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.

Comparison of DLin-MC3-DMA and ALC-0315 for siRNA Delivery to Hepatocytes and Hepatic Stellate Cells.

Ionizable cationic lipids are essential for efficient in vivo delivery of RNA by lipid nanoparticles (LNPs). DLin-MC3-DMA (MC3), ALC-0315, and SM-102 are the only ionizable cationic lipids currently clinically approved for RNA therapies. ALC-0315 and SM-102 are structurally similar lipids used in SARS-CoV-2 mRNA vaccines, while MC3 is used in siRNA therapy to knock down transthyretin in hepatocytes. Hepatocytes and hepatic stellate cells (HSCs) are particularly attractive targets for RNA therapy because they synthesize many plasma proteins, including those that influence blood coagulation. While LNPs preferentially accumulate in the liver, evaluating the ability of different ionizable cationic lipids to deliver RNA cargo into distinct cell populations is important for designing RNA-LNP therapies with minimal hepatotoxicity. Here, we directly compared LNPs containing either ALC-0315 or MC3 to knock-down coagulation factor VII (FVII) in hepatocytes and ADAMTS13 in HSCs. At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2- and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNA-mediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity.

Aortic intimal resident macrophages are essential for maintenance of the non-thrombogenic intravascular state.

Leukocytes and endothelial cells frequently cooperate to resolve inflammatory events. In most cases, these interactions are transient in nature and triggered by immunological insults. Here, we report that in areas of disturbed blood flow, aortic endothelial cells permanently and intimately associate with a population of specialized macrophages that are recruited at birth from the closing ductus arteriosus and share the luminal surface with the endothelium becoming interwoven in the tunica intima. Anatomical changes that affect hemodynamics, like in patent ductus arteriosus, alter macrophage seeding to coincide with regions of disturbed flow. Aortic resident macrophages expand in situ via direct cell renewal. Induced-depletion of intimal macrophages led to thrombin-mediated endothelial cell contraction, progressive fibrin accumulation and formation of microthrombi that, once dislodged, caused blockade of vessels in several organs. Together the findings revealed that intravascular resident macrophages are essential to regulate thrombin activity and clear fibrin deposits in regions of disturbed blood flow.

Hypofibrinogenemia with preserved hemostasis and protection from thrombosis in mice with an Fga truncation mutation.

Genetic variants within the fibrinogen Aα chain encoding the αC-region commonly result in hypodysfibrinogenemia in patients. However, the (patho)physiological consequences and underlying mechanisms of such mutations remain undefined. Here, we generated Fga270 mice carrying a premature termination codon within the Fga gene at residue 271. The Fga270 mutation was compatible with Mendelian inheritance for offspring of heterozygous crosses. Adult Fga270/270 mice were hypofibrinogenemic with ∼10% plasma fibrinogen levels relative to FgaWT/WT mice, linked to 90% reduction in hepatic Fga messenger RNA (mRNA) because of nonsense-mediated decay of the mutant mRNA. Fga270/270 mice had preserved hemostatic potential in vitro and in vivo in models of tail bleeding and laser-induced saphenous vein injury, whereas Fga-/- mice had continuous bleeding. Platelets from FgaWT/WT and Fga270/270 mice displayed comparable initial aggregation following adenosine 5'-diphosphate stimulation, but Fga270/270 platelets quickly disaggregated. Despite ∼10% plasma fibrinogen, the fibrinogen level in Fga270/270 platelets was ∼30% of FgaWT/WT platelets with a compensatory increase in fibronectin. Notably, Fga270/270 mice showed complete protection from thrombosis in the inferior vena cava stasis model. In a model of Staphylococcus aureus peritonitis, Fga270/270 mice supported local, fibrinogen-mediated bacterial clearance and host survival comparable to FgaWT/WT, unlike Fga-/- mice. Decreasing the normal fibrinogen levels to ∼10% with small interfering RNA in mice also provided significant protection from venous thrombosis without compromising hemostatic potential and antimicrobial function. These findings both reveal novel molecular mechanisms underpinning fibrinogen αC-region truncation mutations and highlight the concept that selective fibrinogen reduction may be efficacious for limiting thrombosis while preserving hemostatic and immune protective functions.

Suppression of fibrin(ogen)-driven pathologies in disease models through controlled knockdown by lipid nanoparticle delivery of siRNA.

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.

Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier.

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we report that the clotting factor fibrin is a critical regulator of neutrophil function at the oral mucosal barrier. We demonstrate that commensal microbiota trigger extravascular fibrin deposition in the oral mucosa. Fibrin engages neutrophils through the αMß2 integrin receptor and activates effector functions, including the production of reactive oxygen species and neutrophil extracellular trap formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis in mice and humans. Concordantly, genetic polymorphisms in PLG, encoding plasminogen, are associated with common forms of periodontal disease. Thus, fibrin is a critical regulator of neutrophil effector function, and fibrin-neutrophil engagement may be a pathogenic instigator for a prevalent mucosal disease.

Post-Translational Modifications of Platelet-Derived Amyloid Precursor Protein by Coagulation Factor XIII-A.

The physiological function of amyloid ß precursor protein (APP) in platelets has remained elusive. Upon platelet activation, APP localizes to the platelet surface and is proteolytically processed by proteases to release various metabolites, including amyloid ß (Aß) and soluble APP. Synthetic Aß is a substrate of activated coagulation factor XIII (FXIII-A*), a transglutaminase that is active both inside and on the surface of platelets. Here we tested if platelet APP and its fragments are covalently modified by FXIII-A*. Platelet-derived FXIII-A* and fibrin(ogen) bound to APP, and their bound fractions increased 7- and 11-fold upon platelet activation, respectively. The processing of platelet APP was enhanced when FXIII-A* was inhibited. Soluble APPß was covalently cross-linked by FXIII-A*. This mechanism regulating APP processing is significant, because controlling the processing of APP, such as by inhibiting specific secretases that cleave APP, is a therapeutic target for Alzheimer's disease.

Coagulation factor XII contributes to hemostasis when activated by soil in wounds.

Bleeding is a common contributor to death and morbidity in animals and provides strong selective pressure for the coagulation system to optimize hemostasis for diverse environments. Although coagulation factor XII (FXII) is activated by nonbiologic surfaces, such as silicates, which leads to blood clotting in vitro, it is unclear whether FXII contributes to hemostasis in vivo. Humans and mice lacking FXII do not appear to bleed more from clean wounds than their counterparts with normal FXII levels. We tested the hypothesis that soil, a silicate-rich material abundant in the environment and wounds of terrestrial mammals, is a normal and potent activator of FXII and coagulation. Blood loss was compared between wild-type (WT) and FXII-knocked out (FXII-/-) mice after soil or exogenous tissue factor was applied to transected tails. The activation of FXII and other components of the coagulation and contact system was assessed with in vitro coagulation and enzyme assays. Soils were analyzed by time-of-flight secondary ionization mass spectrometry and dynamic light scattering. Soil reduced blood loss in WT mice, but not FXII-/- mice. Soil accelerated clotting of blood plasma from humans and mice in a FXII-dependent manner, but not plasma from a cetacean or a bird, which lack FXII. The procoagulant activity of 13 soils strongly correlated with the surface concentration of silicon, but only moderately correlated with the ζ potential. FXII augments coagulation in soil-contaminated wounds of terrestrial mammals, perhaps explaining why this protein has a seemingly minor role in hemostasis in clean wounds.

Diatom Frustule Silica Exhibits Superhydrophilicity and Superhemophilicity.

Special surface wettability attracts significant attention. In this study, dramatic differences in wettability are demonstrated for microparticles with the same chemical composition, SiO2. One is natural silica prepared from the diatom, Melosira nummuloides, and the other is synthetic silica. We found that surface properties of synthetic silica are hydro- and hemophobic. However, diatom frustule silica exhibits superhydrophilicity and even superhemophilicity. Interestingly, such superhydrophilicity of natural silica is not solely originated from nanoporous structures of diatoms but from the synergy of high-density silanol anions and the nanoarchitecture. Furthermore, the observation of superhemophilicity of natural silica is also an interesting finding, because not all superhydrophilic surfaces show superhemophilicity. We demonstrate that superhemowettability is a fundamental principle for developing micropowder-based hemostatic materials despite existing hemorrhaging studies using diatoms.