Hepatic plasminogen lowering with a galnac-conjugated siRNA reduces plasmin generation and fibrinolysis but has no effect on thrombin generation in rats and mice Free

Introduction: Plasminogen (PLG) is primarily liver-derived and plays a key role in fibrinolysis. Antifibrinolytics, such as tranexamic acid, have been used clinically for decades to prevent bleeding by inhibiting the activation of PLG into plasmin, thereby reducing plasmin generation and fibrinolysis. Lowering hepatic PLG with an N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (PLG-lowering siRNA) is designed to reduce plasmin generation and fibrinolysis but to leave thrombin generation unchanged. Suppression of PLG activation and/or plasmin activity does not seem to increase venous thrombosis/thromboembolism risk (Sang et al., Blood 2025). By contrast, there is a known association between increased thrombin generation and higher thrombotic risk (Lutsey et al., JTH 2009). Lowering hepatic PLG with siRNA in humans may be a safer approach for treating bleeding disorders as compared to using agents that increase thrombin generation. Here, we evaluated the impact of hepatic PLG lowering on plasmin generation, fibrinolysis assays and thrombin generation in rats and mice.

Methods: Wild Type (WT) rats (Brown Norway) or mice (C57BL/6) were treated with repeated or single subcutaneous injections of a liver targeting (GalNAc-conjugated) PLG-lowering siRNA (0.5 or 10 mg/kg). Rats and mice injected with vehicle (PBS) were used as controls. Rats received 3 repeated doses of PLG-lowering siRNA, each 17 days apart (on Day 0, 17 and 34), and blood was collected before every dose followed by a terminal blood collection on Day 49. Mice received a single PLG-lowering siRNA injection, and blood was collected 7 days later. PLG protein plasma levels were measured by ELISA, and liver PLG mRNA was measured by RT-qPCR. Plasma plasmin generation was quantified using a calibrated fluorescence-based assay. Plasma fibrinolysis was measured using a fibrinolysis assay (Lysis Timer). Whole blood fibrinolysis was evaluated in blood collected from rats using tissue plasminogen activator-modified Rotational Thromboelastometry (tPA-ROTEM). Plasma thrombin generation was quantified using a fluorescence-based assay.

Results: PLG-lowering siRNA treatment in rats resulted in ~80% (0.5 mg/kg dose) and ~99% (10 mg/kg dose) reduction in plasma PLG protein levels measured on Day 17, which were maintained through Day 49 and reflected in liver PLG mRNA levels. The 10 mg/kg dose of PLG-lowering siRNA in mice resulted in ~99% reduction in plasma PLG protein and liver PLG mRNA by Day 7. PLG lowering in rats resulted in a dose-dependent decrease in plasma plasmin generation (detected on Day 17 and maintained through Day 49), with peak plasmin generation shifting from ~57 nM in control animals to ~25 and ~1 nM on Day 49 in the 0.5 and 10 mg/kg PLG-lowering siRNA treated groups, respectively. There was also an inhibitory effect on plasma fibrinolysis in rats (detected on Day 17 and maintained through Day 49), with a prolongation of clot lysis times on Day 49 from ~27 min in the control group to ~93 and 120 min in the 0.5 and 10 mg/kg PLG-lowering siRNA treated groups, respectively. PLG lowering in rats also resulted in a robust antifibrinolytic effect on whole blood tPA-ROTEM (detected on Day 17 and maintained through Day 49) by decreasing maximum lysis (ML) from 100% in the control group to ~16% in both PLG-lowering siRNA groups after running the assay for 2 hours. After 8 hours, control animals still had a ML of 100%, while the 0.5 mg/kg group had a ML of 58%, and the 10 mg/kg group had a ML of 31%, showing that the PLG-lowering siRNA had a dose-dependent and time-dependent antifibrinolytic effect. PLG lowering had no observed effect on thrombin generation in both rats (measured on Day 49) and mice (measured onDay 7) compared to control treated animals.

Conclusions: Hepatic PLG lowering with a GalNAc-conjugated siRNA reduced plasmin generation and plasma and whole blood fibrinolysis in rats, but had no effect on thrombin generation in rats and mice. These results suggest that targeting the fibrinolytic pathway with a liver-directed PLG-lowering siRNA will likely not be associated with thrombotic risk and may be a safer option compared to alternatives that increase thrombin generation. The results support clinical development of ALN-6400, an investigational RNA interference therapeutic for the treatment of bleeding disorders (NCT06659640).