SMTP (Stachybotrys microspora triprenyl phenol) enhances clot clearance in a pulmonary embolism model in rats.

0.73 MB·English

by  Hu, Weimin

Huetal.ThrombosisJournal2012,10:2
http://www.thrombosisjournal.com/content/10/1/2
ORIGINAL BASIC RESEARCH Open Access
Stachybotrys microspora
SMTP ( triprenyl phenol)
enhances clot clearance in a pulmonary
embolism model in rats
Weimin Hu1, Ritsuko Narasaki1, Naoko Nishimura2 and Keiji Hasumi1,2*
Abstract
Background: Stachybotrys microspora triprenyl phenols (SMTPs) are a novel family of small molecules that enhance
both activation and fibrin-binding of plasminogen. While their effects on fibrinolysis have been characterized in
vitro, little is known about their activity in vivo with respect to plasminogen activation and blood clot clearance.
Results: To select a potent SMTP congener for the evaluation of its action in vitro and in vivo, we tested several
SMTP congeners with distinct structural properties for their effects on plasminogen activation. As a result, SMTP-7
(orniplabin) was found to have distinguished activity. Several lines of biochemical evidence supported the idea that
SMTP-7 acted as a plasminogen modulator. SMTP-7 elevated plasma level of plasmin-a -antiplasmin complex, an
2
index of plasmin formation in vivo, 1.5-fold in mice after the intravenous injections at doses of 5 and 10 mg kg-1. In
a rat pulmonary embolism model, SMTP-7 (5 mg kg-1) enhanced the rate of clot clearance ~3-fold in the absence
of exogenous plasminogen activator. Clot clearance was enhanced further by 5 mg kg-1 of SMTP-7 in combination
with single-chain urokinase-type plasminogen activator.
Conclusions: Our results show that SMTP-7 is a superior plasminogen modulator among the SMTP family
compounds and suggest that the agent enhances plasmin generation in vivo, leading to clearance of thrombi in a
model of pulmonary embolism.
Keywords: plasminogen, fibrinolysis, thrombolysis, thromboembolism
Background Lys62)inthePANdomainandalysinebindingsiteinthe
The plasminogen/plasmin system plays a central role in fifthkringledomain[4,5].Thetightconformationofthe
bloodclotlysis[1,2].Plasminogenisasingle-chainglyco- plasminogenmoleculeattenuatesitsactivationandinter-
protein consisting of an N-terminal PAN domain, five actionwithfibrinandcellularreceptors[3,6].Lysineana-
homologous kringle domains, and a trypsin-like serine logssuchas6-aminohexanoicacidbindtolysinebinding
proteasedomain.Plasminogenisconvertedtotheactive sitesinkringledomainsandinducealarge-scaleconfor-
enzyme plasmin by the specific cleavage of the Arg561- mationalchangeinplasminogen[7,8],facilitatingitsacti-
Val562 bond by tissue-type plasminogen activator(t-PA) vation by plasminogen activators. However, lysine
or urokinase-type plasminogen activator (u-PA). The analogsinhibitplasminogenbindingtofibrinorcellsur-
binding of plasminogen to fibrin and cell surfaces, facereceptorsand,therefore,inhibitfibrinolysis.Fibrinis
mediated by kringle domains in plasminogen, localizes not only a substrate of plasmin but also a cofactor of
fibrinolyticactivityonfibrinandcellsurfaces[3].Plasmi- plasminogen activation. Upon binding to fibrin, plasmi-
nogenadoptsa tightconformationdue toanintramole- nogenundergoesconformationalchangetobecomesus-
cular interaction between a lysine residue (Lys50 and/or ceptible to activation [9]. Further, partial degradation of
fibrin by plasmin generatesC-terminal lysines, resulting
in the accumulation of more plasminogen to degrading
*Correspondence:[email protected] fibrintoacceleratefibrinolysis.
1DepartmentofAppliedBiologicalScience,TokyoNokoUniversity,3-5-8
Saiwaicho,Fuchu,Tokyo183-8509,Japan
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Huetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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Thus, conformational regulation of plasminogen is While SMTP-7 moderately elevated the activity of plas-
implicatedinthelocalizationandactivationofplasmino- min (Figure 2D), the magnitude (~3-fold) was smaller
gen.Thismechanismsuggeststhatpharmacologicalmod- than that of plasminogen activation (~100-fold). In size-
ulation of plasminogen conformation will regulate local exclusion chromatography, the molecular elution time
plasminproduction[10].Werecentlyidentifiedaseriesof of plasminogen was slightly shortened in the presence of
small-molecule modulators of plasminogen activation. SMTP-7 (Figure 2E), suggesting that an increase in
These compounds, which are structurally unrelated to apparent molecular volume was induced by SMTP-7.
lysine,enhanceplasminogenactivator-catalyzedplasmino- Taken together, these properties of SMTP-7 are consis-
gen activation. Unlike lysine analogs, these modulators tent with the idea of a plasminogen modulator, which
increaseplasminogen-fibrinbinding[11].SMTPs(Stachy- increases plasminogen activation by affecting the confor-
botrysmicrosporatriprenylphenols)andstaplabinarethe mational status of plasminogen [10].
representatives of the “nonlysine-analog” plasminogen
modulators.[11-20].Inthispaper,weshowthatSMTP-7 SMTP-7 increases plasma Pm-AP level in mice
(orniplabin),oneofthemostpotentcongeners,increases To assess plasminogen activation in vivo, we determined
plasmingenerationinvivoandpromotesclotclearancein the level of Pm-AP as an index of plasmin generation.
aratpulmonaryembolismmodel.Theseactivitiesprovide Pm-AP was determined by fibrinogen zymography, in
basesofthetherapeuticactivityofSMTP-7towardthrom- which human and mouse Pm-AP appeared as lysis
boticcerebralinfarction[21-23]. bands at ~140 and ~130 kDa, respectively (additional
file 2). When SMTP-7 was administered to normal mice
Results (5 and 10 mg kg-1, bolus intravenous injections), the
Activities of the SMTP congeners in vitro level of Pm-AP in plasma was significantly increased
We assessedstructure-activityrelationshipsof SMTPsto (~1.5-fold, P < 0.05) (Figures 3A and 3B). Both anti-
selectacongenertobestudiedindetail.SMTPcongeners plasminogen IgG and anti-a -antiplasmin IgG decreased
2
identified so far are roughly classified into two groups. the intensity of the lysis band, supporting that the lysis
Oneisthesingle-unitcongener,consistingofthecoretri- band represented Pm-AP (Figure 3C and additional file
prenylphenolunitandanN-linkedside-chain,whichisa 2). These results suggest that SMTP-7 enhances plasmin
side-chainofanaminoacid(Figure1A).Theothergroup generation in vivo.
is the two-unit congener, consisting of two core units
bridgedbyadiamine(Figure1A).Amongthe8congeners SMTP-7 enhances clot clearance in a rat pulmonary
tested, two-unit congeners (SMTP-7, -7D, -8, and -8D) embolism model
were more active than single-unitcongeners(SMTP-4D, SMTP-7 was evaluated further in a rat pulmonary
-5D,-6,and-6D)inenhancingu-PA-catalyzedplasmino- embolism model, in which animals were injected with
gen activation (Figure 1B). a-Tocopherol, which has a small particles of 125I-labeled plasma clots. The clots
structureresembling the core unitofSMTP (Figure1A), predominantly distributed over the lungs, and its clear-
wasinactive(Figure 1B). Tocharacterizefurther,3addi- ance was monitored continually as the decay of radioac-
tionaltwo-unitcongeners,SMTP-9, -30, and-31 (Figure tivity in the thorax. Based on the effects of SMTP-7 on
1A),weresynthesized(additionalfile1).SMTP-9and-31, the Pm-AP accumulation in mice (Figure 3), the dose of
whichhadtwocarboxylgroups,werelessactivethancon- 5 mg kg-1 was used. In control animals given saline
geners with one carboxyl group (Figure 1B). SMTP-30, alone, the clearance of 125I-plasma clots occurred slowly
which had no carboxyl group, was essentially inactive (6.4 ± 2.8% per 20 min) (Figure 4A). The treatment with
(Figure 1B). Thus, the number of carboxyl group affects SMTP-7 (5 mg kg-1, bolus intravenous injection) signifi-
theactivityoftwo-unitSMTPcongeners.Basedonthese cantly increased the rate of clot clearance (19.8 ± 2.4%
results,SMTP-7wasselectedfordetailedcharacterization. per 20 min) (Figure 4A). This increase was comparable
to that (19.8 ± 4.1% per 20 min) brought by scu-PA
Characterization of the action of SMTP-7 in vitro (250 U kg-1, bolus intravenous injection) (Figure 4B).
SMTP-7 enhanced t-PA-catalyzed plasminogen activa- The combination of SMTP-7 (5 mg kg-1) and scu-PA
tion as well as the activation catalyzed by u-PA (Figure (250 U kg-1) increased the clot clearance rate to 42.3 ±
2A). In the kinetic determinations of u-PA-catalyzed 4.2% per 20 min (Figure 4B).
plasminogen activation, SMTP-7 markedly increased
Vmax and slightly decreased Km, resulting in a large SMTP-7 does not increase bleeding in normal mice
increase in k (Figure 2B). The increase in plasmin Effects of SMTP-7 on bleeding and rebleeding were
cat
generation was confirmed by SDS-polyacrylamide gel assessedbyatailamputationassayinnormalmice.SMTP-
electrophoresis (Figure 2C). Amidolytic activities of u- 7wastestedatthepharmacologicaldose(5mgkg-1)anda
PA and t-PA were not affected by SMTP-7 (Figure 2D). higherdose(30mgkg-1).Neitherastatisticallysignificant
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A
B Compound (ECM1)0 (Efomldax) (Emax/EPCo1t0e, nfocyld M-1)
300
7D SMTP-4D 167 29 0.17
on 100 9 SMTP-5D 181 28 0.15
atiol) 6 6D SMTP-6 153 44 0.29
ogen activve to contr 1300 7 8 8D 43D1 5D SSSMMMTTTPPP---767DD 1677514 11045269 102...512619
Plasmin(relati 13 30 Toco SSMMTTPP--88D 17082 113430 11..7308
SMTP-9 179 66 0.37
SMTP-30 NAa 1
0
SMTP-31 NAa 6
0 50 100 150 200 250 300
-Tocopherol NAa 1
Concentration( M)
0 0.5 1 1.5 2
Figure1Structure-activityrelationshipsofSMTPcongeners.(A)StructuresofSMTPcongenerstested.ThecoreunitofSMTPcongenersis
representedbyR.(B)TheactivationofplasminogenwasassayedinthepresenceoftheindicatedconcentrationsofSMTPcongeners.Number
incirclerepresentstheSMTPnumber.Toco,a-tocopherol.Eachvaluerepresentsthemean±SDfromtriplicatedeterminations.Relativeto
controlvaluesareshown.Summaryoftheresultsareshownintherightpanel,whereEC representstheconcentration(μM)ofSMTPthat
10
causes10-foldenhancementofplasminogenactivationandE themaximumleveloftheenhancement(foldincreaseinplasminogen
max
activationcomparedwithcontrol).E andthereciprocalofEC areindependentindexesthatrepresentthepotencyofthecompound.The
max 10
ratioE /EC representscomprehensivepotency.NA,notavailable(duetothatenhancementdidnotreach10-foldatconcentrationstested).
max 10
prolongationofbleedingtimenoranincreaseinbleeding analog with no carboxylic acid in the N-linked side-
volumewasobservedatbothdoses(additionalfile3). chain is essentially inactive. Thus, the importance of
both the core triprenyl phenol unit and a carboxylic
Discussion acid group in the N-linked side-chain is demonstrated.
In this study, we demonstrate that SMTP-7, one of the The action of SMTP-7 to enhance plasminogen activa-
two-unit SMTP congeners, is a plasminogen modulator tion conforms to the idea of zymogen modulators [10]
effective in plasminogen activation and clot clearance. based on the following observations: (i) SMTP-7 does
Among the SMTP congeners tested, the two-unit conge- not affect the enzymatic activity of u-PA and t-PA but
ners are more active than the single-unit congeners. Of increases plasminogen activation catalyzed by u-PA and
the two-unit congeners tested, analogs with two car- t-PA; (ii) SMTP-7 alters conformational status of plas-
boxylic acid groups in the N-linked side-chain are minogen, as evidenced by a change in molecular elution
weaker than those with one carboxylic acid group. The time on analytical size-exclusion chromatography.
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A B C
D E
Figure2CharacterizationofSMTP-7invitro.(A)Effectsonplasminogenactivationbyt-PAandu-PA.Plasminogenactivationwasassayed
usingt-PAoru-PAinthepresenceoftheindicatedconcentrationsofSMTP-7.(B)Kineticmeasurementsofu-PA-catalyzedplasminogen
activationinthepresenceofSMTP-7(100μM).Parametersshownwereobtainedfromtriplicatedeterminations.(C)Effectonplasminogen
conversiontoplasmin.u-PA-catalyzed125I-plasminogenconversiontoplasminwasassayedinthepresenceorabsenceofSMTP-7(100μM).
Positionsofplasminogen(Plg)andA-andB-chainsofplasminareshown.(D)Effectsonamidolyticactivitiesofplasmin,t-PA,andu-PA.Activity
ofeachenzymewasdeterminedusingafluorogenicsubstrateinthepresenceoftheindicatedconcentrationsofSMTP-7.(E)Size-exclusion
chromatography.Alexa488-labeledplasminogenwaselutedintheabsenceorpresenceofSMTP-7(120μM).**,P<0.05byStudent’st-test.
ErrorbarsinpanelsA,B,D,andErepresentSDfromtriplicatedeterminations.
SMTP-7 enhances the conversion of plasminogen to the with a -antiplasmin in a purified system (unpublished
2
two-chain plasmin. This activity, along with the aug- observation). Thus, the finding that SMTP-7 increases
mentation of the catalytic activity of plasmin (~3-fold), plasma level of Pm-AP in mice suggests that SMTP-7
results in the apparent increase (up to 100-fold) in plas- increasesplasminogenactivationinvivo.SMTP-7iseffec-
minogen activation assessed by using a chromogenic tiveinpromotingtheclotclearanceintheratpulmonary
substrate. Kinetic data demonstrate that SMTP-7 embolism model. Compared with the spontaneous clot
increases V of u-PA-catalyzed plasminogen activation clearance rate,a 3-fold increase inthe rate is brought by
max
with a slight decrease in Km, suggesting that faster turn- theadministrationofSMTP-7at5mgkg-1,adosethatis
over of the enzyme is allowed on the SMTP-7-modu- effective in elevating plasma Pm-AP level. The plasma
lated substrate compared with a native substrate. concentrationofSMTP-7atthisdosejustaftertheintra-
TheadministrationofSMTP-7tonormalmiceresulted venous injection is expected to be ~100 μM. The rate of
inanincreaseinthelevelofPm-APinplasma.Sinceplas- clotclearanceaftertheSMTP-7injectioniscomparableto
mingeneratedinvivoisrapidlyinactivatedbya -antiplas- theratebroughtbytheinjectionofscu-PAat250Ukg-1.
2
min to afford Pm-AP, the level of Pm-AP represents the SMTP-7 synergistically enhances the clot clearance in
generationofplasmininvivo[24].SMTP-7doesnotaffect combination with scu-PA. Thus, these properties of the
the rate of Pm-AP formation when plasminis incubated SMTP-7actioninvivoconformtoitsactivityinvitro.
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A B
kDa
130
C
kDa
130
Figure3EffectofSMTP-7onplasmalevelofPm-APinnormalmice.MicewereintravenouslyinjectedwithSMTP-7atabolusdoseof5
mgkg-1(n=5)or10mgkg-1(n=5).After60min,bloodwasdrawntodeterminePm-APlevelsinplasmabyfibrinogenzymography.Control
animals(n=5)receivedsaline.(A)Representativezymogram.Plasmasfrom5animalsineachgroupwerecombinedandaliquots(2μl)ofthe
mixturesweresubjectedtozymography.HumanPm-AP(hPm-AP,1.7ng)wasresolvedasthestandard.(B)Thesummaryofthequantificationof
Pm-AP.Plasma(2μl)fromeachofthefivemicewasindependentlysubjectedtozymographicanalysis.Datawerecalibratedbycomparingthe
intensityofeachlysisbandwiththatofthestandardhumanPm-AP.Themean+SDofthedataobtainedfromeachanimalisshown.*,P<
0.05and**,P<0.01comparedwithcontrolbytheDunnett’smultiplecomparisontest.(C)Authenticityconfirmation.Themixtureofplasmain
the5mgkg-1SMTP-7groupwastreatedwithanti-plasminogenIgG(Anti-Plg),anti-a-antiplasminIgG(Anti-AP),orbufferbeforethe
2
zymographicdeterminationofPm-AP.
One possible drawback of SMTP-7 may be systemic inflammation and neuronal cell death by directly inter-
hyperfibrinolysis and following hemorrhage as that acting with low density lipoprotein receptor-related pro-
observed in disseminated intravascular coagulation. tein and N-methyl-D-aspartate receptor [28,29]. In
However, our preliminary studies suggest that these are consistent with these observations, hemorrhagic trans-
unlikely to occur at a pharmacological dose (<10 mg/ formation is reduced by SMTP-7, while it is increased
kg), since bleeding time and rebleeding volume in a by t-PA [22]. Thus, SMTP-7 can be a unique agent that
mouse tail amputation assay were not changed statisti- aid in the treatment of thrombotic complications.
cally by the SMTP-7 administration at the pharmacolo-
gical dose (5 mg kg-1) and a higher dose (30 mg kg-1) Conclusion
(additional file 3). This moderate effect of SMTP-7 on Our results show that SMTP-7 is a superior plasmino-
hemorrhage can be explained by that SMTP-7 is a gen modulator among the SMTP family compounds and
zymogen modulator and its action depends on endogen- suggest that the agent enhances plasmin generation in
ous plasminogen activators, the availability of which is vivo, leading to clearance of thrombi in a model of pul-
physiologically regulated. monary embolism. These results provide mechanistic
Plasminogen activators such as recombinant t-PA are bases for the recent findings that SMTP-7 has a pro-
important drugs treating acute thrombotic stroke and found activity in treating thrombotic stroke in animal
myocardial infarction [25,26]. Recent investigations have models.
demonstrated that SMTP-7 is quite effective in amelior-
ating thrombotic stroke in models of mouse and gerbil Methods
[21-23]. The data in this paper provide bases of the Materials
therapeutic activity of SMTP-7 in these models. It is of Human native plasminogen was isolated by lysine-
note that SMTP-7 is effective in the treatment after 3-6 Sepharose affinity chromatography from frozen citrated
h of the thrombotic stroke induction, whereas t-PA is plasma. The source of the following reagents were: sin-
®
ineffective when treated after 3 h [21,22]. The difference gle-chain u-PA (scu-PA; Thrombolyse ) from Mitsu-
in the therapeutic efficacy between SMTP-7 and t-PA is bishi Pharma (Osaka, Japan); high molecular weight u-
partly explained as that SMTP-7 reduces inflammatory PA (1.47 × 105 IU mg-1) from JCR Pharmaceuticals
and oxidative responses associated with thrombotic (Kobe, Japan); two-chain t-PA (7.0 × 105 IU mg-1) from
ischemia [22,23,27]. t-PA is reported to induce cerebral Biopool (Umeå, Sweden); sheep anti-mouse plasminogen
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A
**
B
**
Figure4EffectofSMTP-7onclotclearanceinaratpulmonaryembolismmodel.Ratswereinjectedintravenouslywith125I-labeledplasma
clots.TheclearanceoftheclotswasmonitoredbeforeandafterthetreatmentswithSMTP-7and/orscu-PA.(A)SMTP-7(5mgkg-1;n=6)was
administeredintravenously~20minaftertheinjectionof125I-labeledplasmaclot.Controlanimals(n=6)receivedsaline.Radioactivityoverthe
thoraxwasmonitored(oneofthe6tracesisshownintheleftpanel)andthefractionalchangeinradioactivitywassummarized(rightpanel).(B)
SMTP-7(5mgkg-1),followedby250Ukg-1ofscu-PA(Thrombolyse®),wasadministeredintravenously~20minaftertheclotinjection(n=6).
OneunitofThrombolyse®iscomparableto~60IUoftcu-PAinfibrinolyticactivity.Incontrolanimals(n=6),scu-PAwasgiven~20minafter
theclotinjection.Arrowdenotestimeatwhichtheadministrationwasmade.**,P<0.01comparedwithcontrolbytheStudent’st-testwith
normallydistributedvariables.
IgG from Haematologic Technologies (Essex Junction, -6D, -7, -7D, -8, and -8D were isolated as described pre-
VT, USA); rabbit anti-mouse a -antiplasmin IgG from viously [15,17]. SMTP-9, -30, and -31 were originally
2
Merdian Life Science (Saco, ME, USA); human fibrino- isolated as described in additional file 1. In experiments
gen and human a-thrombin from Sigma (St. Louis, MO, in vitro, SMTPs were dissolved directly in buffer A (50
USA); carrier-free Na125I from Amersham. Radioiodina- mM Tris-HCl, 100 mM NaCl, and 0.01% Tween 80, pH
tion of fibrinogen and plasminogen was performed 7.4). In animal experiments, SMTP-7 was dissolved in
using the iodine monochloride method. Upon trichlor- saline by adjusting pH ~9 with dilute NaOH.
oacetic acid treatment, more than 95% of radioactivity
in the fibrinogen and plasminogen preparations precipi- Assay for plasminogen activation
tated with protein. When treated with thrombin, Plasminogen activation was determined by measuring
approximately 70% of the radioactivity in 125I-fibrinogen the initial velocity of plasmin generation using H-Val-
was incorporated into the resulting clots. Leu-Lys-p-nitroanilide (Bachem, Bubendorf, Switzer-
land), a chromogenic substrate for plasmin. A reaction
SMTP congeners mixture consisting of 50 nM plasminogen, 50 IU ml-1 u-
All the SMTP congeners used in this study were pro- PA (or 200 IU ml-1 t-PA) and 0.1 mM of the substrate
duced by S. microspora IFO 30018. SMTP-4D, -5D, -6, in 50 μl of buffer A was incubated in a well of a 96-well
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microplate at 37°C for up to 40 min. Absorbance at 405 pH 6.8, 4% (w/v) SDS, 20% (w/v) glycerol, and 0.004%
nm was measured with an interval of 1 to 2 min. From (w/v) bromophenol blue). In some experiments, plasma
the slope of the plots of A nm versus t2, the initial was pretreated with ant-plasminogen IgG (0.6 mg ml-1)
405
velocity of plasmin generation was calculated. In the or anti-a -antiplasmin IgG (3 mg ml-1) for 30 min at
2
experiment to determine kinetic parameters, assays were room temperature before mixing with buffer B. The
performed with varying concentrations of plasminogen mixture (equivalent to 1-2 μl of plasma) was resolved on
(0.5-2 μM) and a fixed concentration of u-PA (50 IU nonreduced SDS-polyacrylamide gel electrophoresis on a
ml-1) in the presence or absence of SMTP-7 (100 μM). 7.5% gel containing fibrinogen (2 mg ml-1) at 4°C. As
Since SMTP-7 at 100 μM enhanced plasmin activity the standard for the calibration of Pm-AP in plasma
toward H-Val-Leu-Lys-p-nitroanilide by 3.26-fold (see samples, 1.7 ng of human Pm-AP (prepared by incubat-
Figure 2D), this factor was taken into account in the ing 120 nM human plasmin and 600 nM human a -
2
process of the calculation of plasmin generation antiplasmin for 30 min at 37°C in buffer A) was resolved
velocities. on the same gel. After electrophoresis, the gel was cut at
Plasminogen activation was alternatively assayed by the position of ~90 kDa, and the upper half was washed
determining the conversion to the two-chain form. 125I- with Triton X-100 (2.5 %, w/v) and incubated in 50 mM
Plasminogen (100 nM) was incubated with u-PA (50 IU Tris-HCl, pH 8.3, and 100 mM glycine for 60 h at 37°C.
ml-1) and aprotinin (100 kallikrein inhibitor units ml-1) (The lower half gave a strong lysis band at ~70 kDa,
in buffer A at 37°C for 30 min. The mixture was possibly due to plasma kallikrein) Gels were stained
resolved on SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue R-250. The lysis zones
under reducing conditions. The gel was stained with due to protease activities appeared as unstained bands
Coomassie Brilliant Blue R-250. on a blue background. Human Pm-AP gave a lysis band
at ~140 kDa, while the mouse counterpart afforded a
Assay for activities of plasmin, u-PA, and t-PA band at ~130 kDa (additional file 2). The scanned image
Amidolytic activities of plasmin (10 nM), u-PA (1 IU of the stained gel was reversed for presentations. The
ml-1), and t-PA (2000 IU ml-1) were determined at 37°C band intensity was determined using Scion image. The
in buffer A using 10 μM of H-Val-Leu-Lys-7-amino-4- amounts of Pm-AP in test samples were calibrated by
methylcoumarin, t-butyloxycarbonyl-Glu-Gly-Arg-7- comparing intensities of lysis bands of samples with that
amino-4-methylcoumarin, and succinyl-Phe-Ser-Arg-7- of the standard, and data were expressed as human Pm-
amino-4-methylcoumarin, respectively. AP equivalent (nM in plasma).
Size-exclusion chromatography Preparation of 125I-plasma clot particles
Size-exclusion chromatography was performed using a Platelet-poor plasma from male Wistar rats was mixed
TSK-Gel G-3000SW column (7.5 × 600 mm, TOSOH, with 125I-fibrinogen (139 μg ml-1, ~2.5 MBq) anda -
Tokyo, Japan) equilibrated with buffer A in the presence thrombin (1 IU ml-1) in the presence of 44 mM CaCl .
2
or absence of SMTP-7 (120 μM). Plasminogen labeled After incubation at 37°C for 120 min, the resulting
®
with Alexa Fluor 488 (Molecular Probes, Eugene, OR, plasma clot was washed thrice with saline and powdered
USA) (10 μg) was resolved at room temperature at a in a mortar under liquid nitrogen, followed by homoge-
rate of 1 ml/min. The elution was monitored using a nization 4 strokes in 2.7 ml saline using a Potter Elveh-
fluorescence detector with an excitation at 495 nm and jem homogenizer with a Teflon pestle (13-mm in
an emission at 520 nm. diameter). The suspension was left at room temperature
for 30 min, and the resulting precipitates were homoge-
Animal Experiments nized again. This operation was repeated once more.
All of the animal protocols were approved by the insti- The combined homogenates were settled for 30 min,
tutional animal care committee of Tokyo Noko Univer- and the resulting supernatant was centrifuged at 20 × g
sity. Male Wistar rats and male ICR mice were obtained for 3 min to obtain pellet consisting of clot particles of
from Japan SLC (Hamamatsu). 10-100 μm in diameter.
Determination of plasmin-a -antiplasmin complex (Pm- Measurement of clot clearance in the lungs
2
AP) Male Wistar rats weighing ~120 g were kept at 22°C
Male ICR mice (7 weeks of age) received intravenous with normal chaw for 1-7 days before the use in experi-
SMTP-7 or saline. After 60 min, blood was collected ments. Rats were anesthetized with urethane and chlora-
from inferior vena cava in 13 mM sodium citrate. lose (750 and 65 mg kg-1, respectively, i.p.), and a probe
Plasma was rapidly prepared by centrifugation and (equipped with a 10-mm collimator) of a model TCS-
mixed with one volume of buffer B (125 mM Tris-HCl, 163 NaI scintillation survey monitor (Aloka, Tokyo,
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Japan) was placed above the thorax. A suspension of
Additionalfile3:SupplementaryFigure2:Bleedingandrebleeding
125I-plasma clot particles (75 μl kg-1; ~1.2 × 107 Bq per followingSMTP-7administrationinmice.(A)Bleedingtimeinmice
animal) was injected simultaneously with heparin (165 wasdeterminedafteranintravenousadministrationof5%mannitol
units kg-1) and NaI (3.3 mg kg-1) into a caudal vein. The (Control)orSMTP-7(5and30mgkg-1)dissolvedinthevehicle.(B)
Followingthedeterminationofbleedingtime,themiceweresubjected
125I-plasma clots predominantly distributed over the tothemeasurementofrebleeding.Themean+SD(n=7forcontrol
lungs [30], and radioactivity over the thorax was mea- and5mgkg-1groupsandn=5for30mgkg-1group)wasshown.
Therewasnostatisticaldifferencebetweenanygroupswithrespectto
suredcontinually.Salinealone(0.5mlperanimal),saline
bothbleedingtimeandrebleedingvolumeasassessedbytheTukey-
containing SMTP-7(5mgkg-1),scu-PA (250Ukg-1),or Kramertest.
bothSMTP-7andscu-PAwasintravenouslyinjected~20
minaftertheembolization.Themonitoringofradioactiv-
ity was continued further for ~20 min. The fractional
Listofabbreviations
change in radioactivity during 20 min after the treat-
t-PA:tissue-typeplasminogenactivator;u-PA:urokinase-typeplasminogen
mentsrepresentedtherateofclotdissolution. activator;scu-PA:single-chainurokinase-typeplasminogenactivator;Pm-AP:
plasmin-α-antiplasmincomplex.
2
Bleeding and rebleeding in mice Acknowledgements
The measurement of bleeding time and rebleeding WethankKeikoHasegawaandHarukiKoideforthepreparationofSMTP
volume (secondary oozing from the bleeding time compounds.Bloodplasmafortheisolationofplasminogenwasdonatedby
theJapaneseRedCrossSociety,Tachikawa.Thisstudywasfinancially
wounds) were performed using male ICR mice (6 weeks
supportedbygrantsfromtheJapanSocietyforthePromotionofScience,
of age) as described previously [31]. Briefly, mice were theJapanScienceandTechnologyAgency,andtheNewEnergyand
anesthetized with 60 mg kg-1 pentobarbital intraperito- IndustrialTechnologyDevelopmentOrganization,Japan.
neally and given bolus injection of 5% mannitol (5 ml
Authordetails
kg-1) or SMTP-7 (5 and 30 mg kg-1) in 5% mannitol via 1DepartmentofAppliedBiologicalScience,TokyoNokoUniversity,3-5-8
a tail vein. Five minutes after the administration, a 5- Saiwaicho,Fuchu,Tokyo183-8509,Japan.2ResearchandDevelopment
Division,TMSCo.,Ltd.,1-32-1-102Fuchucho,Fuchu,Tokyo183-0055,Japan.
mm tail segment was amputated with a razor blade. The
tail was immersed immediately in prewarmed saline at Authors’contributions
37°C, and the time required to stop visual spontaneous WHcarriedouttheanimalstudies.RNcarriedoutthebiochemicalstudies.
NNperformedtheexvivostudiesandthebleedingassays.KHparticipated
bleeding was determined. To evaluate rebleeding, the
inthedesignandcoordinationofthestudyanddraftedthemanuscript.All
tail was then immersed in another 4-mL prewarmed sal- authorsreadandapprovedthefinalmanuscript.
ine (37°C) containing 14 mM trisodium citrate for 60
Authors’information
min. Red blood cells were collected and lysed in water HWiscurrentlyaProfessor,WeifangMedicalUniversity,Shangdong,China.
to measure absorbance at 490 nm, from which blood RNiscurrentlyapostdocfellowatW.M.KeckCenterforTransgene
loss by rebleeding was calculated. Research,UniversityofNotreDame,IN,USA.NNisaresearchscientistof
TMSCo.,Ltd.KHisaProfessor,DepartmentofAppliedBiologicalScience,
TokyoNokoUniversity,andPresidentofTMSCo.,Ltd.
Additional material
Competinginterests
Theauthorsdeclarethattheyhavenocompetinginterests.
Additionalfile1:SupplementaryMethodsandSupplementary
Table1. Received:20August2011 Accepted:9January2012
Additionalfile2:SupplementaryFigure1:Thebasesofthe Published:9January2012
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