Ixonnexin from Tick Saliva Promotes Fibrinolysis by Interacting with Plasminogen and Tissue-Type Plasminogen Activator, and Prevents Arterial Thrombosis

Tick saliva is a rich source of modulators of vascular biology. We have characterized Ixonnexin, a member of the “Basic-tail” family of salivary proteins from the tick Ixodes scapularis. Ixonnexin is a 104 residues (11.8 KDa), non-enzymatic basic protein which contains 3 disulfide bonds and a C-terminal rich in lysine. It is homologous to SALP14, a tick salivary FXa anticoagulant. Ixonnexin was produced by ligation of synthesized fragments (51–104) and (1–50) followed by folding. Ixonnexin, like SALP14, interacts with FXa. Notably, Ixonnexin also modulates fibrinolysis in vitro by a unique salivary mechanism. Accordingly, it accelerates plasminogen activation by tissue-type plasminogen activator (t-PA) with Km 100 nM; however, it does not affect urokinase-mediated fibrinolysis. Additionally, lysine analogue ε-aminocaproic acid inhibits Ixonnexin-mediated plasmin generation implying that lysine-binding sites of Kringle domain(s) of plasminogen or t-PA are involved in this process. Moreover, surface plasmon resonance experiments shows that Ixonnexin binds t-PA, and plasminogen (KD 10 nM), but not urokinase. These results imply that Ixonnexin promotes fibrinolysis by supporting the interaction of plasminogen with t-PA through formation of an enzymatically productive ternary complex. Finally, in vivo experiments demonstrates that Ixonnexin inhibits FeCl3-induced thrombosis in mice. Ixonnexin emerges as novel modulator of fibrinolysis which may also affect parasite-vector-host interactions

Amylin induces hypoglycemia in mice

Amylin is a 37-aminoacid pancreatic protein that exerts control over several metabolic events such as glycemia and lacticemia. Amylin has long been shown to induce increases in arterial plasma glucose. We decided to investigate whether amylin plays additional roles in the glucose metabolism. We evaluated glucose homeostasis using whole blood from the tail tip of fasting, conscious, unrestrained normal and streptozotocyn-induced diabetic mice following subcutaneous administration of mouse amylin. Subcutaneous injection of 1 μg mouse amylin caused a transient decrease in whole blood glucose in both normal and diabetic mice in the absence of insulin. The blood glucose levels were lowest approximately 2 hours after amylin administration, after that they gradually recovered to the levels of the control group. The hypoglycemic effect followed a dose-dependent response ranging from 0.1 to 50 µg / mouse. These results reveal the ability for amylin in the direct control of glycemia at low doses in the absence of insulin

In Vitro Mode of Action and Anti-thrombotic Activity of Boophilin, a Multifunctional Kunitz Protease Inhibitor from the Midgut of a Tick Vector of Babesiosis, Rhipicephalus microplus.

BACKGROUND:Hematophagous mosquitos and ticks avoid host hemostatic system through expression of enzyme inhibitors targeting proteolytic reactions of the coagulation and complement cascades. While most inhibitors characterized to date were found in the salivary glands, relatively few others have been identified in the midgut. Among those, Boophilin is a 2-Kunitz multifunctional inhibitor targeting thrombin, elastase, and kallikrein. However, the kinetics of Boophilin interaction with these enzymes, how it modulates platelet function, and whether it inhibits thrombosis in vivo have not been determined. METHODOLOGY/PRINCIPAL FINDINGS:Boophilin was expressed in HEK293 cells and purified to homogeneity. Using amidolytic assays and surface plasmon resonance experiments, we have demonstrated that Boophilin behaves as a classical, non-competitive inhibitor of thrombin with respect to small chromogenic substrates by a mechanism dependent on both exosite-1 and catalytic site. Inhibition is accompanied by blockade of platelet aggregation, fibrin formation, and clot-bound thrombin in vitro. Notably, we also identified Boophilin as a non-competitive inhibitor of FXIa, preventing FIX activation. In addition, Boophilin inhibits kallikrein activity and the reciprocal activation, indicating that it targets the contact pathway. Furthermore, Boophilin abrogates cathepsin G- and plasmin-induced platelet aggregation and partially affects elastase-mediated cleavage of Tissue Factor Pathway Inhibitor (TFPI). Finally, Boophilin inhibits carotid artery occlusion in vivo triggered by FeCl3, and promotes bleeding according to the mice tail transection method. CONCLUSION/SIGNIFICANCE:Through inhibition of several enzymes involved in proteolytic cascades and cell activation, Boophilin plays a major role in keeping the midgut microenvironment at low hemostatic and inflammatory tonus. This response allows ticks to successfully digest a blood meal which is critical for metabolism and egg development. Boophilin is the first tick midgut FXIa anticoagulant also found to inhibit thrombosis

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

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

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 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