Reciprocal stabilization of coagulation factor XIII-A and -B subunits is a determinant of plasma FXIII concentration.

ABSTRACT: Transglutaminase factor XIII (FXIII) is essential for hemostasis, wound healing, and pregnancy maintenance. Plasma FXIII is composed of A and B subunit dimers synthesized in cells of hematopoietic origin and hepatocytes, respectively. The subunits associate tightly in circulation as FXIII-A2B2. FXIII-B2 stabilizes the (pro)active site-containing FXIII-A subunits. Interestingly, people with genetic FXIII-A deficiency have decreased FXIII-B2, and therapeutic infusion of recombinant FXIII-A2 (rFXIII-A2) increases FXIII-B2, suggesting FXIII-A regulates FXIII-B secretion, production, and/or clearance. We analyzed humans and mice with genetic FXIII-A deficiency and developed a mouse model of rFXIII-A2 infusion to define mechanisms mediating plasma FXIII-B levels. Like humans with FXIII-A deficiency, mice with genetic FXIII-A deficiency had reduced circulating FXIII-B2, and infusion of FXIII-A2 increased FXIII-B2. FXIII-A-deficient mice had normal hepatic function and did not store FXIII-B in liver, indicating FXIII-A does not mediate FXIII-B secretion. Transcriptional analysis and polysome profiling indicated similar F13b levels and ribosome occupancy in FXIII-A-sufficient and -deficient mice and in FXIII-A-deficient mice infused with rFXIII-A2, indicating FXIII-A does not induce de novo FXIII-B synthesis. Unexpectedly, pharmacokinetic/pharmacodynamic modeling of FXIII-B antigen after rFXIII-A2 infusion in humans and mice suggested FXIII-A2 slows FXIII-B2 loss from plasma. Accordingly, comparison of free FXIII-B2 vs FXIII-A2-complexed FXIII-B2 (FXIII-A2B2) infused into mice revealed faster clearance of free FXIII-B2. These data show FXIII-A2 prevents FXIII-B2 loss from circulation and establish the mechanism underlying FXIII-B2 behavior in FXIII-A deficiency and during rFXIII-A2 therapy. Our findings reveal a unique, reciprocal relationship between independently synthesized subunits that mediate an essential hemostatic protein in circulation. This trial was registered at www.ClinicalTrials.com as #NCT00978380.

Fibrinolysis-Mediated Pathways in Acute Liver Injury.

Acute liver injury (ALI), that is, the development of reduced liver function in patients without preexisting liver disease, can result from a wide range of causes, such as viral or bacterial infection, autoimmune disease, or adverse reaction to prescription and over-the-counter medications. ALI patients present with a complex coagulopathy, characterized by both hypercoagulable and hypocoagulable features. Similarly, ALI patients display a profound dysregulation of the fibrinolytic system with the vast majority of patients presenting with a hypofibrinolytic phenotype. Decades of research in experimental acute liver injury in mice suggest that fibrinolytic proteins, including plasmin(ogen), plasminogen activators, fibrinolysis inhibitors, and fibrin(ogen), can contribute to initial hepatotoxicity and/or stimulate liver repair. This review summarizes major experimental findings regarding the role of fibrinolytic factors in ALI from the last approximately 30 years and identifies unanswered questions, as well as highlighting areas for future research.

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.

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.

Factor XIII cross-links fibrin(ogen) independent of fibrin polymerization in experimental acute liver injury.

Intravascular fibrin clot formation follows a well-ordered series of reactions catalyzed by thrombin cleavage of fibrinogen leading to fibrin polymerization and cross-linking by factor XIIIa (FXIIIa). Extravascular fibrin(ogen) deposits are observed in injured tissues; however, the mechanisms regulating fibrin(ogen) polymerization and cross-linking in this setting are unclear. The objective of this study was to determine the mechanisms of fibrin polymerization and cross-linking in acute liver injury induced by acetaminophen (APAP) overdose. Hepatic fibrin(ogen) deposition and cross-linking were measured following APAP overdose in wild-type mice, mice lacking the catalytic subunit of FXIII (FXIII-/-), and in FibAEK mice, which express mutant fibrinogen insensitive to thrombin-mediated fibrin polymer formation. Hepatic fibrin(ogen) deposition was similar in APAP-challenged wild-type and FXIII-/- mice, yet cross-linking of hepatic fibrin(ogen) was dramatically reduced (>90%) by FXIII deficiency. Surprisingly, hepatic fibrin(ogen) deposition and cross-linking were only modestly reduced in APAP-challenged FibAEK mice, suggesting that in the APAP-injured liver fibrin polymerization is not strictly required for the extravascular deposition of cross-linked fibrin(ogen). We hypothesized that the oxidative environment in the injured liver, containing high levels of reactive mediators (eg, peroxynitrite), modifies fibrin(ogen) such that fibrin polymerization is impaired without impacting FXIII-mediated cross-linking. Notably, fibrin(ogen) modified with 3-nitrotyrosine adducts was identified in the APAP-injured liver. In biochemical assays, peroxynitrite inhibited thrombin-mediated fibrin polymerization in a concentration-dependent manner without affecting fibrin(ogen) cross-linking over time. These studies depict a unique pathology wherein thrombin-catalyzed fibrin polymerization is circumvented to allow tissue deposition and FXIII-dependent fibrin(ogen) cross-linking.

Plasmin-mediated cleavage of high-molecular-weight kininogen contributes to acetaminophen-induced acute liver failure.

Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor-dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK-/-) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK-/- mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.

Good cells go bad: immune dysregulation in the transition from acute liver injury to liver failure after acetaminophen overdose.

The role of inflammatory cells and other components of the immune system in acetaminophen (APAP)-induced liver injury and repair has been extensively investigated. Although this has resulted in a wealth of information regarding the function and regulation of immune cells in the liver after injury, apparent contradictions have fueled controversy around the central question of whether the immune system is beneficial or detrimental after APAP overdose. Ultimately, this may not be a simple assignment of "good" or "bad." Clinical studies have clearly demonstrated an association between immune dysregulation and a poor outcome in patients with severe liver damage/liver failure induced by APAP overdose. To date, studies in mice have not uniformly replicated this connection. The apparent disconnect between clinical and experimental studies has perhaps stymied progress and further complicated investigation of the immune system in APAP-induced liver injury. Mouse models are often dismissed as not recapitulating the clinical scenario. Moreover, clinical investigation is most often focused on the most severe APAP overdose patients, those with liver failure. Notably, recent studies have made it apparent that the functional role of the immune system in the pathogenesis of APAP-induced liver injury is highly context dependent and greatly influenced by the experimental conditions. In this review, we highlight some of these recent findings, and suggest strategies seeking to resolve and build on existing disconnects in the literature. Significance Statement Acetaminophen overdose is the most frequent cause of acute liver failure in the United States. Studies indicate that dysregulated innate immunity contributes to the transition from acute liver injury to acute liver failure. In this review, we discuss the evidence for this and the potential underlying causes.

Baat Gene Knockout Alters Post-Natal Development, the Gut Microbiome, and Reveals Unusual Bile Acids in Mice.

Bile acids (BAs) are steroid detergents in bile that contribute to fat absorption, cell signaling, and microbiome interactions. The final step in their synthesis is amino acid conjugation with either glycine or taurine in the liver by the enzyme bile acid-CoA:amino acid N-acyltransferase (BAAT). Here, we describe the microbial, chemical, and physiological consequences of Baat gene knockout. Baat-/- mice were underweight after weaning but quickly exhibited catch-up growth. At three weeks of age, KO animals had increased phospholipid excretion and decreased subcutaneous fat pad mass, liver mass, glycogen staining in hepatocytes, and hepatic vitamin A stores, but these were less marked in adulthood. Additionally, KO mice had an altered microbiome in early life. Their BA pool was highly enriched in cholic acid but not completely devoid of conjugated BAs. KO animals had 27-fold lower taurine-conjugated BAs than wild type in their liver but similar concentrations of glycine-conjugated BAs and higher microbially conjugated BAs. Furthermore, the BA pool in Baat-/- was enriched in a variety of unusual BAs that were putatively sourced from cysteamine conjugation with subsequent oxidation and methylation of the sulfur group mimicking taurine. Antibiotic treatment of KO mice indicated the microbiome was not the likely source of the unusual conjugations, instead, the unique BAs in KO animals were likely derived from the peroxisomal acyltransferases Acnat1 and Acnat2, which are duplications of Baat in the mouse genome that are inactivated in humans. This study demonstrates that BA conjugation is important for early life development of mice.

A high-fat diet delays plasmin generation in a thrombomodulin-dependent manner in mice.

Obesity is a prevalent prothrombotic risk factor marked by enhanced fibrin formation and suppressed fibrinolysis. Fibrin both promotes thrombotic events and drives obesity pathophysiology, but a lack of essential analytical tools has left fibrinolytic mechanisms affected by obesity poorly defined. Using a plasmin-specific fluorogenic substrate, we developed a plasmin generation (PG) assay for mouse plasma that is sensitive to tissue plasminogen activator, α2-antiplasmin, active plasminogen activator inhibitor (PAI-1), and fibrin formation, but not fibrin crosslinking. Compared with plasmas from mice fed a control diet, plasmas from mice fed a high-fat diet (HFD) showed delayed PG and reduced PG velocity. Concurrent to impaired PG, HFD also enhanced thrombin generation (TG). The collective impact of abnormal TG and PG in HFD-fed mice produced normal fibrin formation kinetics but delayed fibrinolysis. Functional and proteomic analyses determined that delayed PG in HFD-fed mice was not due to altered levels of plasminogen, α2-antiplasmin, or fibrinogen. Changes in PG were also not explained by elevated PAI-1 because active PAI-1 concentrations required to inhibit the PG assay were 100-fold higher than circulating concentrations in mice. HFD-fed mice had increased circulating thrombomodulin, and inhibiting thrombomodulin or thrombin-activatable fibrinolysis inhibitor (TAFI) normalized PG, revealing a thrombomodulin- and TAFI-dependent antifibrinolytic mechanism. Integrating kinetic parameters to calculate the metric of TG/PG ratio revealed a quantifiable net shift toward a prothrombotic phenotype in HFD-fed mice. Integrating TG and PG measurements may define a prothrombotic risk factor in diet-induced 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.

The multifaceted role of fibrinogen in tissue injury and inflammation.

The canonical role of the hemostatic and fibrinolytic systems is to maintain vascular integrity. Perturbations in either system can prompt primary pathological end points of hemorrhage or thrombosis with vessel occlusion. However, fibrin(ogen) and proteases controlling its deposition and clearance, including (pro)thrombin and plasmin(ogen), have powerful roles in driving acute and reparative inflammatory pathways that affect the spectrum of tissue injury, remodeling, and repair. Indeed, fibrin(ogen) deposits are a near-universal feature of tissue injury, regardless of the nature of the inciting event, including injuries driven by mechanical insult, infection, or immunological derangements. Fibrin can modify multiple aspects of inflammatory cell function by engaging leukocytes through a variety of cellular receptors and mechanisms. Studies on the role of coagulation system activation and fibrin(ogen) deposition in models of inflammatory disease and tissue injury have revealed points of commonality, as well as context-dependent contributions of coagulation and fibrinolytic factors. However, there remains a critical need to define the precise temporal and spatial mechanisms by which fibrinogen-directed inflammatory events may dictate the severity of tissue injury and coordinate the remodeling and repair events essential to restore normal organ function. Current research trends suggest that future studies will give way to the identification of novel hemostatic factor-targeted therapies for a range of tissue injuries and disease.

Intrahepatic fibrin(ogen) deposition drives liver regeneration after partial hepatectomy in mice and humans.

Platelets play a pivotal role in stimulating liver regeneration after partial hepatectomy in rodents and humans. Liver regeneration in rodents is delayed when platelets are inhibited. However, the exact mechanisms whereby platelets accumulate and promote liver regeneration remain uncertain. Thrombin-dependent intrahepatic fibrin(ogen) deposition was recently reported after partial hepatectomy (PHx) in mice, but the role of fibrin(ogen) deposits in liver regeneration has not been investigated. We tested the hypothesis that fibrin(ogen) contributes to liver regeneration by promoting intrahepatic platelet accumulation and identified the trigger of rapid intrahepatic coagulation after PHx. PHx in wild-type mice triggered rapid intrahepatic coagulation, evidenced by intrahepatic fibrin(ogen) deposition. Intrahepatic fibrin(ogen) deposition was abolished in mice with liver-specific tissue factor deficiency, pinpointing the trigger of coagulation after PHx. Direct thrombin activation of platelets through protease-activated receptor-4 did not contribute to hepatocyte proliferation after PHx, indicating that thrombin contributes to liver regeneration primarily by driving intrahepatic fibrin(ogen) deposition. Fibrinogen depletion with ancrod reduced both intrahepatic platelet accumulation and hepatocyte proliferation after PHx, indicating that fibrin(ogen) contributes to liver regeneration after PHx by promoting intrahepatic platelet accumulation. Consistent with the protective function of fibrin(ogen) in mice, low postoperative plasma fibrinogen levels were associated with liver dysfunction and mortality in patients undergoing liver resection. Moreover, increased intrahepatic fibrin(ogen) deposition was evident in livers of patients after liver resection but was remarkably absent in patients displaying hepatic dysfunction postresection. The results suggest a novel mechanism whereby coagulation-dependent intrahepatic fibrin(ogen) deposition drives platelet accumulation and liver regeneration after PHx.

Von Willebrand factor delays liver repair after acetaminophen-induced acute liver injury in mice.

BACKGROUND & AIM: Acetaminophen (APAP)-induced acute liver failure is associated with substantial alterations in the hemostatic system. In mice, platelets accumulate in the liver after APAP overdose and appear to promote liver injury. Interestingly, patients with acute liver injury have highly elevated levels of the platelet-adhesive protein von Willebrand factor (VWF), but a mechanistic connection between VWF and progression of liver injury has not been established. We tested the hypothesis that VWF contributes directly to experimental APAP-induced acute liver injury. METHODS: Wild-type mice and VWF-deficient (Vwf-/-) mice were given a hepatotoxic dose of APAP (300 mg/kg, i.p.) or vehicle (saline). VWF plasma levels were measured by ELISA, and liver necrosis or hepatocyte proliferation was measured by immunohistochemistry. Platelet and VWF deposition were measured by immunofluorescence. RESULTS: In wild-type mice, VWF plasma levels, high molecular weight (HMW) VWF multimers, and VWF activity decreased 24 h after APAP challenge. These changes coupled to robust hepatic VWF and platelet deposition, although VWF deficiency had minimal effect on peak hepatic platelet accumulation or liver injury. VWF plasma levels were elevated 48 h after APAP challenge, but with relative reductions in HMW multimers and VWF activity. Whereas hepatic platelet aggregates persisted in livers of APAP-challenged wild-type mice, platelets were nearly absent in Vwf-/- mice 48 h after APAP challenge. The absence of platelet aggregates was linked to dramatically accelerated repair of the injured liver. Complementing observations in Vwf-/- mice, blocking VWF or the platelet integrin αIIbß3 during development of injury significantly reduced hepatic platelet aggregation and accelerated liver repair in APAP-challenged wild-type mice. CONCLUSION: These studies are the first to suggest a mechanistic link between VWF, hepatic platelet accumulation, and liver repair. Targeting VWF might provide a novel therapeutic approach to improve repair of the APAP-injured liver. LAY SUMMARY: Patients with acute liver injury due to acetaminophen overdose have highly elevated levels of the platelet-adhesive protein von Willebrand factor. It is not known whether von Willebrand factor plays a direct role in the progression of acute liver injury. We discovered that von Willebrand factor delays repair of the acetaminophen-injured liver in mice and that targeting von Willebrand factor, even in mice with established liver injury, accelerates liver repair.

Dichotomous Role of Plasmin in Regulation of Macrophage Function after Acetaminophen Overdose.

Kupffer cells and monocyte-derived macrophages are critical for liver repair after acetaminophen (APAP) overdose. These cells produce promitogenic cytokines and growth factors, and they phagocytose dead cell debris, a process that is critical for resolution of inflammation. The factors that regulate these dynamic functions of macrophages after APAP overdose, however, are not fully understood. We tested the hypothesis that the fibrinolytic enzyme, plasmin, is a key regulator of macrophage function after APAP-induced liver injury. In these studies, inhibition of plasmin in mice with tranexamic acid delayed up-regulation of proinflammatory cytokines after APAP overdose. In culture, plasmin directly, and in synergy with high-mobility group B1, stimulated Kupffer cells and bone marrow-derived macrophages to produce cytokines by a mechanism that required NF-κB. Inhibition of plasmin in vivo also prevented trafficking of monocyte-derived macrophages into necrotic lesions after APAP overdose. This prevented phagocytic removal of dead cells, prevented maturation of monocyte-derived macrophages into F4/80-expressing macrophages, and prevented termination of proinflammatory cytokine production. Our studies reveal further that phagocytosis is an important stimulus for cessation of proinflammatory cytokine production as treatment of proinflammatory, monocyte-derived macrophages, isolated from APAP-treated mice, with necrotic hepatocytes decreased expression of proinflammatory cytokines. Collectively, these studies demonstrate that plasmin is an important regulator of macrophage function after APAP overdose.

Hemostasis and Liver Regeneration.

The liver is unique in its remarkable regenerative capacity, which enables the use of liver resection as a treatment for specific liver diseases, including removal of neoplastic liver disease. After resection, the remaining liver tissue (i.e, liver remnant) regenerates to maintain normal hepatic function. In experimental settings as well as patients, removal of up to two-thirds of the liver mass stimulates a rapid and highly coordinated process resulting in the regeneration of the remaining liver. Mechanisms controlling the initiation and termination of regeneration continue to be discovered, and many of the fundamental signaling pathways controlling the proliferation of liver parenchymal cells (i.e., hepatocytes) have been uncovered. Interestingly, while hemostatic complications (i.e., bleeding and thrombosis) are primarily thought of as a complication of surgery itself, strong evidence suggests that components of the hemostatic system are, in fact, powerful drivers of liver regeneration. This review focuses on the clinical and translational evidence supporting a link between the hemostatic system and liver regeneration, and the mechanisms whereby the hemostatic system directs liver regeneration discovered using experimental settings.

Direct Amplification of Tissue Factor:Factor VIIa Procoagulant Activity by Bile Acids Drives Intrahepatic Coagulation.

OBJECTIVE: Regulation of TF (tissue factor):FVIIa (coagulation factor VIIa) complex procoagulant activity is especially critical in tissues where plasma can contact TF-expressing cells. One example is the liver, where hepatocytes are routinely exposed to plasma because of the fenestrated sinusoidal endothelium. Although liver-associated TF contributes to coagulation, the mechanisms controlling the TF:FVIIa complex activity in this tissue are not known. Approach and Results: Common bile duct ligation in mice triggered rapid hepatocyte TF-dependent intrahepatic coagulation coincident with increased plasma bile acids, which occurred at a time before observable liver damage. Similarly, plasma TAT (thrombin-antithrombin) levels increased in cholestatic patients without concurrent hepatocellular injury. Pathologically relevant concentrations of the bile acid glycochenodeoxycholic acid rapidly increased hepatocyte TF-dependent procoagulant activity in vitro, independent of de novo TF synthesis and necrotic or apoptotic cell death. Glycochenodeoxycholic acid increased hepatocyte TF activity even in the presence of the phosphatidylserine-blocking protein lactadherin. Interestingly, glycochenodeoxycholic acid and taurochenodeoxycholic acid increased the procoagulant activity of the TF:FVIIa complex relipidated in unilamellar phosphatidylcholine vesicles, which was linked to an apparent decrease in the Km for FX (coagulation factor X). Notably, the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a bile acid structural analog, did not increase relipidated TF:FVIIa activity. Bile acids directly enhanced factor X activation by recombinant soluble TF:FVIIa complex but had no effect on FVIIa alone. CONCLUSIONS: The results indicate that bile acids directly accelerate TF:FVIIa-driven coagulation reactions, suggesting a novel mechanism whereby elevation in a physiological mediator can directly increase TF:FVIIa procoagulant activity.

Plasminogen Activator Inhibitor-1 Reduces Tissue-Type Plasminogen Activator-Dependent Fibrinolysis and Intrahepatic Hemorrhage in Experimental Acetaminophen Overdose.

Acetaminophen (APAP)-induced liver injury in mice is associated with activation of the coagulation cascade and deposition of fibrin in liver. Plasminogen activator inhibitor-1 (PAI-1) is an important physiological inhibitor of tissue-type plasminogen activator (tPA) and plays a critical role in fibrinolysis. PAI-1 expression is increased in both experimental APAP-induced liver injury and patients with acute liver failure. Prior studies have shown that PAI-1 prevents intrahepatic hemorrhage and mortality after APAP challenge, but the downstream mechanisms are not clear. We tested the hypothesis that PAI-1 limits liver-related morbidity after APAP challenge by reducing tPA-dependent fibrinolysis. Compared with APAP-challenged (300 mg/kg) wild-type mice, hepatic deposition of cross-linked fibrin was reduced, with intrahepatic congestion and hemorrhage increased in PAI-1-deficient mice 24 hours after APAP overdose. Administration of recombinant wild-type human PAI-1 reduced intrahepatic hemorrhage 24 hours after APAP challenge in PAI-1-/- mice, whereas a mutant PAI-1 lacking antiprotease function had no effect. Of interest, tPA deficiency alone did not affect APAP-induced liver damage. In contrast, fibrinolysis, intrahepatic congestion and hemorrhage, and mortality driven by PAI-1 deficiency were reduced in APAP-treated tPA-/-/PAI-1-/- double-knockout mice. The results identify PAI-1 as a critical regulator of intrahepatic fibrinolysis in experimental liver injury. Moreover, the results suggest that the balance between PAI-1 and tPA activity is an important determinant of liver pathology after APAP overdose.

Role of the blood coagulation cascade in hepatic fibrosis.

Liver is the primary source of numerous proteins that are critical for normal function of the blood coagulation cascade. Because of this, diseases of the liver, particularly when affiliated with severe complications like cirrhosis, are associated with abnormalities of blood clotting. Although conventional interpretation has inferred cirrhosis as a disorder of uniform bleeding risk, it is now increasingly appreciated as a disease wherein the coagulation cascade is precariously rebalanced. Moreover, prothrombotic risk factors are also associated with a more rapid progression of fibrosis in humans, suggesting that coagulation proteases participate in disease pathogenesis. Indeed, strong evidence drawn from experimental animal studies indicates that components of the coagulation cascade, particularly coagulation factor Xa and thrombin, drive profibrogenic events, leading to hepatic fibrosis. Here, we concisely review the evidence supporting a pathologic role for coagulation in the development of liver fibrosis and the potential mechanisms involved. Further, we highlight how studies in experimental animals may shed light on emerging clinical evidence, suggesting that beneficial effects of anticoagulation could extend beyond preventing thrombotic complications to include reducing pathologies like fibrosis.

Platelets as Modulators of Liver Diseases.

Platelets are key players in thrombosis and hemostasis. Alterations in platelet count and function are common in liver disease, and may contribute to bleeding or thrombotic complications in liver diseases and during liver surgery. In addition to their hemostatic function, platelets may modulate liver diseases by mechanisms that are incompletely understood. Here, we present clinical evidence for a role of platelets in the progression of chronic and acute liver diseases, including cirrhosis, acute liver failure, and hepatocellular carcinoma. We also present clinical evidence that platelets promote liver regeneration following partial liver resection. Subsequently, we summarize studies in experimental animal models that support these clinical observations, and also highlight studies that are in contrast with clinical observations. The combined results of clinical and experimental studies suggest that platelets may be a therapeutic target in the treatment of liver injury and repair, but the gaps in our understanding of mechanisms involved in platelet-mediated modulation of liver diseases call for caution in clinical application of these findings.