Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis

Abstract

Blood coagulation starts immediately after damage to the vascular endothelium. This system is essential for minimizing blood loss from an injured blood vessel but also contributes to vascular thrombosis. Although it has long been thought that the intrinsic coagulation pathway is not important for clotting in vivo, recent data obtained with genetically altered mice indicate that contact phase proteins seem to be essential for thrombus formation. We show that recombinant Ixodes ricinus contact phase inhibitor (Ir-CPI), a Kunitz-type protein expressed by the salivary glands of the tick Ixodes ricinus, specifically interacts with activated human contact phase factors (FXIIa, FXIa, and kallikrein) and prolongs the activated partial thromboplastin time (aPTT) in vitro. The effects of Ir-CPI were also examined in vivo using both venous and arterial thrombosis models. Intravenous administration of Ir-CPI in rats and mice caused a dose-dependent reduction in venous thrombus formation and revealed a defect in the formation of arterial occlusive thrombi. Moreover, mice injected with Ir-CPI are protected against collagen- and epinephrine-induced thromboembolism. Remarkably, the effective antithrombotic dose of Ir-CPI did not promote bleeding or impair blood coagulation parameters. To conclude, our results show that a contact phase inhibitor is an effective and safe antithrombotic agent in vivo.

Figures

Figure 1.

In vitro anticoagulant activity of Ir-CPI. Increasing amounts of Ir-CPI were incubated with human plasma for 2 min. Coagulation aPTT (A) and clot lysis times (B) were determined as described in Materials and methods. Each point represents the mean ± SEM of three independent determinations.

Figure 2.

Effect of Ir-CPI on the thrombin activity profile during the coagulation process induced either by intrinsic or extrinsic coagulation pathway. (A) Human plasma was incubated with various concentrations of Ir-CPI (0, 0.001, 0.003, 0.009, 0.027, 0.081, 0.243, 0.729, 2.187, 6.561, and 9.077 µM), and the mixture was activated with ellagic acid and PL to initiate the intrinsic coagulation pathway. (B) Human plasma was incubated with various concentrations of Ir-CPI (0, 0.729, 2.187, 6.561, and 9.077 µM), and the mixture was activated with 5 pM TF and PL to initiate the extrinsic coagulation pathway. The amidolytic activity of thrombin was determined by adding its specific fluorogenic substrate and recording the increase in fluorescent signal. Results are presented as the mean of three independent experiments.

Figure 3.

Inhibitory effect of Ir-CPI on generation of FXIIa, FXIa, and kallikrein in human plasma and on reconstituted systems. (A) Diluted human plasma was incubated with various concentrations of Ir-CPI (0.0625, 0.125, 0.25, 0.5, and 1 µM), and the mixture was activated with aPTT reagent to initiate the contact system. The amidolytic activities of generated FXIIa, FXIa, and kallikrein were determined by adding their specific chromogenic substrates and recording the increase in absorbance at 405 nm. (B–D) The effect of Ir-CPI was examined in reconstituted systems using purified factors and their specific chromogenic substrates. The activation of a nonactivated factor by an activated factor, in the presence or absence of Ir-CPI, was analyzed in each experiment. (B) Activation of prekallikrein into kallikrein by FXIIa. (C) Activation of FXI into FXIa by FXIIa. (D) Activation of FXII into FXIIa by FXIa Results are presented as the mean ± SEM of three independent determinations.

Figure 4.

Sensorgrams for interactions between targeted factors (kallikrein, FXIa, FXIIa, and plasmin) and immobilized Ir-CPI measured by surface plasmon resonance. Ir-CPI was immobilized on the surface of a CM5 sensor chip at a level of 200 RU. Factors (from 5 to 300 nM) were injected at a flow rate of 70 µl/min in HBS buffer, and association was monitored for 84 s. After return to buffer flow, dissociation was monitored for 150 s. The sensor chip surface was regenerated by a pulse injection of 25 mM NaOH after each experiment. For each interaction, one representative result from three independent determinations is shown.

Figure 5.

Effect of Ir-CPI on pulmonary embolism model. (A) Mortality associated with i.v. of collagen and epinephrine after administration of PBS (vehicle) or Ir-CPI. All PBS-treated mice died within 5 min. Animals alive 30 min after the challenge were considered survivors. Data represent the mean ± SEM of the results obtained with 15 animals per group from two independent experiments. *, P ≤ 0.05. (B) Platelet counts in mice 2 min after infusion of collagen/epinephrine. The control group was not injected with collagen/epinephrine. Data represents the mean ± SEM of results obtained with five animals per group from two independent experiments. Values on the number of circulating platelets in the three groups of injected mice are significantly alike. (C) Number of thrombi in the lungs of mice 2 min after infusion of collagen/epinephrine. Thrombi per visual field were counted at 20×. Data represent the mean for 20 fields per mice (n = 3 per group) from two independent experiments.

Figure 6.

Effect of Ir-CPI on venous thrombosis in rats and mice. (A) Ir-CPI at the indicated doses was administered i.v. 5 min before induction of thrombosis by 10% FeCl3 and complete stasis. The control group received PBS (□) instead of Ir-CPI (▪). Each point represents the mean ± SEM for five or six animals. (B and C) Ir-CPI at the indicated doses was administered i.v. before IVC ligature. The control group received PBS instead of Ir-CPI. The thrombosed IVC fragments were harvested and weighed 24 h later. Results were expressed by dividing the thrombus weight with mouse weight (mg/g; B) or thrombus length (mg/mm; C). (D and E) Time-course experiments of clot formation. Results were expressed by dividing the thrombus weight with mouse weight (mg/g; D) or thrombus length (mg/mm; E). The experiment shown in A is representative of two independent experiments. The data shown in B–E are presented as the mean ± SEM of at least four independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (relative to vehicle); #, P ≤ 0.05 (between 1 and 10 mg/kg Ir-CPI dose).

Figure 7.

Intravital microscopy images from mouse dorsal skinfold window chambers illustrating the effect of Ir-CPI on high light intensity–induced thrombosis. (A) Representative images of microvessels of 1-mg/kg Ir-CPI–treated (bottom) and untreated (top) mice. The vessels after injection of 10 mg/kg of rose bengal but before high light intensity stimulation (time, 0) are shown on the left. The same vessels 2 h after high light intensity excitation (time, 2 h) are shown on the right. Representative images of five experiments are shown. Bars, 100 µm. (B) Bar graph showing changes in blood flow velocity in untreated and Ir-CPI–treated mice (n = 5). Results are expressed as a percentage of initial flow measured in each vessel. Data represent the mean ± SEM of results obtained from five independent experiments. **, P < 0.01.