Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in patients receiving ...

14 Pages·2017·2.21 MB·English

REGULAR ARTICLE
Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in
patients receiving acalabrutinib
AlexanderP.Bye,1AmandaJ.Unsworth,1MichaelJ.Desborough,2,3CatherineA.T.Hildyard,4NiamhAppleby,4,5DavidBruce,4,5
NelineKriek,1SophieH.Nock,1TanyaSage,1CraigE.Hughes,1,*andJonathanM.Gibbins1,*
1InstituteforCardiovascularandMetabolicResearch,UniversityofReading,Reading,UnitedKingdom;2OxfordHaemophiliaandThrombosisCentre,OxfordBiomedical
ResearchCentre,ChurchillHospital,Oxford,UnitedKingdom;3NuffieldDivisionofClinicalLaboratorySciences,UniversityofOxford,Oxford,UnitedKingdom;4Departmentof
Haematology,ChurchillHospital,OxfordUniversityHospitalsNationalHealthServicesFoundationTrust,Oxford,UnitedKingdom;and5DepartmentofOncology,Universityof
Oxford,Oxford,UnitedKingdom
TheBrutontyrosinekinase(Btk)inhibitoribrutinibinducesplateletdysfunctionandcauses
KeyPoints
increasedriskofbleeding.Off-targetinhibitionofTecisbelievedtocontributetoplatelet
(cid:129)Acalabrutinibpreserves
dysfunctionandothersideeffectsofibrutinib.Thesecond-generationBtkinhibitor
Srcfamilykinase activ-
acalabrutinibwasdevelopedwithimprovedspecificityforBtkoverTec.Weinvestigated
ity andavoids dysfunc-
plateletfunctioninpatientswithnon-Hodgkinlymphoma(NHL)receivingibrutinibor
tional platelet thrombus
acalabrutinibbyaggregometryandbymeasuringthrombusformationoncollagenunder
formation caused by
arterialshear.Bothpatientgroupshadsimilarlydysfunctionalaggregationresponsesto
ibrutinib therapy.
collagenandcollagen-relatedpeptide,andcomparisonwithmechanisticexperimentsin
(cid:129)Platelet thrombus
whichplateletsfromhealthydonorsweretreatedwiththeBtkinhibitorssuggestedthatboth
formation is enhanced
drugsinhibitplateletBtkandTecatphysiologicalconcentrations.Onlyibrutinibcaused
by intermediate purity
dysfunctionalthrombusformation,whereassizeandmorphologyofthrombifollowing
factor VIII.
acalabrutinibtreatmentwereofnormalsizeandmorphology.Wefoundthatibrutinibbut
notacalabrutinibinhibitedSrcfamilykinases,whichhaveacriticalroleinplateletadhesion
tocollagenthatislikelytounderpinunstablethrombusformationobservedinibrutinib
patients.Wefoundthatplateletfunctionwasenhancedbyincreasinglevelsofvon
Willebrandfactor(VWF)andfactorVIII(FVIII)exvivobyadditionofintermediatepurity
FVIII(HaemateP)tobloodfrompatients,resultinginconsistentlylargerthrombi.We
concludethatacalabrutinibavoidsmajorplateletdysfunctionassociatedwithibrutinib
therapy,andplateletfunctionmaybeenhancedinpatientswithB-cellNHLbyincreasing
plasmaVWFandFVIII.
Introduction
TheBrutontyrosinekinase(Btk)inhibitoribrutinibwasthefirstofanewclassofdrugforthetreatmentof
indolentnon-Hodgkinlymphomas(NHLs).1Treatmentwithibrutinibhasproventobeefficaciousbutis
associated with side effects, including increased risk of major bleeding.2 Bleeding associated with
ibrutinib is potentially explained by off-target inhibition of other kinases in addition to Btk; a study of
patientswithX-linkedagammaglobulinemia(XLA),whohaveBtkdeficiency,reportednoincreasedrisk
ofbleeding3althoughgeneticdeficiencyofBtkwasfoundtoincreasetimeocclusioninamousemodel
ofcarotidarteryinjury.4Furthermore,studiesperformedusinggeneticallymodifiedmicedemonstrated
redundancybetweenBtkandTecinplateletsignalingbecauseablationofbothkinaseswasrequiredto
Submitted28August2017;accepted7November2017.DOI10.1182/ Thefull-textversionofthisarticlecontainsadatasupplement.
bloodadvances.2017011999. ©2017byTheAmericanSocietyofHematology
*C.E.H.andJ.M.G.contributedequallytothiswork.
2610 12DECEMBER2017xVOLUME1,NUMBER26
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Table1.Patientinformation
Acalabrutinib100mgBD(n58) Ibrutinib480mgOD(n56) Btkinhibitornaı¨ve(n55)
Age,median(range),y 68(53-80) 68(60-77) 78(60-80)
Sex,n(%)
Female 2(25) 3(50) 3(60)
Male 6(75) 3(50) 2(40)
Diagnosis,n(%)
CLL 7(87.5) 6(100) 5(100)
Mantlecelllymphoma 1(12.5) — —
Laboratoryparameters
Hemoglobin,median(range),g/L 139(113-155) 131(109-147) 140(117-148)
Plateletcount,median(range),3109/L 148.5(35-228) 114(58-197) 118(109-274)
Clinicaldetails,n(%)
Concurrentantiplateletdrugs — — —
Concurrentanticoagulants 1(13)* — —
Inheritedbleedingdisorders — — —
DurationofBtkinhibitor,median(range),mo 21.5(19-32) 19(2-39) —
Activebleedingattimeofrecruitment — — —
BleedingonBtkinhibitor,n(%)†
None 3(38) 5(83) —
Grade1 2(25) 1(17) —
Grade2 3(38) — —
ResponsetoBtkinhibitor,n(%)‡
Completeremission 5(63) 3(50) —
Partialremission 3(38) 3(50) —
Stableorprogressivedisease — — —
BD,twicedaily;OD,oncedaily.
*Dabigatran150mgtwicedaily.
†CommonTerminologyCriteriaforAdverseEventsgradingscale.
‡InternationalWorkshoponChronicLymphocyticLeukemiacriteriaforchroniclymphocyticleukemia;positronemissiontomographycomputedtomographycriteriaformantlecell
lymphoma.
renderplateletsfullyinsensitivetocollageninaggregationassays.5 discrepancy between bleeding risk in patients with XLA and
The effect of simultaneous genetic ablation of Btk and Tec on patients receiving ibrutinib has not been characterized, but is
platelet adhesion under shear has not been explored in vivo or in thoughttorelatetooff-targetinhibitionofTec.8However,ibrutinib
vitro.Theoff-targeteffectsofibrutinibonplateletfunctionhaveadded mayalsoinhibit otherimportant plateletkinases such asSFK that
complications to its use, such as risks associated with concurrent phosphorylate several signaling molecules in the GPVI signaling
treatment with anticoagulant or antiplatelet medication, which is pathway.17BtkandTecarethemselvesdependentonSFK-mediated
commonamongpatientswithchroniclymphocyticleukemia(CLL).6 tyrosine phosphorylation to become active.18,19 Inhibition of SFK is
Ibrutinib therapy is interrupted before surgery to reduce the risk of known to cause hemostatic dysfunction because the Src inhibitor,
bleeding, but prolonged dose interruption or dose reduction may dasatinib,isassociatedwithincreasedbleedingrisk.20-22Comparison
reduceefficacyandleadtoaflareinsymptoms.7Acalabrutinibisa ofibrutinibandacalabrutinibtherapythereforerepresentsanimportant
second-generationBtk inhibitorand exhibitsgreater selectivity over opportunitytocharacterizetheirrelativeeffectsandunderstandhow
kinases that are important for platelet function such as Src family improvedkinasespecificityaffectsplateletdysfunction.
kinases(SFKs)and Tec.8 Nomajorbleeding eventswere reported
ThrombocytopeniaisfrequentlyassociatedwithCLLandmaybea
duringaphase2trialofacalabrutinibfortreatmentofCLL,8butitis
contributing factor to bleeding events in combination with drug-
notyetcleariftheselectivityofacalabrutinibissufficienttofullyavoid induced platelet dysfunction.23,24 Treatment options for patients
theplateletdysfunctionassociatedwithibrutinib.
thatareathighriskofbleedingarecurrentlylimitedandtheclinical
Ibrutinib causes platelet dysfunction downstream of the GPVI efficacyofplatelettransfusionhasnotbeenestablished.2Evidence
receptor, GPIb, and integrin a b .9-11 Although XLA is not thatplatelettransfusionmayreverseplateletdysfunctioncausedby
IIb 3
associatedwithincreasedriskofbleeding,deficientexpressionor ibrutinib is based on in vitro data11 with few studies investigating
functionofGPVI,12-14GPIb,15orintegrina b 16isassociatedwith this approach clinically.25 Furthermore, the benefit of platelet
IIb 3
bleedingphenotypesinmiceorhumans.Theprecisereasonforthe transfusion during treatment with other antiplatelet medication is
12DECEMBER2017xVOLUME1,NUMBER26 ACALABRUTINIBAVOIDSSEVEREPLATELETDYSFUNCTION 2611
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A
NR
-3
p>0.05
ml) -4
g/ p>0.05
or
C (M 50 -5 p>0.05
gE p>0.05 Heathy Donors
nist lo -6 Ibrutinib Patient
o
g
A -7 Acalabrutinib Patient
Treatment Naive Patient
-8
-9
ADP U46619 Epinephrine TRAP-6 CRP-XL Collagen
B C
120 120
100
100
n n
missio 80 missio 80
ns ns 60
a a
ht tr 60 ht tr Healthy Donors
g g 40
Max li 40 p < 0.001 Max li Acalabrutinib Patients
% % 20 p < 0.001 Ibrutinib Patients
20 0 Btk Inhibitor Naive Patients
0 -20
-10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5
log [CRP-XL] (g/ml) log [collagen] (g/ml)
D E
Acalabrutinib Ibrutinib
100 100
[Inhibitor]M
80 80 Vehicle
on on 0.1
missi 60 missi 60 0.3
ns ns
a a
ht tr 40 ht tr 40 1
g g
% li % li 3
10
20 20
0 0
-8 -7 -6 -5 -8 -7 -6 -5
log[CRP] (g/ml) log[CRP] (g/ml)
Figure1.AcalabrutinibandibrutinibtherapycausedysfunctionalGPVI-mediatedplateletaggregation.PRPfrompatientsreceivingibrutinib(Ibr;n56),
acalabrutinib(Acal;n57),healthydonors(n59),orBtkinhibitorna¨ıveCLLpatients(n55)wasloadedinto96-wellmicrotiterplatescontaininglyophilizedplateletagonists.
Plateswereshakenfor5minutesat37°Candaggregationwasmeasurebylighttransmissiontogiveanendpointmeasurement.(A)ScatterplotofEC50valuescalculatedfrom
2612 BYEetal 12DECEMBER2017xVOLUME1,NUMBER26
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controversial and may lead to poorer outcomes.26 Investigating centrifuging at 350g for 20 minutes and resuspending the platelet
alternatives to platelet transfusion to treat or prevent bleeding pelletat43108cells/mLinTyrode’sbufferaspreviouslydescribed.9
duringibrutiniboracalabrutinibtherapyisthereforeapriority.One
Plate-basedaggregometry. Formeasurementofaggrega-
alternative to platelet transfusion for bleeding patients, or those
tion using PRP from patients or healthy donors, 96-well plates
undergoingsurgeryandatriskofbleedingfromplateletdysfunction,
(Greiner)containingthefollowingfreeze-driedplateletagonistsata
isadministration of desmopressin,27-29 which stimulates secretion
range of concentrations: ADP, CRP-XL, epinephrine, U46619,
of the contents of Weibel-Palade bodies and results in an acute
collagen, and TRAP-6 were prepared in advance and stored in
increase in plasma von Willebrand factor (VWF) and factor VIII
vacuum-sealedbagsfornomorethan2months.PRPisolatedfrom
(FVIII)levelsoftwo-tosixfold.30Indeed,successfuladministrationof
thebloodofhealthydonorsorpatientswasloadedontoplatesand
desmopressintostopbleedinginpatientssufferingfromthrombo-
shaken at 1200 rpm for 5 minutes at 37°C using a plate shaker
cytopenia has also been reported.31 It may be possible to
(Quantifoil Instruments), as described by Lordkipanidze´ et al;
administer desmopressin to treat bleeding or reduce bleeding risk
absorptionof405nmlightwasmeasuredusingaMultiskanAscent
during treatment with Btk inhibitors, either as an alternative for or
354 Microplate Reader (LabSystems). Aggregation of washed
adjunct to platelet transfusion. However, because ibrutinib inhibits plateletswasperformedin96-wellplatesaspreviouslydescribed.9
GPIb-mediated platelet function,11 it has remained unclear if this
approachmightbebeneficial. Thrombusformationunderflow. Thrombusformationwas
performedusingbloodfromhealthydonorsusingmicrofluidicflow
Methods chips (Vena8, CellixLtd, Dublin, Ireland) coated with 100 mg/mL
type I collagen and measured in real time following incubation of
Materials bloodfromhealthydonorswithibrutinib,acalabrutinib,orvehiclefor
10 minutes, or fixed with 10% formyl saline after 8 minutes of
TypeIcollagenwasobtainedfromNycomed(Munich,Germany)and
perfusionatashearrateof1000s21andimagedatalatertime.
collagen-relatedpeptide(CRP-XL)fromProfessorRichardFarndale
(University of Cambridge, Cambridge, United Kingdom). Adenosine Protein phosphorylation studies. Washed platelets at
59-diphosphate (ADP), arachidonic acid, and U46619 were from 4 3 108 cells per milliliter in the presence of inhibitors to prevent
Sigma(Gillingham,United Kingdom), and TRAP-6 epinephrine was aggregation(100mMMRS2179,1mMcangrelor,10mMindometh-
from Labmedics (Salford, United Kingdom). Acalabrutinib, ibrutinib, acin,and1mMEGTA)wereincubatedwithibrutiniboracalabrutinibat
anddasatinibwerefromSelleckchem(Munich,Germany).Antiphos- concentrationsspecifiedinindividualfiguresorvehiclefor5minutesat
photyrosineantibody4G10wasobtainedfromMillipore(Burlington, 37°Cbeforeadditionof1mg/mLCRP-XL.Sampleswereincubatedfor
MA).PKCsubstrateantibodyandphospho-PLCg2Y759wasfrom 3minuteswithstirringat37°Cusinganaggregometer(Helena)before
NewEnglandBiolabs (Hitchin,UnitedKingdom).Phospho-Src418 additionofreducingLaemmlisampletreatmentbuffer.
antibodyandFura-2AMwasfromLifeTechnologies(Paisley,United Phospho-Tec ELISA. Platelets were prepared and stimu-
Kingdom).Phospho-BtkY223,phospho-LynY396,andphospho-b3
latedasdetailedpreviouslybutlysedinNP40buffer(300mMNaCl,
Y773 antibodies were from Abcam (Cambridge, United Kingdom).
20mMTrisbase,2mMEGTA,2mMEDTA,1mMPMSF,10mg/mL
Actin11CantibodywasfromSantaCruzBiotechnology(Heidelberg,
aprotinin,10mg/mLleupeptin,0.7mg/mLpepstatinA,2mMsodium
Germany). Greiner 96-well plates and DiOC6 were from Thermo
orthovanadate,2%v/vNP-40;pH7.3)andanalyzedusingahuman
FisherScientific(Dartford,UnitedKingdom).
phosphotyrosine Tec enzyme-linked immunosorbent assay (ELISA
Patients. Trough blood samples were obtained from patients kit,RayBio).Absorptionat405nmwasmeasuredusingaNOVOstar
withCLLormantlecellleukemiaandreceivingeitheribrutinib(6patients) platereader(BMGLabTech,Aylesbury,UnitedKingdom).
oracalabrutinib(8patients;Table1).OurcontrolgroupcomprisedBkt- Ca21 measurement. The [Ca21] assay was performed at
inhibitorna¨ıveCLLpatients(5patients).Allpatientsprovidedinformed i
37°Cusingfura-2loadedwashedplateletsinblack96-wellplates
consentinaccordancewiththedeclarationofHelsinki.Ethicsapproval (Greiner). Dual excitation was measured using a Novostar
forthisstudywascoveredundertheOxfordRadcliffeBiobankresearch spectrofluorometer(BMGLabtech)previouslydescribed.9
tissuebankethics,HTALicenseNumber12217,OxfordshireCREC:
09/H0606/515,projectapprovalcode17/A016. Platelet adhesion to collagen. Washed platelets at
23107cells/mLwereexposedtocollagen(100mg/mL)-coated,
Platelet preparation. Blood samples were obtained from 96-wellassayplates,andallowedtoadherefor45minutesat37°C.
healthy donors that had given informed consent and using Nonadherent platelets were washed off before, fixed with 10%
proceduresapprovedbytheUniversityofReadingResearchEthics formyl saline for 10 minutes. The wells were then washed and
Committeeorfrompatientsandcollectedintovacutainerscontaining labeled with DiOC6. Fluorescence images of adherent platelets
3.8%(w/v)sodiumcitrate.Platelet-richplasma(PRP)wasprepared werecapturedwiththe203objectivelensofanImageXpressNano
bycentrifugingwholebloodat100gfor20minutes.Washedplatelets high content imaging system and counted using CellReporterX-
were prepared by adding acid citrate dextrose to PRP and presssoftware(MolecularDevices,Winnersh,UnitedKingdom).
Figure1.(continued)concentrationresponsecurvesforADP,U46619,epinephrine,TRAP-6,CRP-XL,andcollagen.Eachpointrepresentstheresponseofapatientreceiving
acalabrutinib(redtriangle)oribrutinib(greensquare);healthydonor(graycircle)orBtkinhibitorna¨ıveCLLpatient(whitediamond).NR,patientsthatdidnotrespondtoanagonist
atthehighestconcentrationtested.ConcentrationresponsecurvesforGPVIagonists(B)CRP-XLand(C)collagen;eachpointrepresentsthemean%aggregationvalues6
standarderrorofthemean(SEM).Significantdifferencesrelativetohealthydonorsweretestedby2-wayANOVAwiththeTurkeymultiplecomparisonstest.Aggregationofwashed
plateletsfromhealthydonorspre-treatedwitharangeof(D)acalabrutinibor(E)ibrutinibconcentrationsandthenstimulatedwith3to0.01mg/mLCRP-XL.
12DECEMBER2017xVOLUME1,NUMBER26 ACALABRUTINIBAVOIDSSEVEREPLATELETDYSFUNCTION 2613
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A B
Acalabrutinib Ibrutinib
200 200
% vehicle phosphorylation 11550000 SBT(Pert)cck- Y YtYo24ta21l38 % vehicle phosphorylation 11550000
0 0
Vehicle-9 -8 -7 -6 -5 -4 Vehicle-9 -8 -7 -6 -5 -4
log[acalabrutinib] (M) log[ibrutinib] (M)
log [Acalabrutinib] (M) log [Ibrutinib] (M)
C
V -8 -7.5 -7 -6.5 -6 -5.5 -5 V -8 -7.5 -7 -6.5 -6 -5.5 -5
Btk Y223
50
50 Src Y418
50
Actin loading control
D E F G
140 140 140 140
% vehicle Lyn Y396 phosphorylation 11864220000000 % vehicle total phosphotyrosine 11864220000000 % vehicle aggregation 11864220000000 % vehicle adhesion 11864202000000 AIDbcaruastlaaintbiinbriubtinib
0 0 0 0
Vehicle-8 -7 -6 -5 Vehicle-8 -7 -6 -5 Vehicle-8 -7 -6 -5 Vehicle-8 -7 -6 -5
log[inhibitor] (M) log[inhibitor] (M) log[inhibitor] (M) log[inhibitor] (M)
H log [Acalabrutinib] (M) log [Ibrutinib] (M) log [Dasatinib] (M)
V-7.5-7-6.5-6-5.5-5 V-7.5-7-6.5-6-5.5-5 V-7.5-7-6.5-6-5.5-5
Lyn Y396
4G10
Total Phosphotyrosine
Actin
Figure2.SFKactivationandadhesiontocollagenissparedbyacalabrutinibbutnotibrutinib.Washedhumanplateletsfromhealthydonorswerepreincubated
witharangeof(A)acalabrutinibor(B)ibrutinibandthenstimulatedwith1mg/mLCRP-XLfor3minutesat37°C;tyrosinephosphorylationofSrc(Y418)andBtk(Y223)were
measuredbywesternblotusingsite-specificantibodiesandtotallevelsofTectyrosinephosphorylationweremeasurebyELISA.Pointsrepresentmeanlevelsoftyrosine
phosphorylationrelativetovehicle-treatedcontrols6SEM.(C)Representativeimagesofphosphoblots.Washedplateletstreatedwitharangeofacalabrutinib,ibrutinib,or
dasatinibconcentrationsandstimulatedaspreviouslyandblottedfor(D)tyrosinephosphorylationofLyn(Y396)or(E)totalphosphotyrosineusingasite-specificantibodyor
4G10,respectively.Pointsrepresentmeanlevelsoftyrosinephosphorylationrelativetovehicle-treatedcontrols6SEM.(F)Aggregation1mg/mlCRP-XLstimulatedofwashed
plateletswasperformedin96-wellplatesaftertreatmentwitharangeofconcentrationsofacalabrutinib,ibrutinib,ordasatinibconcentrationresponsecurvesaremeanplatelet
2614 BYEetal 12DECEMBER2017xVOLUME1,NUMBER26
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Fluorescence-activated cell sorting measurement of Acalabrutinib is selectivefor SFK but not Tec
fibrinogenbindingandp-selectinexposure. Measurements of
Both ibrutinib and acalabrutinib have been screened for activity
fibrinogen binding and p-selectin exposure were performed
againstapanelofkinasesincell-free,invitroassays.8,33However,
using washed platelets pretreated with inhibitors as described
the estimated potency of the drugs in such assays does not
in figure legends and stimulated with 1 mg/mL CRP in the
correspond closely with measurements performed in cell-based
presence of fluorescein isothiocyanate–conjugated polyclonal
assays. In addition to this, kinase regulation in the platelet is highly
rabbit anti-fibrinogen antibody (Agilent Technologies LDA UK
complexandinvolvespositiveandnegativefeedbackmechanisms.34
Limited, Cheadle, United Kingdom) and PE-Cy5-conjugated
To elucidate the mechanistic differences between ibrutinib and
mouseanti-P-selectin(CD62P)antibody(BD,Berkshire,United
acalabrutinibthatunderpindifferencesinbleedingrisk,wecharacter-
Kingdom), and then incubated for 20 minutes in the dark.
izedtheeffectsofthedrugsonphosphorylationeventsdownstreamof
Platelets were then fixed by addition of filtered formyl saline
GPVIinplateletsfromhealthydonors (Figure2).Phosphorylation of
(0.2% formaldehyde in 0.15M NaCl) and median fluorescence
BtkY223,theautophosphorylationsiteofBtkthatcorrelateswithits
intensitiesweremeasuredfor5000plateletspersampleonan
kinaseactivity,wasinhibitedwithequalpotencybyibrutinib(loghalf
AccuriC6FlowCytometer(BDBiosciences,Berkshire,United
maximal inhibitory concentration [logIC ] 5 27.2M 6 0.30) and
Kingdom). 50
acalabrutinib(logIC 527.1M60.21;Figure2A-B).InhibitionofTec
50
Statistical methods. Statistical testing as described in was harder to quantify because of the lack of site-specific phospho
figure legends and the results section were performed using antibodies, but total phosphotyrosine levels can be measured by
GraphPadPrismSoftware(GraphPad,LaJolla,CA). ELISA. Total Tec phosphotyrosine levels are likely to include the Tec
autophosphorylationsiteY20635aswellastyrosineresiduesphosphor-
Results ylatedbyotherkinasessuchasSFK.WeestimatedthatTecwasmore
potentlyinhibitedbyibrutinib(logIC 526.4M)thanbyacalabrutinib
50
Acalabrutiniband ibrutinib cause dysfunction of (logIC 525.5M;Figure2A-B);however,theconcentration-response
50
GPVI-mediated platelet aggregation profilesofbothibrutinibandacalabrutinibwerecomplex,involvingboth
positiveandnegativecomponentsanddifferentialregulationofmultiple
PlateletaggregationtoGPVIagonistshaspreviouslybeenshownto
sites. Phosphorylation of the autophosphorylation site Y418 of Src
be inhibited in blood samples taken from patients receiving
appeared to be potentiated by both drugs at lower concentrations.
ibrutinib,10,11 but the effect of acalabrutinib on platelet function
However, at higher concentrations, ibrutinib also inhibited Src Y418
has not been reported. Aggregation of PRP was used to phosphorylation(logIC 525.7M60.18),whereasacalabrutinibdid
characterize platelet function of patients treated with ibrutinib or 50
not inhibit Src activity and actually potentiated activation even at high
acalabrutinib (Table 1). One patient receiving acalabrutinib was concentrations(logEC 527.5M60.46;Figure2A-B).Becauseour
excluded from this aspect of the study because of a low platelet 50
data indicated that platelet Btk and Tec were likely to be inhibited
count (,50 3 109/L). The aggregation assay included ADP,
by physiological concentrations of both ibrutinib and acalabrutinib,
U46619, epinephrine, thrombin receptor activator peptide-6
we further investigated inhibition of SFK by comparing both drugs to
(TRAP-6), CRP-XL, and collagen was adapted from the method
the SFK inhibitor dasatinib. Phosphorylation of Lyn Y396 (Figure 2D)
developedandvalidatedbyLordkipanidze´ etal.32Weusedarange was ablated by dasatinib (logIC 5 27.1M 6 0.05) and ibrutinib
of agonist concentrations to enable comparison of patient (logEC 5 25.8M 6 0.08)5b0ut not acalabrutinib, which only
sensitivity to each agonist by estimation of the log half maximal 50
inhibited phosphorylation to 70% of vehicle-treated platelets at
effective concentration (logEC ) by nonlinear regression analysis.
50 the highest concentration tested (10 mM). Total phosphotyrosine
Results were compared with a group of healthy donors and Btk
levels(Figure2E)followedasimilarpatternwherebydasatinibcaused
inhibitorna¨ıveCLL patients (Figure 1A). Aggregationstimulated by near ablation of CRP-XL–evoked tyrosine phosphorylation
ADP, U46619, epinephrine, and TRAP-6 was not significantly (logIC 5 26.9M 6 0.24), whereas ibrutinib was less potent
different in either patient group compared with healthy controls. (logIC505 25.1M 6 0.29) and acalabrutinib only inhibited partially
Responsestocollagenwerecompletelyablatedinallpatientstested 50
(71.7%ofvehicle)at10mM.
forbothpatientgroupsreceivingBtkinhibitors,evenatthehighest
concentrationofcollagen(3mg/mL)(Figure1A-B).Responsestothe
Ibrutinib but not acalabrutinib potently inhibits
GPVI receptor agonist CRP-XL were either less potent (ibrutinib,2
platelet adhesion tocollagen
patients; acalabrutinib, 3 patients) or ablated (ibrutinib, 4 patients;
acalabrutinib, 4 patients) relative to responses of healthy controls Aggregationofwashedplateletsevokedby1mg/mLCRP-XLwas
(logEC 5 27.7M 6 0.30; Figure 1C) and Btk inhibitor na¨ıve completelyablatedbyacalabrutinib(logIC 525.3M60.06)and
50 50
CLL patients (logEC50 5 27.3M 6 0.38). Experiments in which withsignificantlygreaterpotencybyibrutinib(logIC 526.2M6
50
CRP-XL was titrated against ibrutinib and acalabrutinib indicated 0.28)anddasatinib(logIC 526.6M60.6;Figure2F).Wehave
50
that 10mM acalabrutinib or 1mM ibrutinib was required to ablate previouslyreportedthatdiscrepancies arepresentinthewaythat
platelet aggregation of washed platelets from healthy donors ibrutinib inhibits platelet aggregation to immobilized collagen-
(Figure1D-E). coatedsurfaces.9Adhesionofplateletstocollagen(Figure2G)was
Figure2.(continued)aggregation6SEMrelativetovehicle-treatedcontrol.(G)Acalabrutinib,ibrutinib,ordasatinib-treatedwashedplateletsfromhealthydonorswere
allowedadheretocollagen-coatedwellsofa96-wellplateafter45minutesat37°C;graphsplotconcentrationresponsecurvestomeannumbersofadheredplatelets6SEM
relativetovehicletreatment.(H)Representativeimagesofphosphoblotsforplateletstreatedwithtyrosinekinaseinhibitors.
12DECEMBER2017xVOLUME1,NUMBER26 ACALABRUTINIBAVOIDSSEVEREPLATELETDYSFUNCTION 2615
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A B
140 140
120 120
PLC2 (P) Y759 18000 P) PKC substrate 18000
% vehicle 4600 % vehicle ( 4600
Ibrutinib
20 Acalabrutinib 20
0 0
Vehicle -9 -8 -7 -6 -5 -4 Vehicle -9 -8 -7 -6 -5 -4
log[inhibitor] (M) log[inhibitor] (M)
C
log [Acalabrutinib] (M) log [Ibrutinib] (M)
V -8 -7.5 -7 -6.5 -6 -5.5 -5 V -8 -7.5 -7 -6.5 -6 -5.5 -5
PLC2 (Y759)
37
150
100 (P) S/T
75 PKC Substrates
50
37
25
20
50
D E Acalabrutinib
140
120
Vehicle
2+ [Ca] 18000 2+M [Ca]i 000...1303MMM
% vehicle 60 100 n 60s 13MM
40 F Ibrutinib
10M
20
0
Vehicle -9 -8 -7 -6 -5 -4 +]i
log[inhibitor] (M) 2a
C
M [
n
0
0 60s
1
Figure3.AcalabrutinibinhibitssignaltransductiondownstreamofGPVIwithlowerpotencythanibrutinib.Washedhumanplateletsfromhealthydonorswere
preincubatedwitharangeofacalabrutiniboribrutinibconcentrationsandthenstimulatedwith1mg/mLCRP-XLfor3minutesat37°Candphosphorylationof(A)PLCg2
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A B C
140 140 140
120 120 120
% vehicle P-selectin exposure 186400000 % vehicle fibrinogen binding 186400000 % vehicle (P) Y7733 186400000
Acalabrutinib
20 20 20
Ibrutinib
0 0 0
Vehicle-9 -8 -7 -6 -5 -4 Vehicle-9 -8 -7 -6 -5 -4 Vehicle-9 -8 -7 -6 -5 -4
log[inhibitor] (M) log[inhibitor] (M) log[inhibitor] (M)
D
log [Acalabrutinib] (M) log [Ibrutinib] (M)
V -8 -7.5 -7 -6.5 -6 -5.5 -5 V -8 -7.5 -7 -6.5 -6 -5.5 -5
150
- 3 Y773
100
75
50 - Actin loading control
Figure4.AcalabrutinibisalesspotentinhibitorofintegrinaIIbb3activationandgranulesecretionthanibrutinib.Washedhumanplateletsfromhealthydonors
werepreincubatedwitharangeofacalabrutiniboribrutinibconcentrationsandthenstimulatedwith1mg/mLCRP-XLfor20minutes.(A)P-selectinand(B)fibrinogenbinding
wasmeasuredbyfluorescence-activatedcellsorting.(C)Plateletswerestimulatedwith1mg/mLCRP-XLfor3minutesat37°C.Phosphorylationofb3(Y773)was
measuredbywesternblotting.Pointsrepresentmeanresponsesrelativetovehicle6SEM.(D)Representativeb3(Y773)blots.
partiallyinhibited(;40%ofvehicle)byibrutinib(logIC 525.9M6 stimulated downstream of PLCg2 and was also inhibited less
50
0.18) and dasatinib (logIC 5 26.9M 6 0.09), which closely potentlybyacalabrutinib(logIC 526.1M60.08)thanibrutinib
50 50
matchedtheirabilitytoinhibitLynactivity.Acalabrutinibwasaweak (logIC 526.760.07,P,.05,Studentttest).
50
inhibitorofadhesiontocollagenandonlyreducedthenumberof
adheredplateletto60%69.4ofvehicle. Acalabrutinib inhibits granule secretion and integrin
a b activation less potently than ibrutinib
IIb 3
Acalabrutinibinhibits signalingdownstream ofGPVI
Secretionofa-granuleswasinvestigatedbymeasuringexposureof
less potently than ibrutinib
P-selectin on the platelet surface following stimulation with CRP-
To understand the consequences of the differences in kinase XL. P-selectin exposure was more potently inhibited by ibrutinib
specificity of the inhibitors, signaling events downstream of the (logIC 526.5M60.04)thanbyacalabrutinib(logIC 525.9M6
50 50
kinases were measured. We found that phosphorylation of the 0.11; Figure 4A). Binding of fluorescently labeled fibrinogen was
Btk substrate, Y759 of PLCg2, was inhibited by both ibrutinib used as measure of integrin a b activation following stimulation
IIb 3
(logIC 5 26.7M 6 0.18) and acalabrutinib (logIC 5 26.7 6 with CRP-XL (Figure 4B). Fibrinogen binding was inhibited more
50 50
0.26;Figure3B).However,acalabrutinibdidnotcompletelyablate potently by ibrutinib (logIC 5 26.5M 6 0.04) than by
50
phosphorylation of PLCg2 Y759 even at the highest concentra- acalabrutinib (logIC 5 25.8M 6 0.07). Phosphorylation of the
50
tionofacalabrutinibtested.PhosphorylationofPKCsubstrateswas integrinb subunitisacriticaleventintheinitiationofintegrina b
3 IIb 3
inhibitedlesspotentlybyacalabrutinib(logIC 526.2M60.19) outside-insignaling.Phosphorylationofb Y773(correspondingto
50 3
thanbyibrutinib(logIC 526.560.24,P,.05,Student ttest; Y747inmouse)waspotentlyinhibitedbybothibrutinib(logIC 5
50 50
Figure3B).AcalabrutinibinhibitedPKCactivityby52%64.5relative 26.7M 6 0.13) and acalabrutinib (logIC 5 26.6M 6 0.11.
50
tovehicle,significantlylessthanibrutinib,whichinhibitedby72%6 Figure4C).Acalabrutinibappearedtobeonlyapartialinhibitorof
3.3 (P , .05, Student t test) at the highest concentration b Y773phosphorylation,becauseevenatthehighestconcentra-
3
tested. Release of Ca21 from intracellular stores into the cytosol is tiontested,a reductiontoonly27%62.5relative tovehiclewas
Figure3.(continued)(Y759)and(B)PKCsubstrates(S/T)wasmeasuredbywesternblotting.(C)RepresentativewesternblotimagesforPLCg2(Y759)andS/T
phosphorylatedPKCsubstrates.(D)CytosolicCa21followingstimulationwith1mg/mLCRP-XLwasmeasuredinfura-2loadedplateletsinrealtimefor5minutes.Points
representthemeanresponserelativetovehicle6SEM.Representative[Ca21]itracesfollowingincubationwith(E)acalabrutinibor(F)ibrutinib.
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Figure5.Ibrutinib,butnotacalabrutinib,causesthrombus
A
instability.Wholebloodfromhealthydonorswasincubatedwith
vehicle,1mMibrutinib,and1mMacalabrutinibfor20minutesand
e flowedthroughcollagen-coatedmicrofluidicflowchambersata
hicl shearrateof1000s21for6minutes.(A)Representativeimagesof
e
V thrombiformedundereachoftheconditionstestedat2-minute
intervals.(B)Asummaryofeffectsof1mMibrutiniband1mM
acalabrutinibonthrombusformationoncollagenandotherplatelet
b
Mni signalingandfunctionalmeasurementsrelativetovehicle-treated
1bruti control.Barsrepresentthemean6SD;Pvalueswerecalculated
I usingmultipletestswithHolm-Sidakcorrectionformultiple
comparisons.
b
ni
Muti
br
1a
al
c
A
0 2 min 4 min 6 min 8 min
B
250 1M Ibrutinib
p=0.054
1M Acalabrutinib
onse 200 p=0.046 p=0.002
esp 150 p=0.003 p=0.002 p=0.011
hicle r 100 p=0.009 p=0.007
e
% v 50 p=0.814
0
Src (Y418B)tk (Y223)Tec (P-Y) [Ca2+Fi]b binP-disnelg exposuArgegregationAdhmebsiuos nformation
Thro
achieved, whereas ibrutinib caused inhibition to 6% 6 1.0 of importantsignalingandfunctionalmeasurementsaresummarized
vehicle. inFigure5B.
To test whether experiments performed using blood from
Acalabrutinibavoids dysfunctional thrombus
healthy donors matched the effects of drugs at therapeutic
formation caused by ibrutinib
concentrations, we performed measurements of thrombus
Giventhedifferencesintheeffectsofthedrugsonadhesionto formationoncollagenunderarterialshearusingbloodsamples
collagen compared with GPVI-mediated aggregation, we com- from healthy donors, Btk-inhibitor na¨ıve patients, or patients
paredtheeffectsofthedrugsonplateletadhesionandthrombus receiving ibrutinib or acalabrutinib (Table 1). Blood from
formationincollagen-coatedflowchambersunderarterialshear acalabrutinib patients formed morphologically normal thrombi
conditions. After treating whole blood from healthy donors with on collagen (Figure 6A), although heterogeneity in platelet
either 1mM ibrutinib or acalabrutinib for 20 minutes, ibrutinib counts strongly influenced thrombus volume (Figure 6C).
causedreducedthrombusformation(1mMibrutinib:383AU640.1, Thrombusvolumeinacalabrutinibpatientswithplateletcounts
vehicle 585AU 6 67.6, P , .05, Student t test), whereas in .100 3 109/L (78895 mm3 6 16258) were not significantly
thepresenceofvehicleoracalabrutinib,thrombusformationwas different to those of Btk-inhibitor na¨ıve patients (75608 mm3 6
not significantly different (Figure 5A-B). The most striking 19995, P . .05, Student t test). In contrast, thrombus volume
differencebetweenibrutinibandacalabrutinibtreatmentwasthe foribrutinibpatientswashighlyvariableanddidnotcorrelatewith
lack of stable, retracted thrombi after ibrutinib treatment plateletcount(Figure6D).Imagesofthrombimeasuredinsamples
(Figure 5A). In the presence of vehicle or acalabrutinib, platelets from patients with platelet counts .150 3 109/L are presented in
accumulated on collagen fibers while continuously contracting Figure 6A. Samples from 2 of the ibrutinib patients exhibited very
into dense thrombi, whereas ibrutinib prevented retraction of pronounceddysfunctionwherebyplateletsformedaloosecovering
platelet aggregates, which consequently remained unstable, ofthemajorityofthesurfaceoftheflowchamberwithoutforming
appearedloose,andwerepronetodisaggregation(supplemental stable thrombi (“large”; Figure 6A), whereas others formed small
Video1).Theeffectsof1mMibrutinibandacalabrutinibonseveral thrombi(“small”;Figure6A).
2618 BYEetal 12DECEMBER2017xVOLUME1,NUMBER26
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A
(-) Haemate P (+) Haemate P
y
h
alt
e
H
b
hie
nv
k INai
Bt
al
c
A
e
Ibrarg
L
brmall
IS
400m 600m
200m
0
B C D
3m) 300000 (-) Haemate P 3m) 300k 3m) 300000
  
me ( 200000 (+) Haemate P non-zero slope me ( 200k non-zero slope me ( 200000
mbus volu 100000 pp==00..045609 mbus volu 100k pp==00..002461 mbus volu 100000
Thro 0 Thro 0k Thro 0
0 50 100 150 200 250 300 0 50 100 150 200 0 50 100 150 200
Platelet count (x109 / L) Platelet count (x109 / L) Platelet count (x109 / L)
Figure6.Thrombusformationoncollagencorrelateswithplateletcountduringtreatmentwithacalabrutinibbutnotwithibrutinib.(A)Bloodfromhealthy
donors(n56),Btk-inhibitorna¨ıveCLLpatients(n55),patientsreceivingibrutinib(n56)oracalabrutinib(n58)wasperfusedthroughcollagen-coatedmicrofluidicflow
chambersatroomtemperatureatashearrateof1000s21beforebeingfixedwith10%formylsaline.FixedsampleswerestainedwithDioC6andz-stackimagesacquiredto
enableestimationofthrombusvolume.(A)RepresentativeimagesofthrombiinbloodfromahealthydonorandpatientsinthepresenceorabsenceofHaemateP(intermediate
factorVIII)addedexvivo.Thrombusvolumeplottedagainstplateletcountfor(B)Btkinhibitorna¨ıveCLLpatients;patientstreatedwith(C)acalabrutinibor(D)ibrutinibwithor
withoutaddedHaemateP.CorrelationbetweenplateletcountandthrombusvolumeissignificantinthepresenceorabsenceofHaematePforacalabrutinibpatients
(significantlynonzero,P#.05)butnonsignificantforibrutinibpatients(P..05).
Addition ofHaemate Pex vivo topatients’ blood repeated measures and the Sidak multiple comparisons test;
improves platelet function Figure7).
Thrombus formation assays using patients’ blood were per-
Discussion
formedinthepresenceorabsenceof2units/mLofHaemateP.
Correlation of thrombus volume with platelet count was not Ibrutinibhasproventobeaneffectivetreatmentoflow-gradeNHL
significant within the Btk-inhibitor na¨ıve patient group because but is associated with an increased risk of bleeding, whereas the
platelet counts were all .100 3 109/L and thrombus volume effects of the second-generation Btk inhibitor, acalabrutinib, on
was fairly uniform. The correlation was significant in patients platelet signaling and function have not been studied in detail.
treated with acalabrutinib in the presence and absence of Ibrutinibhasbeenshowntoinhibitplateletaggregationevokedby
HaemateP(Figure6B),butnotinpatientstreatedwithibrutinib GPVIreceptoragonists,9-11buttheeffectsofacalabrutinibtherapy
(Figure 6C), despite half of the patients having platelet counts on platelet aggregation have not been reported. We found that
,100 3 109/L, which strongly influences thrombus volume. aggregation evoked by collagen and CRP-XL was either partially
AdditionofHaematePsignificantlyincreasedthrombusvolume or completely inhibited in all patients receiving ibrutinib or
in healthy donors and patients treated with acalabrutinib or acalabrutinib (Figure 1). Peak plasma concentrations of ibrutinib
ibrutinib (P , .05, 2-way analysis of variance (ANOVA) with and acalabrutinib have been estimated at approximately 300 nM
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