Lipid nanoparticles and siRNA targeting plasminogen provide lasting inhibition of fibrinolysis in mouse and dog models of hemophilia A.

Antifibrinolytic drugs are used extensively for on-demand treatment of severe acute bleeding. Controlling fibrinolysis may also be an effective strategy to prevent or lessen chronic recurring bleeding in bleeding disorders such as hemophilia A (HA), but current antifibrinolytics have unfavorable pharmacokinetic profiles. Here, we developed a long-lasting antifibrinolytic using small interfering RNA (siRNA) targeting plasminogen packaged in clinically used lipid nanoparticles (LNPs) and tested it to determine whether reducing plasmin activity in animal models of HA could decrease bleeding frequency and severity. Treatment with the siRNA-carrying LNPs reduced circulating plasminogen and suppressed fibrinolysis in wild-type and HA mice and dogs. In HA mice, hemostatic efficacy depended on the injury model; plasminogen knockdown improved hemostasis after a saphenous vein injury but not tail vein transection injury, suggesting that saphenous vein injury is a murine bleeding model sensitive to the contribution of fibrinolysis. In dogs with HA, LNPs carrying siRNA targeting plasminogen were as effective at stabilizing clots as tranexamic acid, a clinical antifibrinolytic, and in a pilot study of two dogs with HA, the incidence of spontaneous or excess bleeding was reduced during 4 months of prolonged knockdown. Collectively, these data demonstrate that long-acting antifibrinolytic therapy can be achieved and that it provides hemostatic benefit in animal models of HA.

Elimination of fibrin polymer formation or crosslinking, but not fibrinogen deficiency, is protective against diet-induced obesity and associated pathologies.

BACKGROUND: Obesity predisposes individuals to metabolic syndrome, which increases the risk of cardiovascular diseases, non-alcoholic fatty liver disease (NAFLD), and type 2 diabetes. A pathological manifestation of obesity is the activation of the coagulation system. In turn, extravascular fibrin(ogen) deposits accumulate in adipose tissues and liver. These deposits promote adiposity and downstream sequelae by driving pro-inflammatory macrophage function through binding the leukocyte integrin receptor αM ß2 . OBJECTIVES: An unresolved question is whether conversion of soluble fibrinogen to a crosslinked fibrin matrix is required to exacerbate obesity-driven diseases. METHODS: Here, fibrinogen-deficient/depleted mice (Fib- or treated with siRNA against fibrinogen [siFga]), mice expressing fibrinogen that cannot polymerize to fibrin (FibAEK ), and mice deficient in the fibrin crosslinking transglutaminase factor XIII (FXIII-) were challenged with a high-fat diet (HFD) and compared to mice expressing a mutant form of fibrinogen lacking the αM ß2 -binding domain (Fib𝛾390-396A ). RESULTS AND CONCLUSIONS: Consistent with prior studies, Fib𝛾390-396A mice were significantly protected from increased adiposity, NAFLD, hypercholesterolemia, and diabetes while Fib- and siFga-treated mice gained as much weight and developed obesity-associated pathologies identical to wildtype mice. FibAEK and FXIII- mice displayed an intermediate phenotype with partial protection from some obesity-associated pathologies. Results here indicate that fibrin(ogen) lacking αM ß2 binding function offers substantial protection from obesity and associated disease that is partially recapitulated by preventing fibrin polymer formation or crosslinking of the wildtype molecule, but not by reduction or complete elimination of fibrinogen. Finally, these findings support the concept that fibrin polymerization and crosslinking are required for the full implementation of fibrin-driven inflammation in obesity.

Nanoparticle diffusion measures bulk clot permeability.

A clot's function is to achieve hemostasis by resisting fluid flow. Permeability is the measurement of a clot's hemostatic potential. It is sensitive to a wide range of biochemical parameters and pathologies. In this work, we consider the hydrodynamic phenomenon that reduces the mobility of fluid near the fiber surfaces. This no-slip boundary condition both defines the gel's permeability and suppresses nanoparticle diffusion in gel interstices. Here we report that, unlike previous work where steric effects also hindered diffusion, our system-nanoparticles in fibrin gel-was subject exclusively to hydrodynamic diffusion suppression. This result enabled an automated, high-throughput permeability assay that used small clot volumes. Permeability was derived from nanoparticle diffusion using the effective medium theory, and showed one-to-one correlation with measured permeability. This technique measured permeability without quantifying gel structure, and may therefore prove useful for characterizing similar materials (e.g., extracellular matrix) where structure is uncontrolled during polymerization and difficult to measure subsequently. We also report that PEGylation reduced, but did not eliminate, the population of immobile particles. We studied the forces required to pull stuck PEG particles free to confirm that the attachment is a result of neither covalent nor strong electrostatic binding, and discuss the relevance of this force scale to particle transport through physiological clots.

Abnormal plasma clot formation and fibrinolysis reveal bleeding tendency in patients with partial factor XI deficiency.

Individuals with factor XI (FXI) deficiency have a variable bleeding risk that cannot be predicted from plasma FXI antigen or activity. This limitation can result in under- or overtreatment of patients and risk of bleeding or thrombosis. Previously, plasma clot fibrinolysis assays showed sensitivity to bleeding tendency in a small cohort of patients with severe FXI deficiency. Here, we determined the ability of plasma clot formation, structure, and fibrinolysis assays to predict bleeding tendency in a larger, independent cohort of patients with severe and partial FXI deficiency. Patients were characterized as nonbleeders or bleeders based on bleeding after tonsillectomy and/or dental extraction before diagnosis of FXI deficiency. Blood was collected in the absence or presence of the contact pathway inhibitor corn trypsin inhibitor (CTI). Clotting was triggered in platelet-poor plasma with tissue factor, CaCl2, and phospholipids in the absence and presence of thrombomodulin or tissue plasminogen activator. Clot formation and fibrinolysis were assessed by turbidity and confocal microscopy. CTI-treated plasmas from bleeders showed significantly reduced clot formation and decreased resistance to fibrinolysis compared with plasmas from controls or nonbleeders. Differences were enhanced in the presence of CTI. A model that combines activated partial thromboplastin time with the rate of clot formation and area under the curve in fibrinolysis assays identifies most FXI-deficient bleeders. These results show assays with CTI-treated platelet-poor plasma reveal clotting and clot stability deficiencies that are highly associated with bleeding tendency. Turbidity-based fibrinolysis assays may have clinical utility for predicting bleeding risk in patients with severe or partial FXI deficiency.

Dysregulated coagulation associated with hypofibrinogenaemia and plasma hypercoagulability: implications for identifying coagulopathic mechanisms in humans.

Identifying coagulation abnormalities in patients with combined bleeding and thrombosis history is clinically challenging. Our goal was to probe the complexity of dysregulated coagulation in humans by characterizing pathophysiologic mechanisms in a patient with both bleeding and thrombosis. The patient is a 56-year-old female with a history of haematomas, poor wound healing, and thrombosis (retinal artery occlusion and transient cerebral ischaemia). She had a normal activated partial thromboplastin time, prolonged thrombin and reptilase times, and decreased functional and antigenic fibrinogen levels, and was initially diagnosed with hypodysfibrinogenaemia. This diagnosis was supported by DNA analysis revealing a novel FGB mutation (c.656A>G) predicting a Q189R mutation in the mature chain that was present in the heterozygote state. However, turbidity analysis showed that purified fibrinogen polymerisation and degradation were indistinguishable from normal, and Bß chain subpopulations appeared normal by two-dimensional difference in-gel electrophoresis, indicating the mutated chain was not secreted. Interestingly, plasma thrombin generation testing revealed the patient's thrombin generation was higher than normal and could be attributed to elevated levels of factor VIII (FVIII, 163-225%). Accordingly, in an arterial injury model, hypofibrinogenaemic mice (Fgn(+/-)) infused with factor VIII demonstrated significantly shorter vessel occlusion times than saline-infused Fgn(+/-) mice. Together, these data associate the complex bleeding and thrombotic presentation with combined hypofibrinogenaemia plus plasma hypercoagulability. These findings suggest previous cases in which fibrinogen abnormalities have been associated with thrombosis may also be complicated by co-existing plasma hypercoagulability and illustrate the importance of "global" coagulation testing in patients with compound presentations.