -2 did not inhibit ADP- and
thrombin-induced platelet aggregation, but slightly
inhibited that induced by arachidonic acid and U46619,
a thromboxane A2 analog However, this weak
inhibi-tion did not titrate in a concentrainhibi-tion-dependent
man-ner, suggesting that it was an artifact In contrast, both
triplatin-1 and -2 clearly inhibited collagen-induced
aggregation in a dose-dependent manner Maximum
inhibition of 50–60% at micromolar concentrations of
triplatin-1 and -2 were recorded These results
demon-strated that triplatin-1 and -2 are inhibitors of platelet
aggregation induced by collagen
To investigate precisely the effect of triplatin-1 and
-2 on platelet aggregation caused by collagen, an
inhi-bition assay was performed using washed platelets instead of PRP In this assay, both triplatin-1 and -2 completely inhibited platelet aggregation induced by collagen (Fig 4A) Based on these results, IC50values
of triplatin-1 and -2 were calculated at approximately
60 nm and 620 nm, respectively We also examined inhibition of collagen-induced platelet aggregation using an aggregometer and PRP (Fig 4B) After sti-mulation by collagen, the characteristic peak caused by
a change of platelet shape was observed within a few minutes Triplatin-1 completely inhibited collagen-induced platelet aggregation and also attenuated the change of platelet shape
Identification of the target receptor for triplatin-1 and -2
Next, we attempted to identify triplatin-1 and -2 target molecules Collagen-induced platelet aggregation is initiated by the interaction between vWF and
GPIb-IX-V Ristocetin promotes this interaction and aggregates platelets [9–12] Therefore, we first examined the effects
Fig 3 Effect of triplatin on platelet aggregation induced by several different agonists Platelets in platelet rich plasma (PRP) (3.5–3.6 · 10 5
plateletsÆmL)1) were incubated with triplatin at 37 C for 10 min before adding collagen (2.0 lgÆmL)1), ADP (0.5 l M ), arachidonic acid (1.0 m M ) or U46619 (2.0 l M ) For stimulation by 0.1 n M thrombin, washed platelets (2.6 · 10 5
plateletsÆmL)1) were used instead of PRP Light transmittance at 600 nm was measured 10 min after stimulation, and platelet aggregation is represented as the percentage of control aggregation of platelets preincubated in the absence of triplatin Results are the mean ± SE of three experiments.
Trang 4of triplatin-1 and -2 on ristocetin-induced platelet
gation (Fig 4A) No inhibitory effect on platelet
aggre-gation induced by ristocetin was observed, indicating
that GPIb-IX-V is not a target for triplatin
Collagen-related peptide (CRP) contains the specific
GPVI recognition motif and directly activates this
receptor [26–28] Therefore, we next examined whether
triplatin-1 and -2 inhibit CRP-induced platelet
aggrega-tion Triplatin-1 and -2 did inhibit CRP-induced
plate-let aggregation in a dose-dependent manner (Fig 4A)
We also examined inhibition of collagen-induced
plate-let aggregation using an aggregometer (Fig 4B)
Trip-latin-1 almost completely inhibited CRP-induced
platelet aggregation and also attenuated the change of
platelet shape These results demonstrated that
triplat-ins inhibit platelet activation mediated by GPVI
We also assessed the effect of triplatin-1 and -2 on
platelet adhesion to immobilized soluble collagen
(Fig 5), which has been reported to be entirely
dependent on interactions between collagen and a2b1
integrin In control experiments, monoclonal antibody
against integrin a2b1, Gi9, did inhibit platelet adhesion However, triplatins did not inhibit platelet adhesion, even at a concentration of 1.0 lm
These results indicate that triplatins are specific inhibitors of collagen-induced platelet aggregation mediated by GPVI To confirm this finding, we exam-ined the effect of triplatin-1 on phosphorylation of the FcR c-chain [5,13,14] This was achieved by immuno-precipitation, because the binding of GPVI to collagen causes tyrosine phosphorylation of the FcR c-chain, which is coupled to GPVI In the absence of
triplatin-1, phosphorylated FcR c-chain of platelets stimulated
by 10 lgÆmL)1 collagen was clearly detected (Fig 6)
In contrast, in the presence of 1.0 lm triplatin-1, phos-phorylation of FcR c-chain was completely absent
Discussion
GPVI is a surface signaling receptor on platelets and has a central role in platelet activation by collagen [10] Here, we have identified a novel type of platelet
Fig 4 (A) Effect of triplatin on platelet aggregation induced by agonists of collagen receptors Platelets in PRP (3.4 · 10 5 plateletsÆmL)1) incubated with triplatin were stimulated 1.25 mgÆmL)1ristocetin Washed platelets (3.3–3.4 · 10 5 plateletsÆmL)1) incubated with triplatin-1 were stimulated with 2.0 lgÆmL)1collagen or 0.25 lgÆmL)1CRP Further methods are as in Fig 3 legend (B) Anti-aggregatory properties of triplatin on collagen- or CRP-induced platelet aggregation Washed platelets (3.0 · 10 5
plateletsÆmL)1) incubated with triplatin-1 at 37 C for
2 min were induced to aggregate by 2.0 lgÆmL)1of collagen or 0.2 lgÆmL)1of CRP After stimulation, light transmittance was monitored by aggregometer for 10 min.
Trang 5aggregation inhibitor, triplatin-1 and its isoform,
trip-latin-2, which specifically inhibit platelet aggregation
induced by collagen, especially by CRP, a specific
agonist of GPVI [26–28] Furthermore, triplatin-1
inhibited tyrosine phosphorylation of FcR c-chains
induced by collagen, the initial step in the GPVI
signa-ling cascade [13,14] These results strongly suggest that
triplatin-1 and -2 are antagonists for GPVI To the
best of our knowledge, triplatin-1 and -2 are the first
natural inhibitors for GPVI to be identified
The injured arterial wall exposes collagen to the blood and recruits platelets to the injured site In the physiological state, in which shearing has an important role, GPVI is involved in recruitment and subsequent aggregation of platelets It was reported that platelets from GPVI-deficient mice show no adhesion to colla-gen and no aggregation [9] In humans, platelets from GPVI-deficient patients can attach to collagen but nonetheless do not form aggregates These findings suggest that GPVI is crucial for thrombus formation after arterial injury [13] Feeding activities of hemato-phagous arthropods injure the blood vessels of host animals It is likely that triplatin-1 and -2 are injected into the host during T infestans blood feeding and attenuate host hemostasis at the initial phase
To date, some other inhibitors of collagen-induced platelet aggregation have been reported in the saliva
of blood-sucking arthropods Among them, pallidipin and moubatin are similar to triplatin in their inhibitory properties Like triplatin, pallidipin and moubatin inhi-bit platelet aggregation induced by collagen but not by other agonists [25] They also exert potent inhibitory effects on both platelets in plasma and washed plate-lets Furthermore, unlike other inhibitors of collagen-induced platelet aggregation, such as LAPP [19–21] and Calin [22], they do not inhibit the platelet adhe-sion to collagen mediated by a2b1integrin under static conditions [23,24] Although their mechanisms of action are not fully understood, it is possible that pal-lidipin and moubatin are also GPVI antagonists
In summary, we have identified inhibitors of colla-gen-induced platelet aggregation from the saliva of
T infestans Platelet–collagen interactions have an important role in thrombus formation, and GPVI plays a pivotal role therein Further investigations on the inhibitory mechanisms of these insect proteins might lead to development of antiplatelet agents that antagonize thrombus formation at the initial phase
Experimental procedures
Materials
Prostaglandin E1, deoxycholate, sodium orthovanadate and genenase were purchased from Biogenesis Ltd (Poole, UK), Nakarai Tesque, Inc (Kyoto, Japan), ICN Biomedi-cals, Inc (Aurora, OH, USA) and New England Biolabs, Inc (Beverly, MA, USA), respectively Apyrase, phenyl-methylsulfonyl fluoride, leupeptin and aprotinin were pur-chased from Wako Pure Chemical, Ind Ltd (Osaka, Japan) Collagen, ADP and ristocetin were purchased from Chronolog, Corp (Havertown, PA, USA) U46619, thrombin, fibrinogen and Gly-Pro-Arg-Pro peptide were
Fig 5 Effect of triplatin on platelet adhesion to collagen 3.0 · 10 5
washed platelets ⁄ well were incubated with triplatin-1, -2 or
mono-clonal antibody against integrin a2b1(Gi9) for 10 min and introduced
into wells coated with 2.0 lgÆwell)1 collagen After washing, the
adherent platelets were quantified using a protein assay The
relat-ive number of adherent platelets is presented Results are the
mean ± SE of three experiments.
Fig 6 Inhibition of the phosphorylation of FcR c-chain by triplatin.
Anti FcR c-chain immunoprecipitates were prepared from lysates of
platelets stimulated by 10 lgÆmL)1collagen (10 lgÆmL)1) in the
pres-ence or abspres-ence of 1.0 l M triplatin-1 and analyzed by anti-FcR c-chain
(aFcR c-chain) and antiphosphotyrosine (aPY) immunoblotting.
Trang 6purchased from Calbiochem (San Diego, CA) CRP was a
kind gift of H Takayama of Kyoto University
Arachido-nic acid, Nonident P-40, and other chemicals were
pur-chased from Sigma-Aldrich, Inc (St Louis, MD, USA)
Isolation and sequencing of cDNA clones
Salivary glands of T infestans were dissected from thoraces
of unfed adults, and poly A(+) RNA was isolated from 30
pairs of salivary glands using a MicroPrep mRNA isolation
kit (Amersham Pharmacia Biotech, Ltd, Amersham,
Buck-inghamshire, UK) The salivary gland cDNA library was
constructed from this isolated mRNA using the SuperScript
plasmid system (Gibco BRL Life Technologies, Inc.,
Rock-ville, MD, USA) according to the manufacturer’s
instruc-tions From this constructed cDNA library, 550 clones
were randomly selected and their DNA sequences were
determined using ABI PRISM BigDye Terminator cycle
sequencing kits (Applied Biosystems, Foster City, CA,
USA), by ABI 310 genetic analyzer (Applied Biosystems)
The cDNA sequences of triplatin-1 and -2 were deposited
in the DNA Data Bank of Japan (DDBJ) (accession
num-ber, AB 250209 and AB 250210) Sequence similarities and
signal peptide prediction of these clones were carried out
using blast (http://www.blast.genome.ad.jp/) and signal p
programs (http://www.cbs.dtu.dk/services/SignalP/),
respect-ively Sequence alignment was performed using the clustal
wprogram (http://www.clustal.genome.ad.jp/)
Production and purification of the recombinant
protein
Triplatin-1 and -2 recombinant proteins were produced in a
baculovirus-insect cell system Full-length cDNA of
tripla-tin-1 and -2 were cloned into the BamHI site of the
baculo-virus transfer vector, pAcYM1 The Sf9 cells were
cotransfected with the constructed plasmid and linearized
baculovirus DNA, AcRp23, lacZ (Becton Dickinson
Bio-sciences, San Jose, CA, USA) Recombinant proteins from
Tn5 cells infected by the recombinant baculoviruses were
secreted into the culture medium due to their original signal
peptide sequences They were purified by a combination of
cation-exchange chromatography and gel-filtration
chroma-tography Briefly, culture supernatants containing secreted
recombinant proteins were first applied to a PD-10 column
(Amersham Pharmacia Biotech) in 20 mm sodium acetate
buffer, pH 5.2 Next, the eluted samples were applied to a
MONO S column (Amersham Pharmacia Biotech)
equili-brated with 20 mm sodium acetate buffer, pH 5.2 and
eluted with a gradient from 0 to 1 m NaCl at a flow rate of
1 mLÆmin)1 The fractions containing recombinant proteins
were pooled, concentrated by Centricon 10 (Millipore
Corp., Bedford, MA, USA) and applied to a TSK G2000
SW column (Tosoh, Tokyo, Japan) equilibrated with
50 mm Tris⁄ HCl, pH 7.4, containing 150 mm NaCl Purity
of recombinant product was established by 15.0% SDS⁄ PAGE under reducing conditions Protein concentra-tion was determined with a Coomassie protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA) using bovine c-globulin as the standard
Preparation of antibody to triplatin-1
To obtain a large amount of triplatin-1 to use as an immu-nogen, recombinant triplatin-1 was also produced as a maltose-binding protein (MBP)-fusion protein Briefly, a cDNA fragment encoding triplatin-1 without signal peptide was amplified by PCR and cloned into an expression plas-mid, pMAL-c2q (New England Biolabs) The MBP-fused triplatin was produced using this plasmid in Escherichia coli BL21, and was purified by amylose-resin affinity chroma-tography Cleavage of the recombinant protein from MBP was achieved using genenase For antibody production, rats were immunized with the MBP-free triplatin-1 emulsified in TiterMax gold (CytRx Corp., Los Angeles, CA, USA) After the first immunization, booster immunizations were done twice at intervals of 2 weeks After the final immun-ization, whole blood was taken and antisera against tripla-tin-1 were prepared
Western blot analysis
Salivary gland proteins were separated by SDS⁄ PAGE in 5–20% gradient gels and transferred to nitrocellulose mem-branes Blotted membranes were blocked in NaCl⁄ Pi con-taining 5% skimmed milk, and incubated with primary antibody against triplatin-1, and then with alkaline phos-phatase-conjugated antirabbit IgG The signal was detected using nitroblue tetrazolium
To detect the FcR c-chain in platelets, western blot analysis was performed according to Tulasne et al [29] Platelet proteins were separated by 4–20% gradient SDS⁄ PAGE, and transferred to a polyvinylidene difluoride membrane The FcR c-chain was detected with specific antisera (Upstate Biotechnology, Lake Placid, NY, USA)
in a manner similar to that described above Tyrosine phosphorylation was detected by monoclonal antibody 4G10 (Upstate Biotechnology) The signal was detected using an enhanced chemiluminescence detection system (SuperSignal West Pico Chemiluminescent Substrate, Pierce Biotechnology)
Platelet preparation
Blood was collected from healthy human volunteers by veni-puncture on acid–citrate–dextrose anticoagulant PRP was obtained by centrifugation at 110 g for 10 min Platelets were obtained by centrifugation at 1100 g for 20 min, fol-lowing incubation for 10 min with 20 ngÆmL)1prostaglandin
Trang 7E1(PGE1), and washed three times using modified Tyrode’s
buffer (5 mm Hepes buffer, pH 7.4, 134 mm NaCl, 3 mm
KCl, 0.3 mm NaH2PO4, 2 mm MgCl2, 5 mm glucose, 12 mm
NaHCO3, 1 mm EGTA, 3.5 mgÆmL)1 bovine serum
albu-min, 20 ngÆmL)1 PGE1, 20 ngÆmL)1 apyrase) Finally,
washed platelets were resuspended in modified Tyrode’s
buf-fer, substituting 2 mm CaCl2for 1 mm EGTA The number
of platelets was counted using a hematocytometer
Effect of triplatin-1 and -2 on platelet aggregation
and adhesion
Effects of triplatin-1 and -2 on platelet aggregation were
photometrically measured according to Bednar et al [30]
PRP or washed platelets were incubated with triplatin-1
and -2 for 10 min in a 96-well flat-bottom plate
(MICROTESTTM 96, Becton Dickinson Biosciences) in
50 mm Tris⁄ HCl, pH 7.4, containing 150 mm NaCl
Plate-let aggregation was initiated by addition of 2.0 lgÆmL)1
collagen, 0.5 lm ADP, 1.0 mm arachidonic acid, 0.2 lm
U46619, 0.1 nm thrombin, 1.2 mgÆmL)1 ristocetin or
0.25 lgÆmL)1 CRP as platelet activators The reaction
mixture was stirred continuously at 37C for 10 min
Platelet aggregation was monitored by light transmittance
using a microplate reader (MPR-A4i, Tosoh, Tokyo,
Japan)
Effects of triplatin-1 and -2 on platelet aggregation
induced by collagen and CRP were also analyzed using an
aggregometer (C-500, Chronolog) PRP was mixed with
triplatin-1 or -2, and platelet aggregation was started by
addition of either 2.0 lgÆmL)1 collagen or 0.2 lgÆmL)1
CRP Platelet aggregation was monitored by light
transmit-tance in the aggregometer with continuous stirring at
37C
Inhibition of platelet adhesion was examined according
to Keller et al [19] Microtiter plates (Microtiter
Poly-styrene Base Immunoassay Plates, DYNEX Technologies,
Inc., Chantilly, VA, USA) were coated with collagen
(2.0 lgÆwell)1) in 5 mm acetic acid for 1 h at room
tem-perature, followed by addition of 1% bovine serum
albu-min for 1 h at room temperature to block the nonspecific
binding of platelets to the wells After blocking, wells
were washed three times with Hepes-buffered saline,
20 mm Hepes, pH 7.4 containing 0.14 m NaCl and 2 mm
MgCl2 Washed platelets (3.0· 105
cells) and triplatin or Gi9 (Immunotech, Marseille, France) were mixed in
Tyrode’s buffer containing 2 mm CaCl2 and 100 ngÆmL)1
PGE1 and then transferred into wells After 45 min
incu-bation, wells were washed three times with
Hepes-buf-fered saline again The number of platelets adhering to
immobilized collagen was determined using Micro BCA
Protein Assay Kits (Pierce Biotechnology) The percentage
of specifically adherent platelets was calculated on the
basis of a standard curve obtained with known numbers
of platelets
Platelet lysis and immunoprecipitation
of FcR c-chains
Immunoprecipitation was performed according to Ichinohe
et al [16] and Tulasne et al [29] Washed platelets (1· 109 cells) were incubated with 1.0 lm triplatin-1 at 37C for
10 min After incubation, platelets were stimulated by
10 lgÆmL)1 collagen at 37C for 10 min and then lyzed with an equal volume of ice-cold lysis buffer, 20 mm Tris,
300 mm NaCl, 2 mm EDTA, 2% Nonident P-40, 1% deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluor-ide, 2 mm sodium orthovanadate, 10 lgÆmL)1 leupeptin and 10 lgÆmL)1 aprotinin Non-lysed cells and debris were removed by centrifugation Platelet lysate was incubated with 1 mgÆmL)1anti-FcR c-chain antiserum at 4C After overnight incubation, protein A-Sepharose beads were added to the mixture and washed three times with 10 mm Tris, 160 mm NaCl and 0.1% Tween 20 The protein bound
to beads was eluted with Laemmli buffer and applied to SDS⁄ PAGE and western blot analysis
Acknowledgements
We thank H Takayama of Kyoto University for the generous gift of CRP This study was supported by
a grant-in-aid for Scientific Research (13006374) to HI, for Scientific Research on Priority Areas (08281103) to
YC, for Scientific Research (B) (12470060) to MY, and for Exploratory Research (10877043, 11877043 and 12877042) to YC from the Ministry of Education, Sci-ence, Culture and Sports of Japan It was also suppor-ted by a grant-in-aid for Scientific Research, Research
on Health Sciences focusing on Drug Innovation (KH23306) and Young Scientists Fellowship B (16790249) to HI from the Naito Foundation, the Japan Health Sciences Foundation and JSPS, respect-ively, and a grant from the Research for the Future Program from JSPS to YC
References
1 Clemetson KJ & Clemetson JM (2001) Platelet collagen receptors Thromb Haemost 86, 189–197
2 Clemetson JM, Polgar J, Magnenat E, Wells TN & Clemetson KJ (1999) The platelet collagen receptor gly-coprotein VI is a member of the immunoglobulin super-family closely related to FcaR and the natural killer receptors J Biol Chem 274, 29019–29024
3 Miura Y, Ohnuma M, Jung SM & Moroi M (2000) Cloning and expression of the platelet-specific collagen receptor glycoprotein VI Thromb Res 98, 301–309
4 Ezumi Y, Uchiyama T & Takayama H (2000) Molecu-lar cloning, genomic structure, chromosomal localiza-tion, and alternative splice forms of the platelet collagen
Trang 8receptor glycoprotein VI Biochem Biophys Res Commun
277, 27–36
5 Watson SP, Auger JM, McCarty OJ & Pearce AC
(2005) GPVI and integrin aIIbb3signaling in platelets
J Thromb Haemost 3, 1752–1762
6 Coller BS, Beer JH, Scudder LE & Steinberg MH
(1989) Collagen–platelet interactions: evidence for a
direct interaction of collagen with platelet GP Ia⁄ IIa
and indirect interaction with platelet GP IIb⁄ IIIa
medi-ated by adhesive proteins Blood 74, 182–192
7 Berndt MC, Shen Y, Dopheide SM, Gardiner EE &
Andrews RK (2001) The vascular biology of the
glyco-protein Ib–IX–V complex Thromb Haemost 86, 178–188
8 Savage B, Saldivar E & Ruggeri ZM (1996) Initiation
of platelet adhesion by arrest onto fibrinogen or
translocation on von Willebrand factor Cell 84, 289–297
9 Nieswandt B, Brakebusch C, Bergmeier W, Schulte V,
Bouvard W, Mokhtari-Nejad R, Lindhout T,
Heems-kerk JWM, Zirngibl H & Fa¨ssler R (2001) Glycoprotein
VI but not a2b1integrin is essential for platelet
interac-tion with collagen EMBO J 20, 2120–2130
10 Nieswandt B & Watson SP (2003) Platelet–collagen
inter-action: is GPVI the central receptor? Blood 102, 449–461
11 Andrews RK & Berndt MC (2004) Platelet physiology
and thrombosis Thromb Res 114, 447–453
12 Furie B & Furie BC (2005) Thrombus formation in vivo
J Clin Invest 115, 3355–3362
13 Gibbins JM, Okuma M, Farndale R, Barnes M &
Watson SP (1997) Glycoprotein VI is the collagen
recep-tor in platelets which underlies tyrosine phosphorylation
of the Fc receptor c-chain FEBS Let 413, 255–259
14 Tsuji M, Ezumi Y, Arai M & Takayama H (1997) A
novel association of Fc receptor c-chain with
glycopro-tein VI and their co-expression as a collagen receptor in
human platelets J Biol Chem 272, 23528–23531
15 Miura Y, Takahashi T, Jung SM & Moroi M (2002)
Analysis of the interaction of platelet collagen receptor
glycoprotein VI (GPVI) with collagen: a dimeric form of
GPVI, but not the monomeric form, shows affinity to
fibrous collagen J Biol Chem 277, 46197–46204
16 Ichinohe T, Takayama H, Ezumi Y, Arai M,
Yama-moto N, Takahashi H & Okuma M (1977)
Collagen-stimulated activation of Syk but not c-Src is severely
compromised in human platelets lacking membrane
glycoprotein VI J Biol Chem 272, 63–68
17 Ribeiro JM (1987) Role of saliva in blood-feeding by
arthropods Annu Rev Entomol 32, 463–478
18 Basanova AV, Baskova IP & Zavalova LL (2002)
Vascular-platelet and plasma hemostasis regulators from
bloodsucking animals Biochemistry 67, 143–150
19 Keller PM, Schultz LD, Condra C, Karczewski J &
Connolly TM (1992) An inhibitor of collagen-stimulated
platelet activation from the salivary glands of the
Haementetia officinalisleech II Cloning of the cDNA
and expression J Biol Chem 267, 6899–6904
20 Connolly TM, Jacobs JW & Condra C (1993) An inhi-bitor of collagen-stimulated platelet activation from the salivary glands of the Haementetia officinalis leech I Identification, isolation, and characterization J Biol Chem 267, 6893–6898
21 Depraetere H, Kerekes A & Deckmyn H (1999) The collagen-binding leech products rLAPP and calin pre-vent both von Willebrand factor and a2b1(GPIa⁄ IIa)-I-domain binding to collagen in a different manner Thromb Haemost 82, 1160–1163
22 Harsfalvi J, Stassen JM, Hoylaerts MF, van Houtte E, Sawyer RT, Vermylen J & Deckmyn H (1995) Calin from Hirudo medicinalis, an inhibitor of platelet adhesion to col-lagen under static and flow conditions Blood 85, 705–711
23 Waxman L & Connolly TM (1993) Isolation of an inhi-bitor selective for collagen-stimulated platelet aggrega-tion from the soft tick Ornithodoros moubata J Biol Chem 268, 5445–5449
24 Keller PM, Waxman L, Arnold BA, Schultz LD, Con-dra C & Connolly TM (1993) Cloning of the cDNA and expression of moubatin, an inhibitor of platelet aggregation J Biol Chem 268, 5450–5456
25 Noeske-Jungblut C, Kra¨tzschmar J, Haendler B, Alagon
A, Possani L, Verhallen P, Donner P & Schleuning W-D (1994) An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis
J Biol Chem 269, 5050–5053
26 Morton LF, Hargreaves PG, Farndale RW, Young RD
& Barnes MJ (1995) Integrin a2b1-independent activation
of platelets by simple collagen-like peptides: collagen ter-tiary (triple-helical) and quternary (polymeric) structures are sufficient alone for a2b1-independent platelet reactiv-ity Biochem J 306, 337–344
27 Asselin J, Gibbins J, Achison M, Lee YM, Morton LF, Farndale RW, Barnes MJ & Watson SP (1997) A col-lagen-like peptide stimulates tyrosine phosphorylation
of syk and phospholipase C c2 in platelets independent
of the integrin a2b1 Blood 89, 1235–1242
28 Kehrel B, Wierwille S, Clemetson KJ, Anders O, Steiner
M, Knight CG, Farndale RW, Okumura M & Barnes
MJ (1998) Glycoprotein VI is a major collagen receptor for platelet activation: It recognizes the platelet-activat-ing quternary structure of collagen, whereas CD36, glycoprotein IIb⁄ IIIa, and von Willebrand factor do not Blood 91, 491–499
29 Tulasne D, Judd BA, Johansen M, Asazuma N, Best D, Brown EJ, Kahn M, Koretzky GA & Watson SP (2001) C-terminal peptide of thrombospondin-1 induces plate-let aggregation through the Fc receptor c-chain-associ-ated signaling pathway and by agglutination Blood 98, 3346–3352
30 Bednar B, Condra C, Gould RJ & Connolly TM (1995) Platelet aggregation monitored in a 96 well microplate reader is useful for evaluation of pletelet agonists and antagonists Thromb Res 77, 453–463