28_35_2016-04-15 Michelson (Platelet Function Testing in the Clinic)

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28_35_2016-04-15 Michelson (Platelet Function Testing in the Clinic)
Platelet Function Testing in the Clinic
Alan D. Michelson
Professor of Pediatrics and Professor of Medicine
Harvard Medical School
Director, Center for Platelet Research Studies
Director, Thrombosis and Anticoagulation Program
Boston Children’s Hospital, Dana-Farber Cancer Institute
Disclosures
Research grants: GLSynthesis, Lilly, Sysmex
Data monitoring boards: Lilly
Scientific advisory board: AstraZeneca
Platelet Function
• 3
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Platelet Function
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Platelet Function
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Platelet Function
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Platelet Function
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Platelet Function
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Platelet Function
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Platelet Function
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Platelet Function
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Platelet Function
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Antiplatelet Agents
FDA-approved
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Antiplatelet Agents
FDA-approved
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Antiplatelet Agents
FDA-approved
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Antiplatelet Agents
FDA-approved
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Antiplatelet Agents
FDA-approved
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Antiplatelet Agents
FDA-approved
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Congenital Disorders of Platelet Function
Michelson Nature Reviews Drug Discovery 2010;9:154-169
Congenital Disorders of Platelet Function
Bernard-Soulier
syndrome
Platelet-type VWD
GPVI defect
α2β1 defect
Receptors for
Adhesive
Proteins
Glanzmann
thrombasthenia
Modified from Michelson Nature Reviews Drug Discovery 2010;9:154-169
Congenital Disorders of Platelet Function
P2Y12 defect
Receptors for
Soluble
Agonists
TXA2 receptor
defect
Modified from Michelson Nature Reviews Drug Discovery 2010;9:154-169
Congenital Disorders of Platelet Function
α-storage pool
deficiency (gray
platelet syndrome)
α-Granules
Quebec platelet
disorder
Paris-Trousseau
syndrome/11-q
terminal deletion
disorder
Modified from Michelson Nature Reviews Drug Discovery 2010;9:154-169
Congenital Disorders of Platelet Function
δ-storage pool
deficiency
Hermansky-Pudlak
syndrome
Dense
Granules
Chediak-Higashi
syndrome
Modified from Michelson Nature Reviews Drug Discovery 2010;9:154-169
Congenital Disorders of Platelet Function
α-Granules
and Dense
Granules
α,δ-storage pool
deficiency
Modified from Michelson Nature Reviews Drug Discovery 2010;9:154-169
Congenital Disorders of Platelet Function
Signal
Transduction
G proteins
Phospholipase A2
Phospholipase C
COX1
Modified from Michelson Nature Reviews Drug Discovery 2010;9:154-169
Congenital Disorders of Platelet Function
Procoagulant
Phospholipids
Scott syndrome
Stormorken
Modified from Michelson Nature Reviews Drug Discovery 2010;9:154-169
Michelson in PLATELETS (Michelson, 3rd ed, Elsevier/Academic Press, 2013)
Bernard-Soulier Syndrome
Cattaneo in PLATELETS (Michelson, 3rd ed, Elsevier/Academic Press, 2013)
MYH9-Related Disease
Lambert & Poncz in PLATELETS (Michelson, 3rd ed, Elsevier/Academic Press, 2013)
2013;121:250
2013;121:250
Light Transmisssion Aggregometry
Jennings in PLATELETS (Michelson, 2nd ed, Elsevier/Academic Press, 2007)
Measurement of Platelet ATP Release by Lumiaggregometry
PPi = inorganic pyrophosphate
Hayward in PLATELETS (Michelson, 3rd ed, Elsevier/Academic Press, 2013)
Lumiaggregometry
PAR1 100 µM
Dawood Blood 2012;120:5041
WHYHAVEGUIDELINESFORTHEDIAGNOSISOF
INHERITEDPLATELETFUNCTIONDISORDERS(IPFD)?
• Laboratorytestsforplateletfunctiondisordersarenotwell
standardizedandnotaswidelyavailableasbloodclottingassays
• Arationalstepwisediagnosticapproach,goingfromsimple
screeningteststohighlyspecializedlaboratorytechniques,may
becost-effective
• Astepwisediagnosticapproachcanalsoallownon-specialized
centerswithlimitedresourcestoformulatewell-grounded
diagnostichypothesesforsubsequentreferraltospecialized
centers
• GeneticdiagnosisofIFPD,althoughdesirable,iscurrentlyusually
impracticalbecausesinglecandidategenesforconventional
sequencingseldomexistandnextgenerationsequencingisstill
notwidelyavailable,isrelativelyexpensiveandispronetofalsepositives
ISTH SSC Working Party JTH 2015;13:314
ISTH SSC Working Party JTH 2015;13:314
Bleeding ScoreQuestionnaire
• Astandardizedbleedingtoolistobeencouragedto
unifytheassessmentofbleedingseverity
• ThequantitativeISTHVWDBleedingScorecanbe
provisionallyused butitisnotvalidatedfor
inheritedplateletdisorders
ABleedingScoreQuestionaire shouldbespecifically
validatedforIPFD
ISTH SSC Working Party JTH 2015;13:314
Testsexcludedfromthediagnosticflowchart
•
PFA-100® and Template Skin Bleeding Time: not recommended
because of their poor diagnostic accuracy and low sensitivity
•
Impedance aggregometry: not recommended because inaccurate
for milder platelet defects (although it has theoretical advantages
like rapidity, lack of centrifugation, ease of execution)
•
Mepacrine uptake by flow cytometry not recommended for the
study of dense granules: lack of sensitivity/validation
Theymaybeusedasadditionaltestsinindividual
laboratoriesifadequatelystandardized
ISTH SSC Working Party JTH 2015;13:314
DIAGNOSTICWORK-UP
ISTH SSC Working Party
JTH 2015;13:314
FIRSTSTEPTESTS
LTA
(screening)
Bloodsmear
(lumiaggregometry)
Platelet size
S
N
L
normal
altered
All the
remaining
Morphologic
alterations
platelets
GPS
ARC
othercells
Stormorken
CHS
GATA1
GPS
MYH9-RD
BSS
GPS
GT-variant
PT-VWD
PTS
VCF
ARC
GATA1
Filaminopathy
Medich
White
MYH9-RD
α
δ
GPS
QPD
WAS(±)
ARC
Stormorken
GATA1
Scott
WAS
Flowcytometry(FC)
(screening)
Granulesrelease
α,δ
GPIIb/IIIaactivation
δ- SPD
HPS
α-δ SPD
CHS
PSD
WAS(±)
FPDAMLMDS
GT
Stormorken
Platelet GP
GPIIb/IIIa
GPIIIa
GPIb/IX
GPIb
Filaminopathy (±)
EPI(*)
QPD/α 2 receptor(±)/GT/WAS(±)/FPD AML MDS/LADIII/δSPD/PSD/cPLA2/ARC(±)/White(±)
ADP
P2Y12 defect/GPS/GT/WAS(±)/FPD
SPD/PSD/cPLA2/ARC(±)
COLLAGEN
α 2β 1/GPVI/SPD/P2Y12/GT/Stormorken/GPS/WAS/LADIII/PSD/c
PLA2/ARC(±)/FPD AML MDS/GATA1/Filaminopathy(±)
White(±)
AA
COX-1/TP
defect/GT/P2Y12
MDS/ARC(±)/White(±)
RISTOCETIN
PT-VWD/BSS/GATA1
AML
GT
GT
BSS/VC
F
BSS
MDS/LADIII/δ-
defect/δ-SPD/FPD
AML
Total amount of blood required: ~21-28ml
ISTH SSC Working Party
JTH 2015;13:314
DIAGNOSTICWORK-UP
SECONDSTEPTESTS
Flowcytometry
(extension)
LTA
(extension)
α-thr
GPS/GT/LADIII
TRAP-6
GPS/GT/LADIII
U46619
TP
defect/GT/LADIII
GPIa/IIa
α 2β 1
CRP
GPVI/GT/LADIII
GPIV
GPIV
CVX
GPVI/GT/LADIII/
Filaminopathy(±)
GPVI
GPVI
PAR4-ap
GPS/GT/LADIII
PMA
PKC
defect/GT/LADIII
A23187
Ca2+
defects/GT/LADIII
Inhibition
byIloprost
or PGE1
Serum
TxB2
Gsplateletdefect
cPLA2
COX-1
Txsynthase
deficiency
Granulescontent
Platelet
procoagulant
activity
PlateletGP
impaired enhanced
Scott
Stormorken
LTAristocetinenhanced
A) Patient’splasma+control
pltsenhanced
B) Patient’splasma+control
pltsnormal
2B-VWD
GPS
WAS(±)
ARC
Stormorken
GATA1
Clot
retraction
Mixing tests
(LTA/Flowcytometry)
A
α
B
PT-VWD
δ
α,δ
δ- SPD
HPS
CHS
WAS(±)
FPDAMLMDS
Filaminopathy(±)
α-δ SPD
TEM
Granulecontent
ormorphology
Structural
abnormalities
GPS
δ−SPD
αδ-SPD
PTS
Medich
ARC
GT
White
α 2β 1
Stormorken
York
Medich
WAS
White
PTS
Filaminopathy
Total amount of blood required: ~3-15ml
ISTH SSC Working Party
JTH 2015;13:314
DIAGNOSTICWORK-UP
Biochemicalstudies
THIRDSTEPTESTS
Surfaceglycoproteins(WB) /Spreading
assay/Adhesionandthrombusformationunder flow
conditions/Protein phosphorylation (WB,
FC)/Secondmessengers(Ca2+,IP3,cAMP)/
Receptorbindingstudies/WesternBlottingfor
MYH10
Signallingpathwaydefects
Filaminopathy(±)
WAS
Gsplateletdefect
Receptordefects
FPD/AML/MDS
PTS
MYH9-RD
Total amount of blood required: ~3-50ml
Molecular
genetic
diagnosis
ITGA2B,ITGB3
GP1BA,GP1BB,GP9
GP1BA
WAS
NBEAL2,GFI1B
Del11q23 (FLI1)
del22q11.2
CD36
GP6
TMEM16F
PLAU(duplication)
TBXA2R
HPS1,AP32B1,HPS3,
HPS4,HPS5,
HPS6,DTNBP1,
BLOC1S3,PLDN
LYST
P2RY12
GNAS
VPS33B,VIPAS39
STIM1,ORAI1
FERMT3
RUNX1
GATA1
FLNA
TBXAS1
PLA2G4A
MYH9
GT
BSS
PT-VWD
WAS
GPS
PTS
VCF
GPIVdeficiency
GPVIdeficiency
ScottSyndrome
QPD
ThromboxaneA2
HPS
CHS
P2Y12defect
Gsplateletdefect
ARC
Stormorken
LADIII
FPD/AML/MDS
GATA1
Filaminopathy
Txsynthasedeficiency
cPLA2
MYH9-RD
Association of Explanatory Variables Identified as
Significant in Univariate Analysis with Bleeding
Score After Adjustment for the Platelet Count
These tests may be useful markers of future bleeding risk in ITP
Frelinger Blood 2015;126:873
Platelet Function Testing for
Monitoring of Antiplatelet Therapy?
Laboratory tests to monitor P2Y12
inhibition
Test
Light transmission
aggregation (LTA) of
platelet-rich plasma
Principle
Decreased turbidity of
aggregated vs. non-aggregated
platelets
Readout
% aggregation
VerifyNow P2Y12
Co-aggregation of fibrinogencoated beads with platelets in
whole blood
PRU (Platelet
Reactivity Units)
Multiplate MEA
Whole blood impedance
aggregometry
AUC (area under
the curve)
PLT VASP/P2Y12
Changes in VASP
phosphorylation measured by
flow cytometry
PRI (Platelet
Reaction Index)
TEG
PlateletMapping(TM)
Platelet-dependent increase in
clot strength
MA (Maximum
Amplitude, mm)
Published recommendations/guidelines
2011 ACCF/AHA/SCAI Guideline for Percutaneous
Coronary Intervention (J Am Coll Cardiol 2011;58:e44–
122)
Class IIb
Platelet function testing may be considered in patients at
high risk for poor clinical outcomes. (Level of Evidence: C)
In patients treated with clopidogrel with high platelet
reactivity, alternative agents, such as prasugrel or ticagrelor,
might be considered. (Level of Evidence: C)
Class III: No Benefit
The routine clinical use of platelet function testing to screen
patients treated with clopidogrel who are undergoing PCI is
not recommended. (Level of Evidence: C)
Platelet Function Testing for Monitoring of
Antiplatelet Therapy?
Not currently recommended in clinical practice,
because randomized controlled trials of
modification of antiplatelet therapy based on
platelet function testing have not shown benefit:
– GRAVITAS (Price JAMA 2011:305;1097)
– TRIGGER-PCI (Trenk JACC 2012:59;2159)
– ARCTIC (Collet NEJM 2012:367;2100)
Barnard & Michelson N Engl J Med 1995;333:1051