Fibrin clot properties and thrombus composition in cirrhosis

Patients with cirrhosis frequently acquire profound hemostatic alterations, which may affect thrombus quality and composition—factors that determine the susceptibility to embolization and fibrinolysis. In this narrative review, we describe in vitro studies on fibrin clot formation and quantitative and qualitative changes in fibrinogen in patients with cirrhosis, and describe recent findings on the composition of portal vein thrombi in patients with cirrhosis. Patients with mild cirrhosis have increased thrombin generation capacity and plasma fibrinogen levels, which may be balanced by delayed fibrin polymerization and decreased factor XIII levels. With progressing illness, plasma fibrinogen levels decrease, but thrombin generation capacity remains elevated. Fibrinogen is susceptible to posttranslational protein modifications and is, for example, hypersialylated and carbonylated in patients with cirrhosis. Despite changes in thrombin generation, factor XIII levels and the fibrinogen molecule, fibrin fiber thickness, and density are normal in patients with cirrhosis. Paradoxically, fibrin clot permeability in patients with cirrhosis is decreased, possibly because of posttranslational protein modifications. Most patients have normal fibrinolytic potential. We have recently demonstrated that portal vein thrombosis is likely a misnomer as the material that may obstruct the cirrhotic portal vein frequently consists of a thickened portal vein wall, rather than a true thrombus. Patients with cirrhosis often have thrombocytopenia and anemia, which may also affect clot stability and composition, but the role of cellular components in clot quality in cirrhosis has not been extensively studied. Finally, we summarize abstracts on fibrin formation and clot quality that were presented at the ISTH 2022 meeting in London.


| I N T R O D U C T I O N
Patients with liver diseases were historically considered to be at risk of bleeding complications, mainly because of derangements of routine laboratory measures such as prolongation of the prothrombin time, the derived international normalized ratio, and thrombocytopenia.
These tests, however, are only sensitive to deficiencies or defects in procoagulant factors, and neglect the simultaneous decline in anticoagulant factors in patients with liver diseases. Indeed, as a result of a reduced capacity of the liver to produce both pro-and anticoagulant factors, patients with liver diseases are in a rebalanced hemostatic state. [1] Rebalanced hemostasis in liver disease has, for example, been demonstrated by a normal to increased thrombin generation capacity using thrombomodulin-modified calibrated automated thrombinography, including the activation of anticoagulant pathways by the addition of thrombomodulin. [2][3][4] The net effects of the complex hemostatic alterations in liver diseases have been extensively described. [5] However, there has not been much attention to the quality and composition of thrombi in these patients, but this aspect is of great importance because quality and composition of a thrombus affect mechanical properties, [6] which determine the susceptibility to embolization and fibrinolysis. [7] For example, abnormally dense fibrin structures were found in patients with deep vein thrombosis, coronary artery disease, and stroke, and these dense fibrin clots are more resistant to degradation by plasmin and alter mechanical properties by increasing clot stiffness. [8] In this narrative review, we will describe in vitro studies on fibrin clot formation and quantitative and qualitative changes in fibrinogen in patients with liver diseases. In addition, we will discuss changes in coagulation and fibrinolysis and the composition and structure of portal vein thrombi in patients with cirrhosis. Finally, we will shed light on future directions in this field and summarize new data from abstracts on the topic of fibrin clot quality and composition that were presented at the ISTH 2022 annual meeting in London.

| P L A S M A F I B R I N O G E N C O N C E N T R A T I O N I N P A T I E N T S W I T H C I R R H O S I S
Fibrinogen is a soluble 340-kD glycoprotein that is primarily synthesized by hepatocytes and is present in the blood of healthy individuals at concentrations between 1.5 and 4 g/L. Fibrinogen levels are normal to increased in patients with stable liver disease. [9] Its levels decrease with increasing severity of disease and may drop below 1.5 g/L in patients with acutely decompensated cirrhosis (AD) and acute-on-chronic liver failure (ACLF). [10] ACLF is a condition that is characterized by critical illness, with disease complications such as ascites, gastrointestinal bleeding, and hepatic encephalopathy, and is associated with multiorgan failure and increased mortality. [11] In the general population, decreased levels of fibrinogen do not necessarily induce a bleeding risk, but are in fact associated with both bleeding and thrombotic complications. [12] In hospitalized patients with liver disease, low fibrinogen levels were a risk factor for bleeding. [13] However, whether there is a causal relationship between hypofibrinogenemia and bleeding in liver disease is unclear. For example, in a study consisting of critically ill patients with cirrhosis, it was showed that administration of cryoprecipitate to correct low (<1.5 g/L) fibrinogen levels did not affect survival or bleeding complications, which suggested that a low fibrinogen level was an additional marker of severity of illness but is not itself a direct factor of bleeding complications in these patients. [14] Indeed, despite low levels of fibrinogen in patients with AD or ACLF, in vitro-formed clots from the plasma of these patients have normal to thrombogenic properties. For example, normal clot lysis times and decreased clot permeability (a function of clot pore size) were measured in in vitro-formed clots from acutely ill cirrhosis patients, suggesting that factors other than plasma fibrinogen concentration also determine these clot properties. [15] Only when fibrinogen levels were very low (<0.5 U/dL, for example, during liver transplant surgery), and clot stability parameters in ex vivo experiments were severely impaired. [16]

| F I B R I N F O R M A T I O N I N P AT I E N T S W I T H C I R R H O S I S
Fibrinogen is converted by thrombin to fibrin, which forms the scaffold of a thrombus. Fibrinogen consists of 2 sets of 3 different polypeptide chains: 2-Aα, 2-Bβ, and 2-γ chains, which are held together by disulfide bridges. [17] It plays a central role in clot formation and stabilization, and is converted to crosslinked fibrin in several steps [18]: 1) proteolytic cleavage of thrombin causes the release of fibrinopeptides and formation of fibrin monomers; 2) linear association of fibrin monomers results in double-stranded protofibrils; 3) association of protofibrils results in formation of fibrin fibers; and 4) factor (F)XIII facilitates covalent crosslinking of polymerized fibrin.
The process of fibrin clot formation and stabilization and potential changes in the process in patients with liver diseases are outlined in Figure 1 and described hereafter. In the first step, thrombin cleaves fibrinopeptides of the Aα and Bβ chains to produce fibrin monomers.
Thrombin levels affect the fibrin clot, with higher levels of thrombin producing a dense network of relatively thin fibrin strands, resulting in less permeable clots and resistance to fibrinolysis. [19,20] Increased thrombin levels have been shown to affect viscoelastic properties of a clot as assessed with thromboelastography (TEG), for example, increased maximum amplitude (which represents the ultimate strength of a fibrin clot) and increased α-angle (which represents the speed of fibrin formation). [21,22] Patients with cirrhosis have normal to increased thrombin generation, [23] which may suggest that levels of thrombin do not largely affect the fibrin clot structure in patients with liver disease. Most stable cirrhosis patients exhibit normal TEG parameters, and hypo-or hypercoagulable TEG profiles were correlated with platelet counts and plasma fibrinogen levels. [24] The second step of fibrin clot formation is the linear association of fibrin monomers, resulting in double-stranded protofibrils. Fibrin polymerization assays have showed that this step is markedly delayed in patients with liver diseases, with 76% of patients with cirrhosis and 2 of 10 -DRIEVER AND LISMAN 86% of patients with acute liver failure showing abnormal fibrin polymerization rates, [25,26] This delay in polymerization has been explained by increased sialylation of the fibrinogen molecule in patients with liver disease. [27] Sialylation is a posttranslational protein modification, and is a form of glycosylation in which sialic acid is bound at the end of a sugar chain of the protein. It has been suggested that sialic acid inhibits polymerization of fibrin by electrostatic repulsion between fibrin monomers, which impairs the fibrin monomers from interacting with each other. [28] Sialic acid residues are neutralized by calcium, but with increased levels of sialic acid and normal calcium levels, the lowaffinity interaction between sialic acid and calcium is inadequate to neutralize the excessive charge repulsion between hypersialylated fibrin molecules, resulting in delayed fibrin polymerization. [28] In the third step, double-stranded protofibrils are connected to form fibrin fibers. This association can be affected by thrombin concentration and the presence of an alternatively spliced form of fibrinogen, fibrinogen γ ′ . This form of fibrinogen causes a partially impaired protofibril formation, again likely because of electrostatic repulsion. [29] Higher levels of fibrinogen γ ′ have been associated with decreased protofibril packing and less stiff fibrin clots. [30] A study conducted by our group showed normal levels of fibrinogen γ ′ in patients with cirrhosis. In addition, similar fibrin fiber density and fiber diameter between cirrhosis patients and healthy individuals were found, suggesting that the intrafibrillar structure is not altered in cirrhosis. [31] Finally, FXIII, a transglutaminase, is activated by thrombin and becomes FXIIIa, which stabilizes the fibrin clot by forming covalent bonds between fibrin fibers. Thereby, it defines structure, stability, and effector functions of fibrin. [32] Patients with cirrhosis have decreased levels of FXIII, [15] which would theoretically lead to less stable fibrin networks.
In vitro experiments of fibrin clot structure with plasma from healthy controls and patients with AD and ACLF showed very minor effects on clot parameters after the addition of exogenous FXIII concentrate, despite markedly decreased FXIII levels and reduced clot permeability in patients compared to controls. [15] Nevertheless, it was shown that FXIII does contribute to clot stability in these assays because complete inhibition of FXIIIa activity by T101 (a transglutaminase inhibitor) shortened clot lysis time and lowered permeability in pooled normal plasma. In addition, results of a clot retraction assay (in which isolated platelets and red blood cells [RBCs] from a healthy donor are mixed with patient plasma, coagulation is subsequently initiated with tissue factor and calcium, and the red cell extrusion is measured after 2 hours of clot formation [33]) showed a slightly increased clot weight and a reduced percentage of extruded RBCs in patients compared to controls, despite lower FXIII and fibrinogen levels. Again, full inhibition of FXIII in normal plasma decreased clot weight and increased RBC extrusion, showing that the assay is sensitive for FXIII. In this assay, clot weight is determined by the amount of RBCs that retain within the clot, which also determines the clot size. [34] It could be that only very small amounts of FXIII are necessary for clot protective effects in the assays performed in this study. [15]

| P O S T T R A N S L A T I O N A L M O D I F I C A T I O N S O F F I B R I N O G E N I N C I R R H O S I S
Acquired dysfibrinogenemia, a term used for altered functionality of the fibrinogen molecule, is common in patients with liver diseases. [35] F I G U R E 1 Fibrin formation and fibrinogen properties in patients with cirrhosis. Created with BioRender. FXIII, factor XIII DRIEVER AND LISMAN -3 of 10 Dysfibrinogenemia can be a result of posttranslational protein modifications of fibrinogen, of which several are known to affect the function of fibrinogen and therefore affect clot formation and characteristics. [36] Fibrinogen in liver disease is altered compared to healthy individuals with increased sialylation (a form of glycosylation) and oxidation. [37][38][39] Hypersialylation leads to delayed fibrin polymerization rates because of electrostatic repulsion between fibrin monomers, as described in the previous section. [28] A recent study performed by our group showed that, despite delayed fibrin polymerization rates in patients with cirrhosis, fibrin clot permeability was decreased, suggesting that although clot formation is delayed, once these clots form they are more thrombogenic compared to healthy controls. [31] Conversely, visual analysis of fibrin clots of glycosylated fibrinogen from healthy donors showed thinner, more-branched fibrin bundles with a more porous network and decreased turbidity, suggesting that glycosylation results in a visually less thrombogenic clot structure. [40] Interestingly, when fibrin clots from cirrhotic patients were visualized with electron microscopy, fiber thickness and density were similar to those of controls. [31] These findings suggest that sialic acid content of fibrin(ogen) in cirrhosis may affect polymerization rates and results in decreased permeability, but the structure of a matured clot may also be affected by mechanisms other than sialylation of fibrinogen alone. For example, carbonylation and nitration of fibrinogen (which may be increased in patients with cirrhosis because of oxidative stress and subsequent production of reactive oxygen and nitrogen species) have distinct effects on clot structure. Nitration of fibrinogen has, for example, been shown in hepatic fibrin deposits in acetaminopheninduced acute liver failure in mice, and was associated with delayed fibrin polymerization rates and reduced clot turbidity. [41] Nitration of normal donor-fibrinogen has been demonstrated to result in large bundles of thin fibrin fibers with large pores between the fibers in 1 study, [42] but showed more thrombogenic clot properties in another study. [43] Carbonylation (a form of oxidation) of fibrinogen has been proposed to increase the thrombogenicity of fibrin clots, for example, in the context of patients with acute myocardial infarction. [44] Nevertheless, conflicting reports on the effects of fibrinogen carbonylation on fibrin structure and function have appeared in the literature. For example, 1 study demonstrated decreased fibrin polymerization with thinner fibers and resistance to lysis in hypercarbonylated fibrinogen from patients with myocardial infarction, [44] whereas another study showed enhanced polymerization in a similar setting. [45] Interestingly, fibrinogen carbonyl content is increased in patients with cirrhosis, and was inversely related to clot permeability.

| F I B R I N O L Y S I S A N D P E R M E A B I L I T Y O F F I B R I N C L O T S I N C I R R H O S I S
We have studied the stability of clots generated from plasma in patients with chronic liver diseases using 2 distinct assays: a plasma-based clot lysis test and a clot permeability assay. Both tests are sensitive for levels and function of key proteins involved in clot formation and breakdown. [51,52] Historically, patients with cirrhosis were classified as hyperfibrinolytic. [53] A hyperfibrinolytic state in patients with cirrhosis has been linked to decreased levels of antiplasmin and thrombin-activatable fibrinolysis inhibitor or increased levels of tPA, and was described to potentially contribute to bleeding complications in these patients. [54,55] More recent studies used global assays of fibrinolysis, and found a normal fibrinolytic phenotype in patients with compensated or stably decompensated cirrhosis, which was explained by a simultaneous decline in pro-and antifibrinolytic factors. [9,56] Notably, in these studies, individual patients had hypo-or hyperfibrinolytic profiles. [9,56] Others, however, exhibited hyperfibrinolysis on a group level using a similar methodology, who were apparently similar patients. The discrepancies may be explained by differences in methodology and selected patients. [54,[56][57][58][59] For example, in patients with mild cirrhosis caused by nonalcoholic fatty liver disease or cholestatic liver disease, we demonstrated hypofibrinolytic profiles, which were not present in patients with cirrhosis related to alcohol or viral hepatitis. [9,60] Finally, although patients with stable cirrhosis may have hyperfibrinolytic features based on laboratory measures, hyperfibrinolysis-related bleeding complications in these patients are exceedingly rare. [58] Whereas patients with relatively stable liver disease mostly have normal fibrinolytic phenotypes, a recent study by our group has 4 of 10 -DRIEVER AND LISMAN showed that patients with AD and ACLF, with higher disease severity and additional disease complications such as presence of ascites or development of hepatic encephalopathy, have very variable fibrinolytic phenotypes. [61] Patients with AD were primarily hyperfibrinolytic and patients with ACLF were primarily hypofibrinolytic.
Patients with ACLF and hypofibrinolysis often had sepsis. Indeed, sepsis in patients without an underlying liver disease is often accompanied by a hypofibrinolytic state, which can be explained by high levels of plasminogen activator inhibitor-1 in patients with sepsis. [61][62][63] Other comorbidities such as diabetes mellitus: type II or use of drugs, such as anticoagulants or statins, also affect fibrinolysis. [64] How these factors affect fibrinolysis in patients with liver disease has not yet been elucidated and should be subject to further research.
Clot permeability, another key measure of clot structure and function, is decreased in patients with cirrhosis when measured using an experimental setup in which permeation is tested by measuring fluid permeation through the clot by the force of gravity. These thrombogenic clot properties were observed despite the delayed fibrinogen to fibrin conversion, and were present even in patients with decreased plasma fibrinogen levels. Notably, decreased permeability was observed despite unaltered fibrin fiber thickness or pore size within the clot. [31] To better understand these paradoxes, we recently reanalyzed fibrin permeation and compared fibrin clot permeability assessed by the force of gravity with permeability assessed by compressional force, where a clot is formed between 2 parallel plates of a rheometer and fluid is pressed out of the fibrin network by lowering the upper plate. [65] We found that under the force of gravity, permeability decreases with increasing severity of the disease. In contrast, when permeability was assessed using rheometry, no differences in permeability were observed between patients and controls (unpublished data). Ongoing studies are focused on addressing the reason for this discrepancy. We hypothesize that the studies under the force of gravity may show decreased permeability not because the clot is truly more thrombogenic but because the increased negative charge in the clot retains water. In studies under compressional force, the electrostatic repulsion may not be strong enough to retain water in the fibrin network.

| C L O T C O M P O S I T I O N I N C I R R H O S I S
The major determinant of clot stability and quality is fibrin, and the effects of alterations in fibrinogen and fibrin have been extensively studied in patients with liver diseases as discussed in the previous sections. Other components of the thrombus, such as platelets and RBCs, also contribute to clot stability and quality, but these have not been studied in patients with liver diseases yet. Patients with liver diseases are often diagnosed with thrombocytopenia, anemia, and changes in white blood cell counts and function, [66] and these alterations may affect thrombus composition and mechanical characteristics. [67,68] The composition of venous and arterial thrombi has been extensively studied in the general population, [69] but data from patients with liver diseases are not available yet.
Venous thrombi consist mainly of RBCs and fibrin. [69] Upon clot contraction of venous thrombi, a phenomenon driven by activated platelets, RBCs undergo deformation and become polyhedral structures. [70] Contraction of venous thrombi causes volume shrinkage of the thrombus, and determines the degree of vessel obstruction and the likelihood of thrombus mechanical rupture, which may lead to thrombotic embolization. [71,72] The majority of RBCs within a thrombus are of polyhedral shape and are called polyhedrocytes. [70] Recently, we studied the composition of portal vein thrombi in patients with cirrhosis who underwent liver transplantation, [73] and observed RBCs to be of biconcave shape instead of their polyhedral shape in those thrombi (Figure 2). [71,74] This interesting and unexpected finding suggests that the portal vein thrombus in cirrhosis does not have the same features as other venous thrombi. Future studies should investigate whether platelet activation differs in these thrombi or whether RBCs in cirrhotic portal vein thrombi are unable to deform into a polyhedral shape, for example, because of changes in the lipid composition of the red cell membrane. [75,76] Moreover, in this study on the structure and composition of cirrhotic portal vein thrombi, we found that the thrombi lacked a fibrin-rich part in two-thirds of the cases, whereas all thrombi consisted of a thickened, fibrotic vessel wall (Figure 2). In other words, portal vein thrombi often lack "classical" thrombus components that are uniformly present in thrombi isolated from patients with deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. [69,7] Rather, portal vein thrombi appear to consist of intimal fibrosis, which in in some cases were overlaid by a fibrin-rich clot. These observations suggest that the term "portal vein thrombosis" may be a misnomer and that the terms "portal vein stenosis" or "nonmalignant portal vein occlusion" may be more appropriate. We propose 2 possible mechanisms by which portal vein intimal fibrosis develops: 1) an initial fibrin-rich thrombus organizes into a fibrotic structure that re-endothelializes over time, and/or 2) intimal fibrosis develops in the absence of overt initial fibrin formation and is a result of, for example, portal hypertension and vascular endothelial cell stress. These findings may also explain why portal vein thrombi do not always recanalize after treatment with anticoagulant therapy. The current pharmacologic treatment of portal vein thrombosis consists of lowmolecular-weight heparins or direct oral anticoagulants, but this therapy does not always lead to recanalization of the portal vein. [77] Treatment with anticoagulants seems to be most effective in more recently formed thrombi, which may consist of fibrin. A thrombus that only contains fibrotic tissue and no fibrin does not likely respond to anticoagulants.
Future studies should specifically focus on the pathophysiology and the prevention and treatment of portal vein intimal hyperplasia.

| C O N C L U S I O N S A N D F U T U R E D I R E C T I O N S
In summary, the coagulation system of patients with compensated or stably decompensated cirrhosis is in a rebalanced state, characterized by normal to increased thrombin generation and plasma fibrinogen

| I S T H L O N D O N 2 0 2 2 R E P O R T
A number of abstracts on clot quality and composition were presented at the ISTH 2022 congress in London. In this section, we will briefly summarize studies on the topic of fibrin clot stability and composition.
Luyendyk et al. used murine models to investigate intrahepatic microthrombosis in acetaminophen-induced acute liver injury [79]. An abstract that was presented at the ISTH 2022 by Poole et al. [81] describes the effects of acetaminophen overdose in mice with a mutation in the fibrinogen γ-chain, making fibrin(ogen) incapable of interacting with platelet integrin αIIbβ3 (FibγΔ5). It was previously showed that acetaminophen overdose causes accumulation of fibrinogen and platelets in the injured liver, [79,80] but the mechanisms initiating platelet accumulation in the injured liver are not understood.
Poole et al. [81] hypothesized that hepatic platelet accumulation in the acetaminophen-injured liver is mediated by fibrin(ogen) engagement of the platelet integrin αIIbβ3. Surprisingly, FibγΔ5 mice had modestly higher platelet accumulation than wild-type mice after acetaminophen challenge. In addition, the FibγΔ5 mice had enhanced hepatic accumulation of high-molecular-weight crosslinked γ-chain multimers of fibrin, but no γ-γ dimer formation. Fibrin clots from the mutant mice were denser and consisted of thinner fibers compared with the clots from wild-type mice. The effect was independent of platelet integrin αIIbβ3-fibrinogen interactions, as treatment of wild-type mice with an αIIbβ3 inhibitor had no effect on acetaminophen-induced hepatic necrosis. The aberrant fibrinogen crosslinking may exacerbate liver injury following acetaminophen overdose.
More abstracts studied crosslinking of αand γ-chains of fibrin.
These crosslinking events increase clot stiffness and stabilize the final clot. In the study by Feller et al. [82] presented at the ISTH 2022, they described a mouse model in which α-α crosslinking was impaired by the introduction of 4-point mutations in the fibrinogen α-chain. Clots made of plasma obtained from these mice rupture at lower stress and have reduced toughness compared with clots made of plasma from wild-type mice. In another abstract, this research group [83] described their study on the impact of impaired α-crosslinking on venous thromboembolism in the mouse model. The clotting time was delayed, clot firmness and contraction were reduced, and the mutant mice was at a higher risk of embolization. In a previous study, the authors described that in the absence of γ-chain crosslinking, [84] fibrin fibers rupture at lower stress and have reduced toughness, resulting in more frequent embolization of clots in a mouse model of vena cava thrombosis. Based on the results from these studies, the investigators concluded that deficiency in both αand γ-chain crosslinking results in fibers that are more prone to rupture and that αand γ-crosslinking play complementary roles in generating key biochemical properties of fibrin clots for the prevention of embolism.
A study presented by Fish et al. [85] showed the effects of fibrinogen γ-chain knockdown in larval zebrafish and of a large deletion in the zebrafish fibrinogen γ-gene. Knockdown of fibrinogen γ-chain expression prevented laser-induced venous thrombosis, which was assessed by measuring the time until occlusion of the vessel. The authors also generated zebrafish with a deletion in the fibrinogen γ-gene, which showed no blood coagulation or thrombosis after laserinduced injury. The authors proposed that this model can serve as a stable knockout background to study the effects of specific mutations of fibrinogen γ-chain expression.
Several studies described fibrin clot properties such as permeability and susceptibility to lysis in in vitro experimental setups. For example, Klajmon et al. [86] described reduced clot permeability and prolonged clot lysis times in fibrin clots made of plasma from patients with genetically confirmed antithrombin deficiency. The findings suggest a mechanism by which antithrombin deficiency contributes to thrombosis risk. Another study by Smith and Morrissey [87] described that fibrinogen that was incubated with a neutrophil-released enzyme (cathepsin G, for example, released during inflammatory responses) causes cleavage of fibrinogen, which results in faster polymerization rates of fibrin induced by thrombin, higher clot turbidity, weaker clots, and decreased lysis times compared to control. The authors concluded that release of cathepsin G from activated neutrophils may affect clot formation in the presence of an ongoing inflammatory response, with potential consequences for thrombosis.
Another interesting abstract was presented by Risman et al. [88] about clot contraction and its impact on fibrinolysis. By using a combination of mathematical modeling and experimental methodologies to characterize the process of external fibrinolysis, the authors aimed to understand how key structural changes mechanistically drive the decrease in fibrinolysis of contracted blood clots. In their modeling and experimental approach, the densification of fibrin was the most significant determinant of the rate of fibrinolysis, which the authors ascribed to reduced diffusion of tPA in the clot. These findings may potentially be used therapeutically to optimize timing and delivery of lytic agents.

RELATIONSHIP DISCLOSURE
There are no competing interests to disclose.