polyphosphate, platelets, and coagulation - · pdf filemorrissey lab: collaborators: *now ......
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Credits and DisclosuresMorrissey Lab: Collaborators:
*now at Aberdeen University
Funding
Brian Cooley (University of North Carolina)
Roberto Docampo (University of Georgia)
Chuck & Naomi Esmon (Okla. Med. Res. Fndn.)
David Gailani (Vanderbilt University)
Jay Kizhakkedathu (Univ. British Columbia)
Thomas Renné (Karolinska Institute)
Disclosures – I have consulted for:Bayer HealthcareBiogen-IdecrEVO Biologics
Catherine BakerRachel BreitenfeldSharon ChoiCarleigh HebbardNicola Mutch*
Stephanie SmithRichard TraversYan Wang
Polyphosphate (polyP)Linear polymers of inorganic phosphate connected by high-energy phosphoanhydride bonds
PolyP biosynthesis
• PolyP biosynthesis in bacteria is catalyzed by polyphosphate kinase (PPK)
• It costs one high-energy phosphate from an ATP for each phosphate added to polyP
• Reaction is reversible• Ahn & Kornberg. Polyphosphate kinase from Escherichia coli. Purification and
demonstration of a phosphoenzyme intermediate. J Biol Chem 265:11734-11739 (1990).
• Brown & Kornberg. The long and short of it—polyphosphate, PPK and bacterial survival. Trends Biochem Sci 33:284-290 (2008).
PolyP degradation
• PolyP is degraded in vivo by exopolyphosphatases1
and endopolyphosphatases• Reaction is irreversible• Mammalian alkaline phosphatase is a very active
exopolyphosphatase• PolyP half-life in human plasma ~90 minutes2
1. Akiyama, Crooke & Kornberg. An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon. J Biol Chem 268:633-639 (1993).
2. Smith , Mutch, Baskar, Rohloff, Docampo & Morrissey. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci USA 103:903-908 (2006).
PolyP in microbes• Stored in granules (bacteria) or acidocalcisomes (eukaryotes)
• Hundreds to thousands of phosphates long
Docampo et al. Nature Reviews 3:251-261 (2005).
acidocalcisomeacidocalcisome
Agrobacterium tumefaciens
Trypanosoma cruzi
PolyP in human platelets
Stored in dense granules:• About 60-100 phosphates
long
• Abundant: 0.74 nmol polyP per 108 platelets
• Secreted upon platelet activation; will yield ~1 to 3 µM polyP in blood
Ruiz , Lea, Oldfield & Docampo. Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. J Biol. Chem. 279:44250-44257 (2004).
DAPI-stained human platelets(DAPI bound to polyP undergoes a shift in fluorescence emission from blue to yellow-green; micrograph courtesy F. Ruiz & R. Docampo.)
PolyP enhances coagulation
Clot time = 25 minClot time = 25 min
Plasma + uPA
Smith , Mutch, Baskar, Rohloff, Docampo & Morrissey. Proc. Natl. Acad. Sci. USA 103:903-908 (2006).
0
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0 25 50 75 100Time (min)
A405
Clot time = <5 minClot time = <5 min
Plasma + uPA + polyP
0 25 50 75 100Time (min)
A405
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0.2
0.4
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PolyP acts at three points in the clotting cascade:
1. Triggers the contact pathway2. Accelerates factor V activation3. Enhances fibrin clot structure
(thicker fibrils; more resistant to fibrinolysis)
4. Promotes factor XI back-activation by thrombin
PolyP acts at four points in the clotting cascade:
• Mutch, Engel, Uitte de Willige, Philippou, Ariëns. Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin. Blood115:3980-3988, 2010.
• Müller, Mutch, Schenk, Smith, Esterl, Spronk, Schmidbauer, Gahl, Morrissey & Renné. Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo. Cell 139:1143-1156 (2009).
• Choi, Smith & Morrissey. Polyphosphate is a cofactor for the activation of factor XI by thrombin. Blood 118: 6963-6970 (2011).
• Smith, Mutch, Baskar, Rohloff, Docampo & Morrissey. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci USA 103:903-908 (2006).
• Smith & Morrissey. Polyphosphate enhances fibrin clot structure. Blood 112:2810-2816 (2008).
Tissue Factor Pathway Inhibitor (TFPI) added to plasma inhibits clotting
Thanks to George Broze for recombinant TFPI & anti-TFPI antibodies!
Factor Va recapitulates the ability of polyP to abrogate TFPI function
See also: Mast & Broze. Physiological concentrations of tissue factor pathway inhibitor do not inhibit prothrombinase. Blood 87:1845-1850 (1996).
• Fibrinogen + Ca2+ + polyP was clotted with thrombin
• Clots were washed extensively in buffer
• Clots were then stained with toluidine blue, which stains polyP a characteristic purple color (metachromatic staining)
+polyP −polyP
Smith SA and Morrissey JH. Polyphosphate enhances fibrin clot structure. Blood 112:2810-6 (2008). see also:Mutch NJ, Engel R, Uitte de Willige S, Philippou H, and Ariëns RA. Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin. Blood 115:3980-8 (2010).
PolyP is incorporated into fibrin clots
No PolyP 100 µM polyP
500 µM polyP 1 mM polyP
PolyP increases fibrin fiber thickness (SEM)—also slows fibrinolysis
Smith & Morrissey. Polyphosphate enhances fibrin clot structure. Blood 112:2810-2816 (2008).
PolyP:• Reverses effect of several anticoagulant drugs (heparin,
enoxaparin, rivaroxaban, argatroban)• Normalizes clotting times of hemophilic plasma• Shortens plasma clotting times of patients on warfarin• Effect similar to adding FVa to plasma• Effect is additive with FVIIa
Smith SA & Morrissey JH. Polyphosphate as a general procoagulant agent. J Thromb Haemost 6:1750-1756 (2008).
What is the size-dependence of the procoagulant activities of polyP?
PolyP was size‐fractionated (typically by preparative PAGE), resolved on analytical PAGE and detected by DAPI negative staining.
• Smith, Choi, Davis-Harrison, Huyck, Boettcher, Rienstra, & Morrissey. Polyphosphate exerts differential effects on blood clotting, depending on polymer size. Blood 116: 4353-4359 (2010).
• Choi, Smith & Morrissey. Polyphosphate is a cofactor for the activation of factor XI by thrombin. Blood 118: 6963-6970 (2011).
Size-Dependence of PolyP Procoagulant Activities
PolyP acts at three points in the clotting cascade:
• In the classic “water-fall” scheme, FXI is activated by FXIIa
• FXI deficiency is associated with bleeding tendency
— BUT —
• FXII deficiency is not
How is FXI activated in hemostasis?
PolyP acts at three points in the clotting cascade:
• So, in normal hemostasis, FXI must be activated by a protease other than FXIIa
• But which one?
How is FXI activated in hemostasis?
?
PolyP acts at three points in the clotting cascade:
• In 1991, Gailani & Broze1 and Naito & Fujikawa2 showed that thrombin can feed back to activate FXI
• Reaction is relatively slow in the absence of polyanions like dextran sulfate
• Is there a physiologic cofactor for this reaction?
How is FXI activated in hemostasis?
1. Gailani & Broze. Factor XI activation in a revised model of blood coagulation. Science 253:909-912 (1991).
2. Naito & Fujikawa. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces. J Biol Chem266:7353-7358 (1991).
PolyP acts at three points in the clotting cascade:PolyP promotes FXI activation by thrombin
Choi, Smith & Morrissey. Polyphosphate is a cofactor for the activation of factor XI by thrombin. Blood 118:6963-6970 (2011) .
polyP 22merpolyP 65mer
polyP 167mer
polyP 350mer Reaction conditions:30 nM FXI5 nM α-thrombinvarying polyP
PolyP accelerates this rate ~3000‐fold
PolyP clotting activities depend onpolymer length
1. Triggers the contact pathway
2. Abrogates TFPI function & accelerates factor V activation
3. Enhances fibrin clot structure (thicker fibrils; more resistant to fibrinolysis)
4. Promotes factor XI back-activation by thrombin
Factor XI and thrombosis• Severe FXI deficiency = mild bleeding tendency
• Severe FXI deficiency (studied in Ashkenazi Jewish population) protects against:
– venous thromboemolism1
– ischemic stroke2
– (but not myocardial infarction)
• In general population studies, elevated FXI is associated with increased risk of venous thromboembolism & ischemic stroke; relationship to MI less clear
• FXI a tempting target for novel antithrombotics with reduced bleeding side-effects?
1. Salomon et al. Thromb Haemost. 2011;105:269‐273 2. Seligsohn J. Thromb. Haemost. 2009;7 Suppl 1:84‐873. reviewed by He et al. Thromb. Res. 2012;129:541‐550
Hemostasis versus Thrombosis
Hemostasis Thrombosisno bleeding
mild bleeding
severe bleeding
protectedprotected
protected
Effects of clotting factor deficiencies on:
How to abrogate polyP function?
• Alkaline phosphatase destroys polyP, but is slow & can remove phosphates indiscriminately (including from ADP)
• Specific antibodies to polyP unlikely• So, we used high-throughput screen to identify
polyP blockers• Method is based on inhibiting thrombin binding to
immobilized polyP in 384-well plates1
1. Choi, Collins, Smith, Davis-Harrison, Rienstra & Morrissey. Phosphoramidate end labeling of inorganic polyphosphates: Facile manipulation of polyphosphate for investigating and modulating its biological activities. Biochemistry 49:9935-9941 (2010)
Using polyP covalently immobilized onto microplates to quantify the binding
affinities of clotting proteins for polyP
Choi, Collins, Smith, Davis-Harrison, Rienstra & Morrissey. Phosphoramidate end labeling of inorganic polyphosphates: Facile manipulation of polyphosphate for investigating and modulating its biological activities. Biochemistry 49:9935-9941 (2010)
thrombin factor XIa plasma kallikrein
PolyP inhibitors to test ex vivo & in vivo
polymyxin Bgen 1.0 dendrimer
Smith et al. Blood 120:5103-5110 (2012).
Gen 1.0 PAMAM dendrimer, polyethylenimine (PEI) & polymyxin B decrease arterial thrombosis in mice
FeCl3‐induced carotid injury in mice, with blood flow monitored by a Doppler flow probe
vehicle
dendrimer
polymyxinPEI
Smith, Choi, Collins, Travers, Cooley, Morrissey. Inhibition of polyphosphate as a novel strategy for preventing thrombosis and inflammation. Blood 120:5103-5110 (2012).
• All these compounds are toxic
• This limits the dose we can use and hence, their efficacies
Universal Heparin Reversal Agents (UHRA)
• Dendrimer-like scaffolds with very low toxicity
• Heparin-binding groups are tertiary amines shielded by PEG
• Developed by Jay Kizhakkedathu (Univ. British Columbia)
UHRA Compounds
• Dendrimer-like scaffolds with very low toxicity
• Heparin-binding groups are tertiary amines shielded by polyethylene glycol (PEG) moieties
• Could they also be the basis for polyP inhibitors?
UHRA Compounds
• Some of the UHRAs potently inhibited polyP activity
• Potency against polyP was not the same as potency against heparin
• We also tested them in thrombosis models in vivo in mice
UHRA 10was selected for further
testing in vivo
UHRA 10was selected for further
testing in vivo
Inhibition of thrombin
binding to polyP
Travers, Shenoi, Kalathottukaren, Kizhakkedathu & Morrissey. Nontoxic polyphosphate inhibitors reduce thrombosis while sparing hemostasis. Blood 124:3183‐3190, 2014.
FeCl3‐inducedcarotid artery thrombosis
model in mice
Mouse tail bleeding model
Travers, Shenoi, Kalathottukaren, Kizhakkedathu & Morrissey. Nontoxic polyphosphate inhibitors reduce thrombosis while sparing hemostasis. Blood 124:3183‐3190, 2014.
Contributions of polyP to hemostasis, thrombosis and inflammation
• Microbial polyP triggers the contact pathway—host response to pathogens?
• Platelet polyP accelerates clotting, abrogates TFPI, enhances fibrin clot structure, and may explain the role of FXI in hemostasis
• PolyP is a target for novel antithrombotic drugs
• Undiscovered roles for polyP in hemostasis, thrombosis, inflammation and ???