Smith, Stephanie A.; Morrissey, James H.
Inorganic polyphosphate (polyP) is an intensely anionic, linear polymer of orthophosphate units linked by high-energy phosphoanhydride bonds that are widespread in biology (Fig. 1). Polymer sizes vary from about 10 phosphates to thousands of phosphates long, depending on organism and cell type [1,2]. In microorganisms, polyP is synthesized from ATP via a fully reversible enzymatic reaction  and is degraded by endopolyphosphatases (cleavage within chain) and exopolyphosphatases (progressive removal of terminal phosphates) [4▪▪]. Roles for polyP in mammalian systems are rapidly emerging, but polyP has been most intensively studied in prokaryotes and unicellular eukaryotes. Microorganisms store polyP in granules (with varying names, including ‘acidocalcisomes’ ), which typically contain very long-chain polyP, ranging in length from hundreds to thousands of phosphate units . Mammalian cellular compartments identified to contain polyP include lysosomes , platelet dense granules , mitochondria, and nuclei . Tissue extracts from mammalian heart, liver, lung, and kidneys are reported to contain heterogeneous polyP of 50 to 800 phosphate units long, although brain polyP is longer and more narrowly distributed at about 800 phosphates long . Functions ascribed to polyP in mammalian systems include angiogenesis , apoptosis , cell proliferation , energy metabolism , osteoblast function , bone mineralization [14,15], and tumor metastasis [16,17]. Here we review the emerging understanding of the roles of polyP in hematology. Recent studies from our lab and others have revealed that polyP is potently prohemostatic, prothrombotic, and proinflammatory [18–26], primarily via its influences on the blood coagulation and complement cascades (Fig. 2).
POLYP IN PLATELETS
Acidocalcisomes in microorganisms, in addition to polyP, contain divalent metal ions, such as Ca2+, Mg2+, and Zn2+, and are spherical, acidic , and electron dense . Platelet dense granules share these properties and have long been known to contain inorganic phosphate and pyrophosphate . Ruiz et al. reported that platelet dense granules are essentially acidocalcisomes, containing abundant polyP inside the granules. Platelet alpha granules also contain polyP, but at markedly lower concentrations . Platelet polyP is smaller and much less heterodisperse than microbial polyP, with lengths of approximately 60 to 100 phosphate units [7,21]. Dense granule polyP is secreted along with other granule contents when platelets are activated [7,21]. Furthermore, patients diagnosed with platelet dense granule defects have abnormally low levels of polyP (approximately 10% of normal) and experience a bleeding tendency . Knocking out the gene for inositol hexakisphosphate kinase 1 in mice resulted in a 10-fold decrease in polyP accumulation in platelet dense granules, which was associated with deficiencies in hemostasis and thrombosis [32▪▪].
POLYP IN MAST CELLS AND BASOPHILS
Like platelet dense granules, a subset of mast cell and basophil granules share many features with bacterial acidocalcisomes; they are spherical, electron dense (14), and contain phosphorus and cations . These granules contain polyP of about 60 units (similar to the size found in platelets). PolyP colocalizes in mast cell granules with serotonin, but not with histamine, and is released when the cells are stimulated to secrete their granule contents .
POLYP AND OTHER HEMATOLOGIC CELLS
PolyP stimulates apoptosis in plasma cells and myeloma cells, but not in normal B or T lymphocytes, or nonlymphoid cell lines . Myeloma cells contain much higher levels of nuclear polyP than normal plasma cells . In human erythrocytes, polyP is reported to be a component of the Ca2+-ATPase pump [35,36].
ROLES OF POLYP IN COAGULATION
The discovery that polyP was released from platelets  suggested a potential role in coagulation.
Amplification of thrombin generation
The production of a fibrin clot is highly dependent on the ability to generate a burst of thrombin that exceeds a necessary threshold. The rate of thrombin generation is a function of rates of activation of procoagulant factors and cofactors and the inhibitory ability of anticoagulants. PolyP acts at several steps that influence thrombin generation: it enhances the generation of factor Va, it enhances the generation of factor XIa, and it opposes the anticoagulant activity tissue factor pathway inhibitor (TFPI) (Fig. 2). The combined result of these polyP effects is that the time to achieve a thrombin burst is shortened . The relevance of polyP amplification of thrombin generation is demonstrated by the fact that platelets lacking dense granules (from Hermansky–Pudlak syndrome patients) are less able to generate thrombin and form a plasma clot in vitro, but activity can be restored by adding exogenous polyP .
Factor Va is of critical importance in the generation of thrombin, as it serves as the cofactor for factor Xa in the prothrombinase complex. The presence of factor Va enhances activation of prothrombin by several orders of magnitude. Relatively short polyP chains (of the size in platelets)  accelerate the activation of factor V to Va by both factor Xa and thrombin .
Although the classic waterfall cascade of coagulation describes factor XIIa as activating factor XI to factor XIa, recent work has indicated that this reaction, although relevant to clotting in vitro, is of no consequence to hemostasis in vivo. Rather, factor XI is now thought to be activated via a feedback mechanism by thrombin, and this reaction is markedly enhanced by polyP . PolyP also potently accelerates factor XI activation by factor XIa (i.e., factor XI autoactivation) . PolyP of the size secreted by platelets is able to enhance activation of factor XI by either enzyme, and polyP in platelet releasates strongly promotes factor XI activation by thrombin . It is likely that polyP provides a template for assembly of the enzyme/substrate complex in these reactions [18,22,25,38].
TFPI, a protease inhibitor found on endothelial cells, in plasma, and in platelets, targets factor Xa, and then the tissue factor–factor VIIa complex . In-vitro experiments indicate that polyP profoundly abrogates the inhibitory function of TFPI , and polyP in platelet releasates strongly inhibits TFPI function [18,21]. Factor Xa that is already bound to factor Va (i.e., assembled into the prothrombinase complex) is resistant to TFPI, especially in the presence of the substrate, prothrombin . Recent work, however, has revealed that TFPI can still inhibit prothrombinase assembled with partially cleaved versions of FVa (i.e., that still retain portions of the B domain, as may be released from activated platelets), and polyP can block the ability of TFPI to inhibit factor Xa in this version of the prothrombinase [41▪].
Impacts on fibrin clot structure and stability
PolyP influences fibrin clot structure. As compared to clots without polyP, fibrin clots formed in the presence of polyP are more turbid, contain thicker fibrin fibrils, are more resistant to elastic stretching, and are more resistant to fibrinolysis . PolyP appears to become incorporated directly into fibrin clots, although the mechanism is not known. PolyP of the size secreted by activated platelets is just large enough to have an impact on fibrin clot structure, but optimal fibrin enhancement requires longer polyP polymers (>400 mer) . Interestingly, pyrophosphate abrogates the ability of polyP to enhance fibrin clot structure, while having no effect on fibrin clots formed in the absence of polyP . Platelet dense granules also contain pyrophosphate (in quantities greater than polyP) , but little has been discovered about the function of this pyrophosphate.
The mechanism by which fibrin with incorporated polyP is resistant to fibrinolysis is not entirely clear. Anionic polymers other than polyP (e.g., heparin) similarly increase fibrin clot turbidity, but cause a clot that is more susceptible to fibrinolysis . One possibility is that the shift toward earlier thrombin generation allows for more activation of thrombin activatable fibrinolysis inhibitor (TAFI) . Because TAFI modifies binding sites for plasmin on the fibrin molecule, earlier TAFI generation results in less plasmin binding sites and consequently resistance to lysis . The presence of polyP also inhibits the binding of tissue-type plasminogen activator and plasminogen to fibrin .
The contact pathway is triggered when factor XII, prekallikrein, and high molecular weight kininogen assemble on anionic polymers or surfaces. Factor XIIa can then activate factor XI to XIa, which in turn activates factor IX, leading to propagation of the clotting cascade and ultimately thrombin generation. Although this pathway is important for the clotting of blood or plasma ex vivo, it is dispensable for clotting in vivo because complete factor XII deficiency is not associated with a bleeding tendency .
Although the contact pathway is not required for normal hemostasis, recent evidence indicates that contact activation contributes to thrombosis. Clinical studies have determined that elevated plasma factor XII, factor XI, or kallikrein is associated with atherosclerosis  or myocardial infarction [45–47], although patients with severe factor XI deficiency have reduced risk of stroke . In animal models, factor XII deficiency is protective against both arterial and venous thrombus formation [49,50].
The identity of the true (patho)physiologic activator(s) of the contact pathway in vivo has not been definitively determined. Most in-vitro studies have employed artificial anionic surfaces such as glass, kaolin (powdered clay), diatomaceous earth, ellagic acid, or sulfatides. RNA  and misfolded proteins  have been proposed as possible natural activators, but polyP is a very potent activator of the contact pathway in both plasma and purified protein systems [18,21]. PolyP binds with high affinity to the proteins responsible for initiating the contact pathway [18,23,38]. PolyP-mediated contact activation, like polyP-mediated factor XI activation, likely occurs via a template mechanism [18,23].
Contact activation by polyP is profoundly dependent on polymer length, with optimal activity requiring very long polyP polymers . It has long been known that activated human platelets express a weak but measurable ability to trigger the contact pathway in a factor XII-dependent manner . Platelet-derived polyP is able to weakly activate contact factors, but is thousands of time less potent than long-chain polyP . The weak contact activation ability of platelet polyP is consistent with the idea that platelets are much more effective at accelerating coagulation reactions than they are at initiating them.
Recent studies in animal models of thrombosis have suggested a role for polyP in vivo. PolyP administered intravenously leads to lethal pulmonary embolism in normal mice, while mice deficient in factor XII, or those given an inhibitor to factor XIIa, survive . In-vivo activation of platelets via intravenous injection of a platelet agonist similarly causes fatal pulmonary embolism in normal mice, but not when factor XII activity is absent . If an enzyme that degrades polyP is injected at high doses, the mice are more likely to survive. These experiments indicate that polyP is thrombogenic in vivo, and that the thrombogenicity is dependent on factor XII . Proof-of-principal polyP inhibitors have recently been identified, and were shown to be antithrombotic in mouse arterial thrombosis models, while having fewer bleeding side-effects than conventional anticoagulant drugs [54,55].
POLYP IS PROINFLAMMATORY
Although the contact pathway is dispensable for hemostasis, it plays important roles in inflammatory responses (Fig. 3). Activation of the contact pathway (often called the kallikrein–kinin pathway) results in kallikrein-mediated release of bradykinin from high molecular weight kininogen. Bradykinin is a potent vasoactive peptide. When bradykinin binds to its receptors on the endothelial cell, it causes release of prostacyclin, nitric oxide, and endothelium-derived hyperpolarizing factor, resulting in vasodilation . In addition to bradykinin generation, kallikrein has been shown to directly activate complement components C3 and C5 [57,58], while factor XIIa also initiates the classical complement cascade .
The importance of the contact pathway in vivo is demonstrated by the severe clinical manifestations of hereditary angioedema. In this disease, patients are deficient in an important inhibitor of the contact pathway. Unregulated activation of contact factors leads to vasodilation and vascular leakage, resulting in potentially lethal edema and hypotension [60▪]. Another circumstance demonstrating the importance of contact activation in vivo was the severe and fatal adverse reactions reported for pharmaceutical heparin that was contaminated with a potent contact activator [61,62]. Factor XII gene knockout in mice causes defective immune responses to infection , indicating that the contact pathway also contributes to host responses to pathogens. The identification of multiple microbial contact activators (e.g., bacterial surface proteins [64,65], lipopolysaccharide , teichoic or lipoteichoic acid , and long-chain polyP [18,23]) is consistent with this concept.
As noted above, long-chain polyP is an extremely potent trigger of the contact pathway [18,21,23], suggesting that polyP is a proinflammatory mediator. Mast cell-derived polyP  and platelet-derived polyP  are also able to initiate the contact pathway, but more weakly. As with other activators of the contact system, polyP promotes release of bradykinin . In mouse models, subcutaneous injection of polyP causes localized capillary leak [21,54], and intraperitoneal injection leads to a rapid drop in systemic arterial blood pressure and death . On the basis of gene knockout studies, these polyP-mediated effects are dependent on both factor XII and bradykinin.
PolyP additionally contributes to inflammatory processes through mechanisms that are independent of the contact pathway. PolyP substantially enhances the activity of histones, resulting in increased platelet activation and thrombin generation that is independent of factor XII . Extracellular histones have been shown to exhibit potent proinflammatory and procoagulant activities . PolyP also activates nuclear factor κB . PolyP has been shown to induce proliferation and differentiation of mesenchymal stem cells via activation of fibroblast growth factors . Interestingly, long-chain polyP suppresses complement via the terminal pathway by destabilizing C5b,6, thereby reducing the lytic capacity of the membrane attack complex .
OTHER ROLES FOR POLYP
The extent of the contributions of polyP to physiologic processes is just beginning to be explored. PolyP is present in von Willebrand factor isolated from platelet alpha granules or plasma. It binds von Willebrand factor in vitro with high affinity and modulates its interaction with glycoprotein Ib . PolyP accelerates the autoactivation of factor seven-activating protease, a broad spectrum enzyme with possible roles in coagulation, fibrinolysis, vascular biology, atherosclerosis, and autoimmune disease . In a mouse model, polyP blocked metastasis of melanoma cells due to antiangiogenic activity . PolyP stimulates the protein kinase mammalian target of rapamycin in mammary cancer cells, making the cells markedly deficient in their response to mitogens. This suggests that polyP is a regulatory factor in the activation of mammalian target of rapamycin in proliferative signaling pathways . PolyP serves as a molecular chaperone, stabilizing proteins in vivo and protecting them against stress-induced unfolding and aggregation. It binds to unfolding proteins with high affinity and supports refolding once stress conditions are resolved .
The work reviewed here shows that polyP accelerates thrombin generation, reverses the anticoagulant activity of a variety of anticoagulants in vitro (including heparins, direct factor Xa inhibitors, and thrombin inhibitors), and shortens the clotting times of plasma from patients with hemophilia A or B, or patients taking vitamin K antagonists . PolyP or suitable derivatives might consequently have use in the future as parenteral or topical hemostatic agents. Recent work suggests that polyP is a key player in the web of host–pathogen interactions. Long-chain microbial polyP is a potent activator of the blood clotting system via the contact pathway and can trigger thrombosis and inflammation. The activities of polyP in vivo may therefore also represent a novel target for future antithrombotic and/or anti-inflammatory agents. The detailed molecular mechanisms by which polyP acts as modulator of coagulation and inflammation have yet to be definitively described. Further, it is likely that future research will reveal many more physiologic roles for this ancient molecule.
We thank Catherine Baker for assistance with figures. The authors’ studies were supported by grants R01 HL047014 from the National Heart, Lung and Blood Institute of the National Institutes of Health.
The authors are coinventors on patents and pending patent applications on medical uses of polyP.
Conflicts of interest
There are no conflicts of interest.
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