Salivary Thromboxane A2-Binding Proteins from Triatomine Vectors of Chagas Disease Inhibit Platelet-Mediated Neutrophil Extracellular Traps (NETs) Formation and Arterial Thrombosis

<div><p>Background</p><p>The saliva of blood-feeding arthropods contains a notable diversity of molecules that target the hemostatic and immune systems of the host. Dipetalodipin and triplatin are triatomine salivary proteins that exhibit high affinity binding to prostanoids, such as TXA2, thus resulting in potent inhibitory effect on platelet aggregation <i>in vitro</i>. It was recently demonstrated that platelet-derived TXA2 mediates the formation of neutrophil extracellular traps (NETs), a newly recognized link between inflammation and thrombosis that promote thrombus growth and stability.</p><p>Methodology/Principal Findings</p><p>This study evaluated the ability of dipetalodipin and triplatin to block NETs formation <i>in vitro</i>. We also investigated the <i>in vivo</i> antithrombotic activity of TXA2 binding proteins by employing two murine models of experimental thrombosis. Remarkably, we observed that both inhibitors abolished the platelet-mediated formation of NETs <i>in vitro</i>. Dipetalodipin and triplatin significantly increased carotid artery occlusion time in a FeCl₃-induced injury model. Treatment with TXA2-binding proteins also protected mice from lethal pulmonary thromboembolism evoked by the intravenous injection of collagen and epinephrine. Effective antithrombotic doses of dipetalodipin and triplatin did not increase blood loss, which was estimated using the tail transection method.</p><p>Conclusions/Significance</p><p>Salivary TXA2-binding proteins, dipetalodipin and triplatin, are capable to prevent platelet-mediated NETs formation <i>in vitro</i>. This ability may contribute to the antithrombotic effects <i>in vivo</i>. Notably, both molecules inhibit arterial thrombosis without promoting excessive bleeding. Our results provide new insight into the antihemostatic effects of TXA2-binding proteins and may have important significance in elucidating the mechanisms of saliva to avoid host’s hemostatic responses and innate immune system.</p></div

Effect of dipetalodipin and triplatin on the pulmonary embolism model.

<p>(A-B) Kaplan-Meier survival curves. Mortality associated with i.v. injection of collagen (0.8 mg/kg) and epinephrine (60 μg/kg) after administration of PBS, (A) dipetalodipin or (B) triplatin. Animals still alive 30 min after the challenge were considered survivors. **<i>P</i> < 0.01 vs control (log-rank test).</p

Dipetalodipin and triplatin abolish the collagen-mediated acceleration of PRP clotting.

<p>Human citrated-anticoagulated (A) PRP or (B) PPP supplemented with PC/PS was pretreated with dipetalodipin (1 μM) or triplatin (2 μM). Preparations were then incubated with collagen (50 μg/ml, final concentration) or vehicle solvent (control) and activated with 16.6 mM CaCl₂. Mean ± SEM (n = 5); *<i>P</i> < .05; **<i>P</i> < .01; ***<i>P</i> < .001; NS, non-significant; analysis of variance (ANOVA) with Tukey's posttest.</p

Dipetalodipin and triplatin are antithrombotic <i>in vivo</i>.

<p>Thrombosis was induced in the carotid artery of mice via local application with 7.5% FeCl₃. Blood flow was monitored with a perivascular flow probe for 60 min or until stable occlusion occurred. (A) Dipetalodipin or (B) triplatin was injected into the caudal vein 15 min before injury. Each symbol represents one individual. *<i>P</i> < 0.05 vs control, **<i>P</i> < 0.01 vs control; ANOVA with the Dunnett posttest.</p

Dipetalodipin and triplatin inhibit platelet-mediated NET formation.

<p>(A-F) Representative images from the immunofluorescence staining for neutrophil activation. NET formation was visualized via confocal microscopy using antibodies against DNA (blue) and citrullinated histones (green), as described in the Materials and methods section. No NET formation was apparent for treatment with (A) culture medium or (B) resting platelets. (C) PMA (5 nM) was used as positive control for the formation of NETs. (D) Treatment with platelets previously activated by collagen (1.3 μg/mL) elicited the formation of NETs. Neutrophil incubation with platelets previously activated by collagen in the presence of (E) dipetalodipin (1 μM) or (F) triplatin (1 μM) did not elicit the formation of NETs. Scale bar: 20 μm.</p

Bleeding effect by the transection model.

<p>PBS (control), dipetalodipin or triplatin was administered intravenously and allowed to circulate for 15 min. Blood loss was determined as a function of the hemoglobin concentration in the water (absorbance at 540 nm). Each symbol represents one individual (ANOVA with Tukey's posttest).</p

Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin

<div><p>A group of peptides from the salivary gland of the tick <i>Hyalomma marginatum rufipes</i>, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, <i>Haemaphysalis longicornis</i>. We have evaluated the anti-serine protease activity of one of these <i>H</i>. <i>marginatum</i> peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic.</p></div

Hyalomin-1 is a specific thrombin inhibitor.

<p>(A) The activity of 16 serine proteases in the presence of hyalomin-1 (1 μM) relative to their activity in the absence of inhibitor. (B) Coagulation time of human plasma incubated with hyalomin-1 as measured using the APTT (solid line), and PT (dashed line) assay procedures. (C) Conversion of fibrinogen to fibrin by thrombin in the presence of increasing concentrations of hyalomin-1 as indicated by seconds for increase in absorbance to 0.01 at 650 nm. (D) Aggregation of washed platelets induced by thrombin in the presence of various concentrations of hyalomin-1, as measured by an increase in transmittance in an aggregometer. (E) Polyphosphate-activated cleavage of FXI by thrombin in presence of hyalomin-1. FXIa was measured by hydrolysis of the chromogenic substrate S2236. (F) FV cleavage by thrombin in the presence and absence of hyalomin-1 as measured by SDS-PAGE. Lane 1 –FV alone after 60 min incubation. Lane 2 –FV and thrombin after 10 minutes incubation. Lane 3 –FV, thrombin and hyalomin-1 after 10 minutes incubation. Lane 4 –FV and thrombin after 60 minutes incubation. Lane 5 –thrombin alone.</p

Sulfation of hyalomin-1 (13–44) truncated form results in a modest increase in potency.

<p>(A) Conversion of fibrinogen to fibrin by thrombin in the presence of different hyalomin-1 truncated forms (5 μM) as indicated by time in seconds for increase in absorbance of 0.01 at 650 nm. Bars represent mean with SE. Full hyalomin-1 (01–59) completely inhibited fibrinogen clotting during the time assayed. (B) Same assay as in A but at different concentrations of hyalomin-1 (13–44) sulfated. (C) APTT and (D) PT assay procedures for coagulation time of human plasma in the presence of different concentrations of hyalomin -1 (13–44) sulfated (dashed lines), or hyalomin-1 (13–44) solid lines.</p

Hyalomin-1 is a competitive fast binding classical inhibitor of thrombin that is also cleaved by thrombin.

<p>(A) Kinetics of S2238 hydrolysis by thrombin in the presence of increasing concentrations of hyalomin-1 at a sodium chloride concentration of 150 mM. Inhibitor concentrations: 0 nM (filled circles), 10 nM (filled squares), 50 nM (filled triangles), 100 nM (filled inverted triangles), 200 nM (filled diamonds), 400 nM (open circles), 600 nM (open squares). (B) Experiment performed as in panel A but at a sodium chloride concentration of 50 mM. Hyalomin-1 concentrations: 0 nM (filled circles), 1.25 nM (filled squares), 2.5 nM (filled triangles), 5 nM (filled inverted triangles), 10 nM (filled diamonds), 25 nM (open circles), 50 nM (open squares), 100 nM (open triangles). (C) Progress curves of thrombin activity in the presence of hyalomin-1: 0 nM (filled circles), 50 nM (filled squares), 100 nM (filled triangles), 200 nM (filled inverted triangles), 400 nM (filled diamonds), 600 nM (open circles). (D) Measurement of thrombin binding to immobilized hyalomin-1 by SPR. Sensograms were obtained by injection of thrombin at concentrations of 50 nM (1), 25 nM (2), 12.5 nM (3), 6.25 nM (4), 3.125 nM (5) and 1.563 nM (6). Kinetic constants are indicated in the text. (E) Mass spectral analysis of hyalomin-1 cleavage products after incubation with thrombin for 2 h at 37°C. The mass values on the graph correspond to cleavage at the Arg₄₁-Leu₄₂ peptide bond (peptides 01–41 and 42–59 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133991#pone.0133991.g005" target="_blank">Fig 5A</a>). (F) Effect of incubation time at 37°C on the inhibition of thrombin (0.5 nM) by hyalomin-1 (400 nM) in the presence of 50 μM S2238. The activity of thrombin in the absence of hyalomin-1 is shown as filled circles, while activity in the presence of hyalomin-1 is shown as filled squares.</p