Ir-CPI: an original inhibitor of thrombosis and thrombosis-associated with cancer [Abstract ISTH SSC 2018]
Darbousset R.3, Pireaux V.1, Crescence L.2, Demoulin S.1, Panicot-Dubois L.2, Godfroid E.1, Dubois C.2
1Bioxodes, Marche-en-Famenne, Belgium; 2Aix-Marseille University, Center for CardioVascular and Nutrition Research, Marseille, France; 3The University of Sydney, Heart Research Institute, Sydney, Australia.
Background: Ir-CPI, a protein derived from saliva of the tick Ixodes ricinus, is an inhibitor of the activated form of factor XI (FXIa) and XII (FXIIa). In vitro, this molecule inhibits specifically the contact phase pathway.
Aims: Our aims were to assess the efficacy of Ir-CPI in the prevention of thrombosis dependent on the Tissue Factor (TF) pathway in mouse models of thrombosis.
Methods: Two thrombosis models dependent on TF activation were studied. First, we used a laser-injury model on a cremaster muscle arteriole that is strictly dependent on TF and independent of FXII. Second, we used a laser-induced thrombosis model in mice bearing a pancreatic tumor. This model is mainly dependent on TF expressed by cancer cell-derived microparticles. As control, we assessed the efficacy of Ir-CPI in the Deep Vein Thrombosis (DVT) model in which FXII plays a central role in thrombus formation.
Results: As expected, Ir-CPI significantly reduced thrombus weight in the DVT model. Surprisingly, Ir-CPI inhibited thrombus formation, platelet accumulation and fibrin generation in the laser-injury model in normal mice. The effects of Ir-CPI on thrombosis were confirmed in tumor-bearing mice despite their procoagulant state. Since neutrophils play a key role in the activation of the TF dependent pathway in the laser model, the presence and activation of neutrophils were compared in control and Ir-CPI-treated mice. In vivo, Ir-CPI significantly inhibited neutrophil accumulation and their secretion of Neutrophil Elastase at the site of injury. In vitro, Ir-CPI strongly diminished the activation of neutrophils leading to NETs formation.
Conclusion: As expected, Ir-CPI is an effective antithrombotic in the FXII-dependent mouse model. Surprisingly, it is effective in a laser-injury model, even in a prothrombotic tumoral state. The inhibitory effect on neutrophil recruitment in vivo and NETs formation in vitro strongly suggests that Ir-CPI has additional targets and offers new therapeutic opportunities in thrombosis-associated diseases.
Efficacy of a novel contact pathway inhibitor, Ir-CPI, on in vitro clotting induced by PCI catheter segment [Abstract ISTH 2017]
Douxfils J1, Gheldof D1, Derochette S2, Tassignon J2, Meinguet C2, Guyaux M2, Donné J-M1, Godfroid E2
1University of Namur, Pharmacy, Namur, Belgium; 2Bioxodes, Marche-en-Famenne, Belgium
Background: Ir-CPI, a protein derived from the tick Ixodes ricinus salivary, is a serine protease inhibitor of both factor XIa (FXIa) and FXIIa. In patients undergoing percutaneous coronary intervention (PCI), catheter thrombosis may occur as catheters trigger activation of FXII/FXI.
Aims: The aim of this study was to evaluate the effect of Ir-CPI on in vitro clotting induced by PCI catheter segment.
Methods: Catheter segments were pressed flat, shaped into rings and placed around the perimeter of wells (96-well plate), leaving the center of the well unobstructed. To the wells were added serial dilution of Ir-CPI (until 10 µM) with normal pooled plasma (NPP) or plasmas deficient in FXI or FXII. After incubation at 37°C and addition of a CaCl2 solution, clot formation was assessed by monitoring absorbance at 340nm. Time to reach one-half maximal absorbance (IC50) was defined as the clotting time. Thrombin generation test (TGT) was also assessed using catheter segment as trigger of the process. Positive inhibitory controls were used (fondaparinux, enoxaparin).
Results: Presence of the catheter reduced the clotting time of NPP; an effect reversed by the addition of Ir-CPI. At high concentrations (≥ 5µM), Ir-CPI allowed to overpass the clotting time without catheter. On TGT, catheter segments decreased lag time and time to peak while the endogenous thrombin potential (ETP) and the peak were increased. The presence of Ir-CPI allowed the restoration of baseline value, i.e. value of the NPP without exposition to catheter segments, in a concentration-dependent manner. When clotting was triggered with FXII deficient plasma, we confirmed that catheter thrombosis is linked to FXI activation and that clotting can be abrogated with 3µM of Ir-CPI.
Conclusion: Ir-CPI can be used to inhibit the clotting induced by catheter segments and achieve antithrombotic effect. Ir-CPI is a promising agent with a better safety profile than heparins to face the problem of catheter thrombosis during PCI procedures.
Antithrombotic effects of Ir-CPI in an arterio-venous shunt model in the rabbit [Abstract ISTH 2015]
Guyaux M1, Gueret P2,3, Becher F4, Amiral J5, Simon S4, Godfroid E1
1Bioxodes, Marche-en-Famenne, Belgium; 2Hemostasis Unit, University Hospital Rennes; 3GETBO EA3878 Brest, France, 4Laboratoire Etudes et Recherches Immunoanalyse, CEA Saclay, Gif sur Yvette; 5Hyphen BioMed, Neuville sur Oise, France
Background: Ir-CPI is a 67 aa protein derived from the salivary glands of the tick Ixodes ricinus. It is a contact phase inhibitor targeting specifically factors XIa and XIIa.
Aims: The aim of the current study was to evaluate the antithrombotic potential and the pharmacokinetic-pharmacodynamic relationships of Ir-CPI.
Methods: The experiment was performed on New Zealand rabbits (n=34) anesthetized with ketamine-xylazine. An arterio-venous (AV) shunt device containing a silk thread was connected between the femoral artery and vein. Ir-CPI was administered intravenously at variable doses as a bolus alone or followed by a continuous infusion starting 5 min before the opening the AV-shunt. Thrombus weight was measured at the end of a 40 min shunt period. Blood samples were taken 10 min before and 45 min after administration. Plasma was prepared to monitor the activated partial thromboplastin time (aPTT), FXI and FXII activities and Ir-CPI concentration. The aPTT was measured using actin FS as reagent. Factors XI and XII activities were measured using FXI or FXII deficient plasmas and aPTT (Cephen) method. Ir-CPI plasma concentration was determined by mass spectrometry and enzyme-linked immunosorbent assay.
Results: Ir-CPI reduced thrombus weight by 36.8%, 62.1% and 97.2% at 1, 3 and 5 mg/kg, iv and by 90.4% at 3 mg/kg,iv followed by 2.3 mg/kg.h infusion. Ir-CPI plasma concentrations increased proportionally with the dose. Antithrombotic activity (>90 %) corresponded to an Ir-CPI plasma level of 2500 ng/mL, a prolongation of the aPTT of 50%, an inhibition of FXI and FXII activities of 35-40%. The effects on aPTT, FXI and FXII and Ir-CPI exposure level measured in vivo were consistent with the data obtained in vitro using rabbit plasma spiked with Ir-CPI.
Conclusion: These results demonstrate the antithrombotic potential of Ir-CPI in a model of AV-shunt in the rabbit whilst establishing the relationships between the antithrombotic efficacy, the Ir-CPI circulating concentration, the aPTT prolongation and FXI and FXII inhibition.
Efficacy of a novel contact pathway inhibitor, Ir-CPI, in an extracorporeal membrane oxygenator model [Abstract ISTH 2015]
Combe S1,2, Fromes Y3, Krezel C2, Gueret P4,5, Amiral J6, Guyaux M2 and Godfroid E2
1Hopitaux Universitaires Paris Centre, Paris, France; 2Bioxodes, Marche en Famenne, Belgium; 3Universite Pierre et Marie Curie, Paris; 4GETBO EA3878, Brest; 5Hemostasis Unit Hematology Laboratory, University Hospital, Rennes; 6Hyphen Biomed, Neuville sur Oise, France
Background: Ir-CPI, a protein derived from the tick Ixodes ricinus salivary, is a serine protease inhibitor of both FXIIa and FXIa. Heparins remain suboptimal in managing bleeding complications when extracorporeal life support is required.
Aims: The aim of this study was to evaluate whether inhibition of the contact phase of the coagulation cascade might confer antithrombotic activity.
Methods: Twelve Beagle dogs were included. A pediatric ECMO system was connected between the carotid artery and the jugular vein, and a cardiopulmonary bypass was maintained for 90 min at the level of full-flow systemic perfusion. Anticoagulation was performed by UFH (300 IU kg-1) with protamine reversal at the end of the procedure (N = 3). Three investigative groups (3x N = 3) received various doses of Ir-CPI (bolus and infusion) total dose 4.5 to 24 mg kg-1. After disconnection, circuits and cannulae were examined for clot and protein deposits. All animals were followed-up and sampled for determination of Ir-CPI plasma concentration (Mass Spectrometry), aPTT, FXI and FXII residual activities, before necropsy on Day 7.
Results: All 12 animals completed procedure. First Ir-CPI group showed fibrin deposits, increased pressure gradient in the circuit, and subsequent decreased gas exchanges. In further animals perfusion of Ir-CPI was initiated earlier in order to decrease the observed clot initiation and to improve gas exchanges. High dose achieved close to optimal control. Low dose was less satisfactory but perfusion remained possible. A dose-dependent and related increase in Ir-CPI plasma concentration, increase in aPTT ratio and inhibition of factors XI and XII (20% and >70% respectively) were measured over the course of the procedure. No side effects were observed and necropsies confirm the absence of thrombosis or hemorrhage.
Conclusion: Ir-CPI can be used as a single agent to inhibit the intrinsic coagulation pathway and achieve anticoagulation. Efficiency can be improved without increasing bleeding risk.
Evaluation of the effects of a novel contact pathway inhibitor, Ir-CPI, on in vitro platelet function and coagulation [Abstract ISTH 2015]
Jennings LK1,2, Kotha J2, Cardenas J2, Herr M2, Bhal V2, Dixon M2, White MM2, Combe S3 and Godfroid E3
1Vascular Biology/Medicine, University of Tennessee Health Science Center; 2CirQuest Labs, Memphis, USA; 3Bioxodes SA, Marche-en-Famenne, Belgium
Background: Ir-CPI, the tick Ixodes ricinus salivary protein, is a serine protease inhibitor under development as a novel anticoagulant. Ir-CPI inhibits Factors (F) XIIa, XIa, and kallikrein generation, prolongs the activated partial thromboplastin time, and confers antithrombotic activity in preclinical models.
Aims: The study aim was to test Ir-CPI effects on platelet function using light transmission aggregometry (LTA), clot retraction, and Pselectin expression and to characterize Ir-CPI inhibition of the contact activated coagulation pathway by assessing FXa and FIXa activation time and activity.
Methods: Ir-CPI effects on LTA in response to 20 µM ADP and 1.6 mM arachidonic acid (AA) were studied using consented blood donors (n = 5). Platelet P-selectin expression was evaluated using flow cytometry (n = 4) following activation by contact pathway initiation. Ir-CPI effects on PRP clot retraction was measured (n = 5), and FXa and FIXa generation were determined by quantitative chromogenic assays (n = 3).
Results: LTA response as well as clot retraction were unaffected by Ir-CPI pretreatment. However, the percent of P-selectin positive platelets was decreased following Ir-CPI treatment (0.5, 1, and 2 µM) by 5%, 9%, and 18%, respectively, vs. control. FXa activation lag times were prolonged to 704, 752, 826, 1013 s with increasing concentrations of Ir-CPI (1, 2, 4, 8 µM, respectively) vs. control (611 s). Similarly, FIXa activation lag times were 790, 834, 876, and 902 s, respectively, vs. 581 s (control). Also, the mean reaction rates (OD min -1) of FXa and FIXa with added Ir-CPI were decreased ~2.3-fold.
Conclusion: We demonstrated that Ir-CPI does not affect in vitro platelet aggregation response or clot retraction. However, Ir-CPI dose dependently blocked contact pathway mediated platelet P-selectin expression and the rate of FXa and FIXa activation and activity. This study confirms specific effects of Ir-CPI on the contact pathway of coagulation and provides a basis for monitoring Ir-CPI.
Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis
Decrem Y. et al., 2009, J Exp Med. 206(11):2381-95
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.