Plasminogen activators tPA uPA Plasminogen activator inhibitors PAI

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

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

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

February 19, 2004

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

P 1’ P 1 Active PAI-1 Latent PAI-1 February 19, 2004

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

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

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

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

Inactivation of PAI-1 February 19, 2004

Conformational transitions v cti A e Ia + E EIa’ EIc EI s’ E +I c t n e t La I l b Su te a str I s+ E February 19, 2004

Mechanism of inactivaction P 1’ P 1 • Direct interaction with reactive site • Active latent • Active substrate P 1’ P 1 latent February 19, 2004

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

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

% neutralization of PAI-1 activity PAI-1 neutralizing properties of monoclonal antibodies MA-8 H 9 D 4 MA-33 H 1 MA-55 F 4 MA-33 B 8 MA-37 H 7 100 50 0 5 10 molar ratio (MA: PAI-1) 15 t-PA February 19, 2004

PAI-1 neutralizing effect k. D No MA MA 31 C 9 MAMA 55 F 4 56 A 7 C 10 MA 94 Complex PAI-1 /t-PA 67 t-PA 43 non-reactive cleaved PAI-1 30 20 14 February 19, 2004

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

Epitopes Linear Conformational February 19, 2004

MA-33 H 1 F 7 and MA-55 F 4 C 12 neutralizing monoclonal antibodies epitope between Glu 128 and Ala 156 February 19, 2004

February 19, 2004

Species cross-reactivity 1 128 156 379 Human PAI-1 Human Porcine Rabbit Rat Murine 128 -131 154 EVER EMDR DVQR EVER K Q E K K + + + February 19, 2004

A AAAA K EVER February 19, 2004

Affinity constants for PAI-1 mutants Variants Mutated residues(1) KA (1/M) (2) MA-55 F 4 C 12 MA-33 H 1 F 7 PAI-1 -wt none 2. 7 ± 1. 6 109 5. 4 ± 1. 7 109 PAI-1 -EVER E 128 V 129 E 130 R 131 9. 9 ± 6. 0 106 4. 2 ± 4. 4 108 PAI-1 -K K 154 4. 0 ± 3. 1 107 3. 0 ± 2. 0 107 PAI-1 -EVER/K E 128 V 129 E 130 R 131 / K 154 no binding (1) (2) no binding residues mutated into an alanine mean SD (n = 4 to 11) February 19, 2004

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

February 19, 2004

MA-56 A 7 C 10 neutralizing monoclonal antibody specific binding to the active conformation no reactivity with rat PAI-1 February 19, 2004

Human/rat PAI-1 chimeras Human PAI-1 + Rat PAI-1 - 26 Rat 26 hum-PAI-1 - February 19, 2004

Human/rat PAI-1 chimeras 1 379 Human PAI-1 Rat 26 hum-PAI-1 Rat 81 hum-PAI-1 26 81 187 Rat 187 hum-PAI-1 327 Rat 327 hum-PAI-1 327 Hum 327 rat-PAI-1 277 Hum 277 rat-PAI-1 187 Hum 187 rat-PAI-1 Hum 81 rat-PAI-1 81 Rat PAI-1 February 19, 2004

Affinity constants KA (109 1/M) PAI-1 variant MA-33 H 1 F 7 MA-56 A 7 C 10 Human PAI-1 4. 9 0. 06 1. 2 0. 05 Rat PAI-1 2. 7 0. 05 no binding Rat 26 Hum 2. 9 0. 05 0. 61 0. 02 Rat 81 Hum 8. 5 0. 02 1. 0 0. 07 Rat 187 Hum 6. 2 0. 84 1. 3 0. 16 Rat 327 Hum 2. 0 0. 03 0. 61 0. 004 Hum 81 Rat 2. 9 0. 06 no binding Hum 187 Rat 5. 2 0. 70 no binding Hum 277 Rat 4. 6 0. 12 no binding Hum 327 Rat 6. 7 0. 44 no binding Mean SD (n = 2 to 6) February 19, 2004

Affinity of MA-56 A 7 C 10 (109 1/M) MA-56 A 7 C 10 1 379 1. 2 0. 05 no binding 26 0. 61 0. 02 81 1. 0 0. 07 187 327 81 187 1. 3 0. 16 0. 61 0. 004 no binding 277 no binding 327 no binding February 19, 2004

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

Alanine-scanning E 350 February 19, 2004

Alanine-scanning E 350 EKE 242 -4 RR 185 -7 DR 355 -6 HK 190 -1 D 193 February 19, 2004

Affinity constants for PAI-1 mutants Variants Mutated residues(1) KA (1/M) MA-56 A 7 C 10 (2) PAI-1 -wt none 5. 4 ± 1. 7 109 E 350 A E 350 0. 18 ± 0. 01 109 EKE 242 -4 A R 356 A E 242 K 243 E 244 R 356 0. 11 ± 0. 01 109 0. 067 ± 0. 003 109 (1) (2) residues mutated into an alanine mean SD (n = 2 to 6) February 19, 2004

MA-56 A 7 C 10 in the vicinity and comprising Epitope the distal hinge of the reactive site loop Molecular explanation • neutralizing properties • higher affinity for active PAI-1 February 19, 2004

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

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

VH VL

% neutralization of PAI-1 activity Neutralization of PAI-1 by MA-8 H 9 D 4 and sc. Fv-8 H 9 D 4 MA-8 H 9 D 4 sc. Fv-8 H 9 D 4 100 50 0 0 1 2 3 4 5 molar excess February 19, 2004

Paratope of sc. Fv-8 H 9 D 4 February 19, 2004

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

February 19, 2004

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