maintenance of ribosomal protein s19 in plasma by complex formation with prothrombin

Maintenance of ribosomal protein S19 in plasma by complexformation with prothrombinHiroshi Nishiura1, Sumio Tanase2, Kenichi Tsujita3, Seigo Sugiyama3, Hisao Ogawa3, TomohiroNakagaki4, Umeko Semba1, Tetsuro Yamamoto1

1Department of Molecular Pathology, Faculty of Life Science, Kumamoto University Graduate School, Kumamoto; 2Department of Biomedical

Laboratory Sciences, Faculty of Life Science, Kumamoto University Graduate School, Kumamoto; 3Department of Cardiovascular Medicine,

Faculty of Life Science, Kumamoto University Graduate School, Kumamoto; 4Third Production Department, The Chemo-Sero Therapeutic

Research Institute, Kumamoto, Japan

We have reported the presence of ribosomal protein S19

(RP S19) in plasma (1), although the source of plasma

RP S19 is still unclear. During blood coagulation, a

plasma transglutaminase (FXIIIa) that has been acti-

vated by thrombin intermolecularly cross-links RP S19

between Lys122 and Gln137, causing it to oligomerise.

The cross-linking reaction occurs on surface-exposed

phosphatidylserine molecules of platelets activated by

thrombin. For this reaction, interaction between the

phosphatidylserines and the glycosaminoglycan binding

moiety (Lys23–Lys29) of RP S19 is crucial.

The cross-linkage endows RP S19 with the capacity to

bind the C5a receptor and recruit monocytes ⁄macrophag-

es (2–4). To validate a role of RP S19 in plasma, we

recently developed a coagulum resorption model in the

guinea pig peritoneal cavity. When coagulum preformed

in vitro was inserted in the abdominal cavity, mono-

cytes ⁄macrophages were quickly recruited to the coagulum

resulted in a rapid clearance of it (5). The clearance was

prevented by the immunological neutralisation of the RP

S19 oligomer with anti-RP S19 antibodies or by blocking

the functional oligomer formation with the Gln137Asn-

RP S19 mutant. These data indicated a role of the plasma

RP S19 in the coagulum and thrombus resorption (6–8).

RP S19 is usually synthesised as a component of the

small ribosomal subunit. It is intriguing that such a small

protein (16 kDa) stays in plasma and escapes from filtra-

tion into urine in the renal glomeruli. Recently, it was

reported that macrophage migration inhibitory factor

has the ability to form a complex with RP S19 (9).

Our working hypothesis was that RP S19 complexes

with another plasma protein under non-inflammatory

Abstract

We have demonstrated that the cross-linking of ribosomal protein S19 (RP S19) on platelets by activated

factor XIII provides chemotactic potency to monocytes ⁄ macrophages for a resolution of coagulum. Factor

XIII is activated by an active form of prothrombin, thrombin. We here report that RP S19 is present as a

complex with prothrombin in the blood stream. Formation of this complex was blocked by a mutation of

the glycosaminoglycan-binding basic cluster (Lys23–Lys29) in RP S19. Prothrombin–RP S19 interaction was

enhanced by an absence of Ca2+ and the plasma RP S19 concentration was significantly low in the patient

treated with warfarin, indicating participation of the c-carboxyl glutamic acid domain of prothrombin making

a salt bridge with the basic cluster. The complex formation likely explains why a protein as small as RP

S19 can prevent from a filtering system of renal glomeruli at a steady state. The translocation of RP S19

from prothrombin to platelets during blood coagulation seems to be also advantageous for RP S19 from

the perspective of oligomerisation by activated factor XIII, which should have been activated by thrombin.

Key words Gla domain; plasma proteins; prothrombin; ribosomal protein S19; warfarin treatment

Correspondence Hiroshi Nishiura, Department of Molecular Pathology, Faculty of Life Science, Kumamoto University Graduate

School, Honjyo 1-1-1, Kumamoto 860-8556, Japan. Tel: +81(96)373 5306; Fax: +81(96)373 5308; e-mail: [email protected]

Accepted for publication 2 February 2011 doi:10.1111/j.1600-0609.2011.01585.x

ORIGINAL ARTICLE

European Journal of Haematology 86 (436–441)

436 ª 2011 John Wiley & Sons A/S

conditions. In this report, we describe the identification

of this binding partner as prothrombin. For the reasons

described above, the RP S19–prothrombin complex must

facilitate the intermolecular cross-linking of RP S19

during blood coagulation.

Materials and methods

Patient’s plasma

Plasmas of patients treated with or without warfarin at

Department of Cardiovascular Medicine in the Kuma-

moto University Hospital were collected with 3.2% citrate

under an informed consent according to the guideline of

Faculty of Life Science, Kumamoto University.

Regents

Anti-human RP S19 rabbit IgG was coupled to BrCN-

activated Sepharose 4B beads (Pharmacia, Uppsala,

Sweden) (anti-RP S19 IgG beads: 5 mg ⁄mL) (10).

Recombinant wild-type RP S19 and Lys23, 24, 27, 29Ala

mutant RP S19 (mutant RP S19) or Trx ⁄His ⁄S-Taqwild-type RP S19 were prepared using an Escherichia coli

expression system using a pET-11a or a pET32a vector,

respectively (11, 12). Wild-type RP S19 was coupled to

NHS-activated HP beads (RP S19 column: 5 mg ⁄mL) or

to BrCN-activated Sepharose 4B beads (RP S19 beads:

5 mg ⁄mL). Trx ⁄His ⁄S-Taq wild-type RP S19 was also

coupled to BrCN-activated Sepharose 4B beads

(Trx ⁄His ⁄S RP S19 beads: 50 lg ⁄mL).

Partial purification of plasma RP S19-binding protein

To partially purify a plasma RP S19 binding protein(s),

individual plasma samples from four healthy volunteers

containing 1 mm diisopropylfluorophosphate, 4 mm eth-

ylene diamine-N,N,N’,N’,-tetra acetic acid trisodium salt,

0.05 mm pepstatin-A and 0.5 mm N-ethylmaleimide

(Peptide Institute, Osaka, Japan) were subjected to col-

umn chromatography using a blue column and a heparin

column (Pharmacia), in that order. The RP S19 binding

protein in a breakthrough fraction of the blue column

was recovered from an 800 mm NaCl fraction in the

heparin column (data not shown).

SDS-PAGE and western blotting analyses

Plasma proteins eluted from a 5C4 AR-300 column

(Nakarai, Kyoto, Japan) were dried in a VC-960 speed-

vac concentrator (TAITEC, Aichi, Japan). Proteins

eluted from a Superdex 200HR 10 ⁄30 column (Pharma-

cia), from RP S19 beads or from anti-RP S19 IgG beads

were precipitated in cold acetone. Samples were

resuspended the initial volume of loading buffer (10 mm

Tris–HCl containing 9 m urea, 2% sodium deoxycholate

and 2% 2-mercaptoethanol, pH 6.8) and subjected to

SDS-PAGE (13). Proteins in 12.5% polyacrylamide gels

were then transferred to an Immobilon-PSQ membrane

(Millipore, Bedford, MA, USA) using an AE-6677 semi-

dry blotting machine (Atto, Tokyo, Japan). After treat-

ing with 1% Block AceTM (Dainippon Pharmaceutical,

Suita, Osaka, Japan) for 1 h at 22�C, proteins were incu-

bated with a-RP S19 rabbit IgG (12) or with anti-human

prothrombin rabbit IgG (Dako, Tokyo, Japan)

(200 ng ⁄mL) for 1 h at 22�C. After treatment with HRP-

conjugated anti-rabbit IgG goat IgG (50 ng ⁄mL) for

30 min at 22�C, the bound HRP was detected using the

ECL Plus western blotting detection system (Amersham

Biosciences KK, Tokyo, Japan). The concentration of

RP S19 in plasma was sometimes determined using NIH

Image 1.63 image analysis software. Statistical signifi-

cance was calculated by the non-parametric or para-

metric tests offered in the two-way analysis of variance

window, respectively. Values are expressed as

mean ± SD. A P-value <0.05 was considered statisti-

cally significant and shown as **P < 0.01.

NH2-terminal amino acid sequence analysis

Limited NH2-terminal amino acid sequencing was carried

out in duplicate for the initial 20 cycles with a protein

sequencer (477A; Applied Biosystems, Tokyo, Japan)

equipped with a PTH amino acid analyser (120A; Applied

Biosystems, Tokyo, Japan) according to the manufac-

turer’s manual. A homology search for the amino acid

sequence was carried out using BLAST network service at

Swiss Institute of Bioinformatics.

Immunological pull-down of RP S19–prothrombincomplex in plasma

Normal plasma (100 lL) of 3.2% citrate treated or

20 lm hirudine treated (Sigma, St.Louis. MO, USA) was

incubated with anti-RP S19 IgG beads (25 lL) for 3 h at

22�C. Unbound proteins were recovered by centrifuga-

tion. After washing, proteins bound to the beads were

eluted with 100 lL of 50 mm glycine, pH 3.0.

Competitive inhibition of prothrombin–RP S19 beadsinteraction with soluble RP S19 or RP S19 mutant

Prothrombin (0.3 mg ⁄mL, 50 lL) was incubated with

10 lL of RP S19 beads for 1 h at 37�C in the presence

of either wild-type RP S19 or mutant RP S19

(0.3 mg ⁄mL, 50 lL). After centrifugation, proteins

bound to the RP S19 beads were eluted with 100 lL of

50 mm glycine, pH 3.0.

Nishiura et al. RP S19–prothrombin complex in plasma

ª 2011 John Wiley & Sons A/S 437

Results and discussion

Identification of a plasma protein binding to anRP S19 column

A partially purified plasma sample (see Method) was

applied to an RP S19 column, and the bound proteins

were eluted with a NaCl gradient. As shown in Fig. 1A,

the bound protein(s) eluted with a spike at 400 mm NaCl

and a front shoulder at 280 mm NaCl. The protein(s) in

the spike fraction were subjected to reverse-phase column

chromatography with a 5C4 AR-300 column (Fig. 1B).

The protein(s) eluted as a single peak at an acetonitrile

concentration of 41%. The NH2-terminal amino acid

sequence for twenty residues in the peak fraction consti-

tuted a single set that completely overlapped with the

sequence of prothrombin (Fig. 1C). Consistently, the

peak fraction migrated as a single band with an apparent

molecular mass of 70 kDa in SDS-PAGE (Fig. 1D).

Ca2+-regulated complex formation between RP S19and prothrombin

To examine complex formation in the physiological

liquid phase and the effect of Ca2+ on it, prothrombin

(T) was incubated with an excess amount of RP S19 (S)

in 10 mm Tris–HCl containing 100 mm NaCl (pH. 7.5)

with or without 2.5 mm Ca2+ for 10 min at 22�C, andeach mixture containing or not containing Ca2+ was

subjected to gel filtration column chromatography with

the Superdex 200HR 10 ⁄ 30 column in the presence or

absence of Ca2+, respectively. A typical elution pattern

of the proteins in the absence of Ca2+ is shown in

Fig. 2A and 2C. BSA (67 kDa), ovalbumin (OVA;

0.32A B

C D

0.16

015

Time (min)

Binding protein

94 kDa70 kDa67 kDa

43 kDa30 kDa

20 kDa

14 kDa

Prothrombin (44)

ANTFLEEVRK

ANTFLEEVRK

GNLERECVEE

GNLERECVEE

Con

cent

rati

on o

f N

aCl

A23

5

300

0.5

1.0 0.32

0.16

0.000 15

Time (min)

Con

cent

rati

on o

f ac

eton

itri

l

A21

0

30

100

50

0

Figure 1 Identification of ribosomal protein S19

(RP S19)-binding protein(s) in plasma. (A)

Plasma proteins bound to an RP S19 column

equilibrated with 10 mM Tris–HCl buffer contain-

ing 140 mM NaCl (pH 7.5) were eluted with a

NaCl gradient. (n = 4) (B) RP S19-binding pro-

tein(s) bound to a 5C4AR-300 column equili-

brated with 0.1% trifluoroacetic acid containing

5% acetonitrile were eluted with an acetonitrile

gradient. (C) The NH2-terminal amino acid

sequence of the binding protein was deter-

mined with a protein sequencer. (D) The appar-

ent molecular weight of the RP S19 binding

protein was analysed by SDS-PAGE stained

with Coomassie blue.

0.16 0.08 100

50

0

T

0.04

0.000 15 30

Time (min)

RPS19 +Prothrombin +

CaCl2 –

RPS19 +Prothrombin +

CaCl2 –

RPS19

S T T T

ProthrombinCaCl2

++

+++

–++

0.08

0.000 30

Time (min)

A23

5

A21

0

Con

cent

rati

on o

f ac

eton

itri

l

T S

BSA

94 kDa67 kDa43 kDa30 kDa20 kDa

14 kDa

Cyt-COVA

60

B CA

Figure 2 Identification of the ribosomal protein S19 (RP S19) binding moiety in prothrombin. (A) RP S19 (S) mixed with the same molar of pro-

thrombin (T) in 10 mM Tris–HCl buffer containing 100 mM NaCl (pH. 7.5) without Ca2+ for 10 min at 22�C was applied to a Superdex 200HR

10 ⁄ 30 column. BSA (67 kDa), OVA (43 kDa) and Cys-C (12 kDa) denote eluate positions. (n = 3) The molar ratio of RP S19 to prothrombin in the

70-kDa fraction was analysed (B) by SDS-PAGE stained with Coomassie blue or (C) by reverse-phase column chromatography with a 5C4 AR-300

column.

RP S19–prothrombin complex in plasma Nishiura et al.


43 kDa) and cytochrome-C (Cyt-C; 12 kDa) (Sigma)

were used as the molecular weight markers.

The average molar ratio between RP S19 (S) and pro-

thrombin (T) was analysed by SDS-PAGE and by 5C4

AR-300 column chromatography (Fig. 2B–C). In the

latter, RP S19 and prothrombin eluted separately at

38% and 41% acetonitrile, respectively. The RP S19 ⁄prothrombin ratio was 3 ⁄ 1 or 1 ⁄3 in the absence or

presence of Ca2+, respectively (data not shown).

Co-precipitation of prothrombin with RP S19 inplasma

To obtain direct evidence for the RP S19–prothrombin

complex in plasma, we immunoprecipitated plasma RP

S19 with anti-RP S19 IgG beads and examined whether

prothrombin co-precipitated. In this experiment, we used

citrated plasma and hirudine-treated plasma, because the

RP S19–prothrombin interaction was affected by Ca2+

concentration. As shown in Fig. 3, not only RP S19 but

also prothrombin was observed by western blotting in

the fraction precipitated with the antibody beads in

either plasma. In the supernatant, RP S19 was undetect-

able whereas a large amount of prothrombin remained.

These results indicated that almost all of the RP S19

molecules were present as a complex with prothrombin

in blood stream even under the normal plasma concen-

tration of Ca2+.

Decreased plasma concentration of RP S19 in patientstreated with warfarin

The Ca2+ sensitivity of the RP S19–prothrombin com-

plex suggested the involvement of the c-carboxylglutamic

acid (Gla) domain of prothrombin in the interaction with

RP S19. It is known that the mechanism of anti-coagu-

lant therapy with warfarin is to prevent the c-carboxyla-tion of glutamate residues in the Gla domains; therefore,

a batch of prothrombin and its family proteins in the

warfarin-treated patient plasma should be Gla-less or

proteins-produced in vitamin K absence form. In this

case, plasma RP S19 could not interact with prothrom-

bin and others and should be filtrated at renal glomeruli.

As a consequence, the plasma RP S19 concentration of

the warfarin-treated patient would be low. We examined

it by the western blotting analysis of plasma RP S19

comparing cardiovascular disease patients suffering from

ischaemic heart diseases between warfarin-treated and

non-treated groups (n = 6 in each group). Ages of the

patients were matched between the group; 68.5 ± 14.3

and 69.0 ± 8.5, respectively. The sex ratio (male ⁄ female)

slightly differed between the groups; 1 ⁄ 1 and 2 ⁄ 1, respec-tively. Activated partial thromboplastin time did not dif-

fer between the groups; 80.8 ± 32.0 and 83.0 ± 18.7 in

percentage, respectively. However, partial thromboplastin

time significantly differ between the groups; 54.5 ± 33.1

and 100.3 ± 2.3 in percentage, respectively, indicating

significant effect of the warfarin treatment. As shown in

Fig. 4, the band with an apparent molecular weight of

16 kDa in a membrane stained with the anti-RP S19

antibodies is very faint in the warfarin-treated group. We

quantified the density of band by the NIH Image J soft-

ware. The RP S19 densities in the warfarin-treated group

(22.5 ± 5.2) were significantly lower than those in the

non-treated group (115.9 ± 14.5). These results indicate

that RP S19 is stabilised by interacting with prothrombin

in plasma and that the submolecular region of prothrom-

bin interacting with RP S19 is a Gla domain.

αα-prothrombin Ab

Prothrombin

RPS19

PrecipitateCont rabbitIgG-beads

Citrate-treated Hirudine-treated

Human plasma

Precipitateα-RPS19

IgG-beads

Precipitateα-RPS19

IgG-beads

Supernatantα-RPS19

IgG-beads

α-RPS19 Ab

Figure 3 Validation of the ribosomal protein S19 (RP S19)–prothrom-

bin complex in plasma. Plasma proteins pretreated with citrate or hiru-

dine were bound to anti-RP S19 IgG beads or control rabbit IgG beads

for 3 h at 22�C and were eluted in 50 mM glycine buffer, pH 3.0, and

then western blotting was performed with a-RP S19 antibodies or a-

prothrombin antibodies, respectively (n = 3).

αα-RPS19 Ab**

150

100

50

0Warfarin-treated

Patient’s plasmasNon-treatedWarfarin-treated

Patient’s plasmas

16 kDa

Den

sity

of

RP

S19

Non-treated

Figure 4 Depletion of ribosomal protein S19 (RP S19) from patient’s

plasma by warfarin treatment. Patient’s plasma treated with or with-

out warfarin was applied to SDS-PAGE and examined a concentration

of RP S19 by western blotting (n = 6 in each group). A density of an

apparent molecular weight of 16-kDa band reacted with anti-RP S19

antibodies was measured by the NIH IMAGE J software (n = 4). Value

is expressed as mean ± SD. A P-value <0.05 is considered statistically

significant and shown as **P < 0.01.



Binding of factor X to RP S19-immobilised beads

As a rational consequence, it is expected that other Gla

domain family members such as factor X would also

interact with RP S19. To examine it, factor X purified

from normal human plasma demonstrated two bands in

the silver staining with apparent molecular weights of 57

and 68 kDa. We assume these are a less glycosylated

form and a fully glycosylated form of factor X mole-

cules, respectively (14). Trx ⁄His ⁄S-Taq RP S19 possessed

an apparent molecular weight of 32 kDa; 200 lL of fac-

tor X (75 lg ⁄mL) was mixed with 25 lL of the

Trx ⁄His ⁄S RP S19 beads or the control beads for 30 min

at 22�C (Fig. 5). The 57-kDa and 68-kDa bands were

clearly visible in the Trx ⁄His ⁄S RP S19 bead fraction but

not in the control-bead fraction, indicating that factor X

bound to RP S19. This result supports again the involve-

ment of Gla domains in the interaction with RP S19.

However, considering the difference of plasma concentra-

tions between prothrombin (100 lg ⁄mL) and the other

Gla domain family proteins including factor X

(7.5 lg ⁄mL), we assumes that the latter proteins would

not have chance to form complex with RP S19 under the

presence of large excess amount of prothrombin in

plasma at the normal condition.

Identification of a submolecular region of RP S19involved in complex formation with prothrombin

We finally examined the submolecular region of RP S19

as the interaction partner of Gla domain of prothrom-

bin. Because the interaction was enhanced in the absence

of Ca2+, we hypothesised that a salt bridge between the

highly acidic Ca2+-free Gla residue of prothrombin and

a basic residue(s) in a basic cluster region of RP S19 con-

tributed to complex formation. To test this, Lys23, 24,

27, 29Ala-mutant RP S19 as well as wild-type RP S19

was used to competitively inhibit the prothrombin–RP

S19 bead interaction. If soluble mutant RP S19 is capa-

ble of binding to prothrombin, when it is used as a com-

petitor, prothrombin should be recovered as a soluble

complex in the liquid fraction, as is the case when solu-

ble wild-type RP S19 is used as a competitor. As shown

in Fig. 6, unlike the case with wild-type RP S19, pro-

thrombin did not stay in the liquid phase but bound to

the RP S19 beads when the mutant RP S19 was used as

the competitor. This result indicates the involvement of

the basic glycosaminoglycan-binding cluster of RP S19

composed of Lys23, 24, 27, 29 residues in the interaction

with prothrombin.

The present study showed that RP S19 is stably pres-

ent in blood stream because of complex formation with

prothrombin. We have estimated the concentration of

RP S19 oligomers in serum to be about 10)8m from its

chemoattractant capacity to monocytes (15). A concen-

tration range such as 10)8m is not high enough to mea-

sure accurately in plasma by conventional means. We

have estimated RP S19 monomer in plasma to be

500 ng ⁄mL (2.5 · 10)8m) based on the strength of bands

immunoreactive to anti-RP S19 antibodies in western

blotting. On the other hand, the plasma prothrombin

concentration is as high as 140 lg ⁄mL (2 · 10)6m) (16),

indicating that prothrombin is present in plasma at a

100-fold higher molar concentration than is RP S19.

Under physiological salt and Ca2+ concentrations, the

Silver stain

68 kDa57 kDa

32 kDa

Sup beadsControl-

beads

Sup beadsRP S19-

beads

Figure 5 Binding of factor X to the Trx ⁄ His ⁄ S-Taq ribosomal protein

S19 (RP S19) beads. Purified factor X was incubated with control

beads or with the Trx ⁄ His ⁄ S-Taq RP S19 beads. After separation of

the supernatant fractions and the beads fractions, aliquots of these

fractions were analysed in 12% SDS-PAGE stained with silver-staining

solution (n = 3). A less glycosylated and a fully glycosylated factor X

molecules or Trx ⁄ His ⁄ S-Taq RP S19 demonstrated apparent molecular

weights of 57 and 68 kDa or 32 kDa, respectively.

αα-prothrombin Ab

Prothrombin

RPS19-beadsProthrombinMutant RPS19

RPS19-beadsProthrombinWild type RPS19

RPS19

BeadsSup

α-RPS19 Ab

Figure 6 Identification of the prothrombin-binding moiety in ribosomal

protein S19 (RP S19). Prothrombin was incubated with the RP S19

beads in the presence of wild-type RP S19 or mutant RP S19 (n = 4).

The supernatant (Sup) and the bead eluate (beads) were analysed by

western blotting with anti-RP S19 antibodies or anti-prothrombin anti-

bodies.

RP S19–prothrombin complex in plasma Nishiura et al.


complex formed at an RP S19 to prothrombin molar

ratio of one to three (data not shown). Therefore, all RP

S19 molecules in plasma are presumably in the complex.

The fact that one prothrombin molecule of 100 in

plasma forms a complex with RP S19 indicates the irrele-

vance of the complex to prothrombin biology. However,

it must be important from the side of RP S19, which

would be filtered out at renal glomeruli if present in the

free form because of its small molecular size. The com-

plex must also be advantageous for RP S19 from the

perspective of oligomerisation by factor XIIIa, which

should have been activated by thrombin. We assumes

that local Ca2+ concentration would be somehow

increased beside activated platelets at blood coagulation

site thereby the RP S19–prothrombin complex would be

solved.

In the warfarin-treated patients, the plasma RP S19

concentration was significantly low. It is an experience of

vasculo-cardiologists that resorption of subcutaneous

haemorrhagic plaque or haematoma is significantly slow.

We assume that it would be because of a decrement

capacity of the RP S19 oligomer recruiting mono-

cytes ⁄macrophages to the coagulum.

Conclusion

In the circulating blood, RP S19 is present and forms a

complex with prothrombin. This likely explains why a

protein as small as RP S19 can be present in plasma at a

steady state.

Acknowledgements

This work was supported by a Grant-in Aid for Scientific

Research (C) (Nishiura; KAKENHI 22590362) (Semba;

KAKENHI 22590348) (Yamamoto; KAKENHI

21590441) from the Ministry of Education, Culture,

Sports, Science, and Technology, Japan.

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