Polyanions in Coagulation and Thrombosis: Focus on Polyphosphate and Neutrophils Extracellular Traps.

Neutrophil extracellular traps (NETs) and polyphosphates (polyP) have been recognized as procoagulant polyanions. This review summarizes the activities and regulation of the two procoagulant mediators and compares their functions. NETs are composed of DNA which like polyP is built of phosphate units linked by high-energy phosphoanhydride bonds. Both NETs and polyP form insoluble particulate surfaces composed of a DNA/histone meshwork or Ca2+-rich nanoparticles, respectively. These polyanionic molecules modulate coagulation involving an array of mechanisms and trigger thrombosis via activation of the factor XII-driven procoagulant and proinflammatory contact pathway. Here, we outline the current knowledge on NETs and polyP with respect to their procoagulant and prothrombotic nature, strategies for interference of their activities in circulation, as well as the crosstalk between these two molecules. A better understanding of the underlying, cellular mechanisms will shed light on the therapeutic potential of targeting NETs and polyP in coagulation and thrombosis.

Design of Polyphosphate Inhibitors: A Molecular Dynamics Investigation on Polyethylene Glycol-Linked Cationic Binding Groups.

Inorganic polyphosphate (polyP) released by human platelets has recently been shown to activate blood clotting and identified as a potential target for the development of novel antithrombotics. Recent studies have shown that polymers with cationic binding groups (CBGs) inhibit polyP and attenuate thrombosis. However, a good molecular-level understanding of the binding mechanism is lacking for further drug development. While molecular dynamics (MD) simulation can provide molecule-level information, the time scale required to simulate these large biomacromolecules makes classical MD simulation impractical. To overcome this challenge, we employed metadynamics simulations with both all-atom and coarse-grained force fields. The force field parameters for polyethylene glycol (PEG) conjugated CBGs and polyP were developed to carry out coarse-grained MD simulations, which enabled simulations of these large biomacromolecules in a reasonable time scale. We found that the length of the PEG tail does not impact the interaction between the (PEG) n-CBG and polyP. As expected, increasing the number of the charged tertiary amine groups in the head group strengthens its binding to polyP. Our simulation shows that (PEG) n-CBG initially form aggregates, mostly with the PEG in the core and the hydrophilic CBG groups pointing toward water; then the aggregates approach the polyP and sandwich the polyP to form a complex. We found that the binding of (PEG) n-CBG remains intact against various lengths of polyP. Binding thermodynamics for two of the (PEG) n-CBG/polyP systems simulated were measured by isothermal titration calorimetry to confirm the key finding of the simulations that the length PEG tail does not influence ligand binding to polyP.

Neutralizing blood-borne polyphosphate in vivo provides safe thromboprotection.

Polyphosphate is an inorganic procoagulant polymer. Here we develop specific inhibitors of polyphosphate and show that this strategy confers thromboprotection in a factor XII-dependent manner. Recombinant Escherichia coli exopolyphosphatase (PPX) specifically degrades polyphosphate, while a PPX variant lacking domains 1 and 2 (PPX_Δ12) binds to the polymer without degrading it. Both PPX and PPX_Δ12 interfere with polyphosphate- but not tissue factor- or nucleic acid-driven thrombin formation. Targeting polyphosphate abolishes procoagulant platelet activity in a factor XII-dependent manner, reduces fibrin accumulation and impedes thrombus formation in blood under flow. PPX and PPX_Δ12 infusions in wild-type mice interfere with arterial thrombosis and protect animals from activated platelet-induced venous thromboembolism without increasing bleeding from injury sites. In contrast, targeting polyphosphate does not provide additional protection from thrombosis in factor XII-deficient animals. Our data provide a proof-of-concept approach for combating thrombotic diseases without increased bleeding risk, indicating that polyphosphate drives thrombosis via factor XII.

Caveats in studies of the physiological role of polyphosphates in coagulation.

Platelet-derived polyphosphates (polyP), stored in dense granule and released upon platelet activation, have been claimed to enhance thrombin activation of coagulation factor XI (FXI) and to activate FXII directly. The latter claim is controversial and principal results leading to these conclusions are probably influenced by methodological problems. It is important to consider that low-grade contact activation is initiated by all surfaces and is greatly amplified by the presence of phospholipids simulating the procoagulant membranes of activated platelets. Thus, proper use of inhibitors of the contact pathway and a careful choice of materials for plates and tubes is important to avoid artefacts. The use of phosphatases used to degrade polyP has an important drawback as it also degrades the secondary activators ADP and ATP, which are released from activated platelets. In addition, the use of positively charged inhibitors, such as polymyxin B, to inhibit polyP in platelet-rich plasma and blood is problematic, as polymyxin B also slows coagulation in the absence of polyP. In conclusion we hope awareness of the above caveats may improve research on the physiological roles of polyP in coagulation.

Polyphosphate: a target for thrombosis attenuation.

In this issue of Blood, Travers et al report that a number of synthetic polyphosphate (polyP) inhibitors are able to reduce thrombosis in mice without increasing bleeding as much as heparin.

Nontoxic polyphosphate inhibitors reduce thrombosis while sparing hemostasis.

Polyphosphate (polyP) is secreted by activated platelets and has been shown to contribute to thrombosis, suggesting that it could be a novel antithrombotic target. Previously reported polyP inhibitors based on polycationic substances, such as polyethylenimine, polyamidoamine dendrimers, and polymyxin B, although they attenuate thrombosis, all have significant toxicity in vivo, likely due to the presence of multiple primary amines responsible for their polyP binding ability. In this study, we examined a novel class of nontoxic polycationic compounds initially designed as universal heparin reversal agents (UHRAs) to determine their ability to block polyP procoagulant activity and also to determine their utility as antithrombotic treatments. Several UHRA compounds strongly inhibited polyP procoagulant activity in vitro, and 4 were selected for further examination in mouse models of thrombosis and hemostasis. Compounds UHRA-9 and UHRA-10 significantly reduced arterial thrombosis in mice. In mouse tail bleeding tests, administration of UHRA-9 or UHRA-10 was associated with significantly less bleeding compared with therapeutically equivalent doses of heparin. Thus, these compounds offer a new platform for developing novel antithrombotic agents that target procoagulant anionic polymers such as polyP with reduced toxicity and bleeding side effects.

Inhibition of polyphosphate as a novel strategy for preventing thrombosis and inflammation.

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.

Free nitrous acid (FNA) inhibition on denitrifying poly-phosphate accumulating organisms (DPAOs).

Free nitrous acid (FNA) has been identified to be a ubiquitous inhibitor of a wide range of microorganisms, including bacteria involved in wastewater treatment. The FNA-induced inhibition on the anoxic (nitrite as electron acceptor) metabolism of denitrifying poly-phosphate accumulating organisms (DPAOs) was investigated using sludge from a sequencing batch reactor performing carbon, nitrogen, and phosphorus removal from synthetic wastewater. We found that FNA had a much stronger inhibitory effect on phosphorus (P) uptake and glycogen production than on poly-beta-hydroxyalkanoate degradation and nitrite reduction. The intracellular adenosine triphosphate levels decreased sharply during the FNA incubation, and the decreasing rates were positively correlated with increasing FNA concentrations. The electron transport activity of DPAOs when exposed to FNA displayed a similar trend. Further, at FNA concentrations above 0.044 mg HNO(2)-N/L, the anaerobic metabolism of DPAOs was initiated despite of the presence of nitrite, as evidenced by the release of phosphorus and the consumption of glycogen. DPAO metabolism did not recover completely from FNA inhibition in the subsequent FNA-free environment. The recovery rate depended on the concentration of FNA applied in the previous anoxic period. These results suggest that the inhibitory effects are diverse and may be attributable to different mechanisms operating simultaneously.

[Effects of cellobiose lipid B on Saccharomyces cerevisiae cells: K+ leakage and inhibition of polyphosphate accumulation].

Cellobiose lipid B, a natural fungicide produced by the yeast Pseudozyma fusiformata, induces the leakage of K+ and ATP from cells of Saccharomyces cerevisiae. The presence of glucose decreases the effective concentration of cellobiose lipid B. The concentration of cellobiose lipid B was selected that results in a high rate of K+ leakage and a five- to sevenfold decrease in the intracellular ATP content, while the accumulation of acid-soluble polyphosphates decreased only by half. These results indicate the possibility of synthesis of these polymers independently of the ATP level and of the ion gradient on the plasma membrane.