1 / 44

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.

merry
Download Presentation

Plasmin

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related