Long-chain Polyphosphates Research Articles

Long-chain polyphosphates inhibit type I interferon signaling and augment LPS-induced cytokine secretion in human leukocytes.

Inorganic polyphosphates are evolutionarily conserved bioactive phosphate polymers found as various chain lengths in all living organisms. In mammals, polyphosphates play a vital role in the regulation of cellular metabolism, coagulation, and inflammation. Long-chain polyphosphates are found along with endotoxins in pathogenic gram-negative bacteria and can participate in bacterial virulence. We aimed to investigate whether exogenously administered polyphosphates modulate human leukocyte function in vitro by treating the cells with 3 different chain lengths of polyphosphates (P14, P100, and P700). The long-chain polyphosphates, P700, had a remarkable capacity to downregulate type I interferon signaling dose dependently in THP1-Dual cells while only a slight elevation could be observed in the NF-κB pathway with the highest dose of P700. P700 treatment decreased lipopolysaccharide-induced IFNβ transcription and secretion, reduced STAT1 phosphorylation, and downregulated subsequent interferon-stimulated gene expression in primary human peripheral blood mononuclear cells. P700 also augmented lipopolysaccharide-induced secretion of IL-1α, IL-1β, IL-4, IL-5, IL-10, and IFNγ. Furthermore, P700 has previously been reported to increase the phosphorylation of several intracellular signaling mediators, such as AKT, mTOR, ERK, p38, GSK3α/β, HSP27, and JNK pathway components, which was supported by our findings. Taken together, these observations demonstrate the extensive modulatory effects P700 has on cytokine signaling and the inhibitory effects specifically targeted to type I interferon signaling in human leukocytes.

Failure of Micractinium simplicissimum Phosphate Resilience upon Abrupt Re-Feeding of Its Phosphorus-Starved Cultures.

Microalgae are naturally adapted to the fluctuating availability of phosphorus (P) to opportunistically uptake large amounts of inorganic phosphate (Pi) and safely store it in the cell as polyphosphate. Hence, many microalgal species are remarkably resilient to high concentrations of external Pi. Here, we report on an exception from this pattern comprised by a failure of the high Pi-resilience in strain Micractinium simplicissimum IPPAS C-2056 normally coping with very high Pi concentrations. This phenomenon occurred after the abrupt re-supplementation of Pi to the M. simplicissimum culture pre-starved of P. This was the case even if Pi was re-supplemented in a concentration far below the level toxic to the P-sufficient culture. We hypothesize that this effect can be mediated by a rapid formation of the potentially toxic short-chain polyphosphate following the mass influx of Pi into the P-starved cell. A possible reason for this is that the preceding P starvation impairs the capacity of the cell to convert the newly absorbed Pi into a "safe" storage form of long-chain polyphosphate. We believe that the findings of this study can help to avoid sudden culture crashes, and they are also of potential significance for the development of algae-based technologies for the efficient bioremoval of P from P-rich waste streams.

Investigating the Role of Bacterial Long-Chain Polyphosphates in Macrophages during Legionella pneumophilaInfection

Abstract Up to 10% of community-acquired pneumonia cases are identified as Legionnaires’ Disease, caused by the pathogen Legionella pneumophila. L. pneumophila naturally resides in freshwater systems and becomes pathogenic when man-made plumbing systems grow biofilm and amoeba if not maintained. Hosts are accidentally infected by L. pneumophilavia aerosol inhalation into the respiratory system. L. pneumophila primarily reside in human alveolar macrophages, entering and replicating via the Legionella-containing vacuole. This vacuole will also release and recruit hundreds of effector molecules to prevent itself from being phagocytosed and disguise itself as a host cellular component. Polyphosphates are highly conserved molecules found in essentially every cell performing essential functions. Long-chain polyphosphates, containing hundreds of phosphate units, are produced by bacteria to perform functions critical to their survival in hosts. We have previously found that long-chain polyphosphates produced by E. coli interfere with the host innate response against infection. Here, we show that the infection of L. pneumophila and synthetic long-chain bacterial polyphosphates interfere with the inflammatory response in macrophages. We utilized a mouse model for in vitro infection of L. pneumophila in bone-marrow-derived macrophages, showing a modulated inflammatory cytokine response and cell death. Additionally, the addition of S. cerevisiae derived exopolyphosphatase, which degrades long-chain polyphosphates, decreases inflammation in vitro.

Immunomodulation of neutrophil granulocyte functions by bacterial polyphosphates.

Polyphosphates are highly conserved, linear polymers of monophosphates that reside in all living cells. Bacteria produce long chains containing hundreds to thousands of phosphate units, which can interfere with host defense to infection. Here, we report that intratracheal long-chain polyphosphate administration to C57BL/6J mice resulted in the release of proinflammatory cytokines and influx of Ly6G+ polymorphonuclear neutrophils in the bronchoalveolar lavage fluid causing a disruption of the physiologic endothelial-epithelial small airway barrier and histologic signs of lung injury. Polyphosphate-induced effects were attenuated after neutrophil depletion in mice. In isolated murine neutrophils, long-chain polyphosphates modulated cytokine release induced by lipopolysaccharides (LPS) from Gram-negative bacteria or lipoteichoic acid from Gram-positive bacteria. In addition, long-chain polyphosphates induced immune evasive effects in human neutrophils. In detail, long-chain polyphosphates downregulated CD11b and curtailed the phagocytosis of Escherichia coli particles by neutrophils. Polyphosphates modulated the migration capacity by inducing CD62L shedding resulting in CD62Llow and CD11blow neutrophils. The release of IL-8 induced by LPS was also significantly reduced. Pharmacologic blockade of PI3K with wortmannin antagonized long-chain polyphosphate-induced effects on LPS-induced IL-8 release. In conclusion, polyphosphates govern immunomodulation in murine and human neutrophils, suggesting polyphosphates as a therapeutic target for bacterial infections to restore innate immune defense.

Heat-induced changes in molecular biosignatures and the influence of Mars-relevant minerals

AbstractThe search for signs of life is a major objective in the exploration of Mars. Of particular interest are chemical biosignatures such as biomolecules. However, molecular biosignatures are susceptible to extreme environmental conditions such as heat, ionising radiation and strong oxidants. Therefore, a knowledge of the stability of possible biosignature molecules under present and past conditions on Mars is important, as well as the nature of possible alteration products. In the light of the long volcanically active history of Mars, we have studied the thermal behaviour of selected biological compounds, namely, haemin (an iron porphyrin closely related to the haem prosthetic group), cytochrome c (a small protein) and lecithin (a mixture of phospholipids). Samples were exposed to temperatures up to 900°C under an inert atmosphere of nitrogen, either in neat form or in mineral matrices. The matrix materials used were sodium chloride, gypsum (CaSO4 ⋅ 2H2O), Ca-montmorillonite (STx-1b), the Martian regolith simulant JSC Mars-1A and some mixtures thereof. Key results are: (1) The onset of significant decomposition for haemin, cytochrome c and lecithin occurs around 240°C. At slightly higher temperatures the disappearance of all characteristic infrared spectral bands indicates complete decomposition and loss of the primary biosignatures. (2) Haemin stoichiometrically releases CO2 and HCl during the initial thermal decomposition phase, at the end of which the iron porphyrin core is still intact. High-temperature products of haemin include graphite, α-iron and cementite (Fe3C). (3) Neat lecithin forms long-chain polyphosphates at 500°C, whereas lecithin‒NaCl mixtures form diphosphate (pyrophosphate). As these anions are absent and rare, respectively, in minerals, they may potentially serve as secondary biosignatures. (4) Heating a mixture of NaCl and JSC Mars-1A at 800°C in the presence of lecithin produces the aluminosilicate mineral sodalite (Na8[AlSiO4]6Cl2), which however appears to be of limited use as a secondary biosignature.

Macro bead formation based on polyelectrolyte complexation between long-chain polyphosphates and chitosan

Long chain polyphosphates in the presence of monovalent salts can diffuse with time into chitosan droplets to form complexed beads.

One-pot chemo-enzymatic synthesis and one-step recovery of length-variable long-chain polyphosphates from microalgal biomass

Phosphate, an essential ingredient in fertilizers and detergents used daily worldwide, is a finite resource that may be exhausted within 70 years, while improper phosphate waste disposal into aquatic environments will result in eutrophication.

Bacterial polyphosphates induce CXCL4 and synergize with complement anaphylatoxin C5a in lung injury.

Polyphosphates are linear polymers of inorganic phosphates that exist in all living cells and serve pleiotropic functions. Bacteria produce long-chain polyphosphates, which can interfere with host defense to infection. In contrast, short-chain polyphosphates are released from platelet dense granules and bind to the chemokine CXCL4. Here, we report that long-chain polyphosphates induced the release of CXCL4 from mouse bone marrow-derived macrophages and peritoneal macrophages in a dose-/time-dependent fashion resulting from an induction of CXCL4 mRNA. This polyphosphate effect was lost after pre-incubation with recombinant exopolyphosphatase (PPX) Fc fusion protein, demonstrating the potency of long chains over monophosphates and ambient cations. In detail, polyphosphate chains >70 inorganic phosphate residues were required to reliably induce CXCL4. Polyphosphates acted independently of the purinergic P2Y1 receptor and the MyD88/TRIF adaptors of Toll-like receptors. On the other hand, polyphosphates augmented LPS/MyD88-induced CXCL4 release, which was explained by intracellular signaling convergence on PI3K/Akt. Polyphosphates induced Akt phosphorylation at threonine-308. Pharmacologic blockade of PI3K (wortmannin, LY294002) antagonized polyphosphate-induced CXCL4 release from macrophages. Intratracheal polyphosphate administration to C57BL/6J mice caused histologic signs of lung injury, disruption of the endothelial-epithelial barrier, influx of Ly6G+ polymorphonuclear neutrophils, depletion of CD11c+SiglecF+ alveolar macrophages, and release of CXCL4. Long-chain polyphosphates synergized with the complement anaphylatoxin, C5a, which was partly explained by upregulation of C5aR1 on myeloid cells. C5aR1-/- mice were protected from polyphosphate-induced lung injury. C5a generation occurred in the lungs and bronchoalveolar lavage fluid (BALF) of polyphosphate-treated C57BL/6J mice. In conclusion, we demonstrate that polyphosphates govern immunomodulation in macrophages and promote acute lung injury.

Effect of long-chain inorganic polyphosphate treated with wheat phytase on interleukin 8 signaling in HT-29 cells.

ObjectiveThis study was performed to investigate the potential effect of wheat phytase on long-chain inorganic polyphosphate (polyP)-mediated interleukin 8 (IL-8) signaling in an intestinal epithelial cell line, HT-29 cells.MethodsCell viability and the release of the pro-inflammatory cytokine IL-8 in HT-29 cells exposed to polyP1150 (average of 1,150 phosphate residues) treated with or without wheat phytase were measured by the EZ-CYTOX kit and the IL-8 ELISA kit, respectively. Also, the activation of cellular inflammatory factors NF-κB and MAPK (p38 and ERK 1/2) in HT-29 cells was investigated using ELISA kits.ResultsPolyP1150 negatively affected the viability of HT-29 cells in a dose-dependent manner. However, 100 mM polyP1150 dephosphorylated by wheat phytase increased cell viability by 1.4-fold over that of the intact substrate. Moreover, the 24 h exposure of cells to enzyme-treated 50 mM polyP1150 reduced the secretion of IL-8 and the activation of NF-κB by 9% and 19%, respectively, compared to the intact substrate. PolyP1150 (25 and 50 mM) dephosphorylated by the enzyme induced the activation of p38 MAPK via phosphorylation to 2.3 and 1.4-fold, respectively, compared to intact substrate, even though it had little effect on the expression of ERK 1/2 via phosphorylation.ConclusionWheat phytase could attenuate polyP1150-induced IL-8 release in HT-29 cells through NF-κB, independent of MAP kinases p38 and ERK. Thus, wheat phytase may alleviate inflammatory responses including hypercytokinemia caused by bacterial polyP infection in animals. Therefore, wheat phytase has the potential as an anti-inflammatory therapeutic supplement in animal husbandry.

Long-chain polyphosphates impair SARS-CoV-2 infection and replication.

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.

Cross-Talk between the Complement Pathway and the Contact Activation System of Coagulation: Activated Factor XI Neutralizes Complement Factor H.

Complement factor H (CFH) is the major inhibitor of the alternative pathway of the complement system and is structurally related to beta2-glycoprotein I, which itself is known to bind to ligands, including coagulation factor XI (FXI). We observed reduced complement activation when FXI activation was inhibited in a baboon model of lethal systemic inflammation, suggesting cross-talk between FXI and the complement cascade. It is unknown whether FXI or its activated form, activated FXI (FXIa), directly interacts with the complement system. We explored whether FXI could interact with and inhibit the activity of CFH. We found that FXIa neutralized CFH by cleavage of the R341/R342 bonds. FXIa reduced the capacity of CFH to enhance the cleavage of C3b by factor I and the decay of C3bBb. The binding of CFH to human endothelial cells was also reduced after incubating CFH with FXIa. The addition of either short- or long-chain polyphosphate enhanced the capacity of FXIa to cleave CFH. FXIa also cleaved CFH that was present on endothelial cells and in the secretome from blood platelets. The generation of FXIa in plasma induced the cleavage of CFH. Moreover, FXIa reduced the cleavage of C3b by factor I in serum. Conversely, we observed that CFH inhibited FXI activation by either thrombin or FXIIa. Our study provides, to our knowledge, a novel molecular link between the contact pathway of coagulation and the complement system. These results suggest that FXIa generation enhances the activity of the complement system and thus may potentiate the immune response.

Probiotic-Derived Polyphosphate Accelerates Intestinal Epithelia Wound Healing through Inducing Platelet-Derived Mediators

Inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD), is an intractable intestinal inflammation associated with the disruption of the intestinal mucosa. We previously demonstrated that Lactobacillus brevis-derived long-chain polyphosphate (poly P) improved the intestinal barrier function by the upregulation of cell adhesion and relieved intestinal inflammation, thereby exerting a curing effect on colitis in vitro, in vivo, and in an investigator-initiated clinical study of UC. However, how poly P improves mucosal defects induced by intestinal inflammation has not been elucidated. In this study, we detected the accumulation of platelets in inflamed tissues induced by poly P in a dextran sulfate sodium- (DSS-) induced colitis mouse model. A light transmission aggregometry analysis and scanning electron microscopy showed that poly P promoted the platelet aggregation. An SRB assay and ki-67 staining showed that the supernatant of poly P-treated platelet-rich plasma (PRP) increased intestinal epithelial cell growth. A wound healing assay showed that the supernatant of poly P-treated PRP, but not poly P itself, accelerated wound healing. A Western blotting analysis indicated that mitogen-activated protein kinase activation was induced by the supernatant of poly P-treated human PRP in the epithelial cells and its wound healing effect was significantly decreased by the inhibition of ERK signaling. These data suggested that platelet-derived mediators induced by poly P improved intestinal inflammation through the promotion of epithelial cell growth by the activation of the ERK signaling pathway. The mechanism is a novel host-microbe interaction through mammalian platelet-derived mediators induced by bacterial molecules.

A Broad Response to Intracellular Long-Chain Polyphosphate in Human Cells

Polyphosphates (polyPs) are long chains of inorganic phosphates linked by phosphoanhydride bonds. They are found in all kingdoms of life, playing roles in cell growth, infection, and blood coagulation. Unlike in bacteria and lower eukaryotes, the mammalian enzymes responsible for polyP metabolism are largely unexplored. We use RNA sequencing (RNA-seq) and mass spectrometry to define a broad impact of polyP produced inside of mammalian cells via ectopic expression of the E.coli polyP synthetase PPK. We find that multiple cellular compartments can support accumulation of polyP to high levels. Overproduction of polyP is associated with reprogramming of both the transcriptome and proteome, including activation of the ERK1/2-EGR1 signaling axis. Finally, fractionation analysis shows that polyP accumulation results in relocalization of nuclear/cytoskeleton proteins, including targets of non-enzymatic lysine polyphosphorylation. Our work demonstrates that internally produced polyP can activate diverse signaling pathways in human cells.

Immune escape by polyphosphate?

Host Defense Polyphosphates are inorganic linear polymers with diverse biological functions. In mammals, low–molecular weight short-chain polyphosphates are sequestered within platelets and mast cell secretory dense granules and act as signaling molecules on release. Bacteria such as Escherichia coli can also synthesize high–molecular weight long-chain polyphosphates that can serve as, among other things, energy and phosphate reserves. Roewe et al. report that polyphosphates in E. coli impaired survival in a mouse model of sepsis. In vitro, exposure to bacterial polyphosphates hinders lipopolysaccharide (LPS)–induced polarization of macrophages into a proinflammatory M1 phenotype. Instead, an anti-inflammatory M2 signature is favored, in part caused by suppression of LPS-TLR4–induced interferon β production. Although incompletely understood, E. coli polyphosphates antagonize antimicrobial macrophage functions such as phagocytosis, nitric oxide synthesis, and inflammatory cytokine production. Nat. Commun. 11 , 4035 (2020).

Bacterial polyphosphates interfere with the innate host defense to infection

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.

Potential immune-modulatory effects of wheat phytase on the performance of a mouse macrophage cell line, Raw 264.7, exposed to long-chain inorganic polyphosphate.

ObjectiveThis experiment was conducted to find out the immunological effects of wheat phytase when long-chain inorganic polyphosphate (polyP) treated with wheat phytase was added to a macrophage cell line, Raw 264.7, when compared to intact long-chain polyP.MethodsNitric oxide (NO) production of Raw 264.7 cells exposed to P700, a long-chain polyP with an average of 1,150 phosphate residues, treated with or without wheat phytase, was measured by Griess method. Phagocytosis assay of P700 treated with or without phytase in Raw 264.7 cells was investigated using neutral red uptake. The secretion of tumor necrosis factor α (TNF-α) by Raw 264.7 cells with wheat phytase-treated P700 compared to intact P700 was observed by using Mouse TNF-α enzyme-linked immunosorbent assay kit.ResultsP700 treated with wheat phytase effectively increased NO production of Raw 264.7 cells by 172% when compared with intact P700 at 12 h exposure. At 5 mM of P700 concentration, wheat phytase promoted NO production of macrophages most strongly. P700, treated with wheat phytase, stimulated phagocytosis in macrophages at 12 h exposure by about 1.7-fold compared to intact P700. In addition, P700 treated with wheat phytase effectively increased in vitro phagocytic activity of Raw 264.7 cells at a concentration above 5 mM when compared to intact P700. P700 dephosphorylated by wheat phytase increased the release of TNF-α from Raw 264.7 cells by 143% over that from intact P700 after 6 h exposure. At the concentration of 50 μM P700, wheat phytase increased the secretion of cytokine, TNF-α, by 124% over that from intact P700.ConclusionIn animal husbandry, wheat phytase can mitigate the long-chain polyP causing damage by improving the immune capabilities of macrophages in the host. Thus, wheat phytase has potential as an immunological modulator and future feed additive for regulating immune responses caused by inflammation induced by long-chain polyP from bacterial infection.

Bacterial Polyphosphates Modulate Macrophage Responses to Infection

Abstract Polyphosphates are linear polymers of inorganic monophosphate residues (Pi) and these ancient metabolites exist in all living cells. In bacteria, polyphosphate kinases (PPK) and polyphosphatases (PPX) control the accumulation of polyphosphates from ATP as long chains (>300–1000 Pi) for energy storage and prokaryotic homeostasis. Here, we demonstrate that bacterial (long-chain) polyphosphates bound and were internalized by mouse macrophages. Long-chain polyphosphates modulated the expression of more than 1,800 LPS/TLR4-regulated genes in macrophages. This interference by polyphosphates included antagonism of hundreds of interferon-regulated genes as explained by reduced interferon-β release and responsiveness, reduced phosphorylation of STAT1 and diminished major histocompatibility complex II. Polyphosphates also prevented the LPS/TLR4-induced induction of NOS2 and M1 polarization. In C57BL/6J mice infected with live E. coli, PPK deficiency or polyphosphate neutralization using recombinant PPX resulted in better survival rates, amplified macrophage recruitment and improved pathogen clearance. In conclusion, bacterial polyphosphate metabolites interfere with macrophage responses and the manipulation of such interactions could help to restore immunity to infection.

Inorganic Polyphosphate and Physiological Properties of Saccharomyces cerevisiae Yeast Overexpressing Ppn2.

The effect of the yeast endopolyphosphatase Ppn2 overproduction on the metabolism of inorganic polyphosphates in Saccharomyces cerevisiae yeast was studied. Expression of the PPN2 gene under control of the strong constitutive promoter of glyceraldehyde 3-phosphate dehydrogenase gene (PKG1) led to a significant increase in the endopolyphosphatase activity stimulated by cobalt/zinc ions. This activity was present in both soluble and membrane subcellular fractions; it was higher toward long-chain polyphosphates and could be stimulated by ADP. The content of short-chain polyphosphates in the cells of the overexpressing strain was ~2.5 times higher compared to the parent strain. The cells overexpressing Ppn2 were more resistant to peroxide and alkali. The role of short-chain polyphosphates in the adaptation to these stress factors is discussed.

Polyphosphates can stabilize but also aggregate colloids.

Phosphates are well known as dispersants for a variety of colloidal particles. Here however we use rheological measurements to show that high molecular weight polyphosphates (PP) can instead act as a flocculant for LAPONITE® clay platelets. The proposed mechanism is bridging of PP between clay particle edges, leading to highly charged clusters forming a Wigner glass. Dynamic light scattering shows a bimodal cluster size distribution, independent of PP molecular weight, but the highest molecular weight gave the highest viscous and loss moduli for the PP-clay solid. These unique all-inorganic solids may have application in solid-state ionic conducting materials, controlled release fertilizers and biomedical applications.

Plasma Kallikrein Contributes to Coagulation in the Absence of Factor XI by Activating Factor IX.

FXIa (factor XIa) induces clot formation, and human congenital FXI deficiency protects against venous thromboembolism and stroke. In contrast, the role of FXI in hemostasis is rather small, especially compared with FIX deficiency. Little is known about the cause of the difference in phenotypes associated with FIX deficiency and FXI deficiency. We speculated that activation of FIX via the intrinsic coagulation is not solely dependent on FXI(a; activated FXI) and aimed at identifying an FXI-independent FIX activation pathway. Approach and Results: We observed that ellagic acid and long-chain polyphosphates activated the coagulation system in FXI-deficient plasma, as could be demonstrated by measurement of thrombin generation, FIXa-AT (antithrombin), and FXa-AT complex levels, suggesting an FXI bypass route of FIX activation. Addition of a specific PKa (plasma kallikrein) inhibitor to FXI-deficient plasma decreased thrombin generation, prolonged activated partial thromboplastin time, and diminished FIXa-AT and FXa-AT complex formation, indicating that PKa plays a role in the FXI bypass route of FIX activation. In addition, FIXa-AT complex formation was significantly increased in F11-/- mice treated with ellagic acid or long-chain polyphosphates compared with controls and this increase was significantly reduced by inhibition of PKa. We demonstrated that activation of FXII leads to thrombin generation via FIX activation by PKa in the absence of FXI. These findings may, in part, explain the different phenotypes associated with FIX and FXI deficiencies.