Inhibiting thrombosis without generating bleeding risks is a major challenge in medicine. A promising solution may be the inhibition of coagulation factor XII (FXII), because its knockout or inhibition in animals reduced thrombosis without causing abnormal bleeding. Herein, we have engineered a macrocyclic peptide inhibitor of activated FXII (FXIIa) with subnanomolar activity (K i = 370 ± 40 pM) and a high stability (t 1/2 > 5 days in plasma), allowing for the preclinical evaluation of a first synthetic FXIIa inhibitor. This 1899 Da molecule, termed FXII900, efficiently blocks FXIIa in mice, rabbits, and pigs. We found that it reduces ferricchloride-induced experimental thrombosis in mice and suppresses blood coagulation in an extracorporeal membrane oxygenation (ECMO) setting in rabbits, all without increasing the bleeding risk. This shows that FXIIa activity is controllable in vivo with a synthetic inhibitor, and that the inhibitor FXII900 is a promising candidate for safe thromboprotection in acute medical conditions.

No abstract

Background Extracorporeal membrane oxygenation (ECMO) research using large animals requires a significant amount of resources, slowing down the development of new means of ECMO anticoagulation. Therefore, this study developed and evaluated a new rat ECMO model using a 3D-printed mock-oxygenator. Methods The circuit consisted of tubing, a 3D-printed mock-oxygenator, and a roller pump. The mock-oxygenator was designed to simulate the geometry and blood flow patterns of the fiber bundle in full-scale oxygenators but with a low (2.5 mL) priming volume. Rats were placed on arteriovenous ECMO at a 1.9 mL/min flow rate at two different heparin doses (n = 3 each): low (15 IU/kg/h for eight hours) versus high (50 IU/kg/h for one hour followed by 25 IU/kg/h for seven hours). The experiment continued for eight hours or until the mock-oxygenator failed. The mock-oxygenator was considered to have failed when its blood flow resistance reached three times its baseline resistance. Results During ECMO, rats maintained near-normal mean arterial pressure and arterial blood gases with minimal hemodilution. The mock-oxygenator thrombus weight was significantly different (p < 0.05) between the low (0.02 ± 0.006 g) and high (0.003 ± 0.001 g) heparin delivery groups, and blood flow resistance was also larger in the low anticoagulation group. Conclusions This model is a simple, inexpensive system for investigating new anticoagulation agents for ECMO and provides low and high levels of anticoagulation that can serve as control groups for future studies.

Background: Extracorporeal membrane oxygenation (ECMO) research using large animals requires a significant amount of resources, slowing down the development of new means of ECMO anticoagulation. Therefore, this study developed and evaluated a new rat ECMO model using a 3D-printed mock oxygenator.Methods: The circuit consisted of tubing, a 3D-printed mock oxygenator, and a roller pump. The mock oxygenator was designed to simulate the geometry and blood flow patterns of the fiber bundle in full-scale oxygenators but with a low (2.5 mL) priming volume. Rats were placed on arteriovenous ECMO at a 1.9 mL/min flow rate at two different heparin doses (n = 3 each): low (15 IU/kg/h for eight hours) versus high (50 IU/kg/h for one hour followed by 25 IU/kg/h for seven hours). The experiment continued for eight hours or until the mock oxygenator failed. The mock oxygenator was considered to have failed when its blood flow resistance reached three times its baseline resistance.Results: During ECMO, rats maintained near-normal mean arterial pressure and arterial blood gases with minimal hemodilution. The mock oxygenator thrombus weight was significantly different (p < 0.05) between the low (0.02 ± 0.006 g) and high (0.003 ± 0.001 g) heparin delivery groups, and blood flow resistance was also larger in the low anticoagulation group.Conclusions: This model is a simple, inexpensive system for investigating new anticoagulation agents for ECMO and provides low and high levels of anticoagulation that can serve as control groups for future studies.

Background: Heparin anticoagulation increases the bleeding risk during extracorporeal life support (ECLS) and thus must be limited and monitored carefully. This study determined whether factor XII (FXII) silencing using short interfering RNA (siRNA) can provide ECLS circuit anticoagulation, maintain normal tissue coagulation, and reduce the need for careful anticoagulation monitoring. Methods: Adult male, Sprague-Dawley rats were randomized to four groups (n = 3 each) based on anticoagulant:(1) no anticoagulant, (2) heparin, (3) FXII siRNA, or (4) non-targeting siRNA. In group 1, no anticoagulant was administered. In group 2, heparin was administered intravenously before and during ECLS at an activated clotting time of 180-250 seconds. In groups 3 and 4, siRNA was administered intravenously three days before the initiation of ECLS via lipidoid nanoparticles. The rats were placed on pumped, arteriovenous ECLS with a small 3D-printed mock-oxygenator, and blood ow was maintained at 2 mL/min for eight hours or until the blood ow resistance reached three times its baseline resistance.Results: A single dose of 3 mg/kg siRNA led to a reduction of plasma FXII to 26% ± 3% (mean +/-standard error) of baseline levels three days after treatment. Without anticoagulant, mock-oxygenator resistance tripled within 7 ± 2 minutes. The resistance in the heparin group was signi cantly (p < 0.05) elevated in the rst hour of the experiment before returning to a lower, normal value for the rest of the experiment. The resistance in the FXII siRNA group did not change signi cantly with time (p = 0.94). There were no signi cant differences in resistance or mockoxygenator thrombus volume between the FXII siRNA and the heparin groups (p > 0.99). However, the bleeding time in the FXII siRNA group (3.4 ± 0.6 minutes) was signi cantly shorter than that in the heparin group (5.5 ± 0.5 minutes, p < 0.05).Conclusions: FXII silencing using siRNA provided simpler anticoagulation of ECLS circuits with reduced bleeding risk as compared to heparin.