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The contact pathway of blood clotting has received intense interest in recent years as studies have linked it to thrombosis, inflammation, and innate immunity. Because the contact pathway plays little to no role in normal hemostasis, it has emerged as a potential target for safer thromboprotection, relative to currently approved antithrombotic drugs which all target the final common pathway of blood clotting. Research since the mid-2000s has identified polyphosphate, DNA, and RNA as important triggers of the contact pathway with roles in thrombosis, although these molecules also modulate blood clotting and inflammation via mechanisms other than the contact pathway of the clotting cascade. The most significant source of extracellular DNA in many disease settings is in the form of neutrophil extracellular traps (NETs), which have been shown to contribute to incidence and severity of thrombosis. This review summarizes known roles of extracellular polyphosphate and nucleic acids in thrombosis, with an emphasis on novel agents under current development that target the prothrombotic activities of polyphosphate and NETs., Competing Interests: S.V., S.A.S., J.H.M., and J.N.K. are inventors on issued patents and pending patent applications covering novel inhibitors of polyphosphate and NETs, some of which are optioned to TSRL, Inc., for licensing from the University of Michigan and the University of British Columbia. J.H.M. reports grants from the National Institutes of Health, and J.N.K. reports grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Council of Canada. The laboratory of J.H.M. receives some research funding from Kerafast, Inc., through the sales of polyphosphates and polyphosphate derivatives., (Thieme. All rights reserved.)

Abstract: Background: Inorganic polyphosphate (polyP) is a procoagulant polyanion. We assessed the impact of polyP inhibition on thrombin generation after trauma using the novel polyP antagonists, macromolecular polyanion inhibitor 8 (MPI 8), and universal heparin reversal agent 8 (UHRA-8). Methods: Plasma thrombin generation (calibrated automated thrombogram, CAT), in 56 trauma patients and 39 controls +/- MPI 8 and UHRA-8 (50 μg/mL), was expressed as lag time (LT, minutes), peak height (PH, nM), and time to peak (ttPeak, minutes), with change in LT (ΔLT) and change in ttPeak (ΔttPeak) quantified. Results expressed in median and quartiles [Q1, Q3], Wilcoxon matched-pairs testing, P < 0.05 significant. Results: Trauma patients had greater baseline PH than controls (182.9 [121.0, 255.2]; 120.5 [62.1, 174.8], P < 0.001). MPI 8 treatment prolonged LT and ttPeak in trauma (7.20 [5.88, 8.75]; 6.46 [5.45, 8.93], P = 0.020; 11.28 [8.96, 13.14]; 11.00 [8.95, 12.94], P = 0.029) and controls (7.67 [6.67, 10.50]; 6.33 [5.33, 8.00], P < 0.001; 13.33 [11.67, 15.33]; 11.67 [10.33, 13.33], P < 0.001). UHRA-8 treatment prolonged LT and ttPeak and decreased PH in trauma (9.09 [7.45, 11.33]; 6.46 [5.45, 8.93]; 14.02 [11.78, 17.08]; 11.00 [8.95, 12.94]; 117.4 [74.5, 178.6]; 182.9 [121.0, 255.2]) and controls (9.83 [8.00, 12.33]; 6.33 [5.33, 8.00]; 16.67 [14.33, 20.00]; 11.67 [10.33, 13.33]; 55.3 [30.2, 95.9]; 120.5 [62.1, 174.8]), all P < 0.001. Inhibitor effects were greater for controls (greater ΔLT and ΔttPeak for both inhibitors, P < 0.001). Conclusion: PolyP inhibition attenuates thrombin generation, though to a lesser degree in trauma than in controls, suggesting that polyP contributes to accelerated thrombin generation after trauma., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 by the Shock Society.)

The polyanion, inorganic polyphosphate (polyP), is a procoagulant molecule which has become a promising therapeutic target in the development of antithrombotics. Neutralizing polyP's prothrombotic activity using polycationic inhibitors is one of the viable strategies to design new polyP inhibitors. However, in this approach, a fine balance between the electrostatic interaction of polyP and the inhibitor is needed. Any unprotected polycations are known to interact with negatively charged blood components, potentially resulting in platelet activation, cellular toxicity, and bleeding. Thus, designing potent polycationic polyP inhibitors with good biocompatibility is a major challenge. Building on our previous research on universal heparin reversal agent (UHRA), we report polyP inhibitors with a modified steric shield design. The molecular weight, number of cationic binding groups, and the length of the polyethylene glycol (PEG) chains were varied to arrive at the desired inhibitor. We studied two different PEG lengths ( m PEG-750 versus m PEG-350) on the polyglycerol scaffold and investigated their influence on biocompatibility and polyP neutralization activity. The polyP inhibitor with m PEG-750 brush layer, m PEG 750 UHRA-10, showed superior biocompatibility compared to its m PEG-350 analogs by a number of measured parameters without losing its neutralization activity. An increase in cationic binding groups (25 groups in m PEG 750 UHRA-8 and 32 in m PEG 750 UHRA-10 [HC]) did not alter the neutralization activity, which suggested that the m PEG-750 shield layer provides significant protection of cationic binding groups and thus helps to minimize unwanted nonspecific interactions. Furthermore, these modified polyP inhibitors are highly biocompatible compared to conventional polycations that have been previously used as polyP inhibitors (e.g., PAMAM dendrimers and polyethylenimine). Through this study, we demonstrated the importance of the design of steric shield toward highly biocompatible polyP inhibitors. This approach can be exploited in the design of highly biocompatible macromolecular inhibitors.

Background: Platelets secrete many pro-wound healing molecules such as growth factors and cytokines. We found that releasates from activated human platelets induced the differentiation of cultured murine and human fibroblasts into a myofibroblast phenotype. Surprisingly, most of this differentiation-inducing activity was heat-stable, suggesting it was not due to the protein component of the releasates. Inorganic polyphosphate is a major constituent of platelet-dense granules and promotes blood coagulation and inflammation., Objectives: We aim to investigate the contribution of polyphosphate on myofibroblast differentiating activity of platelet releasates., Methods: Using NIH-3T3 cells and primary human fibroblasts, we examined the effect of human platelet releasates and chemically synthesized polyphosphate on fibroblast differentiation and migration., Results: We found that the myofibroblast-inducing activity of platelet releasates was severely attenuated after incubation with a polyphosphate-degrading enzyme, and that fibroblasts responded to platelet-sized polyphosphate by increased levels of α-smooth muscle actin, stress fibers, and collagen. Furthermore, fibroblasts were chemotactic toward polyphosphate., Conclusions: These findings indicate that platelet-derived polyphosphate acts as a cell signaling molecule by inducing murine and human fibroblasts to differentiate into myofibroblasts, a cell type known to drive both wound healing and fibrosing diseases. Polyphosphate therefore not only promotes early wound responses through enhancing fibrin clot formation, but also may play roles in the later stages of wound healing, and, potentially, progression of fibrotic diseases, by recruiting fibroblasts and inducing their differentiation into myofibroblasts., (© 2020 International Society on Thrombosis and Haemostasis.)

Investigation of the biological roles of inorganic polyphosphate has been facilitated by our previous development of a carbodiimide-based method for covalently coupling primary amine-containing molecules to the terminal phosphates of polyphosphate. We now extend that approach by optimizing the reaction conditions and using readily available "bridging molecules" containing a primary amine and an additional reactive moiety, including another primary amine, a thiol or a click chemistry reagent such as dibenzocyclooctyne. This two-step labeling method is used to covalently attach commercially available derivatives of biotin, peptide epitope tags, and fluorescent dyes to the terminal phosphates of polyphosphate. Additionally, we report three facile methods for purifying conjugated polyphosphate from excess reactants., Competing Interests: The authors have declared that no competing interests exist.

Polyphosphates are linear polymers and ubiquitous metabolites. Bacterial polyphosphates are long chains of hundreds of phosphate units. Here, we report that mouse survival of peritoneal Escherichia coli sepsis is compromised by long-chain polyphosphates, and improves with bacterial polyphosphatekinase deficiency or neutralization using recombinant exopolyphosphatase. Polyphosphate activities are chain-length dependent, impair pathogen clearance, antagonize phagocyte recruitment, diminish phagocytosis and decrease production of iNOS and cytokines. Macrophages bind and internalize polyphosphates, in which their effects are independent of P2Y1 and RAGE receptors. The M1 polarization driven by E. coli derived LPS is misdirected by polyphosphates in favor of an M2 resembling phenotype. Long-chain polyphosphates modulate the expression of more than 1800 LPS/TLR4-regulated genes in macrophages. This interference includes suppression of hundreds of type I interferon-regulated genes due to lower interferon production and responsiveness, blunted STAT1 phosphorylation and reduced MHCII expression. In conclusion, prokaryotic polyphosphates disturb multiple macrophage functions for evading host immunity.

Inorganic polyphosphate (polyP) is the polymer of phosphate. Water-soluble polyPs with average chain lengths of 2-40 P-subunits are widely used as food additives and are currently synthesized chemically. An environmentally friendly highly scalable process to biosynthesize water-soluble food-grade polyP in powder form (termed bio-polyP) is presented in this study. After incubation in a phosphate-free medium, generally regarded as safe wild-type baker's yeast (Saccharomyces cerevisiae) took up phosphate and intracellularly polymerized it into 26.5% polyP (as KPO 3 , in cell dry weight). The cells were lyzed by freeze-thawing and gentle heat treatment (10 min, 70°C). Protein and nucleic acid were removed from the soluble cell components by precipitation with 50 mM HCl. Two chain length fractions (42 and 11P-subunits average polyP chain length, purity on a par with chemically produced polyP) were obtained by fractional polyP precipitation (Fraction 1 was precipitated with 100 mM NaCl and 0.15 vol ethanol, and Fraction 2 with 1 final vol ethanol), drying, and milling. The physicochemical properties of bio-polyP were analyzed with an enzyme assay, 31 P nuclear magnetic resonance spectroscopy, and polyacrylamide gel electrophoresis, among others. An envisaged application of the process is phosphate recycling from waste streams into high-value bio-polyP., (© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

Background: Inorganic polyphosphate modulates the contact pathway of blood clotting, which is implicated in thrombosis and inflammation. Polyphosphate polymer lengths are highly variable, with shorter polymers (approximately 60-100 phosphates) secreted from human platelets, and longer polymers (up to thousands of phosphates) in microbes. We previously reported that optimal triggering of clotting via the contact pathway requires very long polyphosphates, although the impact of shorter polyphosphate polymers on individual proteolytic reactions of the contact pathway was not interrogated., Objectives and Methods: We conducted in vitro measurements of enzyme kinetics to investigate the ability of varying polyphosphate sizes, together with high molecular weight kininogen and Zn 2+ , to mediate four individual proteolytic reactions of the contact pathway: factor XII autoactivation, factor XII activation by kallikrein, prekallikrein activation by factor XIIa, and prekallikrein autoactivation., Results: The individual contact pathway reactions were differentially dependent on polyphosphate length. Very long-chain polyphosphate was required to support factor XII autoactivation, whereas platelet-size polyphosphate significantly accelerated the activation of factor XII by kallikrein, and the activation of prekallikrein by factor XIIa. Intriguingly, polyphosphate did not support prekallikrein autoactivation. We also report that high molecular weight kininogen was required only when kallikrein was the enzyme (ie, FXII activation by kallikrein), whereas Zn 2+ was required only when FXII was the substrate (ie, FXII activation by either kallikrein or FXIIa). Activation of prekallikrein by FXIIa required neither Zn 2+ nor high molecular weight kininogen., Conclusions: Platelet polyphosphate and Zn 2+ can promote subsets of the reactions of the contact pathway, with implications for a variety of disease states., (© 2019 International Society on Thrombosis and Haemostasis.)

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress., (© 2018 John Wiley & Sons Ltd.)

PAGE is often used to resolve inorganic polyphosphates (polyP), but unfortunately polyP size ladders are not commercially available. Since several dyes that are commonly used to detect nucleic acids in gels also stain polyP, we examined the utility of commercially available DNA size ladders for estimating polyP polymer lengths by gel electrophoresis. Narrow size fractions of polyP were prepared and their polymer lengths were quantified using NMR. Commercially available DNA ladders and these polyP fractions were then subjected to PAGE to determine the relationship between migration of DNA vs polyP, which was found to be: log 10 (dsDNA length in bp) = 1.66 × log 10 (polyP length in phosphate units) - 1.97. This relationship between DNA and polyP size held for a variety of different polyacrylamide concentrations, indicating that DNA size ladders can readily be employed to estimate polyP polymer lengths by PAGE., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective- Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood promotes thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo. Here, we sought to determine whether presence of long-chain polyP or bacteria in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Approach and Results- Long-chain polyP promoted platelet P-selectin expression, microaggregate formation, and platelet consumption in flowing whole blood in a contact activation pathway-dependent manner. Moreover, long-chain polyP promoted local fibrin formation on collagen under shear flow in a FXI-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the blood flow in a FXI- and FXII-dependent manner. In a murine model, long-chain polyP promoted platelet deposition and fibrin generation in lungs in a FXII-dependent manner. In a nonhuman primate model of bacterial sepsis, pre-treatment of animals with an antibody blocking FXI activation by FXIIa reduced lethal dose 100 Staphylococcus aureus-induced platelet and fibrinogen consumption. Conclusions- This study demonstrates that bacterial-type long-chain polyP promotes platelet activation in a FXII-dependent manner in flowing blood, which may contribute to sepsis-associated thrombotic processes, consumptive coagulopathy, and thrombocytopenia.

Short-chain polyphosphate (polyP) is released from platelets upon platelet activation, but it is not clear if it contributes to thrombosis. PolyP has increased propensity to clot blood with increased polymer length and when localized onto particles, but it is unknown whether spatial localization of short-chain polyP can accelerate clotting of flowing blood. Here, numerical simulations predicted the effect of localization of polyP on clotting under flow, and this was tested in vitro using microfluidics. Synthetic polyP was more effective at triggering clotting of flowing blood plasma when localized on a surface than when solubilized in solution or when localized as nanoparticles, accelerating clotting at 10-200 fold lower concentrations, particularly at low to sub-physiological shear rates typical of where thrombosis occurs in large veins or valves. Thus, sub-micromolar concentrations of short-chain polyP can accelerate clotting of flowing blood plasma under flow at low to sub-physiological shear rates. However, a physiological mechanism for the localization of polyP to platelet or vascular surfaces remains unknown., Competing Interests: S.A.S., D.K., G.D.S., and J.H.M. are co-inventors on pending patent applications covering potential medical uses of SNP-polyP. A.J.D. and Y.L. are co-inventors on a pending patent application related to the therapeutic usage and delivery of NP-polyP. D.K., G.D.S., and J.H.M. declare competing financial interests in this work. The remaining authors declare no competing financial interests.

The contact pathway of the plasma clotting cascade is dispensable for normal hemostasis, but contributes to thrombosis and serves as a bridge between inflammation and coagulation. This pathway is triggered upon exposure of plasma to certain anionic polymers and artificial surfaces. Recently, extracellular nucleic acids and inorganic polyphosphate (polyP) have been implicated as being important (patho)physiologically relevant activators of this pathway. However, mechanistic details regarding how nucleic acids or polyP modulate the individual reactions of the contact pathway have been lacking. In this study, we investigate the ability of RNA homopolymers and polyP to bind the primary constituents of the contact pathway: factor XIa, factor XIIa, and plasma kallikrein, in the presence and absence of high molecular weight kininogen (HK), an important cofactor in this pathway. We examine seven proteolytic activation reactions within the contact pathway and report that polyP greatly enhances the rate of all seven, while RNA is effective in supporting only a subset of these reactions. HK both enhances and suppresses these proteolytic activation reactions, depending on the specific reaction evaluated. Overall, we find that polyP is a potent mediator of contact pathway activation reactions in general, that RNA secondary structure may be important to its procoagulant activity, and that nucleic acids versus polyP may differentially modulate specific enzyme activation events within the contact pathway., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Introduction: Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin., Methods and Results: Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma., Conclusions: Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP., Competing Interests: A. Gruber, E.I. Tucker, and Oregon Health & Science University have a significant financial interest in Aronora Inc., a company that may have a commercial interest in the results of this research. This potential conflict of interest has been reviewed and managed by the Oregon Health & Science University Conflict of Interest in Research Committee. J. Morrissey and S. Smith have a patent: Polyphosphate-functionalized inorganic nanoparticles as hemostatic compositions and methods of use (9,186,417). These do not alter our adherence to PLOS ONE policies on sharing data and materials. The remaining authors declare no competing financial interests.

The complement system plays a key role in innate immunity, inflammation, and coagulation. The system is delicately balanced by negative regulatory mechanisms that modulate the host response to pathogen invasion and injury. The serpin, C1-esterase inhibitor (C1-INH), is the only known plasma inhibitor of C1s, the initiating serine protease of the classical pathway of complement. Like other serpin-protease partners, C1-INH interaction with C1s is accelerated by polyanions such as heparin. Polyphosphate (polyP) is a naturally occurring polyanion with effects on coagulation and complement. We recently found that polyP binds to C1-INH, prompting us to consider whether polyP acts as a cofactor for C1-INH interactions with its target proteases. We show that polyP dampens C1s-mediated activation of the classical pathway in a polymer length- and concentration-dependent manner by accelerating C1-INH neutralization of C1s cleavage of C4 and C2. PolyP significantly increases the rate of interaction between C1s and C1-INH, to an extent comparable to heparin, with an exosite on the serine protease domain of the enzyme playing a major role in this interaction. In a serum-based cell culture system, polyP significantly suppressed C4d deposition on endothelial cells, generated via the classical and lectin pathways. Moreover, polyP and C1-INH colocalize in activated platelets, suggesting that their interactions are physiologically relevant. In summary, like heparin, polyP is a naturally occurring cofactor for the C1s:C1-INH interaction and thus an important regulator of complement activation. The findings may provide novel insights into mechanisms underlying inflammatory diseases and the development of new therapies., (© 2016 by The American Society of Hematology.)

Granular platelet-sized polyphosphate nanoparticles (polyP NPs) were encapsulated in sterically stabilized liposomes, forming a potential, targeted procoagulant nanotherapy resembling human platelet dense granules in both structure and functionality. Dynamic light scattering (DLS) measurements reveal that artificial dense granules (ADGs) are colloidally stable and that the granular polyP NPs are encapsulated at high efficiencies. High-resolution scanning transmission electron microscopy (HR-STEM) indicates that the ADGs are monodisperse particles with a 150 nm diameter dense core consisting of P, Ca, and O surrounded by a corrugated 25 nm thick shell containing P, C, and O. Further, the ADGs manifest promising procoagulant activity: Detergent solubilization by Tween 20 or digestion of the lipid envelope by phospholipase C (PLC) allows for ADGs to trigger autoactivation of Factor XII (FXII), the first proteolytic step in the activation of the contact pathway of clotting. Moreover, ADGs' ability to reduce the clotting time of human plasma in the presence of PLC further demonstrate the feasibility to develop ADGs into a potential procoagulant nanomedicine.

Platelet-sized polyphosphate (polyP) was functionalized on the surface of gold nanoparticles (GNPs) via a facile conjugation scheme entailing EDAC (N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride)-catalyzed phosphoramidation of the terminal phosphate of polyP to cystamine. Subsequent reduction of the disulfide moiety allowed for anchoring to the colloidal surface. The ability of the synthesized polyP-GNPs to initiate the contact pathway of clotting in human pooled normal plasma (PNP) was then assayed by quantifying changes in viscous, mechanical, and optical properties upon coagulation. It is revealed that the polyP-GNPs are markedly superior contact activators compared to molecularly dissolved, platelet-sized polyP (of equivalent polymer chain length). Moreover, the particles' capacity to mobilize Factor XII (FXII) and its coactivating proteins appear to be identical to very-long-chain polyP typically found in bacteria. These data imply that nanolocalization of anionic procoagulants on colloidal surfaces, achieved through covalent anchoring, may yield a robust contact surface with the ability to sufficiently cluster active clotting factors together above their threshold concentrations to cease bleeding. The polyP-GNPs therefore serve as a promising foundation in the development of a nanoparticle hemostat to treat a range of hemorrhagic scenarios.

Inorganic polyphosphate (polyP), a linear polymer of phosphates, is present in many infectious microorganisms and is secreted by mast cells and platelets. PolyP has recently been shown to accelerate blood clotting and slow fibrinolysis, in a manner that is highly dependent on polymer length. Very long-chain polyP (of the type present in microorganisms) is an especially potent trigger of the contact pathway, enhances the proinflammatory activity of histones, and may participate in host responses to pathogens. PolyP also inhibits complement, providing another link between polyP and inflammation/innate immunity. Platelet-size polyP (which is considerably shorter) accelerates factor V activation, opposes the anticoagulant action of tissue factor pathway inhibitor, modulates fibrin clot structure, and promotes factor XI activation. PolyP may have utility in treating bleeding. It is also a potential target for the development of antithrombotic drugs with a novel mechanism of action and potentially fewer bleeding side effects compared with conventional anticoagulants., (© 2015 International Society on Thrombosis and Haemostasis.)

While we have understood the basic outline of the enzymes and reactions that make up the traditional blood coagulation cascade for many years, recently our appreciation of the complexity of these interactions has greatly increased. This has resulted in unofficial 'revisions' of the coagulation cascade to include new amplification pathways and connections between the standard coagulation cascade enzymes, as well as the identification of extensive connections between the immune system and the coagulation cascade. The discovery that polyphosphate is stored in platelet dense granules and is secreted during platelet activation has resulted in a recent burst of interest in the role of this ancient molecule in human biology. Here we review the increasingly complex role of platelet polyphosphate in hemostasis, thrombosis, and inflammation that has been uncovered in recent years, as well as novel therapeutics centered on modulating polyphosphate's roles in coagulation and inflammation., (© 2015 John Wiley & Sons Ltd.)

Background: Inorganic polyphosphate (polyP) elicits pro-inflammatory signaling responses in endothelial cells through interaction with two receptors, RAGE and P2Y1 . It is known that polyP activates mTOR signaling in breast cancer cells., Objectives: The objective of this study is to understand the mechanism of the polyP-mediated signaling pathway in endothelial cells and to determine whether polyP exerts its pro-inflammatory effect through activation of mTOR., Methods: mTOR activation by polyP or platelet releasates in cellular and animal models was monitored in the absence and presence of pharmacological inhibitors and/or siRNA knockdown of specific signaling molecules., Results: PolyP effectively induced phosphorylation of mTOR complex 1 (mTORC1) substrate, p70S6K, in endothelial cells by an AKT-dependent but ERK-independent mechanism. The siRNA knockdown of both RAGE and P2Y1 or specific inhibitors of the PI3K/PLC/PKC/Ca(2+) signaling axis inhibited polyP-mediated p70S6K phosphorylation. Moreover, either rapamycin or siRNA knockdown of raptor (mTORC1-specific component) abrogated polyP-mediated phosphorylation of p70S6K. By contrast, the siRNA knockdown of rictor (mTOR complex 2-specific component) but not raptor eliminated the barrier-disruptive effect of polyP. Specific NF-κB inhibitors abrogated polyP-mediated phosphorylation of p70S6K and rapamycin suppressed polyP-induced activation of NF-κB. Finally, specific inhibitors of the mTOR signaling network eliminated polyP-mediated vascular leakage and leukocyte recruitment in animal models., Conclusions: PolyP, through interaction with RAGE and P2Y1 , activates both the mTORC1 and mTORC2 signaling network. Both the pro-inflammatory and mTOR signaling functions of polyP are linked., (© 2015 International Society on Thrombosis and Haemostasis.)

We present a silica nanoparticle (SNP) functionalized with polyphosphate (polyP) that accelerates the natural clotting process of the body. SNPs initiate the contact pathway of the blood-clotting system; short-chain polyP accelerates the common pathway by the rapid formation of thrombin, which enhances the overall blood-clotting system, both by accelerating fibrin generation and by facilitating the regulatory anticoagulation mechanisms essential for hemostasis. Analysis of the clotting properties of bare SNPs, bare polyP, and polyP-functionalized SNPs in plasma demonstrated that the attachment of polyP to SNPs to form polyP-SNPs creates a substantially enhanced synergistic effect that lowers clotting time and increases thrombin production at low concentrations. PolyP-SNP even retains its clotting function at ambient temperature. The polyP-SNP system has the potential to significantly improve trauma-treatment protocols and outcomes in hospital and prehospital settings., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Factor Va enhances the rate of prothrombin activation by factor Xa by four to five orders of magnitude. Production of initiating levels of factor Va from its precursor, factor V, is a critical event early in haemostasis, as factor V exhibits negligible cofactor activity. While thrombin is the most potent physiological back-activator of factor V, the first prothrombinase complexes require a source of factor Va prior to thrombin generation. A recent study by Whelihan et al. (J Thromb Haemost 2010; 8:1532-1539) identified factor XIa as a candidate for the initial thrombin-independent activation of factor V, although this reaction was slow and required relatively high concentrations of factors V and XIa. Activated platelets secrete polyphosphate, which we previously showed to be potently procoagulant. We now report that polyphosphate greatly accelerates factor V activation by factor XIa, and that this is supported by polyphosphate polymers of the size secreted by activated human platelets. This finding provides additional evidence that factor XIa-mediated generation of factor Va may contribute to the initiation of haemostasis.

Size-controlled granular polyphosphate (PolyP) nanoparticles were synthesized by precipitation in aqueous solutions containing physiological concentrations of calcium and magnesium. We demonstrate using dynamic light scattering (DLS) that the solubility is correlated inversely with PolyP chain length, with very long chain PolyP (PolyP1000+, more than 1000 repeating units) normally found in prokaryotes precipitating much more robustly than shorter chains like those found in human platelet dense granules (PolyP80, range 76-84 repeating units). It is believed that the precipitation of PolyP is a reversible process involving calcium coordination to phosphate monomers in the polymer chain. The particles are stable in aqueous buffer and albumin suspensions on time scales roughly equivalent to catastrophic bleeding events. Transmission electron microscopy images demonstrate that the PolyP nanoparticles are spherical and uniformly electron dense, with a particle diameter of 200-250 nm, closely resembling the content of acidocalcisomes. X-ray elemental analysis further reveals that the P/Ca ratio is 67:32. The granular nanoparticles also manifest promising procoagulant effects, as measured by in vitro clotting tests assaying contact pathway activity.

Purpose of Review: Polyphosphate (polyP) is an inorganic polymer that has recently been shown to be secreted by activated platelets. It is a potent modulator of the blood clotting and complement systems in hemostasis, thrombosis, and inflammation., Recent Findings: This review focuses on what is currently known about which blood cells secrete polyP, and the roles that polyP plays in modulating the blood clotting and complement systems in health and disease., Summary: PolyP is a novel player in normal hemostasis and likely plays roles in thrombotic diseases and also in host responses to pathogens. It is also potentially a drug target, as its contributions to hemostasis appear to be to accelerate blood clotting but are not required for blood clotting to happen.

Polyphosphate, synthesized by all cells, is a linear polymer of inorganic phosphate. When released into the circulation, it exerts prothrombotic and proinflammatory activities by modulating steps in the coagulation cascade. We examined the role of polyphosphate in regulating the evolutionarily related proteolytic cascade complement. In erythrocyte lysis assays, polyphosphate comprising more than 1000 phosphate units suppressed total hemolytic activity with a concentration to reduce maximal lysis to 50% that was 10-fold lower than with monophosphate. In the ion- and enzyme-independent terminal pathway complement assay, polyphosphate suppressed complement in a concentration- and size-dependent manner. Phosphatase-treated polyphosphate lost its ability to suppress complement, confirming that polymer integrity is required. Sequential addition of polyphosphate to the terminal pathway assay showed that polyphosphate interferes with complement only when added before formation of the C5b-7 complex. Physicochemical analyses using native gels, gel filtration, and differential scanning fluorimetry revealed that polyphosphate binds to and destabilizes C5b,6, thereby reducing the capacity of the membrane attack complex to bind to and lyse the target cell. In summary, we have added another function to polyphosphate in blood, demonstrating that it dampens the innate immune response by suppressing complement. These findings further establish the complex relationship between coagulation and innate immunity.

Background: Conversion of factor XI (FXI) to FXIa is enhanced by polymers of inorganic phosphate (polyP). This process requires FXI to bind to polyP. Each FXIa subunit contains anion-binding sites (ABSs) on the apple 3 (A3) and catalytic domains that are required for normal heparin-mediated enhancement of FXIa inhibition by antithrombin., Aims: To determine the importance of FXI ABSs to polyP enhancement of FXI activation., Methods: Recombinant FXI variants lacking one or both ABSs were tested in polyP-dependent purified protein systems, plasma clotting assays, and a murine thrombosis model., Results: In the presence of polyP, activation rates for FXI lacking either ABS were reduced compared with wild-type FXI, and FXI lacking both sites had an even greater defect. In contrast to heparin, polyP binding to FXIa did not enhance inhibition by antithrombin and did not interfere with FXIa activation of FIX. FXI lacking one or both ABSs does not reconstitute FXI-deficient plasma as well as wild-type FXI when polyP was used to initiate coagulation. In FXI-deficient mice, FXI lacking one or more ABSs was inferior to wild-type FXI in supporting arterial thrombus formation., Conclusions: The ABSs on FXIa that are required for expression of heparin's cofactor activity during protease inhibition by antithrombin are also required for expression of polyP cofactor activity during FXI activation. These sites may contribute to FXI-dependent thrombotic processes., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Inorganic polyphosphates (polyP), which are secreted by activated platelets (short-chain polyP) and accumulate in some bacteria (long-chain polyP), support the contact activation of factor XII (FXII) and accelerate the activation of FXI., Objectives: The aim of the present study was to evaluate the role of FXI in polyP-mediated coagulation activation and experimental thrombus formation., Methods and Results: Pretreatment of plasma with antibodies that selectively inhibit FXI activation by activated FXII (FXIIa) or FIX) activation by activated FXI (FXIa) were not able to inhibit the procoagulant effect of long or short-chain polyP in plasma. In contrast, the FXIIa inhibitor, corn trypsin inhibitor, blocked the procoagulant effect of long and short polyP in plasma. In a purified system, long polyP significantly enhanced the rate of FXII and prekallikrein activation and the activation of FXI by thrombin but not by FXIIa. In FXI-deficient plasma, long polyP promoted clotting of plasma in an FIX-dependent manner. In a purified system, the activation of FXII and prekallikrein by long polyP promoted FIX activation and prothombin activation. In an ex vivo model of occlusive thrombus formation, inhibition of FXIIa with corn trypsin inhibitor but not of FXI with a neutralizing antibodies abolished the prothrombotic effect of long polyP., Conclusions: We propose that long polyP promotes FXII-mediated blood coagulation bypassing FXI. Accordingly, some polyp-containing pathogens may have evolved strategies to exploit polyP-initiated FXII activation for virulence, and selective inhibition of FXII may improve the host response to pathogens., (© 2013 International Society on Thrombosis and Haemostasis.)

Inorganic polyphosphates are linear polymers of orthophosphate that modulate blood clotting and inflammation. Polyphosphate accumulates in infectious microorganisms and is secreted by activated platelets; long-chain polyphosphate in particular is an extremely potent initiator of the contact pathway, a limb of the clotting cascade important for thrombosis but dispensable for hemostasis. Polyphosphate inhibitors therefore might act as novel antithrombotic/anti-inflammatory agents with reduced bleeding side effects. Antipolyphosphate antibodies are unlikely because of polyphosphate's ubiquity and simple structure; and although phosphatases such as alkaline phosphatase can digest polyphosphate, they take time and may degrade other biologically active molecules. We now identify a panel of polyphosphate inhibitors, including cationic proteins, polymers, and small molecules, and report their effectiveness in vitro and in vivo. We also compare their effectiveness against the procoagulant activity of RNA. Polyphosphate inhibitors were antithrombotic in mouse models of venous and arterial thrombosis and blocked the inflammatory effect of polyphosphate injected intradermally in mice. This study provides proof of principle for polyphosphate inhibitors as antithrombotic/anti-inflammatory agents in vitro and in vivo, with a novel mode of action compared with conventional anticoagulants.

Inorganic polyphosphate is widespread in biology and exhibits striking prohemostatic, prothrombotic, and proinflammatory effects in vivo. Long-chain polyphosphate (of the size present in infectious microorganisms) is a potent, natural pathophysiologic activator of the contact pathway of blood clotting. Medium-chain polyphosphate (of the size secreted from activated human platelets) accelerates factor V activation, completely abrogates the anticoagulant function of tissue factor pathway inhibitor, enhances fibrin clot structure, and greatly accelerates factor XI activation by thrombin. Polyphosphate may have utility as a hemostatic agent, whereas antagonists of polyphosphate may function as novel antithrombotic/anti-inflammatory agents. The detailed molecular mechanisms by which polyphosphate modulates blood clotting reactions remain to be elucidated.

Factor XI deficiency is associated with a bleeding diathesis, but factor XII deficiency is not, indicating that, in normal hemostasis, factor XI must be activated in vivo by a protease other than factor XIIa. Several groups have identified thrombin as the most likely activator of factor XI, although this reaction is slow in solution. Although certain nonphysiologic anionic polymers and surfaces have been shown to enhance factor XI activation by thrombin, the physiologic cofactor for this reaction is uncertain. Activated platelets secrete the highly anionic polymer polyphosphate, and our previous studies have shown that polyphosphate has potent procoagulant activity. We now report that polyphosphate potently accelerates factor XI activation by α-thrombin, β-thrombin, and factor XIa and that these reactions are supported by polyphosphate polymers of the size secreted by activated human platelets. We therefore propose that polyphosphate is a natural cofactor for factor XI activation in plasma that may help explain the role of factor XI in hemostasis and thrombosis.

Polyphosphate, a linear polymer of inorganic phosphate, is secreted by activated platelets and accumulates in many infectious microorganisms. We recently showed that polyphosphate modulates the blood coagulation cascade at 3 steps: it triggers the contact pathway, it accelerates factor V activation, and it enhances fibrin polymerization. We now report that polyphosphate exerts differential effects on blood clotting, depending on polymer length. Very long polymers (≥ 500mers, such as those present in microorganisms) were required for optimal activation of the contact pathway, while shorter polymers (∼ 100mers, similar to the polymer lengths released by platelets) were sufficient to accelerate factor V activation and abrogate the anticoagulant function of the tissue factor pathway inhibitor. Optimal enhancement of fibrin clot turbidity by polyphosphate required ≥ 250mers. Pyrophosphate, which is also secreted by activated platelets, potently blocked polyphosphate-mediated enhancement of fibrin clot structure, suggesting that pyrophosphate is a novel regulator of fibrin function. In conclusion, polyphosphate of the size secreted by platelets is very efficient at accelerating blood clotting reactions but is less efficient at initiating them or at modulating clot structure. Microbial polyphosphate, which is highly procoagulant, may function in host responses to pathogens.

Polyphosphates, linear polymers of inorganic phosphates linked by phosphoanhydride bonds, are widely present among organisms and play diverse roles in biology, including functioning as potent natural modulators of the human blood clotting system. However, studies of protein-polyphosphate interactions are hampered by a dearth of methods for derivatizing polyphosphate or immobilizing it onto solid supports. We now report that EDAC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide) efficiently promotes the covalent attachment of a variety of primary amine-containing labels and probes to the terminal phosphates of polyphosphates via stable phosphoramidate linkages. Using (31)P NMR, we confirmed that EDAC-mediated reactions between primary amines and polyphosphate result in phosphoramidate linkages with the terminal phosphate groups. We show that polyphosphate can be biotinylated, labeled with fluorophores, and immobilized onto solid supports, that immobilized polyphosphate can be readily used to quantify protein binding affinities, that covalently derivatized or immobilized polyphosphate retains its ability to trigger blood clotting, and that derivatizing the ends of polyphosphate with spermidine protects it from exopolyphosphatase degradation. Our findings open up essentially the entire armamentarium of protein chemistry to modifying polyphosphate, which should greatly facilitate studies of its biological roles.

Platelets play a central role in thrombosis, hemostasis, and inflammation. We show that activated platelets release inorganic polyphosphate (polyP), a polymer of 60-100 phosphate residues that directly bound to and activated the plasma protease factor XII. PolyP-driven factor XII activation triggered release of the inflammatory mediator bradykinin by plasma kallikrein-mediated kininogen processing. PolyP increased vascular permeability and induced fluid extravasation in skin microvessels of mice. Mice deficient in factor XII or bradykinin receptors were resistant to polyP-induced leakage. PolyP initiated clotting of plasma via the contact pathway. Ablation of intrinsic coagulation pathway proteases factor XII and factor XI protected mice from polyP-triggered lethal pulmonary embolism. Targeting polyP with phosphatases interfered with procoagulant activity of activated platelets and blocked platelet-induced thrombosis in mice. Addition of polyP restored defective plasma clotting of Hermansky-Pudlak Syndrome patients, who lack platelet polyP. The data identify polyP as a new class of mediator having fundamental roles in platelet-driven proinflammatory and procoagulant disorders.

Background: Polyphosphate is secreted by activated platelets and we recently showed that it accelerates blood clotting, chiefly by triggering the contact pathway and promoting factor (F) V activation., Results: We now report that polyphosphate significantly shortened the clotting time of plasmas from patients with hemophilia A and B and that its procoagulant effect was additive to that of recombinant FVIIa. Polyphosphate also significantly shortened the clotting time of normal plasmas containing a variety of anticoagulant drugs, including unfractionated heparin, enoxaparin (a low molecular weight heparin), argatroban (a direct thrombin inhibitor) and rivaroxaban (a direct FXa inhibitor). Thromboelastography revealed that polyphosphate normalized the clotting dynamics of whole blood containing these anticoagulants, as indicated by changes in clot time, clot formation time, alpha angle, and maximum clot firmness. Experiments in which preformed FVa was added to plasma support the notion that polyphosphate antagonizes the anticoagulant effect of these drugs via accelerating FV activation. Polyphosphate also shortened the clotting times of plasmas from warfarin patients., Conclusion: These results suggest that polyphosphate may have utility in reversing anticoagulation and in treating bleeding episodes in patients with hemophilia.

Polyphosphate, a linear polymer of inorganic phosphate, is present in platelet dense granules and is secreted on platelet activation. We recently reported that polyphosphate is a potent hemostatic regulator, serving to activate the contact pathway of blood clotting and accelerate factor V activation. Because polyphosphate did not alter thrombin clotting times, it appeared to exert all its procoagulant actions upstream of thrombin. We now report that polyphosphate enhances fibrin clot structure in a calcium-dependent manner. Fibrin clots formed in the presence of polyphosphate had up to 3-fold higher turbidity, had higher mass-length ratios, and exhibited thicker fibers in scanning electron micrographs. The ability of polyphosphate to enhance fibrin clot turbidity was independent of factor XIIIa activity. When plasmin or a combination of plasminogen and tissue plasminogen activators were included in clotting reactions, fibrin clots formed in the presence of polyphosphate exhibited prolonged clot lysis times. Release of polyphosphate from activated platelets or infectious microorganisms may play an important role in modulating fibrin clot structure and increasing its resistance to fibrinolysis. Polyphosphate may also be useful in enhancing the structure of surgical fibrin sealants.

PAGE is commonly used to identify and resolve inorganic polyphosphates (polyP). We now report highly sensitive and specific staining methods for polyP in polyacrylamide gels based on the fluorescent dye, 4',6-diamidino-2-phenylindol (DAPI). DAPI bound to polyP in gels fluoresced yellow while DAPI bound to nucleic acids or glycosaminoglycans fluoresced blue. Inclusion of EDTA prevented staining of glycosaminoglycans by DAPI. We also identified conditions under which DAPI that was bound to polyP (but not nucleic acids or other anionic polymers) rapidly photobleached. This allowed us to develop an even more sensitive and specific negative staining method that distinguishes polyP from nucleic acids and glycosaminoglycans. The lower LOD using DAPI negative staining was 4 pmol (0.3 ng) phosphate per band, compared to conventional toluidine blue staining with a lower LOD of 250 pmol per band.

Inorganic polyphosphate is an abundant component of acidocalcisomes of bacteria and unicellular eukaryotes. Human platelet dense granules strongly resemble acidocalcisomes, and we recently showed that they contain substantial amounts of polyphosphate, which is secreted upon platelet activation. We now report that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway and promoting the activation of factor V, which in turn results in abrogation of the function of the natural anticoagulant protein, tissue factor pathway inhibitor. Polyphosphate was also found to delay clot lysis by enhancing a natural antifibrinolytic agent, thrombin-activatable fibrinolysis inhibitor. Polyphosphate is unstable in blood or plasma, owing to the presence of phosphatases. We propose that polyphosphate released from platelets or microorganisms initially promotes clot formation and stability; subsequent degradation of polyphosphate by blood phosphatases fosters inhibition of clotting and activation of fibrinolysis during wound healing.