Determination of intracellular tenofovir-diphosphate and emtricitabine-triphosphate concentrations in dried blood spots for pre-exposure prophylaxis adherence.

OBJECTIVES: We measured the intracellular concentrations of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) in dried blood spots (DBS) for pre-exposure prophylaxis (PrEP) adherence using sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: A total of 191 DBS were obtained from 85 participants who were receiving tenofovir disoproxil fumarate (TDF; 300 mg) and emtricitabine (FTC; 200 mg) as PrEP at the Sexual Health Clinic, National Center for Global Health and Medicine, Tokyo, Japan. DBS punch (3 mm) added to 25 µL of 50% methanol and 400 µL of internal standard solution was used for solid phase extraction. Chromatographic separation was achieved on an Atlantis Premier BEH C18 AX Column (50 mm × 2.1 mm i.d.; particle size 1.7 µm) using gradient elution (flow rate: 0.6 mL/min); injection volume: 7 µL and run time: 5.5 min. Calibration curves for the two drugs were linear in the range 0.05-12.5 ng/punch. RESULTS: We determined the intracellular TFV-DP and FTC-TP concentrations in 191 DBS obtained from 85 patients administered with TDF and FTC as PrEP. The analytical performance data (calibration curve and QC samples) for all the analytical runs met the acceptance criteria. Intracellular concentrations of TFV-DP and FTC-TP in the DBS remained stable for at least 24 h after oral administration. CONCLUSIONS: A multiplex LC-MS/MS method was successfully developed for DBS, which can be useful for monitoring the levels of TFV-DP and FTC-TP in individuals receiving PrEP.

Interferon lambda1/IL-29 and inorganic polyphosphate are novel regulators of neutrophil-driven thromboinflammation.

Neutrophils and neutrophil-released meshwork structures termed neutrophil extracellular traps (NETs) are major mediators of thromboinflammation and emerging targets for therapy, yet the mechanisms and pathways that control the role of neutrophils in thromboinflammation remain poorly understood. Here, we explored the role of IFN-λ1/IL-29, a major antiviral cytokine recently shown to suppress the neutrophil migratory capacity, in prothrombotic and proNETotic functions of neutrophils. In an ex vivo human experimental setting of acute ST-segment elevation myocardial infarction (STEMI), we show that IFN-λ1/IL-29 hinders NET release and diminishes the amount of cytoplasmic TF in neutrophils. Since platelet-neutrophil interaction plays a major role in NET-induced thromboinflammation, we further studied how IFN-λ1/IL-29 may interrupt this interaction. In this context, we identified inorganic polyphosphate (polyP) as a platelet-derived NET inducer in STEMI. In arterial STEMI thrombi, polyP was present in platelets and in close proximity to NET remnants. PolyP release from activated platelets was dependent on thrombin present in infarcted artery plasma, resulting in NET formation by promoting mTOR inhibition and autophagy induction. The effect of polyP on mTOR inhibition was counteracted by IFN-λ1/IL-29 treatment, leading to inhibition of NET formation. Consistently, we show in an in vivo model of FeCl3 -induced arterial thrombosis that IFN-λ2/IL-28A exerts strong antithrombotic potential. Taken together, these findings reveal a novel function of IFN-λ1/IL-29 in the suppression of thromboinflammation. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The polyphosphate-factor XII pathway drives coagulation in prostate cancer-associated thrombosis.

Cancer is a leading cause of thrombosis. We identify a new procoagulant mechanism that contributes to thromboembolism in prostate cancer and allows for safe anticoagulation therapy development. Prostate cancer-mediated procoagulant activity was reduced in plasma in the absence of factor XII or its substrate of the intrinsic coagulation pathway factor XI. Prostate cancer cells and secreted prostasomes expose long chain polyphosphate on their surface that colocalized with active factor XII and initiated coagulation in a factor XII-dependent manner. Polyphosphate content correlated with the procoagulant activity of prostasomes. Inherited deficiency in factor XI or XII or high-molecular-weight kininogen, but not plasma kallikrein, protected mice from prostasome-induced lethal pulmonary embolism. Targeting polyphosphate or factor XII conferred resistance to prostate cancer-driven thrombosis in mice, without increasing bleeding. Inhibition of factor XII with recombinant 3F7 antibody reduced the increased prostasome-mediated procoagulant activity in patient plasma. The data illustrate a critical role for polyphosphate/factor XII-triggered coagulation in prostate cancer-associated thrombosis with implications for anticoagulation without therapy-associated bleeding in malignancies.

Putting polyphosphates to the test: evidence against platelet-induced activation of factor XII.

The recent claim that stimulated platelets activate the intrinsic pathway of coagulation by the release of polyphosphates has been considered a breakthrough in hemostasis research. In little more than 3 years, the original publication by Müller et al has been cited >100 times. However, none of the citing articles has sought to independently validate this potentially paradigm-shifting concept. To this end, we performed extensive experimentation in vitro and in vivo in an attempt to verify the claim that factor XII (FXII) is primarily activated by stimulated platelets. In contrast to the original assertion, platelet-derived polyphosphates were found to be weak activators of FXII, with a FXIIa-generating activity of <10% compared with equivalent concentrations of kaolin. Using different coagulation assays, it was shown that platelet contribution to whole blood coagulation was unrelated to the generation of activated FXII in vitro. Additionally, key results used to verify the hypothesis in the original study in vivo were found to be irreproducible. We conclude that platelet-derived polyphosphates are not physiologically relevant activators of FXII.

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.

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, synthesis and studies of triphosphate analogues for the production of anti AZT-TP antibodies.

Triphosphates anabolites are the active chemical species of nucleosidic reverse transcriptase inhibitors in HIV-therapy. Herein, we describe (i) the design of stable triphosphate analogues of AZT using molecular modelling, (ii) their synthesis and (iii) their use for producing anti AZT-TP antibodies in the aim of developing an immunoassay for therapeutic drug monitoring.

Polyphosphate, Zn2+ and high molecular weight kininogen modulate individual reactions of the contact pathway of blood clotting.

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 Zn2+ , 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 Zn2+ was required only when FXII was the substrate (ie, FXII activation by either kallikrein or FXIIa). Activation of prekallikrein by FXIIa required neither Zn2+ nor high molecular weight kininogen. CONCLUSIONS: Platelet polyphosphate and Zn2+ can promote subsets of the reactions of the contact pathway, with implications for a variety of disease states.

Simultaneous quantification of intracellular lamivudine and abacavir triphosphate metabolites by LC-MS/MS.

Nucleoside reverse transcriptase inhibitors (NRTIs) require intracellular phosphorylation to active triphosphate (TP) nucleotide metabolites before they can inhibit the HIV reverse transcriptase. However, monitoring these pharmacologically active TP metabolites is challenging due to their instability and their low concentrations at the pg/ml levels in blood and tissues. The combination of lamivudine (3TC) and abacavir (ABC) is one of the first lines for HIV therapy. Therefore, a sensitive, selective, accurate, and precise LC-MS/MS method was developed and validated for the simultaneous quantification of 3TC- and ABC-TP metabolites in mouse blood and tissues. Calibration curves were linear over the range of 10-100,000 pg/ml for 3TC-TP and 4-40,000 pg/ml for carbovir-TP (CBV-TP; phosphorylated metabolite of ABC). This corresponds to 2.1-21,322 fmol/106 cells for 3TC-TP and 0.8-8000 fmol/106 cells for CBV-TP. Accuracy and precision were less than 15% for all quality control sample (QCs), and absolute extraction recovery of were >65% for 3TC-TP and >90% for CBV-TP. The method was optimized to ensure stability of TP samples and standards during sample collection, preparation, analysis, and storage conditions. This method has enhanced sensitivity and requires smaller amounts of blood and tissue samples compared to previous LC-MS/MS methods for 3TC- and CBV-TP quantification. The developed method was successfully applied to characterize the pharmacokinetic profile of TP metabolites in mouse peripheral blood mononuclear cells (PBMCs), spleen, lymph nodes, and liver cells. In addition, another direct, simple, and high-throughput method for the quantification of TP standards was developed and used for the analysis of stability samples.

Contact system revisited: an interface between inflammation, coagulation, and innate immunity.

The contact system is a plasma protease cascade initiated by factor XII (FXII) that activates the proinflammatory kallikrein-kinin system and the procoagulant intrinsic coagulation pathway. Anionic surfaces induce FXII zymogen activation to form proteolytically active FXIIa. Bacterial surfaces also have the ability to activate contact system proteins, indicating an important role for host defense using the cooperation of the inflammatory and coagulation pathways. Recent research has shown that inorganic polyphosphate found in platelets activates FXII in vivo and can induce coagulation in pathological thrombus formation. Experimental studies have shown that interference with FXII provides thromboprotection without a therapy-associated increase in bleeding, renewing interest in the FXIIa-driven intrinsic pathway of coagulation as a therapeutic target. This review summarizes how the contact system acts as the cross-road of inflammation, coagulation, and innate immunity.

Identification and quantification of diadenosine polyphosphate concentrations in human plasma.

OBJECTIVE: Diadenosine polyphosphates have been demonstrated to be involved in the control of vascular tone as well as the growth of vascular smooth muscle cells and hence, possibly, in atherogenesis. In this study we investigated the question of whether diadenosine polyphosphates are present in human plasma and whether a potential source can be identified that may release diadenosine polyphosphates into the circulation. METHODS AND RESULTS: Plasma diadenosine polyphosphates (ApnA with n=3 to 6) were purified to homogeneity by affinity-, anion exchange-, and reversed phase-chromatography from deproteinized human plasma. Analysis of the homogeneous fractions with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) revealed molecular masses ([M+H]+) of 757, 837, 917, and 997 d. Comparison of the postsource decay matrix-assisted laser desorption/ionization mass spectrometry mass spectra of these fractions with those of authentic diadenosine polyphosphates revealed that these isolated substances were identical to Ap3A, Ap4A, Ap5A, and Ap6A. Enzymatic analysis showed an interconnection of the phosphate groups with the adenosines in the 5'-positions of the ribose moieties. The mean total plasma diadenosine polyphosphate concentrations (micromol/L; mean +/- SEM) in cubital veins of normotensive subjects amounted to 0.89+/-0.59 for Ap3A, 0.72+/-0.72 for Ap4A, 0.33+/-0.24 for Ap5A, and 0.18+/-0.18 for Ap6A. Cubital venous plasma diadenosine polyphosphate concentrations from normotensives did not differ significantly from those in the hypertensive patients studied. There was no significant difference between arterial and venous diadenosine polyphosphate plasma concentrations in 5 hemodialysis patients, making a significant degradation by capillary endothelial cells unlikely. Free plasma diadenosine polyphosphate concentrations are considerably lower than total plasma concentrations because approximately 95% of the plasma diadenosine polyphosphates were bound to proteins. There were no significant differences in the diadenosine polyphosphate plasma concentrations depending on the method of blood sampling and anticoagulation, suggesting that platelet aggregation does not artificially contribute to plasma diadenosine polyphosphate levels in significant amounts. The ApnA (with n=3 to 6) total plasma concentrations in adrenal veins were significantly higher than the plasma concentrations in both infrarenal and suprarenal vena cava: adrenal veins: Ap3A, 4.05+/-1.63; Ap4A, 6.18+/-2.08; Ap5A, 0.53+/-0.28; Ap6A, 0.59+/-0.31; infrarenal vena cava: Ap3A, 1.25+/-0.66; Ap4A, 0.91+/-0.54; Ap5A, 0.25+/-0.12; Ap6A, 0.11+/-0.06; suprarenal vena cava: Ap3A, 1.40+/-0.91; Ap4A, 1.84+/-1.20; Ap5A, 0.33+/-0.13; Ap6A, 0.11+/-0.07 (micromol/L; mean +/- SEM; each P<0.05 (concentration of adrenal veins versus infrarenal or suprarenal veins, respectively). CONCLUSIONS: The presence of diadenosine polyphosphates in physiologically relevant concentrations in human plasma was demonstrated. Because in adrenal venous plasma significantly higher diadenosine polyphosphate concentrations were measured than in plasma from the infrarenal and suprarenal vena cava, it can be assumed that, beside platelets, the adrenal medulla may be a source of plasma diadenosine polyphosphates in humans.

Polyphosphate, platelets, and coagulation.

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.

Polyphosphate accelerates factor V activation by factor XIa.

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.

Polyphosphate as modulator of hemostasis, thrombosis, and inflammation.

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.

Novel components and enzymatic activities of the human erythrocyte plasma membrane calcium pump.

The plasma membrane Ca2+ pump is essential for the maintenance of cystolic calcium ion concentration levels in eukaryotes. Here we show that the Ca2+-ATPase, purified from human erythrocytes, contains two homopolymers, poly(3-hydroxybutyrate) (PHB) and inorganic polyphosphate (polyP), which form voltage-activated calcium channels in the plasma membranes of Escherichia coli and other bacteria. Furthermore, we demonstrate that the plasma membrane Ca2+-ATPase may function as a polyphosphate kinase, i.e. it exhibits ATP-polyphosphate transferase and polyphosphate-ADP transferase activities. These findings suggest a novel supramolecular structure for the functional Ca2+-ATPase, and a new mechanism of uphill Ca2+ extrusion coupled to ATP hydrolysis.

In vivo activation and functions of the protease factor XII.

Combinations of proinflammatory and procoagulant reactions are the unifying principle for a variety of disorders affecting the cardiovascular system. Factor XII (FXII, Hageman factor) is a plasma protease that initiates the contact system. The biochemistry of the contact system in vitro is well understood; however, its in vivo functions are just beginning to emerge. The current review concentrates on activators and functions of the FXII-driven contact system in vivo. Elucidating its physiologic activities offers the exciting opportunity to develop strategies for the safe interference with both thrombotic and inflammatory diseases.