Báo cáo khoa học: Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans ppt

Identification and characterization of a collagen-induced
platelet aggregation inhibitor, triplatin, from salivary
glands of the assassin bug, Triatoma infestans
Akihiro Morita
1
, Haruhiko Isawa
2
, Yuki Orito
1
, Shiroh Iwanaga
3
, Yasuo Chinzei
1
and Masao Yuda
1
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 a
2
b
1
integrin [6] bind to col-
lagen directly, and GPIb-V-IX [7] and a

IIb
b
3
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 a
2
b
1
and
a
IIb
b
3
integrins and secretion of platelet agonists such
as ADP and thromboxane A
2
, 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:
(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; PGE
1
, prostaglandin E
1
; PRP, platelet-rich
plasma; vWF, von Willebrand factor.
FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS 2955
and 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 officinalis and Hirudo medicinalis, respect-
ively. They prevent both the binding to collagen of
a
2
b
1
and 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. infestans saliva.
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 conserved 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 recombinant 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).
Identification and characterization of triplatin A. Morita et al.
2956 FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS
Inhibition 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 A
2
analog. However, this weak inhibi-
tion did not titrate in a concentration-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, IC
50
values
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 l gÆmL
)1
), ADP (0.5 l M), arachidonic acid
(1.0 m
M) or U46619 (2.0 lM). For stimulation by 0.1 nM 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.
A. Morita et al. Identification and characterization of triplatin
FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS 2957
of triplatin-1 and -2 on ristocetin-induced platelet aggre-
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 a
2
b

1
integrin. In control experiments, monoclonal antibody
against integrin a
2
b
1
, 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
)1
ristocetin. Washed platelets (3.3–3.4 · 10
5
plateletsÆmL
)1
) incubated with triplatin-1
were stimulated with 2.0 lgÆmL
)1
collagen or 0.25 lgÆmL
)1
CRP. 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
)1
of collagen or 0.2 lgÆmL
)1
of CRP. After stimulation, light transmittance was monitored by
aggregometer for 10 min.
Identification and characterization of triplatin A. Morita et al.
2958 FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS
aggregation 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 a
2
b
1
integrin 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 E
1
, 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 a
2
b
1
(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
)1
collagen (10 lgÆmL
)1
) in the pres-
ence or absence of 1.0 l
M triplatin-1 and analyzed by anti-FcR c-chain
(aFcR c-chain) and antiphosphotyrosine (aPY) immunoblotting.
A. Morita et al. Identification and characterization of triplatin
FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS 2959
purchased 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 ( and signal p
programs ( respect-
ively. Sequence alignment was performed using the clustal
w program ( />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
1mLÆmin
)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 ⁄ P
i
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
)1
prostaglandin
Identification and characterization of triplatin A. Morita et al.
2960 FEBS Journal 273 (2006) 2955–2962 ª 2006 The Authors Journal compilation ª 2006 FEBS
E
1
(PGE
1
), 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 NaH
2
PO
4
,2mm MgCl
2
,5mm glucose, 12 mm
NaHCO
3

,1mm EGTA, 3.5 mgÆmL
)1
bovine serum albu-
min, 20 ngÆmL
)1
PGE
1
,20ngÆmL
)1
apyrase). Finally,
washed platelets were resuspended in modified Tyrode’s buf-
fer, substituting 2 mm CaCl
2
for 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
(MICROTEST
TM
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 37 °C 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
37 °C.
Inhibition of platelet adhesion was examined according
to Keller et al. [19]. Microtiter plates (MicrotiterÒ Poly-
styrene Base Immunoassay Plates, DYNEX Technologies,
Inc., Chantilly, VA, USA) were coated with collagen
(2.0 lgÆwell
)1
)in5mm 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
MgCl
2
. Washed platelets (3.0 · 10
5
cells) and triplatin or
Gi9 (Immunotech, Marseille, France) were mixed in
Tyrode’s buffer containing 2 mm CaCl
2
and 100 ngÆmL
)1
PGE
1
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 · 10
9
cells) were incubated with 1.0 lm triplatin-1 at 37 °C for
10 min. After incubation, platelets were stimulated by
10 lgÆmL
)1

collagen at 37 °C 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
)1
anti-FcR c-chain antiserum at 4 °C. 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.
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