ATVB in Focus
2013 Scientific Sessions Sol Sherry Distinguished
Lecture in Thrombosis
Polyphosphate: A Novel Modulator of Hemostasis and Thrombosis
Stephanie A. Smith, James H. Morrissey
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Abstract—Polyphosphate is a highly anionic, linear polymer of inorganic phosphates that is found throughout biology,
including in many infectious microorganisms. Recently, polyphosphate was discovered to be stored in a subset of
the secretory granules of human platelets and mast cells, and to be secreted on activation of these cells. Work from
our laboratory and others has now shown that polyphosphate is a novel, potent modulator of the blood clotting and
complement systems that likely plays roles in hemostasis, thrombosis, inflammation, and host responses to pathogens.
Therapeutics targeting polyphosphate may have the potential to limit thrombosis with fewer hemorrhagic complications
than conventional anticoagulant drugs that target essential proteases of the blood clotting cascade. (Arterioscler Thromb
Vasc Biol. 2015;35:00-00. DOI: 10.1161/ATVBAHA.115.301927.)
Key Words: blood coagulation ◼ blood platelets ◼ hemostasis ◼ polyphosphates ◼ thrombosis
I
to hemostasis, thrombosis, and inflammation. In this article,
we review our current understanding of the prohemostatic
and prothrombotic roles of polyphosphate and discuss the
possibility of targeting polyphosphate to develop novel antithrombotic drugs.
norganic polyphosphate is a linear polymer of orthophosphate units linked by high-energy phosphoanhydride bonds
(Figure 1). Polyphosphate is widespread throughout biology,
with polymer lengths that can vary from just a few phosphates to
many hundreds or even thousands of phosphates long, depending on organism and cell type.1–3 It is intensely anionic because
each phosphate unit carries a negative charge at physiological
pH. In many organisms, polyphosphate is stored in intracellular
organelles in a highly condensed state together with metal ions.
Although these acidic, polyphosphate-rich, and calcium-rich
compartments have different names in different organisms, a
general term of acidocalcisomes has been proposed.4
In 2004, the laboratory of Roberto Docampo reported
that polyphosphate is a major constituent of the dense
granules of human platelets, and further noted that platelet dense granules share several features in common with
acidocalcisomes in unicellular eukaryotes—namely, both
are acidic, electron-dense, and contain abundant stores of
both polyphosphate and divalent metal ions.5 As expected
for a dense granule payload, polyphosphate is secreted on
platelet activation. Dr Docampo alerted us to his discovery
before publication, prompting us to work with him to investigate whether polyphosphate secreted from platelets might
contribute to blood clotting. In 2006, we published the first
report that polyphosphate is indeed a potent modulator of
the blood clotting system, both accelerating the clotting cascade and slowing fibrinolysis.6 Our initial report has since
been followed by a series of studies from our laboratory and
others exploring the unique contributions of polyphosphate
Background: Biological Roles
of Polyphosphate
Metabolism of polyphosphate and its biological roles have
been studied most extensively in microorganisms. In bacteria, polyphosphate is synthesized enzymatically from ATP7
and is typically heterodisperse, ranging in size from a few
phosphates to hundreds or even thousands of phosphates
long. Polyphosphate can be enzymatically degraded by specific endopolyphosphatases (which cleave internal phosphoanhydride bonds in polyphosphate) and exopolyphosphatases
(which release terminal phosphates from polyphosphate),
as well as some nonspecific phosphatases, such as alkaline
phosphatase. As noted above, most microorganisms store
polyphosphate in intracellular granules,4 although Neisseria
bacteria express long-chain polyphosphate on their capsule.8,9
Proposed roles for polyphosphate in microbes include serving as a storage molecule for phosphate and a backup energy
source for ATP, sequestering metal ions to reduce metal
toxicity, regulating gene transcription and enzymatic activity, biofilm development, quorum sensing, virulence, stress
responses, growth and development.3
Although functions of polyphosphate in multicellular
organisms have been studied less extensively, roles for this
Received on: March 30, 2015; final version accepted on: April 10, 2015.
From the Department of Biochemistry, University of Illinois at Urbana-Champaign.
Correspondence to James H. Morrissey, PhD, Department of Biochemistry, University of Illinois at Urbana-Champaign, 323 Roger Adams Lab MC-712,
600 S Mathews Ave, Urbana, IL 61801. E-mail [email protected]
© 2015 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
1
DOI: 10.1161/ATVBAHA.115.301927
2 Arterioscler Thromb Vasc Biol June 2015
Nonstandard Abbreviations and Acronyms
FV
FX
TFPI
factor V
factor X
tissue factor pathway inhibitor
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polymer are now being identified. A variety of mammalian
cell types are reported to contain polyphosphate, including platelets,5 mast cells,10 myeloma cells,11 erythrocytes,12,13
and astrocytes.14 Extracts from mammalian heart, liver, lung,
and kidneys contain polyphosphate of varying size ranges,
with brain reported to have relatively long-chain polyphosphate (≈800 phosphates in length).15 In contrast, polyphosphate secreted by activated platelets5 and mast cells10 has a
rather narrow size distribution of ≈60 to 100 phosphates long.
Subcellular compartments in mammalian cells described to
contain polyphosphate include lysosomes,16 platelet dense
granules,5 serotonin-containing secretory granules of mast
cells,10 mitochondria,17 nucleoli,11 and nuclei.15 Polyphosphate
in organelles is often in close association with metal ions, such
as Ca2+, Mg2+, and Zn2+, or with organic cations, such as polyamines and basic amino acids.18 Polyphosphate decays with a
half-life of ≈1.5 to 2 hours in human blood or plasma in vitro.6
Mammalian alkaline phosphatase is a potent exopolyphosphatase,6,19,20 although in most cases, the enzymes responsible for
degrading polyphosphate in mammals have not been extensively studied. Crude extracts that from a variety of mammalian tissues can degrade polyphosphate, and brain has 10- to
30-fold more degradation capability than other tissues.21
Functions ascribed to polyphosphate in mammalian systems include roles in angiogenesis,22 apoptosis,23 cell proliferation,24 energy metabolism,25 tumor metastasis,26,27
osteoblast activity,28 and bone mineralization.29,30 In the
rest of this article, we focus on the emerging understanding of roles of polyphosphate in hemostasis, thrombosis, and
inflammation.
Polyphosphate Triggers Clotting and
Accelerates Thrombin Generation
Polyphosphate acts at multiple steps in blood coagulation in
a manner that is dependent on polymer length. Figure 2 gives
an overview of the plasma clotting cascade, depicting the 5
points at which polyphosphate acts. The contributions of polyphosphate are procoagulant, shortening the time to achieve a
thrombin threshold.6 It is likely that polyphosphate released
from platelets is physiologically relevant because platelets
Figure 1. Inorganic polyphosphate (polyP) is a linear, anionic
polymer of orthophosphates held together by the same type
of high-energy phosphoanhydride bonds found in ATP. PolyP
secreted by activated platelets and mast cells is ≈60 to 100
phosphate units long,5,10 whereas microbial polyP ranges in size
from a few phosphates to many hundreds or even thousands of
phosphates long.3
Figure 2. Overview of the plasma clotting cascade, showing the
principle points at which polyphosphate (polyP) acts. The clotting cascade can be triggered via either the contact pathway or
the tissue factor pathway. The contact pathway is initiated by
a combination of factor (F) XII autoactivation and the reciprocal
activation of FXII by kallikrein and of prekallikrein by FXIIa. The
tissue factor pathway is initiated by the cell-surface complex of
tissue factor (TF) and FVIIa. PolyP acts by (1) triggering the contact pathway; (2) accelerating FV activation; (3) abrogating the
anticoagulant activity of TF pathway inhibitor; (4) greatly accelerating the activation of FXI by thrombin (and also promoting FXI
autoactivation—not shown here); and (5) enhancing the structure
of fibrin fibrils, rendering them more resistant to fibrinolysis.
These actions of polyP are dependent on polyP polymer length,
as described in detail in the text.
lacking dense granules are less able to support thrombin generation, whereas this activity can be restored by adding exogenous polyphosphate.31
Initiation of the Contact Pathway
The contact pathway is triggered when plasma comes into
contact with a variety of artificial surfaces, such as glass,
clay, and diatomaceous earth.32 For years, investigators have
sought to identify the true (patho)physiological activators of
this pathway. We recently showed that polyphosphate is a
potent activator of the contact pathway (Figure 2, point 1),
and therefore may represent at least one of the long-sought,
biologically relevant activators of this pathway of blood
clotting.6,31 Polyphosphate binds kallikrein with high affinity,33 and polyphosphate-mediated contact activation likely
occurs via a template mechanism.6,34 Interestingly, zymogen factor XII has enzymatic activity when in complex with
polyphosphate.35
Initiation of the contact pathway by polyphosphate requires
long polyphosphate polymers—of the size range typically
present in microbes (ie, hundreds of phosphates long).34 One
of the outcomes of triggering the contact pathway is the proteolytic release of the highly vasoactive peptide, bradykinin,
from high molecular weight kininogen. Perhaps more accurately termed the plasma kallikrein/kinin system, the contact
pathway is dispensable for hemostasis but does contribute to
thrombosis, and likely plays roles in inflammation and host
response to pathogens.32,36
Polyphosphate of the size released from activated platelets has only a weak ability to activate the contact pathway
of clotting, with a specific activity that is thousands of times
less potent than that of long-chain polyphosphate.34 This is
Smith and Morrissey Polyphosphate in Hemostasis and Thrombosis 3
consistent with the idea that platelets are good at accelerating
clotting, but are poor at triggering clotting.
is the missing cofactor that explains how FXI contributes to
hemostasis.
Activation of Factor V
Polyphosphate Enhances Fibrin Clot
Structure and Inhibits Fibrinolysis
Factor V (FV) circulates in plasma as a single-chain protein (procofactor) with a molecular weight of 330 kDa.37,38
Limited proteolysis39,40 removes the B domain and generates
mature FVa,37,38,41,42 the essential protein cofactor for factor
Xa (FXa). The complex of these 2 proteins on a suitable
membrane is termed the prothrombinase complex, cleaving prothrombin to release thrombin.43–46 Polyphosphate
enhances the rate of FV activation by FXa,6 thrombin,6 and
FXIa (Figure 2, point 2).47 Short polyphosphate polymers
(<60 phosphates long) have minimal ability to enhance generation of FVa,34 but platelet-sized polymers have robust
ability to promote FV activation.
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Abrogation of Tissue Factor Pathway Inhibitor
Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor found on endothelial cells, in plasma, and in
platelets.48 TFPI targets the active sites of FXa and factor VIIa
in the tissue factor–factor VIIa complex. In vitro experiments
indicate that both exogenous polyphosphate and polyphosphate in platelet releasates profoundly abrogate the inhibitory
function of TFPI (Figure 2, point 3).6,31
One mechanism by which polyphosphate seems to abrogate TFPI inhibition is by accelerating the rate of FVa
generation.6 This is because, once FXa assembles into the
prothrombinase complex, it becomes highly resistant to inhibition by TFPI, especially in the presence of its substrate,
prothrombin.49
Conditions exist in which FVa is incompletely cleaved and
thus retains portions of the B domain. This happens when FXa
activates FV, and also when FV is secreted by activated platelets. Interestingly, TFPI can inhibit the prothrombinase complex when FXa binds to these partially activated versions of
FVa, and furthermore, polyphosphate can abrogate the anticoagulant function of TFPI toward these incompletely activated
versions of prothrombinase.50
FXI Activation by Thrombin
Coagulation FXI circulates in plasma as a homodimer with a
molecular weight of 160 kDa. Limited proteolysis by FXIIa or
thrombin51,52 generates active FXIa. FXIa then activates factor
IX to propagate the clotting cascade.
Although FXIIa can activate FXI, it has long been known
that this reaction is not relevant to hemostasis in vivo
because FXII deficiency is not associated with bleeding.53
Nevertheless, severe FXI deficiency in humans is associated
with clinical bleeding diatheses.54 The source of FXIa in vivo
rather seems to be back-activation of FXI by thrombin.51,52
Early studies described this reaction as being slow in the
absence of an artificial anionic surface to act as a template.55
We recently reported that platelet polyphosphate enhances the
rate of thrombin-catalyzed FXIa generation by ≈3000-fold
(Figure 2, point 4).56,57 In addition, polyphosphate potently
accelerates both FXI autoactivation56 and FV activation by
FXIa.47 Therefore, it is possible that platelet polyphosphate
Thrombin, the last protease in the clotting cascade, removes
2 small, inhibitory peptides from fibrinogen, converting it to
fibrin. These fibrin monomers then spontaneously polymerize
into fibrin polymers. The structure of the fibrin clot influences
both its mechanical properties and susceptibility to fibrinolysis. Alterations in the structure of fibrin fibrils can, therefore,
influence hemostasis and thrombosis.58–60 In 2008, we showed
that polyphosphate enhances fibrin clot structure (Figure 2,
point 5).61
Changing the rate of thrombin generation influences
clot structure,60 so one mechanism by which polyphosphate can affect clot properties is through accelerating the
thrombin burst.6 In addition, the presence of polyphosphate
during clotting of plasma also directly modifies the structure of the resulting fibrin clot.61,62 Polyphosphate seems to
become incorporated directly into fibrin clots, resulting in
thicker fibrin fibrils with greater elastic strength and resistance to fibrinolysis.34,61,62 Inorganic pyrophosphate, which is
also present in platelet dense granules,5 abrogates the ability of polyphosphate to enhance fibrin clot structure, while
having no effect on fibrin clots formed in the absence of
polyphosphate.34
Fibrin clots are ultimately removed in vivo via digestion
by plasmin, which is generated when plasminogen is activated by either tissue-type or urokinase-type plasminogen
activator. For optimal rates of plasmin generation, these
proteins bind to fibrin via C-terminal lysine residues. A
circulating carboxypeptidase (termed thrombin-activatable
fibrinolysis inhibitor or thrombin-activatable fibrinolysis
inhibitor; also known as procarboxypeptidase B2, R, or
U) removes these C-terminal lysines from fibrin, thereby
decreasing binding of fibrinolytic proteins and inhibiting
fibrinolysis. We showed that polyphosphate, by shortening
the lag time to the thrombin burst, results in earlier activation of thrombin-activatable fibrinolysis inhibitor, giving
this carboxypeptidase more time to modify fibrin and thus
render the clot resistant to fibrinolysis.6 Furthermore, Mutch
et al62 showed that the presence of polyphosphate attenuates the binding of both plasminogen and tissue-type plasminogen activator to partially degraded fibrin (possibly by
masking access to C-terminal lysines), which additionally
attenuates fibrinolysis.
Polyphosphate in Hemostasis
The potential contributions of polyphosphate to hemostasis are just beginning to be elucidated. Patients with
Hermansky–Pudlak syndrome and other platelet dense
granule defects experience bleeding tendencies, but it is
unknown whether the reduction of platelet polyphosphate
in these patients contributes to their bleeding diatheses.63
In a mouse model, knocking out the gene for inositol
hexakisphosphate kinase 1 led to a substantial reduction in
accumulation of polyphosphate in platelet dense granules.
4 Arterioscler Thromb Vasc Biol June 2015
As with humans with similarly reduced polyphosphate levels in their platelet dense granules, these mice experienced
a bleeding tendency.64 Administering polyphosphate inhibitors (cationic compounds, polymers or dendrimers, that
block the procoagulant activity of polyphosphate) either had
no measurable effect on hemostasis in mice65 or had much
reduced antihemostatic effects compared with conventional
anticoagulant drugs.66
Polyphosphate in Thrombosis
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Recent studies of mechanisms underlying thrombosis have
led to a new appreciation that thrombosis is not always
triggered by the same activators of blood clotting that
function in normal hemostasis.67 Such prothrombotic activators include proteins in the contact pathway of coagulation (also known as the plasma kallikrein–kinin system36);
products released from inflammatory cells and necrotic
cells such as polyphosphate, extracellular histones, and
nucleic acids; and other inflammatory mediators such as
the complement cascade.
Triggering of the plasma clotting system via the contact
pathway, while irrelevant for hemostasis, can contribute
to thrombosis. Studies of patients with atherosclerosis68
or myocardial infarction,69–71 have demonstrated associations with elevated plasma levels of FXII, FXI, or prekallikrein. Furthermore, patients with severe FXI deficiency
have reduced risk of ischemic stroke72 and deep vein
thrombosis.73 In animal models, FXII deficiency is protective against arterial and venous thrombosis,74,75 whereas
anti-FXII antibodies inhibit thrombus formation in primate
models.76 Real-time fluorescent imaging of clot formation
in vitro and in vivo indicates that excessive contact factor activation and release of dense granule contents during platelet activation contribute to thrombosis and vessel
occlusion.77
The identity of the true (patho)physiological activator(s) of
the contact pathway in vivo has yet to be definitively determined, but extracellular nucleic acids,78 misfolded proteins,79
and polyphosphate 6,31 have been proposed. The ability of
polyphosphate to activate the contact pathway in vitro is profoundly dependent on polymer length, with optimal activity
requiring long polyphosphate polymers (of the size that accumulates in infectious microorganisms).34
The procoagulant and proinflammatory activities of polyphosphate may contribute to thrombosis. Polyphosphate
substantially enhances the procoagulant activity of extracellular histones,80 which activate platelets via toll-like receptors.
Polyphosphate also activates nuclear factor-κB81 and fibroblast growth factors to induce differentiation of mesenchymal
cells.82 Activation of the contact pathway leads to generation
of kallikrein and release of the vasoactive peptide, bradykinin from high molecular weight kininogen. When bradykinin
binds to its receptors on the endothelial cell, it causes release of
nitric oxide and endothelium-derived hyperpolarizing factor,
resulting in vasodilation.83 Activation of the contact pathway
can also activate the complement cascade: FXIIa initiates the
classical pathway84 and kallikrein directly activates complement components C3 and C5.85,86 Interestingly, polyphosphate
destabilizes C5b,6 in the terminal pathway of complement,
reducing the lytic capacity of the membrane attack complex.87
This latter finding indicates that polyphosphate can also be
anti-inflammatory.
Research using animal models has suggested that polyphosphate may contribute to thrombosis in vivo. When administered intravenously, polyphosphate leads to lethal pulmonary
embolism in mice, the occurrence of which is dependent on
FXII.31 A separate study demonstrated that mice were resistant
to thrombosis if their FXI was mutated to have decreased ability to interact with polyphosphate.57
Polyphosphate-Based Therapeutics
Although many hemostatic agents have been developed to
treat bleeding in surgery, trauma, and patients with congenital clotting disorders, substantial room for improvement
still exists. The intriguing possibility that the procoagulant
nature of polyphosphate could be harnessed to treat bleeding warrants further investigation. In fact, recent studies
have shown that addition of polyphosphate enhances the
hemostatic action of chitosan;88 that polyphosphate/silica
nanoparticles have increased hemostatic function compared
with silica particles or polyphosphate alone;89 and that
addition of polyphosphate to fibrin sealants might increase
their effectiveness.61 Adding soluble polyphosphate to
plasma reverses the anticoagulant activity of heparins and
of direct inhibitors of thrombin or FXa. Polyphosphate
also shortens the prolonged clotting times of plasma from
patients with hemophilia A or B or patients taking vitamin
K antagonists.90
On the contrary, the contributions of polyphosphate to
thrombosis suggest that polyphosphate could be a novel
target for antithrombotic therapy. Proof-of-principle studies
have recently demonstrated that certain cationic polymers,
dendrimers, and other compounds can neutralize polyphosphate procoagulant activity in vitro and attenuate both
venous and arterial thrombosis in mice, with minimal bleeding side effects.65,91 The compounds used in these initial
studies have significant toxicity, however. To address this,
we recently reported the use of highly biocompatible, nontoxic, dendrimer-like scaffolds that are also highly effective
polyphosphate inhibitors.66 These compounds were as effective as heparin in protecting mice against arterial thrombosis, while having far less bleeding side effects compared
with heparin.
Conclusions
Polyphosphate is a newly recognized regulator of the blood
clotting that is secreted by activated platelets and mast cells,
and which is present in infectious microorganisms. It acts
in a strongly procoagulant manner that modulates blood
clotting at 4 principal steps (Figure 2) although the precise contributions of polyphosphate to the plasma clotting
system depend on the polyphosphate polymer length. The
contributions of polyphosphate to hemostasis and thrombosis suggest that this molecule may be a useful therapeutic target in coagulation disorders. It is, therefore, possible
that some form of polyphosphate might be useful in the
Smith and Morrissey Polyphosphate in Hemostasis and Thrombosis 5
future as a parenteral or topical hemostatic agent, provided
its proinflammatory activity can be adequately controlled.
On the contrary, polyphosphate inhibitors may have utility
in preventing or treating thrombosis. Many of the detailed
molecular mechanisms by which polyphosphate contributes
to hemostasis, thrombosis, and inflammation remain to be
determined.
Sources of Funding
This study was supported by grant R01 HL047014 from the National
Heart, Lung and Blood Institute of the National Institutes of Health.
Disclosures
The authors are a coinventors on patents and pending patent applications on medical uses of polyphosphate and of inhibitors of
polyphosphate.
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Significance
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Polyphosphate is secreted by activated human platelets and mast cells and is also present in many infectious microorganisms. In 2006,
our laboratory first showed that polyphosphate is a potent modulator of the blood clotting system. Further work from our group and others
has now revealed that polyphosphate is strongly procoagulant and likely plays roles in normal hemostasis, thrombotic diseases, inflammation, and host responses to pathogens. Polyphosphate is a potential drug target for the development of antithrombotic, anti-inflammatory
treatments that may have fewer bleeding side effects compared with conventional anticoagulants. It seems likely that there are still many
contributions of polyphosphate to human biology that remain to be elucidated.
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2013 Scientific Sessions Sol Sherry Distinguished Lecture in Thrombosis: Polyphosphate:
A Novel Modulator of Hemostasis and Thrombosis
Stephanie A. Smith and James H. Morrissey
Arterioscler Thromb Vasc Biol. published online April 23, 2015;
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