Hyperactive Platelets Investigation of Platelet Function

Ravi Sarode, MD Director, Transfusion Medicine and Hemostasis Reference Laboratory Professor of Pathology UT Southwestern Medical Center Dallas, TX Disclosure: Sponsored by Chronolog Corporation Platelet Disorders

Bleeding - mucocutaneous • Quantitative - thrombocytopenia • Qualitative - thrombasthenia ¾ Congenital – rare ¾ Acquired - common • Drugs, herbs, fish oil, • Autoimmune Thrombosis – arterial • Thrombocytosis • Hyperactive platelets Investigation of Platelet Function

Personal and family history Thorough drug and dietary history Screening tests • Platelet count • Peripheral Blood Smear • PFA-100® • Bleeding time obsolete Diagnostic • Aggregation study • Electron Microscopy Platelet Aggregation Study

Light Transmission Aggregometry (LTA) by Optical method • Platelet Rich Plasma (PRP) Impedance method • Whole blood aggregation (WBA) CLSI published “Platelet Function testing by Aggregometry; Approved Guidelines” - Nov 2008 Specimen Handling

Non-traumatic venepuncture Minimum stasis 3.2% Sodium Citrate Evacuated tube or plastic syringe 19-21G needle Gentle mixing Room temperature transport – upright Pneumatic tubing not recommended PRPPRP -- OpticalOptical MethodMethod

FirstFirst introducedintroduced byby BornBorn inin 19621962 ConsideredConsidered ““thethe ((g)oldg)old standardstandard”” inin plateletplatelet functionfunction evaluationevaluation PlateletPlatelet aggregationaggregation isis measuredmeasured byby LTALTA ATPATP secretionsecretion measurementmeasurement--fireflyfirefly luciferinluciferin-- luciferaseluciferase usedused asas chemiluminescencechemiluminescence substratesubstrate forfor ATPATP releaserelease detection;detection; ATPATP secretionsecretion measuredmeasured inin nmolnmol LTALTA -- PRPPRP methodmethod

WBWB centrifugedcentrifuged atat 170g170g xx 1515’’ forfor PRPPRP RemainingRemaining samplesample rere--centrifugedcentrifuged atat 1500g1500g xx 1515’’ toto obtainobtain PPPPPP PlateletPlatelet countcount inin PRPPRP shouldshould bebe inin 200200--300300 KK rangerange ¾¾ AdjustedAdjusted byby addingadding PPPPPP oror NSNS (preferred)(preferred) PRPPRP keptkept inin aggregometeraggregometer wellswells atat 3737°°CC forfor 55 minmin beforebefore studystudy MagneticMagnetic stirstir barbar inin PRPPRP cuvettecuvette maintainsmaintains plateletsplatelets inin suspensionsuspension PRPPRP -- OpticalOptical MethodMethod

BaselineBaseline lightlight transmissiontransmission throughthrough PPPPPP (clear(clear plasma)plasma) isis setset atat 100%100% BaselineBaseline lightlight transmissiontransmission throughthrough PRPPRP (opaque(opaque plasma)plasma) isis setset atat 0%0% AddAdd anan agonistagonist (ADP,(ADP, EPI,EPI, AA,AA, Coll,Coll, Risto)Risto) PlateletsPlatelets aggregateaggregate -- allowsallows increasedincreased lightlight transmittancetransmittance throughthrough plateletplatelet suspensionsuspension ResultsResults reportedreported asas percentagepercentage (%)(%) ofof aggregationaggregation NormalNormal rangesranges areare establishedestablished inin eacheach lablab PRPPRP -- OpticalOptical MethodMethod

PPP PRP ADP

Light

Baseline light transmission Increased light PRPPRP -- NormalNormal ControlControl

Risto 0.25 mg = 2%

AA 0.5 mM = 62%

Coll 2 μg = 70% Coll 1 mg = 54%

Risto 1.25 mg = 91% ADP 10 μM= 73%

AA 0.5 mM = 1.5nm

Coll 2 μg = 1.0nm Coll 1 mg = 54% WBAWBA-- ImpedanceImpedance MethodMethod

FirstFirst introducedintroduced byby CardinalCardinal andand FlowerFlower inin 19801980 MeasuresMeasures electricalelectrical resistanceresistance betweenbetween twotwo electrodeselectrodes immersedimmersed intointo wholewhole bloodblood samplesample SimultaneousSimultaneous ATPATP secretionsecretion measurementmeasurement-- fireflyfirefly luciferinluciferin--luciferaseluciferase usedused asas aa chemiluminescencechemiluminescence WBAWBA-- ImpedanceImpedance MethodMethod

WBWB diluteddiluted 1:11:1 withwith 0.90.9 %% salinesaline (450(450 µµLL each)each) SamplesSamples withwith stirstir barsbars keptkept inin wellswells atat 3737°°CC forfor 55’’ ElectrodeElectrode assemblyassembly consistsconsists ofof twotwo wireswires ¾ Reusable = palladium and Disposable – gold plated ACAC voltagevoltage inin millivoltmillivolt rangerange appliedapplied toto circuitcircuit DuringDuring equilibriumequilibrium monolayersmonolayers ofof plateletsplatelets formform ¾ Stable impedance value established AddAdd agonistsagonists (ADP,(ADP, AA,AA, CollColl andand Risto)Risto) PlateletPlatelet aggregateaggregate onon wireswires -- increasesincreases electricalelectrical resistanceresistance toto flowflow ofof electricelectric currentcurrent AggregationAggregation measuredmeasured inin OhmsOhms ((ΩΩ)) WBAWBA-- ImpedanceImpedance MethodMethod

ADP + - More aggregation Less current flow

Increased resistance

Expressed as Ω WBAWBA-- NormalNormal ControlControl

Risto 0.25 mg = 0 Ω ADP 10 μM=12 Ω

Risto 1 mg = 8 Ω

AA 0.5 mM=24 Ω Coll 2 μg=24 Ω

AA 0.5 mM=1.60 nm

Coll 2 μg = 0.66 nm

Thrombin 1 U = 0.75 nm

ComparisonComparison BetweenBetween WBAWBA andand PRPPRP

Impedance method Optical method

Blood volume 6 cc >12 cc

Test milieu Physiologic, RBCs & Platelets only WBCs present Centrifugation Not required Required ;activation, exhaustion of platelets Large platelets Present Lost

Platelet count 100-1000K 200-300K

Icteric, lipemic, Can be used Cannot be used hemolyzed ≈ 60 min ≈ 110-125 min Total test time

CaseCase 11

AA 66--yearyear oldold boyboy onon valproicvalproic acidacid forfor seizuresseizures PresentedPresented withwith plateletplatelet typetype bleedingbleeding tendencytendency NormalNormal plateletplatelet countcount andand PFAPFA--100100® PRP=PRP= ArachidonicArachidonic acidacid (63%)(63%) andand ADPADP (64%)(64%) normalnormal maximummaximum aggregationaggregation WBAWBA == therethere waswas nono responseresponse toto thesethese agonistsagonists CaseCase--11

Patient = 0 Ω Patient =63%

Control = 14 Ω

Control 76%

WBA = AA 0.5mM PRP = AA 0.5mM CaseCase--11

Patient = 0 Ω

Control = 5 Ω

Patient =64%

Control 66%

WBA = ADP 10 µM PRP= ADP 10 µM CaseCase--22

4949--WFWF--tennistennis playerplayer PresentsPresents withwith worseningworsening bruisingbruising SpontaneousSpontaneous largelarge bruisesbruises whilewhile onon fishfish oiloil SimilarSimilar largelarge bruisesbruises whenwhen onon 81mg81mg ASAASA ThreeThree vaginalvaginal deliveriesdeliveries withwith excessiveexcessive postpartumpostpartum hemorrhagehemorrhage MostMost significantsignificant hemorrhagehemorrhage followingfollowing hysterectomyhysterectomy HbHb droppeddropped fromfrom 1414 toto 88 g/dLg/dL BleedingBleeding afterafter neuromaneuroma removalremoval fromfrom footfoot ScheduledScheduled forfor extensiveextensive pelvicpelvic floorfloor surgerysurgery 0.5 = Ø Ω ↓

0.25 = 3 Ω ↓ 1 μg= 4 Ω 1 mg = 0 Ω 0.5 mg = 0 Ω 0.25 mg = 0 Ω 2 μg = 17 Ω AA Coll Risto

0.35 = nm 0.38 = nm

0.20 = nm 0.20 = nm

Case – 2

20 = 7 Ω PFA-100 10 = 7 Ω

ATP ADP ADP = 90 (49-115) Thromb = 0.43 nm ↓ EPI = 132 (83-164) Repeat Study 6 months later

0.5 = 0 Ω ↓

1μg= 22 Ω AA Coll 2μg = 28 Ω

2μg = 0.31 nm ↓ 0.5 = 0.31 nm ↓ 1μg = 0.31 nm

Risto 1 mg = 2 Ω Case – 2

0.5 = 20 Ω 2 μg = 25 Ω AA Coll

2 μg = 0.58 nm Case – 2 0.5 = 0.33 nm ↓

Post- DDAVP 20 μM= 6Ω 0.25 = 0 Ω

10 μM= 3 Ω 0.5 = 4 Ω 1 mg = 10 Ω

ADP Risto CaseCase--22 Post-DDAVP 77--33--0606 11--55--0707 11--99--0707 ThrombinThrombin ↓↓ 0.430.43 ↓↓ 0.590.59 ↓↓ 0.540.54 (0.73(0.73--1.751.75 nMnM)) AAAA 0.50.5 mM(17mM(17--26)26) ↓↓0/0.350/0.35 ↓↓0/0.310/0.31 ↓↓ 88 CollColl 22µµgg (20(20--30)30) ↓↓ 1717 /0.38/0.38 28/0.6128/0.61 2525 /0.25/0.25 ADPADP 1010 µµMM (8(8--18)18) ↓↓ 77 ↓↓ 00 ↓↓ 66 ADPADP 2020 µµMM (9(9--20)20) ↓↓ 77 ↓↓ 22 ↓↓ 33 RistoRisto 1.01.0 mgmg (>5)(>5) ↓↓ 00 ↓↓ 22 1010 RistoRisto 0.250.25 mgmg (0)(0) 00 00 00 CaseCase 22

ReceivedReceived 0.3ug/Kg0.3ug/Kg DDAVPDDAVP halfhalf anan hourhour beforebefore surgerysurgery UnderwentUnderwent 44 hourhour extensiveextensive pelvicpelvic reconstructionreconstruction surgerysurgery NoNo significantsignificant bleeding!bleeding! DiagnosisDiagnosis ¾ ? Mild global dysfunction SheShe stillstill bruisesbruises offoff andand onon –– muchmuch lessless thanthan fishfish oiloil oror 8181 mgmg ASAASA CaseCase--33

3333--yryr--WFWF presentedpresented withwith flankflank pain,pain, hematuria,hematuria, painfulpainful lesionslesions onon LL--foot,foot, transienttransient antiphospholipidantiphospholipid antibodiesantibodies EpistaxisEpistaxis offoff andand onon TheseThese bleedingbleeding symptomssymptoms startedstarted recentlyrecently NoNo priorprior bleedingbleeding historyhistory EmotionallyEmotionally disturbeddisturbed MedicalMedical SalesSales rep!rep! HadHad transienttransient pancytopeniaspancytopenias AA 0.25 = 0 Ω AA 0.5 = 1 Ω Coll 1μg= 2Ω ADP10=6Ω

ADP 20 = 7 Ω Coll 2 = 7 Ω Risto 1 = 6 Ω

WBA

AA 0.5 = 0.33 nm Thro = 0.59 nm AA = 0.17 nm Coll = 0.12 nm Coll 2 = 0.26 nm

Case – 3 Agonist WBA PRP AA 0.5 = 8% AA ↓ ↓ Coll 2 = 39% PRP ADP 20 = 66% Coll ↓ ↓ ADP N N Risto 1.25 = 68% Risto N N ATP sec ↓ ↓ CaseCase--33

Interpretation:Interpretation: AspirinAspirin likelike defectdefect HyperactiveHyperactive PlateletsPlatelets

AlsoAlso calledcalled ““stickysticky plateletplatelet syndromesyndrome”” PersistentPersistent elevationelevation ofof FVIII/VWFFVIII/VWF andand fibrinogenfibrinogen -- thrombophiliathrombophilia riskrisk factorsfactors SimilarlySimilarly –– increasedincreased plateletplatelet reactivityreactivity couldcould causecause thrombosisthrombosis ¾ Millions of patients on anti-platelets drugs ¾ Aspirin and clopidogrel resistance (20-30%) is well described DetectedDetected byby increasedincreased aggregationaggregation responsesresponses toto submaximalsubmaximal concentrationsconcentrations ofof agonistsagonists HyperactiveHyperactive PlateletsPlatelets

““ThrombosisThrombosis andand bleedingbleeding inin MPD:MPD: identificationidentification ofof atat riskrisk ptspts withwith WBAWBA”” ¾ Manoharan et al, Br J Hematol 1999

““AggregometryAggregometry detectsdetects plateletplatelet hyperactivityhyperactivity inin healthyhealthy individualsindividuals”” ¾ Paul Bray’s group – Blood 2005 WBAWBA -- HyperactiveHyperactive PlateletPlatelet StudyStudy

SpontaneousSpontaneous aggregationaggregation ADPADP == 10,10, 5,5, 2.52.5 andand 1.251.25 µµMM AAAA == 0.5,0.5, 0.25,0.25, 0.1250.125 andand 0.06250.0625 mMmM CollColl == 2.0,2.0, 1.0,1.0, 0.50.5 andand 0.250.25 µµg/mLg/mL ¾¾ EstablishEstablish normalnormal rangesranges ¾¾ DoDo notnot useuse ATPATP secretionsecretion CaseCase -- 44

A 47 year-old female with history of SLE, MI and multiple CVAs despite being on daily 81mg ASA Diagnosed with Hyperactive Platelet Syndrome in the past CaseCase--44

ADP Collagen ADP 2.5 µM; 0 Ω 0.5 µg/ml; 0 Ω AA 5 µM; 8 Ω AA

0.25 mM; 26 Ω 0.125 mM; 24 Ω Collagen (normal = 3-14 ) (normal = 0-10 ) 1 µg/ml; 20 Ω (normal = 7-15 )

ADP AA Hyperactivity 10 µM; 22 Ω 0.063 mM; 0 Ω (normal = 0-3) AA 0.5 mM; 21 Ω with AA and (normal = 9-14 ) Collagen 2 µg/ml; 23 Ω Collagen (normal = 10-17 ) CaseCase -- 55

5555 yryr malemale withwith aa diagnosisdiagnosis ofof EssentialEssential ThrombocythemiaThrombocythemia PlateletPlatelet countscounts betweenbetween 500500--700K700K onon chemotherapychemotherapy DevelopsDevelops suddensudden SOBSOB andand chestchest painpain ¾¾ ChestChest CTCT –– PEPE ThrombophiliaThrombophilia workwork--upup negativenegative HyperactiveHyperactive plateletplatelet studystudy Case - 5

32 Ohms 5 Ohms

30 Ohms 36 Ohms AspirinAspirin andand ClopidogrelClopidogrel EffectEffect

WBAWBA detectsdetects antiplateletantiplatelet drugdrug effectseffects reliablyreliably andand quicklyquickly AgonistsAgonists usedused ArachidonicArachidonic acidacid andand collagencollagen forfor ASAASA ADPADP forfor clopidogrelclopidogrel (ticlopidine(ticlopidine andand dipyridamoledipyridamole)) DetectionDetection ofof ClopidogrelClopidogrel EffectEffect

1717 volunteersvolunteers givengiven 7575 mg/daymg/day xx 10days10days PlateletPlatelet functionfunction atat baselinebaseline andand onon dayday 1111 PRP,PRP, WBAWBA andand PFAPFA--100100 WBAWBA moremore sensitivesensitive toto detectdetect clopidogrelclopidogrel inhibitioninhibition byby ADPADP FollowFollow--upup showedshowed PRPPRP normalizesnormalizes beforebefore WBAWBA RBCsRBCs knownknown toto modulatemodulate plateletplatelet functionfunction duedue toto itsits ADPADP contentcontent

Korpanty et al, Platelets, 2007 PostPost--clopidogrelclopidogrel PlateletPlatelet InhibitionInhibition

ADPADP (n=16)(n=16) MethodMethod Pre Post %↓ WBA 10 μM 18 ± 6 5 ± 6 70 ± 35* (Ω)

PRP 10 μM 80 ± 11 53 ± 20 36 ± 25 (%)

PFA-100® 86 ± 13 101 ± 25 17 ± 23 (Sec.)

*P = <0.001 Korpanty et al, Platelets, 2007 DetectionDetection ofof AspirinAspirin EffectEffect

ComparedCompared WBAWBA withwith VerifyNowVerifyNow AspirinAspirin 5050 volunteersvolunteers givengiven 325325 mgmg xx 3days3days BaselineBaseline andand dayday 44 evaluationevaluation byby WBAWBA (AA(AA andand Coll)Coll) andand VerifyNowVerifyNow AspirinAspirin 12/5012/50 (24%)(24%) nonnon--respondersresponders byby WBAWBA AllAll goodgood respondersresponders (<550(<550 ARU)ARU) byby VerifyNowVerifyNow ¾¾ SeemsSeems thethe cutcut offoff shouldshould bebe muchmuch lowerlower thanthan 550550 ARU.ARU.

Korpanty et al, Thromb Res, 2006 CaseCase -- 66

5555--yryr--WFWF withwith aa diagnosisdiagnosis ofof basilarbasilar arteryartery aneurysmaneurysm ofof brainbrain ScheduledScheduled forfor stentstent assistedassisted coiledcoiled embolizationembolization ToTo preventprevent thrombosisthrombosis atat thethe sitesite ofof coilcoil embolizationembolization plateletplatelet functionfunction hashas toto bebe inhibitedinhibited completelycompletely GivenGiven ASAASA 325325 mgmg andand loadingloading dosedose ofof clopidogrelclopidogrel 300300 mgmg AntiAnti--plateletplatelet effecteffect 44 hourshours laterlater –– nono effecteffect DoubledDoubled ASAASA andand ClopidogrelClopidogrel –– adequateadequate effecteffect Case – 6

AA 0.5 = 6 Ω All = 0 Ω

10 μM ADP = 13 Ω

Coll 1mg = 14 Ω

I. ASA/Plavix - No Effect 2. ASA/Plavix – Adequate

Summary PRPPRP -- LTALTA

PRPPRP methodmethod isis ¾ Labor-intensive ¾ Time consuming ¾ Injurious to platelets (centrifugation) Giant,Giant, hyperhyper-- oror hypoactivehypoactive--plateletsplatelets ¾ Lost during centrifugation ¾ Excluded from evaluation PRPPRP milieumilieu devoiddevoid ofof ¾ RBCs and ¾ WBCs that influence platelet function WBWB--ImpedanceImpedance--basedbased AggregationAggregation

OffersOffers ¾ Rapid evaluation of platelet function ¾ No sample processing ¾ No centrifugation injury to platelets MoreMore physiologicphysiologic milieu,milieu, RBCsRBCs && WBCsWBCs includedincluded SmallerSmaller quantityquantity ofof bloodblood requiredrequired ¾ Advantage in young children ¾ Patients with difficulties in drawing blood MoreMore suitablesuitable andand sensitivesensitive methodmethod forfor ¾ Assessment of anti-platelet therapy ¾ Platelet hyperactivity