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ESC Basic Science Summer School, Sophia Antipolis, 2017
Control of thrombosis and fibrinolysis in vivo: implications for new therapies Nicola J Mutch
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Haemostasis Thrombosis
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Thrombosis artery vein
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Holy grail: An antithrombotic drug that reduces thrombosis with minimal or no bleeding complications
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- - - - - contact pathway fibrin formation extrinsic pathway
TF VIIa VII Xa Va extrinsic pathway PT common pathway V IX XIII fibrinogen PK - XIIa HK - - XII - XI - contact pathway XIa fibrin formation IX XI XI intrinsic pathway fibrin IXa VIIIa VIII XIIIa XL-fibrin tPA thrombin fibrinolysis plasmin D-dimer uPA plasminogen TAFIa
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Contact pathway activation
Natural surfaces Polyphosphate Smith SA*, Mutch NJ* et al. PNAS; 2006; 103: 903-8 DNA/RNA Kannemeier C et al. PNAS; 2007; 104: Neutrophill extracellular traps (NETs) Gould TJ et al. ATVB; 2014; 34: Collagen van der Meijer PE. Blood; 2009; 114: Misfolded proteins Maas C et al. J Clin Invest Sep;118(9): - XI - - Ca2+ - PK HK XIIa - XIa - XII - - - Zn2+ XI - - HK - - - - -
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Platelet polyphosphate (polyP)
DAPI stain Bright field = 10 mm Ruiz, F. A. et al. J. Biol. Chem. 2004; 279: Müller, F*, Mutch NJ* et al, Cell. 2009; 139:
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PolyP enhances coagulation & inhibits fibrinolysis
Smith SA*, Mutch NJ* et al. PNAS; 2006; 103:
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FXII deficiency attenuates thrombosis but not haemostasis
Renné et al, JEM. 2005; 202:
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PolyP stimulates contact activation
Activation by kallikrein Autoactivation DS DS polyP polyP no surface no surface Müller, F*, Mutch NJ* et al, Cell. 2009; 139:
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PolyP initiates FXII activation
Hermansky-Pudlak Syndrome Müller, F*, Mutch NJ* et al, Cell. 2009; 139:
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Taken from Weitz J & Fredenburg J, Front Med 2017; 4:19
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FXI/FXII deficiency in humans….
FXI deficiency protected from VTE and ischemic stroke higher levels associated with increased risk of VTE and ischemic stroke FXII deficiency no differences in VTE or ischemic stroke
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Contact-driven coagulation on artificial surfaces
XI - - - Ca2+ - - PK HK - XIIa - XIa - - XII - - Ca2+ - - IX IXa VIIIa VIII - Zn2+ XI - - Ca2+ HK X Xa - Va - - Ca2+ V - II IIa - Smith SA*, Mutch NJ* et al. PNAS; 2006; 103: Smith SA et al. Blood; 2012; 116: Choi S et al, Blood 2011;118:
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~50% fail due to thrombosis
Medical Device Thrombosis ~50% fail due to thrombosis coagulation proteins and platelets readily adhere to artificial surfaces inducing thrombus formation
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Which is the better target – FXI or FXII?
Epidemiological data Strong Weak Risk of bleeding Low None Evidence for crucial role in thrombosis Phase 2 Preclinical Potential for bypassing inhibition Back activation of FXI by thrombin Potential for off-target effects May modulate inflammation via BK
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Indicators for FXI & FXII targeted therapies
Rationale Primary VTE prophylaxis Simple and safe single-dose regimens Extended VTE treatment Safer than current therapies for extended VTE treatment Prevention of ischemia after ACS Safer anticoagulant platform on top of antiplatelet therapy Medical devices Prevent clotting on heart valves, grafts, central venous catheters, ECMO
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Summary FXII- and FXI-directed anticoagulant strategies are a new era in anticoagulation that will minimise thrombosis with minimal risk of bleeding risk.
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Fibrinolysis
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Fibrin is a cofactor in plasminogen activation
a2AP Plgn tPA fibrin Fibrin stimulates tPA-mediated plasminogen activation 1250-fold i.e. mediates its own destruction
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Plasminogen activation on cells
Plgn-/- mice are unable to stimulate scuPA activation platelets scuPA uPA solution Plasmin Plgn Baeten KM et al. J Thromb Haemost; 2010; 8: 1313.
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Crosstalk between different surfaces
Taken from Dejouvencel T et al. Blood; 2010; 115:
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Platelets act as a focus for fibrin formation
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Fibrinolysis – nature’s own ‘brakes’
Annexin Plgn Merged Plasminogen on spread platelets Thrombin/collagen Plasminogen on procoagulant platelets Whyte CS, Swieringa F, van Der Meijden P, Heemskerk J, & Mutch NJ, Blood 2015; 125: 2568 2 min 7 min 0 min Can we harness nature’s own ‘brake’ service to develop novel antithrombotic drugs?
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Thrombolytic drugs Current uses:
Acute ischaemic stroke – within 4 h window Acute myocardial infarction Peripheral arterial thrombosis Massive pulmonary embolism & DVT Occluded haemodialysis shunts Based on tPA
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Current thrombolytic drugs drive plasminogen activation
Plgn tPA Thrombolytic drugs uPA Pln fibrin FDPs
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Novel thrombolytics – targeted approach
RBC-targeted pro-urokinase zymogen Zaitsev S et al. Blood 2012;119: ,
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Novel thrombolytics – targeted approach αIIbβ3-targeted microplasmin
Bonnard T et al. J Am Heart Assoc 2017;6:e004535
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Targeting Inhibitors fibrin FDPs PAI-1 C1Inh Plgn HRG tPA uPA 2AP Pln
FXIIIa fibrin FDPs TAFIa
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a2AP & FXIII deficient plasma lyse at strikingly similar rates
TG inhibitor has no impact on lysis of a2AP-depleted thrombi Lysis of FXIII and α2AP-depleted thrombi is normalized by PNP a2AP depleted plasma FXIII depleted plasma a2AP ± TG inhibitor, P>0.5 PNP + TG inhibitor, P<0.005 PNP Fraser et al, 2011 Blood
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Novel thrombolytics – targeting inhibitors
Diabody approach Targets PAI-1 and TAFIa simultaneously Small size allows penetrance into thrombi Denorme F et al. Stroke 2016; 47:
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Summary Current thrombolytics exhibit limited efficacy and are associated with bleeding complications. Novel therapies strive to target thrombi directly to facilitate clearance without bleeding.
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Not one size fits all…. - - - - - contact pathway fibrin formation
X TF VIIa VII Xa Va extrinsic pathway PT common pathway V IX XIII fibrinogen PK - XIIa HK - - XII - XI - contact pathway XIa fibrin formation IX XI XI Not one size fits all…. intrinsic pathway fibrin IXa VIIIa VIII XIIIa XL-fibrin tPA thrombin fibrinolysis plasmin D-dimer uPA plasminogen TAFIa
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Acknowledgements Prof Johan Heemskerk Frauke Swieringa
Dr Paola van Der Meijden Tom Mastenbroek Microscopy and Histology Core Facility The Iain Fraser Cytometry Centre
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