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.

Mice expressing nonpolymerizable fibrinogen have reduced arterial and venous thrombosis with preserved hemostasis.

ABSTRACT: Elevated circulating fibrinogen levels correlate with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. Here, we analyzed the impact of nonpolymerizable fibrinogen on arterial and venous thrombosis as well as hemostasis in vivo using FgaEK mice that express normal levels of a fibrinogen that cannot be cleaved by thrombin. In a model of carotid artery thrombosis, FgaWT/EK and FgaEK/EK mice were protected from occlusion with 4% ferric chloride (FeCl3) challenges compared with wild-type (FgaWT/WT) mice, but this protection was lost, with injuries driven by higher concentrations of FeCl3. In contrast, fibrinogen-deficient (Fga-/-) mice showed no evidence of occlusion, even with high-concentration FeCl3 challenge. Fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content were similar in FgaWT/WT, FgaWT/EK, and FgaEK/EK mice, consistent with preserved fibrinogen-platelet interactions that support arterial thrombosis with severe challenge. In an inferior vena cava stasis model of venous thrombosis, FgaEK/EK mice had near complete protection from thrombus formation. FgaWT/EK mice also displayed reduced thrombus incidence and a significant reduction in thrombus mass relative to FgaWT/WT mice after inferior vena cava stasis, suggesting that partial expression of nonpolymerizable fibrinogen was sufficient for conferring protection. Notably, FgaWT/EK and FgaEK/EK mice had preserved hemostasis in multiple models as well as normal wound healing times after skin incision, unlike Fga-/- mice that displayed significant bleeding and delayed healing. These findings indicate that a nonpolymerizable fibrinogen variant can significantly suppress occlusive thrombosis while preserving hemostatic potential in vivo.

Altered fibrinogen γ-chain cross-linking in mutant fibrinogen-γΔ5 mice drives acute liver injury.

BACKGROUND: Hepatic deposition of cross-linked fibrin(ogen) occurs alongside platelet accumulation as a hallmark of acetaminophen (APAP)-induced liver injury. OBJECTIVES: We sought to define the precise role of the fibrinogen γ-chain C-terminal integrin αIIbß3 binding domain in APAP-induced liver injury. METHODS: Mice expressing mutant fibrinogen incapable of engaging integrin αIIbß3 due to a C-terminal fibrinogen γ-chain truncation (mutant fibrinogen-γΔ5 [FibγΔ5] mice) and wild-type mice were challenged with APAP (300 mg/kg, intraperitoneally). RESULTS: We observed an altered pattern of fibrin(ogen) deposition in the livers of APAP-challenged FibγΔ5 mice. This led to the unexpected discovery that fibrinogen γ-chain cross-linking was altered in the livers of APAP-challenged FibγΔ5 mice compared with that in wild-type mice, including absence of γ-γ dimer and accumulation of larger molecular weight cross-linked γ-chain complexes. This finding was not unique to the injured liver because activation of coagulation did not produce γ-γ dimer in plasma from FibγΔ5 mice or purified FibγΔ5 fibrinogen. Sanger sequencing predicted that the fibrinogen-γΔ5 γ-polypeptide would terminate at lysine residue 406, but liquid chromatography tandem mass spectrometry analysis revealed that this critical lysine residue was absent in purified fibrinogen-γΔ5 protein. Interestingly, hepatic deposition of this uniquely aberrantly cross-linked fibrin(ogen) in FibγΔ5 mice was associated with exacerbated hepatic injury, an effect not recapitulated by pharmacologic inhibition of integrin αIIbß3. CONCLUSION: The results indicate that fibrinogen-γΔ5 lacks critical residues essential to form γ-γ dimer in response to thrombin and suggest that hepatic accumulation of abnormally cross-linked fibrin(ogen) can exacerbate hepatic injury.

Fibrinogen γ' promotes host survival during Staphylococcus aureus septicemia in mice.

BACKGROUND: Staphylococcus aureus is a common gram-positive bacterium that is the causative agent for several human diseases, including sepsis. A key virulence mechanism is pathogen binding to host fibrinogen through the C-terminal region of the γ-chain. Previous work demonstrated that FggΔ5 mice expressing mutant fibrinogen γΔ5 lacking a S. aureus binding motif had significantly improved survival following S. aureus septicemia. Fibrinogen γ' is a human splice variant that represents about 10% to 15% of the total fibrinogen in plasma and circulates as a fibrinogen γ'-γ heterodimer (phFibγ'-γ). The fibrinogen γ'-chain is also expected to lack S. aureus binding function. OBJECTIVE: Determine if human fibrinogen γ'-γ confers host protection during S. aureus septicemia. METHODS: Analyses of survival and the host response following S. aureus septicemia challenge in FggΔ5 mice and mice reconstituted with purified phFibγ'-γ or phFibγ-γ. RESULTS: Reconstitution of fibrinogen-deficient or wildtype mice with purified phFibγ'-γ prior to infection provided a significant prolongation in host survival relative to mice reconstituted with purified phFibγ-γ, which was superior to that observed with heterozygous FggΔ5 mice. Improved survival could not be accounted for by quantitative differences in fibrinogen-dependent adhesion or clumping, but phFibγ'-γ-containing mixtures generated notably smaller bacterial aggregates. Importantly, administration of phFibγ'-γ after infection also provided a therapeutic benefit by prolonging host survival relative to administration of phFibγ-γ. CONCLUSION: These findings provide the proof-of-concept that changing the ratio of naturally occurring fibrinogen variants in blood could offer significant therapeutic potential against bacterial infection and potentially other diseases.

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.

Fibrin(ogen) engagement of S. aureus promotes the host antimicrobial response and suppression of microbe dissemination following peritoneal infection.

The blood-clotting protein fibrin(ogen) plays a critical role in host defense against invading pathogens, particularly against peritoneal infection by the Gram-positive microbe Staphylococcus aureus. Here, we tested the hypothesis that direct binding between fibrin(ogen) and S. aureus is a component of the primary host antimicrobial response mechanism and prevention of secondary microbe dissemination from the peritoneal cavity. To establish a model system, we showed that fibrinogen isolated from FibγΔ5 mice, which express a mutant form lacking the final 5 amino acids of the fibrinogen γ chain (termed fibrinogenγΔ5), did not support S. aureus adherence when immobilized and clumping when in suspension. In contrast, purified wildtype fibrinogen supported robust adhesion and clumping that was largely dependent on S. aureus expression of the receptor clumping factor A (ClfA). Following peritoneal infection with S. aureus USA300, FibγΔ5 mice displayed worse survival compared to WT mice coupled to reduced bacterial killing within the peritoneal cavity and increased dissemination of the microbes into circulation and distant organs. The failure of acute bacterial killing, but not enhanced dissemination, was partially recapitulated by mice infected with S. aureus USA300 lacking ClfA. Fibrin polymer formation and coagulation transglutaminase Factor XIII each contributed to killing of the microbes within the peritoneal cavity, but only elimination of polymer formation enhanced systemic dissemination. Host macrophage depletion or selective elimination of the fibrin(ogen) ß2-integrin binding motif both compromised local bacterial killing and enhanced S. aureus systemic dissemination, suggesting fibrin polymer formation in and of itself was not sufficient to retain S. aureus within the peritoneal cavity. Collectively, these findings suggest that following peritoneal infection, the binding of S. aureus to stabilized fibrin matrices promotes a local, macrophage-mediated antimicrobial response essential for prevention of microbe dissemination and downstream host mortality.

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.

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.

Bleeding is increased in amyloid precursor protein knockout mouse.

BACKGROUND: Amyloid precursor protein (APP) is highly expressed in platelets. APP is the precursor to amyloid beta (Aß) peptides that accumulate in cerebral amyloid angiopathy and plaques in Alzheimer disease. APP and its metabolites interact with many components of the coagulation system, and have both anticoagulant and procoagulant properties, but it is unclear if APP contributes to hemostasis in vivo. OBJECTIVES: To determine whether APP contributes to hemostasis in mice, including when inhibitors of coagulation are administered. METHODS: Blood loss in APP knockout (KO) mice was measured in liver laceration and tail transection models of hemorrhage. Blood loss was also measured following tail transection in mice given an inhibitor of coagulation factor Xa (apixaban), platelet inhibitors (aspirin + clopidogrel), tissue-type plasminogen activator (t-PA), or the antifibrinolytic tranexamic acid (TXA). RESULTS AND DISCUSSION: Blood loss from liver lacerations was similar between APP KO mice and wild-type (WT) mice, but APP KO mice bled more from tail transections. When mice were challenged with aspirin + clopidogrel, the difference in bleeding between APP KO and WT mice was abrogated. In contrast, a difference in bleeding between the strains persisted when mice were treated with apixaban, t-PA, or TXA. Blood collected from APP KO mice and analyzed with thromboelastography had longer clotting times, and the clots were less stiff and more susceptible to fibrinolysis compared to blood from WT mice. CONCLUSIONS: The absence of APP measurably increases bleeding in mice, which is consistent with a role for platelet-derived APP and Aß peptides in hemostasis.

Coagulation factor XIII-A and activated FXIII-A decrease in some deep vein thrombosis patients following catheter-directed thrombolysis.

: The objective of the study was to assess the effect of tissue plasminogen activator administered during catheter-directed thrombolysis (CDT) on coagulation factor XIII (FXIII). Thrombolytic therapy carries significant risks, such as life-threatening bleeds. The mechanisms responsible for major bleeds and intracerebral hemorrhages during thrombolysis are not fully understood. Activated FXIII (FXIII-A) lies at the intersection of coagulation and fibrinolysis. Using purified proteins and blood collected from nine deep vein thrombosis patients undergoing CDT, the stability of FXIII-A and FXIII were measured immediately before, immediately after and 1-day post thrombolysis. We found that purified tissue plasminogen activator directly degraded FXIII-A. During CDT, FXIII levels were decreased by more than 40% in five of nine patients and FXIII-A levels were decreased by more than 85% in two patients when it was activated. FXIII-A and FXIII-A can decrease during CDT in some patients, warranting further research into the role of FXIII-A in bleeding from thrombolysis.

Coagulation factor XIIIa cross-links amyloid ß into dimers and oligomers and to blood proteins.

In cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD), the amyloid ß (Aß) peptide deposits along the vascular lumen, leading to degeneration and dysfunction of surrounding tissues. Activated coagulation factor XIIIa (FXIIIa) covalently cross-links proteins in blood and vasculature, such as in blood clots and on the extracellular matrix. Although FXIIIa co-localizes with Aß in CAA, the ability of FXIIIa to cross-link Aß has not been demonstrated. Using Western blotting, kinetic assays, and microfluidic analyses, we show that FXIIIa covalently cross-links Aß40 into dimers and oligomers (kcat/Km = 1.5 × 105 m-1s-1), as well as to fibrin, platelet proteins, and blood clots under flow in vitro Aß40 also increased the stiffness of platelet-rich plasma clots in the presence of FXIIIa. These results suggest that FXIIIa-mediated cross-linking may contribute to the formation of Aß deposits in CAA and Alzheimer's disease.

Coagulation factor XIIIa is inactivated by plasmin.

Coagulation factor XIIIa (FXIIIa) is a transglutaminase that covalently cross-links fibrin and other proteins to fibrin to stabilize blood clots and reduce blood loss. A clear mechanism to describe the physiological inactivation of FXIIIa has been elusive. Here, we show that plasmin can cleave FXIIIa in purified systems and in blood. Whereas zymogen FXIII was not readily cleaved by plasmin, FXIIIa was rapidly cleaved and inactivated by plasmin in solution (catalytic efficiency = 8.3 × 10(3) M(-1)s(-1)). The primary cleavage site identified by mass spectrometry was between K468 and Q469. Both plasma- and platelet-derived FXIIIa were susceptible to plasmin-mediated degradation. Inactivation of FXIIIa occurred during clot lysis and was enhanced both in plasma deficient in fibrinogen and in plasma treated with therapeutic levels of tissue plasminogen activator. These results indicate that FXIIIa activity can be modulated by fibrinolytic enzymes, and suggest that changes in fibrinolytic activity may influence cross-linking of blood proteins.