BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 242, 250–255 (1998)
ARTICLE NO. RC977917

A Novel Anti-Platelet Monoclonal Antibody Induces
Mouse Platelet Aggregation through an Fc
Receptor-Independent Mechanism1
Yukinari Kato,* Satoko Hori,* Naoya Fujita,* and Takashi Tsuruo*,†,2*Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku,
Tokyo 113, Japan; and †Cancer Chemotherapy Center, Japanese Foundation for Cancer Research,
1-37-1, Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan

Received November 25, 1997

Platelets are nonproliferative and terminally differentiated cells. Platelets offer an attractive model system to study the various biochemical events leading tostructural and functional alterations in activated cells.
When platelets are exposed to stimuli, they are activated, undergo a dramatic shape change, adhere toeach other, and aggregate. Several monoclonal antibodies (mAbs) that recognize CD9, GPIIb/IIIa (aIIbb3 integrins), or GPIV are known to stimulate human platelet
aggregation. However, no mAbs able to induce aggregation of mouse platelets have been reported. We haveestablished an anti-mouse platelet mAb (AIP21) thatcan promote mouse platelet aggregation by itself. Because mouse platelets did not express the Fc receptor(FcR, CD32) on their surfaces and because AIP21 is an
IgM subclass, AIP21 might promote platelet aggregation through an FcR-independent mechanism. We could
not identify the antigen recognized by AIP21, but flow
cytometric analysis revealed that it was not identicalto CD9, GPIV, or integrins (i.e., aIIb , av , a5 , a6 , b1 , andb3 integrins). During the aggregation of mouse platelets
mediated by AIP21, several 50-68-kDa proteins are rapidly phosphorylated at tyrosine residues. This phosphorylation by AIP21 was dose-dependent and did not
require plasma components. We identified the 52-kDa
phosphorylated protein as Shc. These results indicatethat AIP21 could be useful for investigating the mechanisms of mouse platelet aggregation. q 1998 Academic Press

1
This study was supported in part by a special grant for Advanced
Research on Cancer and a Grant-in-Aid for Cancer Research from
the Ministry of Education, Culture and Science, Japan, and a Special
Coordination Fund of the Science and Technology Agency, Japan.
2
To whom correspondence should be addressed. Fax: /81-3-38163592. E-mail: ttsuruo@imcbns.iam.u-tokyo.ac.jp.
Abbreviations used: mAb, monoclonal antibody; FcR, Fc receptor;
NR IgM, normal rat IgM; ADP, adenosine diphosphate; PRP, platelet-rich plasma.

Human platelets are stimulated to aggregate by ligating monoclonal antibodies (mAbs) that recognize platelet
surface proteins. However, most mAbs do not exhibit
this aggregating ability when the Fc fragment has been
removed (1-6). Therefore, the platelet Fc receptor (FcRII,
CD32) expressed on human platelets might be involvedin the mAb-mediated platelet aggregation.
Some mAbs have been reported to possess the ability
to stimulate human platelet aggregation through an
FcR-independent mechanism because their F(ab*)2
fragment promoted the aggregation (7-10). The antigens of these mAbs were reported to be CD9 (1, 11),
GPIIb/IIIa (aIIbb3 integrins) (2-4, 7-10, 12), or GPIV
(CD36) (5). The ligation of these mAbs on human platelets resulted not only in aggregation but also in phospholipase C activation, inositol 1,4,5-trisphosphate anddiacylglycerol generation, intraplatelet Ca2/ level rise,and ATP secretion (1, 2, 13).
In contrast, no mAbs were reported to induce aggregation of mouse platelets. Hence, it is still unclear whichmolecules were associated with the FcR-independent aggregation of mouse platelets. We here report that one of
our established anti-mouse platelet mAbs, termed
AIP21, could stimulate mouse platelet aggregation in adose-dependent manner. AIP21 induced platelet aggregation even in washed platelets, indicating that plasmacomponents were not required. AIP21 might stimulatemouse platelets to aggregate through an FcR-independent mechanism because mouse platelets did not express FcR on their surfaces, and AIP21 is an IgM subclass. To our knowledge, AIP21 is an unique mAb thatpossessed the ability to induce aggregation of mouseplatelets. Using the AIP21, we examined the undefinedmechanisms of mouse platelet aggregation.
MATERIALS AND METHODS
Preparation of mouse platelet-rich plasma and washed platelets.
Platelet-rich plasma (PRP) was prepared from fresh heparinized

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FIG. 1. Recognition of mouse platelet surface protein by AIP21 mAb. Washed mouse platelets were inactivated by freezing for 30 min
before ligating mAbs. Then platelets were treated with AIP21, APB2, or NR IgM for 1 h at 47C and reacted with FITC-conjugated anti-rat
IgM. The fluorescence intensity was analyzed using a FACScan. The bold curves indicate platelets treated with AIP21 (A) or APB2 (B).
The dotted curves indicate platelets treated with NR IgM (A, B).

blood drawn from 8- to 12-week-old female BALB/c mice (Charles
River, Kanagawa, Japan) by cardiac puncture, then centrifuged at
230 1 g for 7 min at 237C. When preparing washed platelets, PRP
was centrifuged at 400 1 g for 5 min. Platelets were washed oncewith PBS and then resuspended in PBS (14).
Cell lines and culture conditions. Mouse melanoma B16F10 cells(15) and mouse colon adenocarcinoma NL-17 cells (16) were cultured
in RPMI1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan)
supplemented with 10% heat-inactivated FBS (Gibco Laboratories,
Grand Island, NY) (RPMI growth medium). The mouse bone-derived
endothelial cell line KN-3 (17) was cultured in DMEM (Nissui) supplemented with 10% heat-inactivated FBS (Gibco). These cells were
cultured at 377C in a humidified atmosphere of 95% air and 5% CO2 .
Hybridoma production and purification of mAbs. Three SD rats
were immunized with 8F11 mAb, which can recognize gp44 antigen
and suppress gp44-mediated platelet aggregation (14). The spleencells were fused with mouse myeloma P3U1 cells, and the culture
supernatants of the hybridomas were screened by the MAbs’ ability
to recognize mouse platelets. The mAbs were purified using an antirat IgG column (Sigma Chemical Co., St. Louis, MO).
Flow cytometry. Washed mouse platelets inactivated by freezingfor 30 min before ligating mAbs. Thymocytes and splenocytes wereprepared from 5-week-old female BALB/c mice (Charles River).
Washed mouse platelets, B16F10 cells, KN-3 cells, thymocytes,
splenocytes, or peritoneal macrophages were incubated with AIP21,
APB2, or control normal rat IgM (NR IgM; Chemicon International
Inc., Temecula, CA) or with anti-mouse CD9, aIIb integrin (CD41),
av integrin (CD51), a5 integrin (CD49e), a6 integrin (CDw49f), b1
integrin (CD29), b3 integin (CD61), or FcR (CD32) (Pharmingen, San
Diego, CA; or Serotec, Oxford, UK) for 1 h at 47C. After washing with
PBS, cells were reacted with FITC-conjugated second antibodies.
Fluorescence data were collected using a Becton Dickinson FACScan(San Jose, CA).
Analysis of phosphorylation of the platelet proteins. After incubation of PRP or washed mouse platelets with AIP21, APB2, NR IgM,
adenosine diphosphate (ADP), or NL-17 cells at 377C, platelets weresuspended in a lysis buffer, containing 0.5% Triton X-100, 10 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 100

units of aprotinin, 50 mM sodium fluolide, 50 mM sodium m-vanadate and 30 mM sodium pyrophosphate on ice for 30 min. Aftercentrifugation at 15,000 1 g for 30 min, the supernatants (20 mg)
were subjected to SDS-PAGE in a 10-20% gradient gel. The electrophoresed proteins were transblotted onto a nitrocellulose membrane.
The membrane was preincubated in a blocking buffer [4% skim milkin 25 mM Tris-HCl (pH 8.0), 125 mM NaCl, 0.1% Tween 20, 0.1%
sodium azide] for 1 h at 237C. The membrane was then incubatedwith 1 mg/ml of anti-phosphoserine (PSer) mAb (clone PSR-45; BioMakor, Rehovot, Israel), anti-phosphothreonine (PThr) mAb (clone
PTR-8; BioMakor), or anti-phosphotyrosine (PTyr) mAb (clone
25.2G4) (18) in a blocking buffer for 2 h. After washing, the membrane was incubated with peroxidase-conjugated anti-mouse IgG(Amersham, Buckinghamshire, UK) for 1 h. The membrane was developed using an enhanced chemiluminescence detection system(Amersham) and Kodak X-Omat AR film.
Immunoprecipitation and Western-blot analysis. Washed mouseplatelets were incubated with AIP21 at 377C for 3 min, then they
were suspended in lysis buffer, as described above, for 30 min on ice,followed by centrifugation at 15,000 1 g for 30 min. The Shc protein
was immunoprecipitated from the platelet lysate using protein GSepharose 4B gel beads (Zymed, San Francisco, CA) coated withan anti-Shc mAb (Transduction Laboratories, Lexington, KY). Afterelectrophoresing under reducing conditions using a 10-20% gradientgel polyacrylamide, the immunoprecipitated Shc proteins were transblotted onto a nitrocellulose membrane. The membrane was incubated with either 1 mg/ml of anti-Shc mAb or anti-PTyr mAb in a
blocking buffer for 1 h. After washing, the membrane was incubatedwith peroxidase-conjugated anti-mouse IgG. Then the membrane
was developed as described above.
Estimation of platelet aggregation. Platelet aggregation was measured turbidometrically using an aggregometer (NKK HEMA Tracer
I, Niko Bioscientific Co., Tokyo, Japan). A 200-ml aliquot of PRP or
washed platelets was incubated with AIP21 or NR IgM in a cuvetteat 377C under constant stirring in the aggregometer. In some experiments, washed mouse platelets were preincubated with appropriate
concentrations of EDTA for 5 min at 377C before the AIP21 wasadded. The changes in light transmittance were monitored for 10
min (16).

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FIG. 2. Mouse platelet aggregation induced by the AIP21 mAb. (A and B), PRP (A) or washed platelets (B) from mice were incubatedwith 25 mg/ml of AIP21 (a), APB2 (b), or NR IgM (c) in a cuvette at 377C under constant stirring in an NKK HEMA Tracer I, as describedin Materials and Methods. (C), washed mouse platelets were incubated with 50 mg/ml, 5 mg/ml, or 0.5 mg/ml of AIP21 (curves a-c, respectively).
(D), washed mouse platelets were preincubated with 0 mM, 5 mM, 50 mM, or 250 mM EDTA for 5 min (curves a-d, respectively). Then theplatelets were stimulated to aggregate by adding 25 mg/ml of AIP21. The changes in light transmittance were monitored for 10 min.

RESULTS AND DISCUSSION
Establishment of a mouse platelet aggregation-inducing mAb. We established several anti-mouse mAbs ofthe IgM subclass (i.e., AIP21 and APB2), during thestudy of cancer-mediated platelet aggregation (14). As
shown in Fig. 1, both AIP21 and APB2 recognizedmouse platelet-surface molecules. When AIP21 wasadded to mouse PRP, platelets were immediately activated to aggregate (Fig. 2A, curve a). Aggregation was
also observed when AIP21 was added to washed platelets (Fig. 2B, curve a), indicating that plasma components were not essential for AIP21-mediated platelet
aggregation. Fig. 2C shows that this aggregation occurred in a dose-dependent manner. In contrast to
AIP21, ADP induced platelet aggregation in PRP but
not in washed platelets (data not shown). APB2, theother control anti-mouse platelet mAb of the IgM subclass, could not induce platelet aggregation (Figs. 2A
and 2B, curve b), though it did bind a platelet surface
molecule (Fig. 1B). Therefore, AIP21 could be a novel

mouse platelet aggregation-inducing mAb. The aggregation promoted by AIP21 was inhibited in the presence of EDTA (Fig. 2D). However, flow cytometric analysis revealed chelating Ca2/ with EDTA did not reducethe binding activity of AIP21 to mouse platelets (datanot shown). Therefore, Ca2/ was required for the
AIP21-mediated platelet aggregation.
Induction of mouse platelet aggregation by AIP21
through an FcR-independent mechanism. Many mAbs
to human platelets have been reported to induce activation (1-5, 7-12). Most of these mAbs, however, stimulate
human platelets to aggregate through an FcR-dependent mechanisms because their F(ab*)2 fragments cannot promote the aggregation (1-6). It is still unclear
whether mouse platelets expressed FcR on their surfaces. We, therefore, examined the expression of FcR
on mouse platelets using an anti-FcR mAb. The mAb
could not recognize mouse platelets (Fig. 3A), but it
could bind mouse macrophages that expressed FcR(Fig. 3B), indicating that mouse platelets did not ex-

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FIG. 3. The absence of FcR expression in mouse platelets. Washed mouse platelets were inactivated by freezing for 30 min before
ligating mAbs. Mouse platelets (A) and peritoneal macrophages (B) were treated with an anti-FcR (CD32) mAb (bold curves) or nothing(dotted curves) for 1h at 47C and then reacted with FITC-conjugated anti-rat IgG. The fluorescence intensity was analyzed using a FACScan.

press FcR on their surfaces. For this reason, no mAbs
have been reported that could induce aggregation ofmouse platelets.
We have established a mouse platelet aggregationinducing mAb, AIP21, that is mAb of the IgM subclass(data not shown). Hence, AIP21 induced mouse platelet
aggregation through an FcR-independent mechanism.
When we made the F(ab*)2 fragment of AIP21, it promoted mouse platelets to aggregate (data not shown).
This result also suggests that AIP21 stimulates platelet aggregation by directly binding to the platelet surface antigen. However, the aggregation induced by theF(ab*)2 fragment of AIP21 was weaker than that induced by intact AIP21. Moreover, Fab fragment of
AIP21 could not induce platelet aggregation (data notshown). Therefore, aggregation of the AIP21 antigen
or agglutination of platelets is also involved in AIP21mediated mouse platelet aggregation.
In human platelets, some antigens were known to
be associated with the aggregation through an FcRindependent mechanism (i.e., CD9, GPIIb/IIIa (aIIbb3
integrins), or GPIV (CD36)) (1-5, 7-12). Platelets werealso known to express several integrins, such as av , a5 ,
a6 , and b1 integrins. When we examined the reactivityof AIP21 by flow cytometry, the AIP21 antigen was
expressed on platelets, B16F10 cells, and KN-3 cells
but not on thymocytes or splenocytes (Table 1). CD9
was expressed on both thymocytes and splenocytes; integrins, such as av , a5 , a6 , b1 , and b3 integrins, wereexpressed on either thymocytes or splenocytes. The aIIb

TABLE 1

Distribution of Antigens Recognized by Anti-Mouse Platelet mAbs

AIP21
Anti-CD9
Anti-aIIb (CD41)
Anti-b3 (CD61)
Anti-GPIV (CD36)
Anti-b1 (CD29)
Anti-av (CD51)
Anti-a5 (CD49e)
Anti-a6 (CD49f)

PlateletThymocyte

Splenocyte

B16F10

KN-3

/
/
/
/
/
/
/
/
/

0
/
0
0
N.D.
/
0
/
/

0
/
0
/
{a
0
/
0
0

/
/
0
/
N.D.
/
/
/
/

/
{
0
/
N.D.
/
{
{
/

Note. Mouse washed platelets were frozen for 30 min before the ligation of mAbs. Mouse platelets, thymocytes, splenocytes, B16F10 cells,
or KN-3 cells were incubated with AIP21, anti-mouse CD9, anti-aIIb , anti-mouse b3 , anti-mouse GPIV, anti-b1 , anti-av , anti-a5 , or anti-a6
for 1 h at 47C. After washing with PBS, platelets were reacted with the appropriate second antibodies. Fluorescence data were collectedusing a Becton–Dickinson FACScan.
aReference 20.

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FIG. 4. Phosphorylation of platelet proteins at tyrosine residues
during AIP21-mediated platelet aggregation. (A), washed mouseplatelets were incubated with vehicle (Lane 1), NL-17 cells (1 1 106
cells/ml; Lane 2), or AIP21 (10 mg/ml; Lane 3) for 5 min at 377C.
The platelet lysates were electrophoresed and transblotted onto a
nitrocellulose membrane. The membrane was immunoblotted using
an anti-PTyr mAb (left panel), anti-PSer mAb (middle panel), or antiPThr mAb (right panel). (B), washed mouse platelets were incubatedwith NR IgM (10 mg/ml; Lane 1), AIP21 (10 mg/ml; Lane 2), or APB2
(10 mg/ml; Lane 3) for 5 min at 377C. PRP were also incubated with
AIP21 (10 mg/ml; Lane 4) or ADP (20 mM; Lane 5) for 5 min at 377C.
The platelet lysates were electrophoresed and transblotted onto a
nitrocellulose membrane. The membrane was immunoblotted using
anti-PTyr mAb. (C), washed mouse platelets were incubated with 0
mg/ml (Lane 1), 1 mg/ml (Lane 2), 3 mg/ml (Lane 3), 10 mg/ml (Lane4), or 30 mg/ml (Lane 5) of AIP21 for 1 min at 377C. The platelet
lysates were electrophoresed and transblotted onto a nitrocellulose
membrane. The membrane was immunoblotted using the anti-PTyrmAb. After washing, the membrane was incubated with peroxidaseconjugated second antibody and was developed with an enhanced
chemiluminescence detection system.

integrin was specifically expressed on platelets. Although we could not obtain the anti-GPIV mAb, GPIV
has been reported to express weakly on splenocytes(19). These results indicate that the AIP21 antigen isdifferent from CD9, GPIV, or integrins such as aIIb , b3 ,b1 , av , a5 , and a6 integrins and that it may be a novel
antigen.
We tried to identify molecules recognized by AIP21
and APB2 by immunoprecipitation and/or Western blot
analysis. However, we could not detect the specificband in the lysates from platelets, B16F10 cells, or KN3 cells (data not shown). Identifying the antigen willbe the subject of future study.
Phosphorylation of platelet proteins after ligation of
AIP21. Previous studies have shown that several
platelet proteins were phosphorylated at tyrosine residues when platelets undergo aggregation induced bysome agonists and mAbs (20, 21). Several 50-68-kDa
proteins were phosphorylated at tyrosine residues after
the coating of washed mouse platelets with AIP21 (Fig.
4A, left panel, Lane 3). Likewise, when the platelets
were treated with platelet aggregation-inducing NL-17
cells (Fig. 4A, left panel, Lane 2) (16) and when PRP
were treated with a platelet aggregation-inducing factor, ADP (Fig. 4B, Lane 5), these same proteins weretyrosine phosphorylated. However, platelet proteins
were not phosphorylated at serine or threonine residues by the ligation of AIP21 (Fig. 4A, middle and rightpanels, Lane 3). The ligation of APB2 and NR IgM had
no effects on the phosphorylation of platelet proteins(Fig. 4B, Lanes 1 and 3, respectively). The tyrosine
phosphorylation induced by AIP21 occurred in a dosedependent manner (Fig. 4C).
Identifying platelet proteins that were tyrosine phosphorylated in response to the ligation of AIP21 would
be essential for understanding the mechanisms of signal transduction in mouse platelets. In human platelets, the 52-kDa Shc protein was reported to be tyrosine
phosphorylated after thrombopoietin binding to its receptor, c-mpl (21). As the 52-kDa protein was also tyrosine phosphorylated after the ligation of AIP21 tomouse platelets (Fig. 4A), we assumed the 52-kDa protein was Shc protein. Then, the immunoprecipitated
Shc protein was electrophoresed and immunoblotted

FIG. 5. Phosphorylation of the Shc protein during AIP21-mediated platelet aggregation. Washed mouse platelets were incubatedwith PBS alone (Lane 1) or AIP21 (50 mg/ml; Lane 2) for 5 min at
377C. Shc protein was immunoprecipitated from the platelet using
a specific anti-Shc mAb. Immunocomplexes were electrophoresed ina 10-20% gradient gel. After blotting onto a nitrocellulose membrane,
the membrane was immunoblotted using an anti-PTyr mAb (A) oran anti-Shc mAb (B). After washing, the membrane was incubatedwith peroxidase-conjugated second antibody and was developed withan enhanced chemiluminescence detection system.

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with an anti-PTyr mAb. As shown in Fig. 5A, we founda dramatic increase in tyrosine phosphorylation of the
52-kDa Shc protein in mouse platelets after AIP21 ligation. This result suggests that the Shc protein may
be associated with the AIP21-mediated mouse platelet
aggregation.
In conclusion, we here report a mouse platelet aggregation-inducing mAb, AIP21. This mAb induced mouseplatelet aggregation without the need for plasma components and through an FcR-independent mechanism.
We identified that the 52-kDa Shc protein was involvedin the signal transduction of the platelet aggregation.
Although we could not identify the antigen of AIP21,
identification of the antigen, in future, would contribute to the understanding of the undefined mechanismsof mouse platelet aggregation. Because mouse platelets
did not express FcR on their surface, analysis of antimouse platelet mAbs that induced mouse platelet aggregation would also contribute to the understandingof FcR-independent platelet aggregation.
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