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

Abstract

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.

Figures

Figure 1

Examples of plots of inhibition of thrombin binding to immobilized polyP for 4 selected inhibitors. The percent inhibition of thrombin binding to polyP is plotted for the following inhibitors that encompassed a range of IC50 values: low MW polyethyleneimine (▿), generation 1.0 PAMAM dendrimer (■), polymyxin B (□), and spermidine (▴). The dotted line represents 50% inhibition. Data are mean ± SE (n = 3).

Figure 2

Relative potencies of polyP inhibitors. (A) Inhibitor concentrations resulting in 50% reduction of thrombin binding to immobilized polyP (IC50) are plotted for the 21 most potent substances tested, expressed in terms of mass (left) and molarity (right). Inhibitors that were also used in panels B and C are numbered in parentheses. Data are mean ± SE (n = 3). (B) Plot of IC50 values of the 11 numbered substances from panel A for inhibition of heparin-mediated inactivation of factor Xa by antithrombin (y-axis) versus inhibition of thrombin binding to immobilized polyP (x-axis). Dotted line represents equivalent potency. Data are mean ± bidirectional SE (although error bars are within the symbols; n = 3). (C) Effectiveness of polyP inhibitors in prolonging clotting. Clotting of human plasma was initiated by long-chain polyP (P), polyguanylic acid (RNA), kaolin (K), diatomaceous earth (Dia), or tissue factor (TF). Data are mean inhibitor concentrations that doubled the clotting time relative to no inhibitor (ECdouble) ± SE (n = 4). Horizontal dotted lines indicate that the clotting time with that initiator was either unaffected by the inhibitor or was not prolonged sufficiently to reach a doubling point, even at 100 μg/mL inhibitor.

Figure 3

Chemical structure of the generation 1.0 cationic PAMAM dendrimer used in this study.

Figure 4

Generation 1.0 dendrimer and polymyxin B inhibit clotting of whole human blood initiated by polyP but not by tissue factor. Thromboelastometry (ROTEM) profiles are given for clotting of freshly drawn, nonanticoagulated whole human blood initiated by long-chain polyP (A-B) or tissue factor (C-D), in the presence of generation 1.0 dendrimer (A,C) or polymyxin B (B,D). The x-axis represents time from addition of clotting trigger; and y-axis, amplitude of clot strength.

Figure 5

Thrombin generation. (A) PPXbd and (B) generation 1.0 dendrimer delay thrombin generation in human plasma containing activated platelets. Real-time thrombin generation in plasma was quantified using calibrated automated thrombogram assays (Thrombinoscope; Diagnostica Stago). PPXbd (500 μg/mL), 20 μg/mL dendrimer, or saline was added to freshly drawn, citrated human blood, from which platelet-rich plasma was prepared and the platelet concentration adjusted to 150 000/μL. To some platelet-rich plasmas, TRAP was added at 10μM to activate platelets. After 5 minutes, FluCa reagent (fluorogenic substrate + CaCl2) was added and thrombin generation was quantified. Parallel assays were performed on the same platelet-rich plasmas not pretreated with TRAP, but in which clotting was triggered using FluCa reagent that also contained 5pM tissue factor (TF). Thrombin generation parameters are plotted as mean ± SE (for 5 donors assayed in triplicate). Indicated P values are from paired t tests with and without inhibitor.

Figure 6

PolyP inhibitors reverse the ability of platelet releasates to accelerate factor XI activation by thrombin. Initial rates of activation of 30nM human factor XI by 20nM human α-thrombin were determined in the presence of releasate prepared from TRAP-stimulated human platelets as described, normalized to the rate of factor XI activation without any added polyP inhibitor. Percent inhibition is plotted versus inhibitor concentration for the following: low MW polyethyleneimine (▴); generation 1.0 dendrimer (■); spermine (▵); PPXbd (●); or polymyxin B (□). Data are mean ± SE (n = 4). IC50 values calculated from these curves are given in the text. In the second stage of the assay, factor XIa levels were quantified, as previously described, by monitoring the rate of cleavage of the chromogenic substrate, L-Pyr-Pro-Arg-p-nitroanilide. At the concentrations used, none of the inhibitors altered the rate of hydrolysis of this substrate by factor XIa.

Figure 7

In vivo antithrombotic and anti-inflammatory efficacies of polyP inhibitors. (A-B) Murine model of venous thrombosis. Inhibitors were administered intravenously to mice before initiation of electrolytic injury of the femoral vein (time = 0 in the graphs). Data are mean relative intensities for accumulation of fluorescently labeled fibrin-specific antibodies (A) or labeled platelets (B) in the developing thrombus for mice receiving: red circles, 4 μg/g generation 1.0 dendrimer (n = 10); blue squares, 2 μg/g polymyxin B (n = 8); orange inverted triangles, 100 units/kg unfractionated heparin (n = 5); or open triangles, vehicle only (n = 14). Bars represent 1 SE. (C) Murine model of arterial thrombosis, with Kaplan-Meier curves showing percentage of mice with patent arteries. Inhibitors were injected retro-orbitally 10 minutes before ferric chloride injury to the carotid artery. Blood flow was monitored by Doppler, with occlusion defined as no flow for 1 minute. Log-rank analyses indicated that median patency time was significantly longer for mice injected with 8 μg/g generation 1.0 dendrimer (P < .01, n = 8), 4 μg/g polymyxin B (P < .01, n = 10), or 5 μg/g low MW polyethyleneimine (P < .01, n = 8) versus mice injected with vehicle (n = 11). (D) Murine model of polyP-induced vascular leakage. Mice were given separate retro-orbital injections with Evans blue dye and either a polyP inhibitor (48 μg/g generation 1.0 dendrimer or 20 μg/g polymyxin B) or vehicle. After 40 minutes, saline, bradykinin, and polyP were injected intradermally at 3 respective sites on the back. After an additional 30 minutes, mice were killed and dye was extracted from skin biopsies for quantification. Plots show median (central horizontal lines), mean (triangles), 25th-75th percentile (top and bottom of boxes), and 10th-90th percentile (whiskers) concentrations of extracted dye. Dendrimer administration resulted in significantly less dye leakage at the site of polyP injection compared with control animals (P < .001). Each group (no inhibitor, dendrimer, and polymyxin B) contained 15 mice.