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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

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

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


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


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November 4, 2010, VanCity Theatre, Vancouver

10:45 - 11:45

3:15 - 4:15



,

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


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%)


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


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)


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


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


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


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


+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


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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