Polyphosphate, Platelets, and Coagulation

Jim Morrissey

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

0.2

0.4

0.6

0 25 50 75 100Time (min)

A405

Clot time = <5 minClot time = <5 min

Plasma + uPA + polyP

0 25 50 75 100Time (min)

A405

0

0.2

0.4

0.6

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).

Accelerating factor V activation has interesting consequences for

blood clotting & fibrinolysis

Tissue Factor Pathway Inhibitor (TFPI) added to plasma inhibits clotting

Thanks to George Broze for recombinant TFPI & anti-TFPI antibodies!

PolyP completely abrogatesthe anticoagulant action of TFPI

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

Revised clotting cascade?

(for hemostasis)

Revised clotting cascade?

(for thrombosis)

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 ???