Jean AMIRAL - June 2005

ASSAY METHODS FOR THE ASSAY METHODS FOR THE

EXPLORATION OF FIBRINOLYSISEXPLORATION OF FIBRINOLYSIS

Jean AMIRAL, PresidentJean AMIRAL, President

HYPHEN BioMed (France)HYPHEN BioMed (France)

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Jean AMIRAL - June 2005

Fibrinolysis FunctionsFibrinolysis Functions

FIBRINOLYSISFIBRINOLYSIS

Neurology (brain)Neurology (brain)

MalignancyMalignancy (metastasis)(metastasis)

ThrombosisThrombosis

FertilityFertility

Cell RemodellingCell Remodelling

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Jean AMIRAL - June 2005

Fibrinolysis ActionsFibrinolysis Actions

tPA

IntraIntra--vascularvascular

ExtraExtra--vascularvascular

α2AP/PlgHRGP

Pm-α2AP

uPATAFI PAI-1

PAI-1

uPAR

Plg

Pm

MMPs PAI-1

tPA

Clot

TIMPs

uPA

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Jean AMIRAL - June 2005

Yin and Yan effect of tPA in brainYin and Yan effect of tPA in brain

tPA

Matrix degradation (-)

Reperfusion( )

tPA

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FIBRINOLYSISFIBRINOLYSIS

DdimerDdimerfdp/FDPfdp/FDP

IIa

Pro-fibrinolytic

Anti-fibrinolytic

Endo

thel

ial

Endo

thel

ial c

ell

cell

ClotClotFibrinFibrin αα2AP2AP

PAI

PAI--11

tPAtPA

tPAtPA--PAIPAI--11

uPAuPA--PAIPAI--11

C1C1--INHINH

ScuScu--PAPA

uPAuPA

TrombomodulinTrombomodulinHRGPHRGP

TAFITAFI

αα2AP2APPlasminogenPlasminogen

TAFIaTAFIa

IIaIIa

PlasminPlasmin

ContactContactsystemsystem

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Jean AMIRAL - June 2005

Analytes involved in FibrinolysisAnalytes involved in Fibrinolysis

TriggersTriggers:: tPA, uPAtPA, uPAProteasesProteases:: Plasminogen Plasminogen Plasmin, MMPsPlasmin, MMPsRegulators of clot degradationRegulators of clot degradation:: TAFI, TAFI, αα22APAPReg. of Plg binding to clotReg. of Plg binding to clot:: αα22APAP, HRGP, HRGPInhibitorsInhibitors:: PAIPAI--1, 1, αα22AP, (PAIAP, (PAI--2), TIMPs, 2), TIMPs, ……

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Jean AMIRAL - June 2005

Major diagnostic analytes for fibrinolysisMajor diagnostic analytes for fibrinolysis

IntraIntra--vascularvascular(plasma)(plasma)

tPAtPAPAIPAI--11uPA (?)uPA (?)

ExtraExtra-- vascularvascular

uPAuPAuPAuPA--RRPAIPAI--11MMPs/TIMPs (?)MMPs/TIMPs (?)

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Jean AMIRAL - June 2005

Assay Methods for FibrinolysisAssay Methods for Fibrinolysis

Functional: Plg, TAFI, Anti-Plasmin, tPA, PAI-1, etc…Immunoassays: Plg, TAFI, Anti-Plasmin, tPA, PAI-1, tPA- or uPA- PAI-1 complexes, PAI-2, PAI-3, MMPs, TIMPs, etc…Global Assays for Fibrinolytic Potential

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Jean AMIRAL - June 2005

Functional Assays for tPA or PAIFunctional Assays for tPA or PAI--11

tPA:tPA:Specimen + Fibrin Monomers (Eq.) + PlasminogenSpecimen + Fibrin Monomers (Eq.) + PlasminogenGeneration of PlasminGeneration of PlasminChromogenic substrate for Plasmin (OD 405)Chromogenic substrate for Plasmin (OD 405)

PAIPAI--1:1:Specimen + tPA (or uPA)Specimen + tPA (or uPA)Activator + Plasminogen + SubstrateActivator + Plasminogen + SubstrateOD 405OD 405 (Measurement of tPA or uPA in excess)(Measurement of tPA or uPA in excess)

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Jean AMIRAL - June 2005

Reactivity of Antigen Assays for PAIReactivity of Antigen Assays for PAI--1, 1, tPA, uPAtPA, uPA

Should measure homogeneously all the Should measure homogeneously all the protein whether the presentation is:protein whether the presentation is:

PAIPAI--1 (Active, Bound to Vitronectin, Latent, Complexed 1 (Active, Bound to Vitronectin, Latent, Complexed to tPA or uPA, etcto tPA or uPA, etc……))tPA (Free or Complexed with PAItPA (Free or Complexed with PAI--1, etc 1, etc ……))uPA (Free or Complexed with PAIuPA (Free or Complexed with PAI--1, etc 1, etc ……))

Importance of the selection of the MoAb Importance of the selection of the MoAb pair used for designing the assays.pair used for designing the assays.

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Jean AMIRAL - June 2005

Two Site ELISA for PAITwo Site ELISA for PAI--1 (tPA):Antigen1 (tPA):Antigen

ELISA plate

ELISA plate

ELISA plate

PAI-1 (tPA)

MoAb Anti-PAI-1 (Anti-tPA)

Testedspecimen

TMB

MoAb Anti-PAI-1 (Anti-tPA)-Perox

OD 450 nm

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Jean AMIRAL - June 2005

BioBio--ImmunoImmuno--Assay for PAIAssay for PAI--1: Activity1: Activity

ELISA plate

ELISA plate

ELISA plate

Active PAI-1

Recombinant tPA

Testedspecimen

TMB

MoAb Anti-PAI-1 Perox

OD 450 nm

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Jean AMIRAL - June 2005

tPA concentration in the microtPA concentration in the micro--environmentenvironment and in blood circulationand in blood circulation

PLT

PAI-1 (liver)

PAI-1(IN)

tPAPAI-1

α2APα2M

C1-INH

tPA-PAI-1

tPA

α2AP, α2M, C1-INH

TraceAmountsFree tPA

ClotClot

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Jean AMIRAL - June 2005

PAIPAI--1 in blood vessels1 in blood vessels

uPA tPAPAI-1 PAI-1

tPA-PAI-1uPA-PAI-1

Latent PAI-1PAI-1 (VTN) (liver)

PAI-1 (IN)PLT

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Jean AMIRAL - June 2005

Specimen collectionSpecimen collection

Citrate, CTAD or EDTA anticoagulated plasma: Citrate, CTAD or EDTA anticoagulated plasma: avoid blood activation exavoid blood activation ex--vivo (PAIvivo (PAI--1 release 1 release from platelets).from platelets).

Clean venipuncture.Clean venipuncture.

Avoid tourniquet (tPAAvoid tourniquet (tPA--release).release).

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Jean AMIRAL - June 2005

Standards for tPA, uPA or PAIStandards for tPA, uPA or PAI--11

NIBSC International Standards Available:NIBSC International Standards Available:Defined by ActivityDefined by ActivityAntigen Amount Antigen Amount ±± well defined (acceptable for tPA, well defined (acceptable for tPA, discussable for PAIdiscussable for PAI--1)1)Are dependent on Assays used for their evaluationAre dependent on Assays used for their evaluation

Practically:Practically:Established International Standards for Activity (UI) Established International Standards for Activity (UI) High difficulties to standardize and harmonize antigen High difficulties to standardize and harmonize antigen concentrationsconcentrations

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Jean AMIRAL - June 2005

Available NIBSC Int. Stds. For FibrinolysisAvailable NIBSC Int. Stds. For Fibrinolysis

tPA: tPA: Human, Recombinant 3rd IS (98/714), 1999, Human, Recombinant 3rd IS (98/714), 1999, 10,000 IU per ampoule.10,000 IU per ampoule.Urokinase HMW: Urokinase HMW: 1st IS (87/594), 1989, 4,300 IU 1st IS (87/594), 1989, 4,300 IU per ampoule.per ampoule.PAIPAI--1, Plasma Human: 1, Plasma Human: 1st IS, 1995 (92/654), 27.5 1st IS, 1995 (92/654), 27.5 IU (tPA neutralization) or 7.0 IU (uPA IU (tPA neutralization) or 7.0 IU (uPA neutrlization) per ampoule.neutrlization) per ampoule.Others: Others: Plasmin, SK (All activities), NIBSC Res. Plasmin, SK (All activities), NIBSC Res. Reagent (tPA:Ag, Plasma, 25 ng/ml).Reagent (tPA:Ag, Plasma, 25 ng/ml).

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Jean AMIRAL - June 2005

Other ways to Establish StandardsOther ways to Establish Standards

Highly purified protein preparations:Highly purified protein preparations:High purity grade (> 99%)High purity grade (> 99%)Exact protein level (Lowry, BCA/Bradford, AA Exact protein level (Lowry, BCA/Bradford, AA sequence, etcsequence, etc……))Native (difficult) or Recombinant (Wild Type)Native (difficult) or Recombinant (Wild Type)

Remaining Issues:Remaining Issues:Matrix effect (?), milieu incidence Matrix effect (?), milieu incidence Assay reactivity with the various presentationsAssay reactivity with the various presentations

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Jean AMIRAL - June 2005

PAIPAI--1 Normal ranges1 Normal ranges

0 0 ––25 ng/ml25 ng/ml (4 (4 ––43 ng/ml)43 ng/ml)

MoAb/PoAbMoAb/PoAbZymutestZymutest

4 4 –– 43 ng/ml43 ng/mlMoAb/MoAbMoAb/MoAbTintelizeTintelize

4 4 –– 43 ng/ml43 ng/mlMoAb/MoAbMoAb/MoAbImulyseImulyse

4 4 –– 43 ng/ml43 ng/mlMoAb/MoAbMoAb/MoAbImubindImubind

40 40 ±± 29 ng/ml29 ng/mlMoAb/MoAbMoAb/MoAbCoalizaCoaliza

< 50 ng/ml< 50 ng/mlMoAb/MoAbMoAb/MoAbStagoStago

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Jean AMIRAL - June 2005

PAIPAI--1 Ag with various assays (ng/ml)1 Ag with various assays (ng/ml) (Declerck et al. Thromb Haem 70 (5), 1993)(Declerck et al. Thromb Haem 70 (5), 1993)

20202020171762623232884.04.060604.54.517176.26.27710109.29.28.48.431311313660.70.70.60.60.20.22.22.21.51.5551.61.63.13.14.14.18.58.53.83.844282821212020474745453383836868535311011011711722121210108.68.62828161611

TintelizeTintelizeImulyseImulyseImubindImubindCoalizaCoalizaStagoStagoSampleSample

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Jean AMIRAL - June 2005

Normal ranges for Fibrinolysis proteinsNormal ranges for Fibrinolysis proteins

0 0 –– 5 ng/ml5 ng/mluPAuPA

0 0 –– 10 ng/ml10 ng/mltPAtPA

0 0 ––25 ng/ml25 ng/ml (4(4-- 43 ng/ml)43 ng/ml)PAIPAI--11

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Jean AMIRAL - June 2005

ZYMUTEST PAIZYMUTEST PAI--1 :Ag normal range1 :Ag normal range

N=N= 5757Mean:Mean: 6.58 ng/ml6.58 ng/mlS.D. :S.D. : 5.21 ng/ml5.21 ng/mlMin.:Min.: 1.19 ng/ml1.19 ng/mlMax:Max: 25.28 ng/ml25.28 ng/ml

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Jean AMIRAL - June 2005

Correlation between PAICorrelation between PAI--1 Ag and Activity (N=253)1 Ag and Activity (N=253)

Y = 8,39x + 5,20R = 0,87

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

90,00

0,00 2,00 4,00 6,00 8,00 10,00

PAI-1 Act ng/ml

PAI-1

Ag

ng/m

l

PAI-1 Ag PAI-1 Act

Mean (ng/ml) 14.23 1.08

SD (ng/ml) 12.31 1.28

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Jean AMIRAL - June 2005

Correlation between tPA and PAICorrelation between tPA and PAI--1 Ag (N=253)1 Ag (N=253)

Y = 2,34x - 0,798R = 0,50

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

90,00

0,00 5,00 10,00 15,00 20,00

tPA - AG ng/ml

PAI-1

Ag

ng/m

l

PAI-1: Ag tPA:Ag

Mean ng/ml

14.23 6.42

SD (ng/ml) 12.31 2.63

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Jean AMIRAL - June 2005

Evaluating BodyEvaluating Body’’s Fibrinolytic Potentials Fibrinolytic Potential

Can we Evaluate the Global Fibrinolysis Potential in Can we Evaluate the Global Fibrinolysis Potential in Body?Body?

Fibrinolysis is Initiated, Regulated and Inhibited in the Fibrinolysis is Initiated, Regulated and Inhibited in the MicroMicro--Environment.Environment.

Its activity is delayed, then stimulated and finally Its activity is delayed, then stimulated and finally stopped.stopped.

Is there any Plasma Assay linked to bodyIs there any Plasma Assay linked to body’’s capacity?s capacity?Form AH102 03-2009

Jean AMIRAL - June 2005

Global Fibrinolytic Capacity (GFC)Global Fibrinolytic Capacity (GFC)

Plasma

Fibrin tablet

1 hour at 37°C

Measurement of generated DDimer

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Jean AMIRAL - June 2005

Global Fibrinolytic Capacity assayGlobal Fibrinolytic Capacity assayPlasma Thrombin, silica, Ca++

CLOT FORMATIONCLOT FORMATION

tPA

Record of clot degradationRecord of clot degradation Form AH102 03-2009

Jean AMIRAL - June 2005

AssaysAssays for global for global fibrinolyticfibrinolytic capacitycapacity

StrongStrong correlationcorrelation withwith cardiovascularcardiovascular riskriskfactorsfactors ((obesityobesity, , triglyceridestriglycerides, , bloodblood pressure, pressure, LDLLDL-- CholesterolCholesterol, glucose,, glucose,……), and type II ), and type II diabetesdiabetes or Xor X--syndrome.syndrome.

StrongStrong contribution of PAIcontribution of PAI--1.1.

Inverse Inverse relationshiprelationship withwith tPAtPA concentration.concentration.Form AH102 03-2009

Jean AMIRAL - June 2005

ClinicalClinical applications of Fibrinolysis applications of Fibrinolysis

MetabolicMetabolic Syndrome (XSyndrome (X--Syndrome)Syndrome)

DiabetesDiabetes, Type II (not , Type II (not affectedaffected by Type I)by Type I)

CardiovascularCardiovascular diseasesdiseases ((predictivitypredictivity of of tPAtPA?, ?, PAIPAI--1?, 1?, ……))

MalignancyMalignancy ((BreastBreast Cancer, Cancer, ……), ), etcetc ……Form AH102 03-2009

Jean AMIRAL - June 2005

ConclusionsConclusionsFibrinolysis Fibrinolysis isis a a keykey system in life, system in life, probablyprobably stillstill underunder--evaluatedevaluated..

Important (but Important (but occultoccult?) ?) functionfunction in in regulatingregulating manymanybiologicalbiological functionsfunctions..

Diagnostic and Diagnostic and PrognosticPrognostic Value for the Value for the keykey parametersparametersinvolvedinvolved in Fibrinolysis (in Fibrinolysis (tPAtPA, PAI, PAI--1, 1, uPAuPA, , ……).).

Diagnostic Diagnostic PotentialPotential of of OtherOther FactorsFactors (TAFI, PAI(TAFI, PAI--2, 2, MMPsMMPs, , TIMPsTIMPs, , ……)?)?

Form AH102 03-2009