Targeting the Contact Phase – potential for betteranticoagulants?

Thomas RennéInstitute for Clinical Chemistry and Laboratory Medicine

University Medical Center Hamburg-Eppendorf

The contact pathway

ThrombocytesEndothelial cells Cancer CellsLeukocytes

PKHK

Bradykinin

HKFXII

FXI

B2R

Signalling cascades

Coagulation

FXII

Renné et al, Blood (2018)

ActivatorInflammation

Thromboembolic disease

MI

DVT/PE

stroke

The plasma coagulation system

TF

TFFVII

--

FXII

FXI

Fibrin

Thrombin

ContactInjury

aPTT

5 billionaPTT assays

annually

Ratnoff & Colopy, J Clin Invest (1955)

FXII deficiency - normal haemostasis

John Hageman

TF

TFFVII

FXII

FXI

Fibrin

Thrombin

ContactInjury

FXII has no function in coagulation ?

Naito & Fujikawa, J Biol Chem (1981); Gailani & Broze, Science (1981)

aPTTprolonged

20

40

60

80

aPTT [s]

WT + Heparin

0

300

600

900

1200

> 1200

bleeding time [s]

WT + Heparin

WT FXII-/- FXII-/-

FXII-/- mice have a prolonged aPTT butnormal haemostasis

Pauer et al, Thromb Haemost (2004)

WT

Renné et al, J Exp Med (2005); Kleinschnitz et al, J Exp Med (2006)

Defective thrombosis in FXII-/-micewild-type

FXII-/-

0

20

40

60

*

FXI-/-wt

isch

emic

volu

me

[mm

3 ]

FXI-/-wt FXII-/-

+ + +- - -

fibrin

Impaired pathological fibrin formation in FXI-/-mice

fibrin GP Ib merge

Targeting FXII activity

Antibody 3F7 (3F12) selectively blocks FXII activity

ECMO: Extra Corporeal Membrane Oxygenation

Highly thrombogenic

Needs anticoagulation

Targeting FXIIa provides thromboprotection in ECMO

Saline

3F7

Heparin

D Pressure[mm Hg]

Larsson et al, Science Trans Med (2014)

Heparin

Bleeding timeskin [sec]

Blood losscuticle [ml/10 min]

300

600

NaCl0

3F7

2,5

5,0

0HeparinNaCl 3F7

Targeting FXII activity does not interfere with haemostasis

Targeting FXII in baboons and FXI activity in rabbitsprotects against thrombosis sparing hemostasis

David et al, Science Transl Med (2016)Matafonov et al, Blood (2014)

Effect of FXI-ASO on the Coagulation System.

Büller et al, N Engl J Med (2014)

FXI knockdown using ASO provides safe anticoagulationin patients after knee surgery

Thrombosis

Maas & Renné, Blood (2018)

HaemostasisBleeding

Thrombus

Sealing ofwounds

FXII-drivencoagulation

Revision of the classical coagulation balance

FXII-driven coagulation in thrombosis

Haemostasis Thombosis

Kenne et al, J Int Med (2015)

FXII-/-WT

accelerated clotting time [x-fold]

4

3

2

platelet activator +-+-

5

Castaldi et al, Nature (1965); Walsh & Griffin, Blood (1981); Johne et al, BC (2006); Bäck et al, BBRC (2010)

Activated platelet-driven coagulation is defective inFXII-deficient plasma

• glass• silica• dextran sulfate• kaolin

• DNA/RNA• collagen• lipopolysaccaride• glycosaminoglycans

artificial natural

FXII contact activators

Maas et al, Sem Thromb Hemost (2011)

throm

bin

+

+ p

hosp

hatas

e

[bp]

2550

100200

w/o trap6

throm

bin

Platelet polyphosphate (polyP)

Müller, Mutch et al, Cell (2009)

Platelet polyP initiates FXII-driven coagulation

thrombin [nM]200

100

FVIIai + polyP

0 10 20 30 40time [min]

+ 4.8 µg/ml

+ 1.2 µg/ml

w/o

FXII-def.0

thrombin

TF FVIIa

FVIIai/ASISFXIIa

FXIa

Müller, Mutch et al, Cell (2009)

2/15 10/1513/15

WT WT +PGE1

FXII-/-

FXII-/-mice are protected from polyP- and activated platelet-driven thrombosis

Purified platelet polyP Activated platelets

Inherited deficiency in platelet polyP protectsIp6K1-/-mice from thrombosis

Gosh et al, Blood (2013)

Platelet polyP promotes thrombosis

Modified from Caen & Wu, J Thromb Haemost (2010)Walker et al, Br J Haematol (2007)

n

Activated platelets release polyP into the supernatant…

Labberton et al, Nat Commun (2016)

polyP[nM Pi/108 platelets]

…however the majority of polyP is retained at the platelet surface

Labberton et al, Nat Commun (2016)

polyP[nM Pi/108 platelets]

Insoluble polyP on platelet surfaces

Verhoef et al, Blood (2017)

polyPExtracellular polyPTotal polyP

polyP

Wijeyewickrema et al, Blood (2016)

polyP

solublesupernatant

insolublecell surface

Platelets have two pools of polyP: long chain/insoluble and short chain/soluble polymers

Verhoef et al, Blood (2017)

Platelet polyP form Ca2+-rich microparticles

DensityVerhoef et al, Blood (2017)

Chain-lengthindependent

Disassembly of platelet polyP microparticles impairstheir FXII activating potential

+EDTA

Surface-associated(insoluble)

Verhoef et al, Blood (2017)

soluble

FXII

activ

ity

Platelet-derived polyP microparticles drive thrombosisin a FXII-dependent manner

Nickel et al., ATVB (2017)

Hemostasis Thrombosis

Analysis of polyP nanoparticles on platelet surfaces

Labberton et al, Flow Cytometry (2017)

Targeting the polyP/contact pathway in

cancer-associated thrombosis

Prostate cancer exposes polyP that activates FXII

polyP

FXIIa

overlay

+ Ph

osph

atas

eProstate cancer

polyP

Fibrin

Cancer patient plasmacells MP tissue

Nickel et al, Blood (2015)

Targeting FXII interferes with prostate cancer-triggered PE

The polyP/contact pathway contributes to thrombosis in cancer

Nickel et al, Thromb Res (2016)

Targeting polyP

Degradation of polyP ablates their procoagulat activity

E. coli exopolyphosphatase(PPX)

Targeting polyP blocks procoagulant platelet activity

FXII-/-WT

accelerated clotting time [x-fold]

3

2

1

platelet activator + ++-

4

WT +PPX

Labberton et al, Nat Commun (2016)

Targeting polyP interferes with platelet-driven thrombosis without affecting hemostasis

Labberton et al, Nat Commun (2016)

Regulation of platelet polyP

Pi polyP

Systems biology identified XPR1 as the majorPi exporter in platelets

Mailer, Allend et al, (in revision 2020)

Platelet Xenotropic and polytropic retrovirus receptor 1 (XPR1)

Merge

XPR1

Bright field

Mailer, Allende et al, BLOOD (2020)

Pi

intracellular

extracellular

SPX

PDZbm

N

C

Receptordomain

retrovirus

Subcellular localization of XPR1 in platelets

XPR1 expression inversely regulates polyP in cellsMeg01HEK293

XPR1

40

pCHIX-XPR1 (μg)

84210.5Mock

70

MW(kDa)

VASP

Mock

70

2 4 8 16 32

pCHIX-XPR1 (μg)

40

XPR1

VASP

Mw(kDa)

overexpression

Knock-down

Pharmacology: Recombinant XPR1 inhibitors

XVDL

259XLV-Env

1

6xHis RBD

TM

554478

CT

644

RBD

PRL

Mw(kDa)

35

25

40

PVDL XVDL XVDLPVDLPRL

35

25

40

Mw(kDa)

PVDL

262PLV-Env

1

6xHis RBD

TM

620439

CT

641

RBD

Mailer, Allende et al, BLOOD (2020)

active inhibitor inactive control

Inhibition of XPR1 increases soluble and insoluble polyP

soluble

insoluble

total

Targeting XPR1 increases procoagulant platelet activity

Targeting XPR1 increases thrombosis under flow

PPX 0 2 20 200 PPX 0 2 20 200

arterial venous

Targeting XPR1 increases coagulation and platelet polyP

Fibrin GPIb MergeBright field

0XV

DL

(μg/

ml)

Fibrin GPIb MergeBright field

020

0

0XV

DL

μg/m

l 020

0

polyPBright fieldpolyPBright field

200

200

XVD

L (μ

g/m

l)XV

DL

(μg/

ml)

arterial venous

Platelet-specific ablation of XPR1 in mice

Platelet XPR1 knockout PolyP[mM Pi/108 pl]

MW(kDa)70 XPR1

Platelets

43 β-actin

Xpr1fl/fl Xpr1fl/fl

Pf4-CreXpr1fl/+

Pf4-Cre

Mailer, Allende et al, BLOOD (2020)

Increased arterial occlusion in platelet XPR deficient mice

Increased platelet-driven venous thrombosis but normal haemostasis in platelet XPR1-deficient mice

XPR1 regulates procoagulant platelet polyP

Graphical abstract of Mailer, Allende et al, BLOOD (2020)

Factor XII as a target for treatment of thrombosis

§ FXII contact activation is essential for thrombosis but FXII is dispensable for haemostasis

§ PolyP is an in vivo FXII contact activator triggers the „intrinsic“ coagulation pathway on platelets and cancer cell surfaces in vivo

§ PolyP forms insoluble Ca2+-rich microparticles that activate FXII in vivoindependently of the chain length of the individual poly

§ Targeting polyP or FXII (or FXI) provides safe thromboprotection(“Safe Anticoagulants“) and provides additional anti-inflammatory activities

§ The Pi exporter XPR1 regulates polyP in platelets (and other cells) suggesting that Pi have a role in thrombosis

Clément Naudin

MarcoHeestermans

• Lynn Butler, Tromsø• Christian Drouet, Grenoble• Tobias Fuchs, Hamburg• Dave Gailani, Nashville• Johan Heemskerk, Maastricht• Christoph Kleinschnitz, Essen• Nigel Key, Chapell Hill• Florian Langer, Hamburg• Nigel Mackman, Chapell Hill• Joost Meijers, Amsterdam• James Morrissey, Ann Arbor• Werner Müller-Esterl, Frankfurt• Nicola Mutch, Aberdeen

Jenny Björkqvist Coen Maas

Anna Sala

Thank you very very much!• Gunnar Nielsson, Stockholm• Benhard Nieswandt, Würzburg• Markus Noethen, Bonn• Kosta Panousis, Melbourne• Klaus Preissner, Giessen• Huge ten Cate, Mastricht• Albert Sickmann, Dortmund• Alvin Schmaier, Cleveland• Henri Spronk, Maastricht• Evi Stavrou, Cleveland• Guido Stoll, Würzburg• Ulrich Walter, Mainz• Walter Wuillemin, Luzern

Katrin Nickel

Special thank to the people who did the work!

Mikel AllendeLinda Labberton Katrin NickelReiner Mailer