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doi:10.1182/blood-2010-02-270918
Prepublished online Jul 15, 2010;2010 116: 3981-3989
Gruber and David Gailani András White-Adams, Stephanie A. Smith, Stephen R. Hanson, Owen J. T. McCarty, Thomas Renné, Qiufang Cheng, Erik I. Tucker, Meghann S. Pine, India Sisler, Anton Matafonov, Mao-fu Sun, Tara C.
in vivo A role for factor XIIa m ediated factor XI activation in thrombus formation bloodjournal.hematologylibrary/cgi/content/full/116/19/3981Updated information and rvices can be found at:
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THROMBOSIS AND HEMOSTASIS
A role for factor XIIa–mediated factor XI activation in thrombus formation in vivo
Qiufang Cheng,1Erik I.Tucker,2Meghann S.Pine,3India Sisler,3Anton Matafonov,1Mao-fu Sun,1Tara C.White-Adams,2 Stephanie A.Smith,4Stephen R.Hanson,2Owen J.T.McCarty,2Thomas Renne´,5Andra´s Gruber,2and David Gailani1,6
1Department of Pathology,Vanderbilt University,Nashville,TN;2Division of Biomedical Engineering and Hematology/Oncology,Oregon Health and Sciences University,Portland,OR;3Department of Pediatrics,Vanderbilt University,Nashville,TN;4Department of Internal Medicine,College of Medicine,University of Illinois at Urbana-Champaign,Urbana,IL;5Department of Molecular Medicine and Surgery,Karolinska Institutet,Stockholm,Sweden;and6Department of Medicine,Vanderbilt University,Nashville,TN
Mice lacking factor XII(fXII)or factor XI (fXI)are resistant to experimentally–induced thrombosis,suggesting fXIIa ac-tivation of fXI contributes to thrombus formation in vivo.It is not clear whether this reaction has relevance for thrombo-sis in primates.In2carotid artery injury models(FeCl3and Ro Bengal/lar), fXII-deficient mice are more resistant to thrombosis than fXI-or factor IX(fIX)–deficient mice,raising the possibility that fXII and fXI function in distinct pathways.Antibody14E11binds fXI from a variety of
mammals and interferes with fXI activa-
tion by fXIIa in vitro.In mice,14E11pre-
vented arterial occlusion induced by FeCl3
to a similar degree to total fXI deficiency.
14E11also had a modest beneficial effect
in a tissue factor–induced pulmonary em-
bolism model,indicating fXI and fXII con-
tribute to thrombus formation even when
factor VIIa/tissue factor initiates thrombo-
劳务用工合同
sis.In baboons,14E11reduced platelet-
rich thrombus growth in collagen-coated
grafts inrted into an arteriovenous
shunt.The data support the hypothesis
that fXIIa-mediated fXI activation contrib-
utes to thrombus formation in rodents
入党初衷and primates.Since fXII deficiency does
not impair hemostasis,targeted inhibi-
tion of fXI activation by fXIIa may be a
uful antithrombotic strategy associ-
ated with a low risk of bleeding complica-
tions.(Blood.2010;116(19):3981-3989)
Introduction
Initiation offibrin formation by contact activation requires proteo-lytic conversion of plasma factor XII(fXII)to the protea factor XIIa(fXIIa)on a surface.1-3FXIIa activates the next zymogen in the coagulation cascade,factor XI(fXI),to factor XIa(fXIa), which in turn converts factor IX(fIX)to factor IXa(fIXa).This ries of reactions,referred to as the intrinsic pathway of coagula-tion,drives thrombin generation andfibrin formation in the activated partial thromboplastin time(aPTT)assay ud by clinical laboratories.A role for fIX in hemostasis is not in question,as its deficiency caus the vere bleeding disorder hemophilia B. However,the importance of the intrinsic pathway,as a whole,to clot formation and stability at a site of injury is probably limited,as fXII deficiency is not associated with abnormal bleeding,1,2and fXI-deficient patients have a variable hemorrhagic disorder with milder symptoms than hemophiliacs.2,4Current models of throm-bin generation address the phenotypic differences by incorporat-ing additional mechanisms for protea activation.Thus,fIX is activated by the factor VIIa/tissue factor complex in addition to fXIa,3,5while fXI can be activated by thrombin.3,6
Mice lacking fXII,like their human counterparts,do not have a demonstrable bleeding abnormality,7supporting the premi that fXIIa activation of fXI is not required for hemostasis.8Given this,it was surprising to obrve that mice lacking fXII9or fXI10were resistant to arterial thrombotic oc
爱学习教师端clusion.While this suggested contact activation might play an important role in pathologic coagulation,if not hemostasis,it was not clear that fXIIa was mediating its prothrombotic effect through fXI.We developed an antibody against mou fXI(14E11)that prolongs time to clot formation in plasma by interfering with fXI activation by fXIIa. Bad on the phenotypes of fXII-and fXI-deficient mice,we postulated that14E11would inhibit thrombus formation in vivo, despite lectively interfering with a reaction not required for hemostasis.Here we show that14E11affects fXIIa-dependent coagulation in plasmas from multiple species and report on its effects in mou and primate thrombosis models.
Methods
Reagents
Pooled normal and fXII-deficient plasmas were from George King Bio-Medical.fIX,fXI,and fXIa were from Haematologic Technologies.fXIIa, high-molecular-weight kininogen(HK),and corn trypsin inhibitor(CTI) were from Enzyme Rearch Laboratories.Z-Gly-Gly-Arg-AMC was from Bachem.Dioleoylphosphatidylcholine:dioleoylphosphatidylrine(7:3w/w) was from Avanti Polar Lipids.S-2366was from Diapharma.Bovine rum albumin(BSA),rabbit brain cephalin(RBC),kaolin,and O-phenylenedi-amine(OPD)were from Sigma-Aldrich.
Monoclonal antibodiesit的宾格
FXI-deficient Balb-C mice were immunized with25g of recombinant mou fXI11diluted1:1with Freund adjuvant(200L total)by intraperito-neal injection.Two25-g booster dos in incomplete Freund adjuvant were given3and7weeks later,and hybridomas were generated by standard protocols.Media were tested for capacity to recognize mou fXI by enzyme-linked immunosorbent assay and to prolong the aPTT of mou
Submitted February21,2010;accepted June28,2010.Prepublished online as Blood First Edition paper,July15,2010;DOI10.1182/blood-2010-02-270918. The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment.Therefore,and solely to indicate this fact,this article is hereby marked‘‘advertiment’’in accordance with18USC ction1734.
©2010by The American Society of Hematology
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and human plasmas.Clones of interest were subcloned twice by limiting dilution.Clone 14E11was expanded in a CL1000bioreactor (Integra Biosciences),and immunoglobulin G (IgG)was purified by cation ex-change and thiophilic agaro chromatography.14E11was biotinylated using an EZ-link sulfo-NHS-biotinylation kit (Thermo Scientific).Genera-tion and characterization of monoclonal IgG O1A6,which binds to the A3domain of human fXI and interferes with activation of factor IX by fXIa,has been described.6,12
14E11binding to fXI and fXIa
FXI or fXIa (2g/mL,100L/well)in 50mM Na 2CO 3pH 9.6was incubated overnight at 4°C in Immulon 2HB microtiter plates (Thermo Scientific).Wells were blocked with 150L of phosphate-buffered saline (PBS)with 2%BSA for 1hour at room temperature (RT).One hundred microliters of bio
tinylated 14E11(100to 10Ϫ7M)in 90mM HEPES (N-2-hydroxyethylpiperazine-N Ј-2-ethanesulfonic acid)pH 7.2,100mM NaCl,0.1%BSA,0.1%Tween-20(HBS)were added,with incubation for 90minutes at RT.After washing with PBS-0.1%Tween-20(PBST),100L of strepavidin-horradish peroxida (HRP;Thermo Scientific,1:8000dilution in HBS)was added,with incubation at RT for 90minutes.After washing,100L of substrate solution (12mL of 30mM citric acid,100mM Na 2HPO 4pH 5.0,one tablet OPD,12L of 30%H 2O 2)was added.Reactions were stopped after 10minutes with 50L of 2.5M H 2SO 4.Absorbance at 495nm was measured on a SpectroMax 340microplate reader (Molecular Devices).14E11binding site on fXI
Biotinylated 14E11was the primary antibody in Western blots of recombi-nant fXI with individual apple domains replaced with corresponding domains from human prekallikrein (PK),13,14or individual fXI apple domains attached to tissue plasminogen activator (tPA).15Detection was with strepavidin-HRP and chemiluminescence.Effect of 14E11on plasma coagulation
aPTT assays.Plasma (50L)anticoagulated with 0.38%sodium citrate was mixed with 50L of PBS (with or without 14E11[0to 320nM])and 50L of aPTT reagent (aPTT-ES;Helena Laboratories)at 37°C for 3minutes.Fifty microliters of CaCl 2(20mM)was added and time to clot formation determined on an Amelung KC4Analyzer (Sigma-Aldrich).Modified fXIa PTT.FXIa (30nM)in 25mM Tris-HCl pH 7.
4,100mM NaCl (TBS)with 0.1%BSA (TBSA)was incubated at RT for 30minutes,with or without 14E11(300nM).FXIa (65L)was mixed with 65L of fXI-deficient plasma and 65L of RBC at 37°C.After 30conds,25mM CaCl 2(65L)was added and time to clot formation measured.
Modified fXIIa PTT.65L of fXIIa (1-100nM)in TBSA was mixed with 65L of normal plasma (with or without 14E11[300nM]),and 65L of RBC at 37°C.After 30conds,25mM CaCl 2(65L)was added and time to clot formation determined.FIX activation by fXIa
FIX (100nM)was incubated with fXIa (1nM)in TBS with 5mM CaCl 2at RT with or without 200nM 14E11or O1A6.At intervals,20-L samples were placed in sodium dodecyl sulfate (SDS)sample buffer.Samples were size-fractionated on 10%nonreducing SDS-polyacrylamide gels and trans-ferred to polyvinylidene fluoride membranes.Chemiluminescent Western blotting was performed using goat anti-human fIX IgG (Affinity Biologicals).HK-fXI binding assay
Immulon 2HB microtiter plates were coated with HK (30nM)in 50mM carbonate buffer pH 9.6(100L per well)overnight at 4°C.Wells were blocked with 150L of PBS-2%BSA for 1hour at RT.Biotinylated fXI (10nM)and unlabeled 14E11(25pM to 1M)were added to the wells (total volume 100L)and incubated at RT for 90minutes.After washing with PBST,100L of strepavidin-HRP (1:8000in HBS)was added and incubated at RT for 90minutes.After washing,100L of Substrate
Solution was added.Reactions were stopped after 10minutes with 50L of
2.5M H 2SO 4,and absorbance at 495nm measured.Chromogenic assay for fXI activation
FXI (170nM)in PBS with 1mg/mL BSA was incubated at 37°C for 15minutes with or without 600nM 14E11.At time zero,fXIIa,HK,and kaolin,all in PBS with 1mg/mL BSA,were added so that final concentra-tions were fXI (85nM),fXIIa (17nM),HK (70nM),and kaolin (0.5mg/mL).At various time points,50-L of aliquots were supplemented with CTI (600nM final concentration)and then mixed with an equal volume of 1.0mM S-2366.Changes in absorbance at 405nm were measured.Thrombin generation assay
Thrombin generation was measured by following cleavage of Z-Gly-Gly-Arg-AMC (415M)in fXII-deficient plasma on a Thrombinoscope (Throm-binoscope BV),6in the prence of 14E11(300nM)or vehicle.Plasma (80L)was mixed with 20L of Tyrode buffer pH 7.4containing PC:PS vesicles (30M)and either tissue factor (2.3pM)or fXIIa (10or 100nM).CTI was added to a final concentration of 4M for reactions with tissue factor (TF).Final concentrations:5M PC:PS vesicles,0.23pM TF or 1-10nM fXIIa.Clotting was started with 20L of 20mM HEPES pH 7.4,100mM CaCl 2,6%BSA,and fluorescence was monitored (emission 460nm).Assays were run twice in triplicate,and endogenous thrombin potential (ETP)was determined with Thrombinoscope Analysis software,version 3.0.
Platelet adhesion assay
Glass coverslips were incubated with fXI,fXIa,or fibrinogen (50g/mL)for 1hour at RT.16Surfaces were blocked with denatured fatty acid–free BSA (5mg/mL)for 1hour,then incubated with vehicle,14E11or nonspecific IgG (20g/mL)for 20minutes.Purified human platelets (2ϫ107/mL)were incubated on protein-coated coverslips for 45minutes at 37°C and imaged using Ko ¨hler illuminated Nomarski differential interference contrast optics with a Zeiss 63ϫoil immersion 1.40NA plan-apochromat lens on a Zeiss Axiovert 200M microscope (Carl Zeiss).Numbers of adherent platelets was computed using ImageJ software (National Institutes of Health).16
一瞥所见
Immunoprecipitation of plasma fXI
Plasma anticoagulated with sodium citrate or EDTA was obtained from the following species:African elephant,(Loxodonta africana ),Amur tiger (Panthera tigris altaica ),baboon (Papio anubis ),cattle (Bos taurus ),chicken (Gallus gallus domesticus ),dog (Canis lupus familiaris ),domestic cat (Felis silvestris catus ),giant anteater (Myrmecophaga tridactyla ),hor (Equus ferus caballus ),llama (Lama glama ),pig (Sus scrofa domestica ),rabbit (Oryctolagus cuniculus ),and raccoon (Procyon lotor ).Fifty microli-ters of Affigel-10beads (BioRad)bound with 14E11(3mg/mL)were rocked at RT for
2hour with 500L of plasma and 500L of TBS.Beads were washed with 1mL of TBS and then eluted with 50L of SDS-nonreducing sample buffer.Eluates were size fractionated on 7.5%SDS-polyacrylamide gels,followed by Western blotting using biotinylated 14E11antibody.
Mou carotid artery thrombosis models
Procedures for mice were approved by the Institutional Animal Care and U Committee of Vanderbilt University.C57Bl/6mice deficient in fIX,17fXI,18or fXII 7were described.Mice with combined fXI and fXII deficiencies were generated from the lines.Anesthesia was with 50mg/kg intraperitoneal pentobarbital.The right common carotid artery was fitted with a Doppler flow probe (Model 0.5VB;Transonic System).14E11(1.0mg/kg)was infud into the internal jugular vein 15minutes before injury.In the ferric chloride model,10thrombus formation was induced by applying two 1ϫ1.5-mm filter papers (GB003;Schleicher &Schuell)saturated with FeCl 3(2.5%to 15%solution)to opposite sides of the artery for 3minutes,followed by rinsing with normal saline.Flow was monitored
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for 30minutes.For the lar injury model,19Ro Bengal (75mg/kg)was infud through the internal jugular vein,and the carotid artery was illuminated with a 1.5-mW 540-nm lar (Melles Griot)positioned 6cm from the vesl.Flow was monitored for 120minutes.Mou pulmonary embolism model
Mice anesthetized with 50mg/kg intraperitoneal pentobarbital were fitted with an MP100transducer (AD Instruments)to monitor respiration,and 14E11(1.0mg/kg)or vehicle was infud into the internal jugular vein.After 15minutes,100L of a 1:4dilution of STA-Neoplastin CI Plus (ϳ1nM TF;Diagnostica Stago)was infud into the inferior vena cava over 30conds.20Time to cessation of breathing was determined.Baboon thrombosis model
Nonterminal thrombosis studies were performed on a male baboon (Papio anubis )with an exteriorized femoral arteriovenous shunt.11,21Protocols were approved by the Institutional Animal Care and U Committee of the Oregon Health and Sciences University.Briefly,thrombus formation
东巴纸was initiated by introducing a 20-mm long by 4-mm diameter vascular graft (polytetrafluorethylene [Gore-Tex];Gore &Co.)coated with collagen into the shunt.Flow rate through the shunt was restricted to 100mL/min (mean shear rate 265/s).Thrombus formation was assd by quantitative imaging of 111In-labeled platelet accumulation within,and downstream of the graft (10cm),using a GE-400T gamma scintillation camera interfaced with a NuQuest InteCam computer system.Fibrin deposition in the graft was determined by direct measurement of 125I-labeled fibrinogen as described.21
Results
Antibody 14E11
Antibodies were raid against mou fXI (Figure 1A)in fXI-deficient mice and screened for ability to prolong the aPTT of mou plasma.The IgG 14E11was lected for further study becau it also prolonged the aPTT of human plasma.On Western blots,14E11recognizes a single band of appropriate size (ϳ160kDa)in normal mou (Figure 1B)and human (Figure 1C)plasma,but not in fXI-deficient plasma.14E11has a high affinity for mou and human fXI,and human fXIa in a solid pha binding assay (apparent K d ϳ2-3pM;Figure 1D).fXI is a dimer of 80-kDa subunits,each containing 4apple domains (A1-A4)and a protea domain.PK,a monomeric homolog of fXI,has an identical str
ucture.Previously,we ud human fXI/PK chimeras to show that the A2domain is required for 14E11binding to fXI (Figure 1E).6Western blots using individual fXI apple domains indicate the 14E11binding site is likely located entirely within A2(Figure 1F).
Anticoagulant properties of 14E11
复古海报14E11prolonged the aPTT of mou and human plasma (Figure 2)with maximum inhibition at 25-50nM.This is similar to the plasma fXI concentration in humans (20-45nM),2and probably in mice.12,18The prolongation of the clotting times were similar to tho of fXI-deficient plasmas.The aPTT of fXI-deficient mou plasma,
or
Figure 1.Binding properties of 14E11.(A)Coomassie blue–stained 10%polyacrylamide gel of human (H)and mou (M)recombinant fXI.(B-C)Western blots of
nonreducing 10%polyacrylamide gels of mou (B)and human (C)normal (N)and fXI-deficient (XI Ϫ/Ϫ)plasmas,using biotinylated-14E11for detection.rXI in panel B indicates recombinant mou fXI control.(D)Binding of biotinylated-14E11to immobilized mou fXI (E ),human fXI (F ),or human fXIa (Ⅺ),as described in “Methods.”(E)Western blot of nonreducing 10%polyacrylamide gel of human fXI (hXI),human prekallikrein (PK),and human fXI in which the A1,A2,A3,or A4domain has been replaced with the corresponding domain from PK.Position for fXI dimer is indicated to the right by “D,”and for monomeric PK by “M.”Note fXI with the PK A4domain is a monomer becau A4mediates fXI dimer formation.(F)Western blot (left panel)of a nonreducing 10%polyacrylamide gel of human fXI (hXI)and individual human fXI apple domains linked to tPA.The right panel is a stained gel showing the recombinant apple domain-tPA chimeras.Note the A4chimera forms a dimer.For panels A-C and E,positions of molecular mass standards in kDa are at the left of the figures,and for panel F at the right.
FACTOR XIIa ACTIVATION OF FACTOR XI
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plasma from a wild-type mou treated with 14E11is 48-70conds.This modest prolongation,relative to human fXI-deficient plasma,is a consistent finding.14E11did not affect the prothrombin time of
human plasma.In an assay using fXIa to trigger clotting,14E11did
not affect clotting times,indicating it does not interfere with fXIa activation of fIX.This was confirmed by Western blot analysis in a purified protein system (Figure 3A).In contrast,fIX activation is reduced by antibody O1A6(Figure 3A),which binds to the A3domain 6near an area required for fIX activation.13Cumulatively,the data indicate 14E11interferes with fXI activation,but not fXIa activity.This was supported by studies in which clotting was triggered by addition of fXIIa.Here,14E11prolonged clotting times to an extent consistent with 90%inhibition of fXI activation by fXIIa.
We previously reported that 14E11effects fXI activation by fXIIa in a purified protein system (rate reduced ϳ50%),6but does not completely explain its potent effect in the aPTT assay.fXI forms a complex in plasma with HK.14,22,23In the aPTT,HK enhances fXI activation by fXIIa by facilitating fXI binding to the contact surface.1,2In a solid pha competition assay,14E11inhibited binding of fXI to immobilized HK (apparent K i of ϳ10-20nM;Figure 3B).A modification of the method of Baglia et al 24was ud to measure activation of fXI by fXIIa in the prence of HK on a kaolin (aluminum silicate)surface.14E11significantly reduced the rate of fXI activation in this HK-dependent system (Figure
3C).
Figure 2.Effect of 14E11on the aPTT assay.Mou (E )or human (F )plasma supplemented with 14E11(10Ϫ5to 100M)were tested in an aPTT assay,as described in
“Methods.”
Figure 3.Activities of antibody 14E11.(A)Nonreduc-ing Western blots showing fIX (100nM)activation by fXIa (1nM)in the prence of control vehicle (top),200nM 14E11(middle),or 200nM O1A6(bottom).Blots were developed with a polyclonal anti-human fIX IgG.Arrows to the right of each blot indicate zymogen fIX (IX)and fIXa (IXa).Time of incubation in minutes is shown at the bottom,and positions of molecular mass standards are at the left of the figure.(B)Competition binding assay in which biotinylated fXI is allowed to bind to immobilized HK in the prence of 14E11(10Ϫ5to 100M).Bound fXI was detected with strepavidin-HRP ,as described in “Methods.”Each symbol is the average of results for duplicate experiments.(C)Factor XI (85nM)was acti-vated by fXIIa (17nM)in the prence of HK (70nM and kaolin (0.5mg/mL)in the prence (F )or abnce (E )of 300nM 14E11.At various time points,samples were removed from the reaction into CTI,and fXIa generated was determined with a chromogenic substrate,as de-scribed in “Methods.”Each symbol is the average of results for duplicate experiments.(D)Thrombin genera-tion in fXII deficient plasma initiated by addition of Ca 2ϩand fXIIa (10nM curves 1and 3,or 1nM curves 2and 4)in the abnce (curves 1and 2)or prence (curves 3and 4)of 300nM 14E11.(E)Thrombin generation in fXII deficient plasma initiated by addition of Ca 2ϩin the abnce (curve 1)or prence (curvess 2,3,and 4)of tissu
e factor (0.23pM).Reaction for curves 2,3,and 4included control vehicle,300nM 14E11,or 300nM O1A6,respectively.
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