ASSAY METHODS FOR THE EXPLORATION OF FIBRINOLYSIS

Jean AMIRAL, President HYPHEN BioMed (France)

Form AH102 03-2009 Jean AMIRAL - June 2005

Fibrinolysis Functions
Neurology (brain)

Fertility

FIBRINOLYSIS
Cell Remodelling

Malignancy (metastasis)

Thrombosis

Form AH102 03-2009 Jean AMIRAL - June 2005

Fibrinolysis Actions
TIMPs MMPs PAI-1 Pm uPA Plg uPAR tPA

Extra-vascular
Clot

tPA

PAI-1

2AP/Plg HRGP TAFI PAI-1

Pm-2AP uPA

Intra-vascular
Form AH102 03-2009 Jean AMIRAL - June 2005

Yin and Yan effect of tPA in brain

Matrix degradation tPA

(-)

tPA

Reperfusion(

Form AH102 03-2009 Jean AMIRAL - June 2005

FIBRINOLYSIS
uPA-PAI-1 tPA-PAI-1 uPA C1-INH
PA I-1
Anti-fibrinolytic

Scu-PA

Pro-fibrinolytic

Endothelial cell

Contact system HRGP

TAFI

IIa

IIa Trombomodulin

Plasminogen 2AP

TAFIa tPA Plasmin

2AP

Fibrin Clot

Ddimer fdp/FDP

Form AH102 03-2009 Jean AMIRAL - June 2005

Analytes involved in Fibrinolysis

Triggers: Proteases: tPA, uPA Plasminogen Plasmin, MMPs

Regulators of clot degradation: TAFI, 2AP Reg. of Plg binding to clot: 2AP, HRGP Inhibitors: PAI-1, 2AP, (PAI-2), TIMPs,
Form AH102 03-2009 Jean AMIRAL - June 2005

Major diagnostic analytes for fibrinolysis Intra-vascular (plasma) tPA PAI-1 uPA (?) Extra- vascular uPA uPA-R PAI-1 MMPs/TIMPs (?)
Form AH102 03-2009 Jean AMIRAL - June 2005

Assay Methods for Fibrinolysis

Functional: Plg, TAFI, Anti-Plasmin, tPA, PAI-1, etc Immunoassays: Plg, TAFI, AntiPlasmin, tPA, PAI-1, tPA- or uPA- PAI-1 complexes, PAI-2, PAI-3, MMPs, TIMPs, etc Global Assays for Fibrinolytic Potential
Form AH102 03-2009 Jean AMIRAL - June 2005

Functional Assays for tPA or PAI-1

tPA:
Specimen + Fibrin Monomers (Eq.) + Plasminogen Generation of Plasmin Chromogenic substrate for Plasmin (OD 405)

PAI-1:
Specimen + tPA (or uPA) Activator + Plasminogen + Substrate OD 405 (Measurement of tPA or uPA in excess)
Form AH102 03-2009 Jean AMIRAL - June 2005

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

Should measure homogeneously all the protein whether the presentation is:
PAI-1 (Active, Bound to Vitronectin, Latent, Complexed to tPA or uPA, etc) tPA (Free or Complexed with PAI-1, etc ) uPA (Free or Complexed with PAI-1, etc )

Importance of the selection of the MoAb pair used for designing the assays.
Form AH102 03-2009 Jean AMIRAL - June 2005

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

Tested specimen
ELISA plate PAI-1 (tPA) MoAb Anti-PAI-1 (Anti-tPA)

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

ELISA plate

TMB OD 450 nm
ELISA plate
Form AH102 03-2009 Jean AMIRAL - June 2005

Bio-Immuno-Assay for PAI-1: Activity

Tested specimen
ELISA plate Active PAI-1 Recombinant tPA

MoAb Anti-PAI-1 Perox

ELISA plate

TMB OD 450 nm
ELISA plate
Form AH102 03-2009 Jean AMIRAL - June 2005

tPA concentration in the micro-environment and in blood circulation

Clot PAI-1 PLT PAI-1 (liver) PAI-1 (IN) tPA

2AP 2M C1-INH

tPA-PAI-1 Trace Amounts Free tPA tPA 2AP, 2M, C1-INH

Form AH102 03-2009 Jean AMIRAL - June 2005

PAI-1 in blood vessels

uPA PAI-1 (VTN) (liver)

tPA

PAI-1

PLT

PAI-1 (IN)

PAI-1 tPA-PAI-1 uPA-PAI-1 Latent PAI-1

Form AH102 03-2009 Jean AMIRAL - June 2005

Specimen collection
Citrate, CTAD or EDTA anticoagulated plasma: avoid blood activation ex-vivo (PAI-1 release from platelets). Clean venipuncture. Avoid tourniquet (tPA-release).
Form AH102 03-2009 Jean AMIRAL - June 2005

Standards for tPA, uPA or PAI-1

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

Practically:
Established International Standards for Activity (UI) High difficulties to standardize and harmonize antigen concentrations
Form AH102 03-2009 Jean AMIRAL - June 2005

Available NIBSC Int. Stds. For Fibrinolysis

tPA: Human, Recombinant 3rd IS (98/714), 1999, 10,000 IU per ampoule. Urokinase HMW: 1st IS (87/594), 1989, 4,300 IU per ampoule. PAI-1, Plasma Human: 1st IS, 1995 (92/654), 27.5 IU (tPA neutralization) or 7.0 IU (uPA neutrlization) per ampoule. Others: Plasmin, SK (All activities), NIBSC Res. Reagent (tPA:Ag, Plasma, 25 ng/ml).
Form AH102 03-2009 Jean AMIRAL - June 2005

Other ways to Establish Standards

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

Remaining Issues:
Matrix effect (?), milieu incidence Assay reactivity with the various presentations
Form AH102 03-2009 Jean AMIRAL - June 2005

PAI-1 Normal ranges

Stago Coaliza Imubind Imulyse Tintelize Zymutest
MoAb/MoAb

< 50 ng/ml 40 29 ng/ml 4 43 ng/ml 4 43 ng/ml 4 43 ng/ml 0 25 ng/ml (4 43 ng/ml)

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MoAb/MoAb

MoAb/MoAb

MoAb/MoAb

MoAb/MoAb

MoAb/PoAb

Jean AMIRAL - June 2005

PAI-1 Ag with various assays (ng/ml)

(Declerck et al. Thromb Haem 70 (5), 1993)

Sample

Stago

Coaliza Imubind Imulyse Tintelize

1 2 3 4 5 6 7 8

16 117 45 3.8 1.5 13 6.2 32

28 110 47 8.5 2.2 31 17 62

8.6 53 20 4.1 0.2 8.4 4.5 17

10 68 21 3.1 0.6 9.2 60 20

12 83 28 1.6 0.7 10 4.0 20

Form AH102 03-2009 Jean AMIRAL - June 2005

Normal ranges for Fibrinolysis proteins

PAI-1 0 25 ng/ml (4- 43 ng/ml) 0 10 ng/ml

tPA

uPA

0 5 ng/ml

Form AH102 03-2009 Jean AMIRAL - June 2005

ZYMUTEST PAI-1 :Ag normal range

N= S.D. : Min.: Max: 57 5.21 ng/ml 1.19 ng/ml 25.28 ng/ml
Form AH102 03-2009 Jean AMIRAL - June 2005

Mean: 6.58 ng/ml

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

Y = 8,39x + 5,20 R = 0,87
90,00 80,00 70,00 PAI-1 Ag ng/ml 60,00 50,00 40,00 30,00 20,00 10,00 0,00 0,00 2,00 4,00
Mean (ng/ml) SD (ng/ml) PAI-1 Ag 14.23 12.31 PAI-1 Act 1.08 1.28

6,00

8,00

10,00

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PAI-1 Act ng/ml

Jean AMIRAL - June 2005

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

Y = 2,34x - 0,798 R = 0,50
90,00 80,00 70,00 PA A ng/ml I-1 g 60,00 50,00 40,00 30,00 20,00 10,00 0,00 0,00 5,00 10,00 tPA - AG ng/ml 15,00 20,00
PAI-1: Ag Mean ng/ml SD (ng/ml) 14.23 12.31 tPA:Ag 6.42 2.63

Form AH102 03-2009

Jean AMIRAL - June 2005

Evaluating Bodys Fibrinolytic Potential

Can we Evaluate the Global Fibrinolysis Potential in Body? Fibrinolysis is Initiated, Regulated and Inhibited in the Micro-Environment. Its activity is delayed, then stimulated and finally stopped. Is there any Plasma Assay linked to bodys capacity?
Form AH102 03-2009 Jean AMIRAL - June 2005

Global Fibrinolytic Capacity (GFC)

Plasma Fibrin tablet

1 hour at 37C

Measurement of generated DDimer

Form AH102 03-2009 Jean AMIRAL - June 2005

Global Fibrinolytic Capacity assay

Plasma tPA Thrombin, silica, Ca++

CLOT FORMATION

Record of clot degradation

Form AH102 03-2009 Jean AMIRAL - June 2005

Assays for global fibrinolytic capacity

Strong correlation with cardiovascular risk factors (obesity, triglycerides, blood pressure, LDL- Cholesterol, glucose,), and type II diabetes or X-syndrome. Strong contribution of PAI-1. Inverse relationship with tPA concentration.
Form AH102 03-2009 Jean AMIRAL - June 2005

Clinical applications of Fibrinolysis

Metabolic Syndrome (X-Syndrome) Diabetes, Type II (not affected by Type I) Cardiovascular diseases (predictivity of tPA?, PAI-1?, ) Malignancy (Breast Cancer, ), etc
Form AH102 03-2009 Jean AMIRAL - June 2005

Conclusions
Fibrinolysis is a key system in life, probably still underevaluated. Important (but occult?) function in regulating many biological functions. Diagnostic and Prognostic Value for the key parameters involved in Fibrinolysis (tPA, PAI-1, uPA, ). Diagnostic Potential of Other Factors (TAFI, PAI-2, MMPs, TIMPs, )?
Form AH102 03-2009 Jean AMIRAL - June 2005