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Dissolving the Fibrinolysis Paradigm Ed Pryzdial, Ph.D. Canadian Blood Services

Dissolving the Fibrinolysis Paradigm Ed Pryzdial, Ph.D. Canadian Blood Services Research and Development Dept. University of British Columbia Centre for Blood Research Dept. Pathology and Laboratory Medicine. “…it's not the nicotine patch, it's the blood clots moving

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Dissolving the Fibrinolysis Paradigm Ed Pryzdial, Ph.D. Canadian Blood Services

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  1. Dissolving the Fibrinolysis Paradigm Ed Pryzdial, Ph.D. Canadian Blood Services Research and Development Dept. University of British Columbia Centre for Blood Research Dept. Pathology and Laboratory Medicine

  2. “…it's not the nicotine patch, it's the blood clots moving through his body." Dr. Gregory House Dissolving Clots Must be Important… Objectives • To review the prevailing model of fibrinolysis • To shift the fibrinolysis paradigm by adding our latest data

  3. Alteplase (Tissue Plasminogen Activator): The Blockbuster Clot Buster • One of the first blockbuster recombinant protein therapeutics • Has saved or improved the lives of hundreds of thousands • Has a billion dollar annual global market

  4. Advertisement for Locals November 4, 2010, VanCity Theatre, Vancouver 10:45 - 11:45 3:15 - 4:15

  5. Clots Don’t Dissolve Easily

  6. , PAI-1 α2AP Pg Pg Pn Pn tPA tPA K tPA, tissue plasminogen activator; Pg, plasminogen; Pn, plasmin; K, C-terminal lysine; PAI-1, plasminogen activator inhibitor type-1; α2AP, alpha-2-antiplasmin The Fibrinolysis Paradigm Two phases of fibrin as the ONLY tPA cofactor II Va Xa Prothrombinase IIa [low] [high] Intact Fibrin Phase 1 Primed Fibrin Phase 2 Degraded Fibrin

  7. F G Kringle 1 Kringle 2 Protease tPA is NOT a Perfect Therapeutic tPA domain structure and function Phase 1 binding site Phase 2 binding site • Rapid clearance requires very high therapeutic dose (given as a front-loaded infusion) • Can cause cerebral hemorrhage (1-3%)

  8. tPA is NOT a Perfect Therapeutic (cont’d) • >40% of patient clots are resistant - not understood • Must be administered within 3-5 hours of clinical event • Only ~4% of potential recipients benefit

  9. F G Kringle 1 Kringle 2 Protease Kringle 2 Protease Is there more to the fibrinolysis paradigm? tPA is NOT a Perfect Therapeutic (cont’d) Engineered tPA has NOT been significant after $BILLIONS Phase 1 binding site Phase 2 binding site P1 binding P2 binding Patency +++ +++ +++ Tissue Plasminogen Activator (Front-loaded infusion) + +++ +++ Reteplase (Bolus)

  10. Xa Pg Pg Pn Pn tPA tPA K What Else in the Clot Vicinity has C-Terminal Lysine? II Va Prothrombinase IIa [low] [high] Intact Fibrin Phase 1 Primed Fibrin Phase 2 Degraded Fibrin

  11. Xa FXa-Enhances Pn Generation and Correlates to Xa33/13 Production Pg Xa33/13 100 FXa tPA FXa FXa FXa/ 75 Xa40 Xa33 Plasmin (nM) 50 Xa33 Pn tPA alone 25 Chromo- substrate 0 0 2 6 10 15 20 30 40 0 2 6 10 15 20 30 40 0 10 20 30 40 Minutes

  12. 125I Pg FXa Binds Plasminogen FXa Enhances Pn Generation FXa derivative Xa33/13 Binds Pg Protein Stain 125I-Pg Binding Xa FXa- Pn FXa- FXa33 K Xa -Pn +Pn -Pn +Pn PVDF Cleavage of FXa by Pn 13 33 FXa FXa Xa33/13

  13. light light Fast assay excludes involvement of phase 1 of fibrinolysis Research Laboratory Fibrinolysis Assay Pg .4 tPA .3 fibrinolysis Clot Amount (Turbidity A405) coagulation .2 Pn .1 0 0 30 60 90 120 Minutes

  14. +FXa/ 0 0 500 500 1000 1000 1500 1500 2000 2000 Xa light light FXa and Xa33/13 Accelerate Fibrin Clot Lysis Pg 1 M tPA 75 M tPA + 500 M PAI-1 0.3 no addition no addition tPA +FXa/ 0.2 Clot Amount Turbidity (A405) Pn +Xa33/13 +Xa33/13 0.1 0.0 Minutes

  15. FXa is more Effective than Xa33/13 and best when Present During Clot Formation All added with clotting tPA added after clotting Added after clotting with tPA 1550 1550 1100 Xa33/13 1200 1200 800 Xa33/13 Xa33/13 850 50% Lysis (min) 850 FXa/ 500 FXa/ 500 500 FXa/ 150 150 200 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 [FXa/ or Xa33/13] (nM)

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