ASSAY METHODS FOR THE EXPLORATION OF FIBRINOLYSIS Jean AMIRAL, President HYPHEN BioMed (France)
Fibrinolysis Functions Neurology (brain) Fertility Cell Remodelling FIBRINOLYSIS Malignancy (metastasis) Thrombosis
Fibrinolysis Actions TIMPs MMPs PAI-1 Pm Plg upa upar tpa Extra-vascular tpa Clot PAI-1 α2ap/plg HRGP Pm-α2AP TAFI PAI-1 upa Intra-vascular
Yin and Yan effect of tpa in brain Matrix degradation (-) tpa tpa Reperfusion( )
FIBRINOLYSIS tpa-pai PAI-1 upa-pai PAI-1 Scu-PA Anti-fibrinolytic Pro-fibrinolytic upa C1-INH Endothelial cell PAI PAI-1 tpa HRGP Plasminogen TAFI α2ap Plasmin Contact system IIa Trombomodulin TAFIa IIa Clot Fibrin α2ap Ddimer fdp/fdp
Analytes involved in Fibrinolysis Triggers: Proteases: tpa, upa Plasminogen Plasmin, MMPs Regulators of clot degradation: TAFI, α 2 AP Reg. of Plg binding to clot: α 2 AP,, HRGP Inhibitors: PAI-1, α 2 AP, (PAI-2), TIMPs,
Major diagnostic analytes for fibrinolysis Intra-vascular (plasma) tpa PAI-1 upa (?) Extra- vascular upa upa-r PAI-1 MMPs/TIMPs (?)
Assay 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
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)
Reactivity of Antigen Assays for PAI-1, tpa, upa Should measure homogeneously all the protein whether the presentation is: PAI-1 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.
Two Site ELISA for PAI-1 1 (tpa):antigen Tested specimen PAI-1 (tpa) MoAb Anti-PAI-1 (Anti-tPA) ELISA plate MoAb Anti-PAI-1 (Anti-tPA)-Perox ELISA plate TMB OD 450 nm ELISA plate
Bio-Immuno Immuno-Assay for PAI-1: Activity Tested specimen Active PAI-1 Recombinant tpa ELISA plate MoAb Anti-PAI-1 Perox ELISA plate TMB OD 450 nm ELISA plate
tpa concentration in the micro-environment and in blood circulation Clot tpa-pai-1 PLT PAI-1 (liver) PAI-1 (IN) tpa α2ap α2m C1-INH PAI-1 Trace Amounts Free tpa tpa α2ap, α2m, C1-INH
PAI-1 1 in blood vessels upa PAI-1 (VTN) (liver) tpa PLT PAI-1 PAI-1 (IN) PAI-1 tpa-pai-1 upa-pai-1 Latent PAI-1
Specimen collection Citrate, CTAD or EDTA anticoagulated plasma: avoid blood activation ex-vivo (PAI-1 1 release from platelets). Clean venipuncture. Avoid tourniquet (tpa-release).
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
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).
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
PAI-1 1 Normal ranges Stago Coaliza Imubind Imulyse MoAb/MoAb MoAb/MoAb MoAb/MoAb MoAb/MoAb < 50 ng/ml 40 ± 29 ng/ml 4 43 ng/ml 4 43 ng/ml Tintelize Zymutest MoAb/MoAb MoAb/PoAb 4 43 ng/ml 0 25 ng/ml (4 43 ng/ml)
PAI-1 1 Ag with various assays (ng/ml) (Declerck et al. Thromb Haem 70 (5), 1993) Sample 1 2 3 4 5 6 7 8 Stago 16 117 45 3.8 1.5 13 6.2 32 Coaliza 28 110 47 8.5 2.2 31 17 62 Imubind 8.6 53 20 4.1 0.2 8.4 4.5 17 Imulyse 10 68 21 3.1 0.6 9.2 60 20 Tintelize 12 83 28 1.6 0.7 10 4.0 20
Normal ranges for Fibrinolysis proteins PAI-1 0 25 ng/ml (4-43 ng/ml) tpa 0 10 ng/ml upa 0 5 ng/ml
ZYMUTEST PAI-1 1 :Ag normal range N= 57 Mean: S.D. : Min.: Max: 6.58 ng/ml 5.21 ng/ml 1.19 ng/ml 25.28 ng/ml
Correlation between PAI-1 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 PAI-1 Ag PAI-1 Act Mean (ng/ml) 14.23 1.08 SD (ng/ml) 12.31 1.28 0,00 0,00 2,00 4,00 6,00 8,00 10,00 PAI-1 Act ng/ml
Correlation between tpa and PAI-1 1 Ag (N=253) Y = 2,34x - 0,798 R = 0,50 90,00 PAI-1: Ag tpa:ag Mean 14.23 6.42 ng/ml SD (ng/ml) 12.31 2.63 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 5,00 10,00 15,00 20,00 tpa - AG ng/ml
Evaluating Body s s 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 body s s capacity?
Global Fibrinolytic Capacity (GFC) Plasma Fibrin tablet 1 hour at 37 C Measurement of generated DDimer
Global Fibrinolytic Capacity assay Plasma tpa Thrombin, silica, Ca ++ CLOT FORMATION Record of clot degradation
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. X Strong contribution of PAI-1. Inverse relationship with tpa concentration.
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
Conclusions Fibrinolysis is a key system in life, probably still under- evaluated. Important (but occult?) function in regulating many biological functions. Diagnostic and Prognostic Value for the key parameters involved in Fibrinolysis (tpa( tpa,, PAI-1, upa, ). Diagnostic Potential of Other Factors (TAFI, PAI-2, MMPs, TIMPs, )? )?