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Plasmin

Plasminogen activators (t-PA, u-PA ). Plasminogen activator inhibitors (PAI). Plasminogen activator inhibitors (PAI). ProMMP. ECM. Plasminogen. Plasmin. MMP. Circulation. Fibrin degradation products. Fibrin. February 19, 2004. Plasminogen activator inhibitors.

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Plasmin

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  1. Plasminogen activators (t-PA, u-PA) Plasminogen activator inhibitors (PAI) Plasminogen activator inhibitors (PAI) ProMMP ECM Plasminogen Plasmin MMP Circulation Fibrin degradation products Fibrin February 19, 2004

  2. Plasminogen activator inhibitors • Plasminogen activator inhibitor 1 (endothelial cell type) • Plasminogen activator inhibitor 2 (placental type) • Plasminogen activator inhibitor 3 (protein C inhibitor) February 19, 2004

  3. P1’ P1 Molecular properties of PAI-1 • Member of the SERPIN superfamily February 19, 2004

  4. February 19, 2004

  5. Molecular properties of PAI-1 • Member of the SERPIN superfamily • Labile (active latent) February 19, 2004

  6. P1’ P1 P1’ P1 Active PAI-1 Latent PAI-1 February 19, 2004

  7. Molecular properties of PAI-1 • Member of the SERPIN superfamily • Labile (active latent) • Inhibits t-PA and u-PA • Active PAI-1 binds to vitronectin February 19, 2004

  8. Occurrence of PAI-1 • Plasma • Platelets • Extracellular matrix [vitronectin !] • Cells February 19, 2004

  9. % of total PAI-1 in blood 90 Antigen Activity 10 Plasma Platelets • Plasma: mainly active PAI-1 • Platelets: mainly latentPAI-1 • Extracellular matrix: only active PAI-1 February 19, 2004

  10. Pathophysiological role of PAI-1 • increased levels are associated with thrombotic disorders • role in development and progression of atherosclerosis • causative role in cancer is still controversial (dual effects) • role in tissue fibrosis • circulating vs localized ! February 19, 2004

  11. Inactivation of PAI-1 February 19, 2004

  12. I + E EI EI ’ EIc a a a I l I + E EI EI E +I ’ c s s s Conformational transitions Active Latent Substrate February 19, 2004

  13. P1’ P1 P1’ P1 Mechanism of inactivaction • Direct interaction with reactive site • Active latent • Active substrate • Active substrate latent February 19, 2004

  14. Inactivation of PAI- 1 • Polyclonal antibodies • Monoclonal antibodies • “Low molecular weight” inhibitors February 19, 2004

  15. Monoclonal Antibodies • Detailed characterization • Epitope localizaton • Cloning of smaller fragments ( scFv) February 19, 2004

  16. PAI-1 neutralizing properties ofmonoclonal antibodies MA-8H9D4 MA-33H1 100 MA-55F4 MA-33B8 MA-37H7 % neutralization of PAI-1 activity 50 0 5 10 15 t-PA molar ratio (MA:PAI-1) February 19, 2004

  17. No MA MA- 31C9 MA- 55F4 MA- 56A7C10 kD MA Complex PAI-1 /t-PA 94 t-PA non-reactive 67 43 cleaved PAI-1 30 20 14 PAI-1 neutralizing effect February 19, 2004

  18. Monoclonal Antibodies • Detailed characterization • Epitope localizaton • Cloning of smaller fragments ( scFv) February 19, 2004

  19. Linear Conformational Epitopes February 19, 2004

  20. MA-33H1F7 and MA-55F4C12 neutralizing monoclonal antibodies epitope between Glu128 and Ala156 February 19, 2004

  21. February 19, 2004

  22. 1 128 156 379 Human PAI-1 128-131 154 E V E R E M D R D V Q R E V E R E V E R K Q E K K Human + Porcine - Rabbit - Rat + Murine + Species cross-reactivity February 19, 2004

  23. K A AAAA EVER February 19, 2004

  24. Variants Mutated residues(1) KA (1/M) (2) MA-55F4C12 MA-33H1F7 PAI-1-wt none2.7 ± 1.6 109 5.4 ± 1.7 109 (1)residues mutated into an alanine (2) mean  SD (n = 4 to 11) Affinity constants for PAI-1 mutants PAI-1-EVER E128V129E130R131 9.9 ± 6.0 106 4.2 ± 4.4 108 PAI-1-K K1544.0 ± 3.1 107 3.0 ± 2.0 107 PAI-1-EVER/K E128V129E130R131 / K154 no bindingno binding February 19, 2004

  25. hinge region between -helix F and main part of PAI-1 Epitope Molecular explanation neutralizing properties mobility of the hinge is restricted t-PA not locked in covalent complex February 19, 2004

  26. February 19, 2004

  27. MA-56A7C10  neutralizing monoclonal antibody specific binding to the active conformation  no reactivity with rat PAI-1 February 19, 2004

  28. + Human PAI-1 - Rat PAI-1 26 - Rat26hum-PAI-1 Human/rat PAI-1 chimeras February 19, 2004

  29. 1 379 Human PAI-1 26 Rat26hum-PAI-1 81 Rat81hum-PAI-1 187 Rat187hum-PAI-1 327 Rat327hum-PAI-1 327 Hum327rat-PAI-1 277 Hum277rat-PAI-1 187 Hum187rat-PAI-1 81 Hum81rat-PAI-1 Rat PAI-1 Human/rat PAI-1 chimeras February 19, 2004

  30. PAI-1 variant MA-33H1F7 MA-56A7C10 Mean  SD (n = 2 to 6) Affinity constants KA (109 1/M) Human PAI-1 4.9  0.06 Rat PAI-1 2.7  0.05 1.2  0.05 no binding Rat 26 Hum 2.9  0.05 Rat 81 Hum 8.5  0.02 Rat 187 Hum 6.2  0.84 Rat 327 Hum 2.0  0.03 Hum 81 Rat 2.9  0.06 Hum 187 Rat 5.2  0.70 Hum 277 Rat 4.6  0.12 Hum 327 Rat 6.7  0.44 0.61  0.02 1.0  0.07 1.3  0.16 0.61  0.004 no binding no binding no binding no binding February 19, 2004

  31. 1 379 327 no binding no binding no binding no binding 277 187 81 Affinity of MA-56A7C10 (109 1/M) MA-56A7C10 1.2  0.05 no binding 26 0.61  0.02 1.0  0.07 1.3  0.16 0.61  0.004 81 187 327 February 19, 2004

  32. 1 379 327 Human PAI-1 350 Human + Porcine + Rat - Murine - E E T T Species cross-reactivity February 19, 2004

  33. Alanine-scanning E350 February 19, 2004

  34. Alanine-scanning E350 RR185-7 DR355-6 EKE242-4 HK190-1 D193 February 19, 2004

  35. Affinity constants for PAI-1 mutants Variants Mutated residues(1) KA (1/M) MA-56A7C10 (2) PAI-1-wt none5.4 ± 1.7 109 E350A E3500.18 ± 0.01 109 EKE242-4A E242K243E2440.11 ± 0.01 109 R356A R3560.067 ± 0.003 109 (1) residues mutated into an alanine (2)mean  SD (n = 2 to 6) February 19, 2004

  36. in the vicinity and comprising the distal hinge of the reactive site loop Epitope • neutralizing properties Molecular explanation MA-56A7C10 • higher affinity for active PAI-1 February 19, 2004

  37. Active PAI-1 Latent PAI-1 Higher affinity for PAI-1 in the active form February 19, 2004

  38. Monoclonal Antibodies • Detailed characterization • Epitope localizaton • Cloning of smaller fragments ( scFv) February 19, 2004

  39. VH VL

  40. Neutralization of PAI-1 by MA-8H9D4 andscFv-8H9D4 MA-8H9D4 scFv-8H9D4 100 % neutralization of PAI-1 activity 50 0 0 1 2 3 4 5 molar excess February 19, 2004

  41. Paratope of scFv-8H9D4 February 19, 2004

  42. General conclusions • PAI-1 is a putative therapeutic target • PAI-1 can be targeted at various positions February 19, 2004

  43. February 19, 2004

  44. General conclusions • PAI-1 is a putative therapeutic target • PAI-1 can be targeted at various positions • Molecular and structural information on epitopes and paratopes allows further rational design of PAI-1 neutralizing compounds February 19, 2004

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