Continues With the Conversion of Fibrinogen Into Fibrin

Fibrinogen Synthesis

Enhancement of fibrinogen synthesis and incorporation of the labelled amino acid at greater rate and concentration is accomplished by artificial defibrination.

From: Thrombosis and Bleeding Disorders , 1971

Physiology and Biochemistry of Fibrinolysis

NILS U. BANG M. D. , ... F.K. BELLER , in Thrombosis and Bleeding Disorders, 1971

Principle

A high intensity gamma emitter ⁷⁵Se as seleno methionine is used as the labelling precursor. ⁷⁵Se has the added advantage of a 120-day half-life.

Enhancement of fibrinogen synthesis and incorporation of the labelled amino acid at greater rate and concentration is accomplished by artificial defibrination. Urinary loss of the radioactive amino acid is prevented by nephrectomizing the injected animals.

In order to provide a labelled amino acid pool during the entire period of new fibrinogen formation a constant infusion of seleno methionine is preferred.

An in vivo labelled gamma-emitting fibrinogen of relatively high specific activity and with a long half-life can thus be harvested by conventional purification procedures (see chapter VI).

Materials and Methods

Mongrel dogs and retired Holtzman breeder rats are used for all experiments. The rats are fed Purina rat chow and the dogs a Standard laboratory formulation.

Surgical Procedures

Bilateral nephrectomies are carried out under Nembutal anesthesia through small midline incisions.

Defibrination

Defibrination of the rats is obtained by a single injection of 2500 units of bovine thrombin into the peritoneal cavity. Defibrination in the dogs is accomplished through a two-hour constant I.V. infusion of 2500 units of bovine thrombin.

Labelling Material

L-⁷⁵seleno-methionine is used as the labelled amino acid in all procedures. Material of a specific activity of 1.0 to 1.9 mc/mg methionine is recommended. Prior to its use, radioactivity of this material is determined in a well counter. Amounts used in the rat range from 50–200 microcuries per animal, and in the dog 900 microcuries are used. Slow, continuous infusions are carried out with the use of a Harvard infusion pump.

Determination of Radioactivity

Radioactivity of 1 ml aliquots of each blood sample is determined in a well counter with a single-channel-analyzer unit. Each sample is counted to a total of 5000 counts and background is subtracted. This results in a counting error of ± 1.4 percent at one standard deviation.

Expected Results in Typical Experiments

According to Gans et al. (1967), the slow intravenous infusion of a hundred μc ⁷⁵Se methionine over a 17-hour period into four nephrectomized thrombin-defibrinated rats resulted in a fibrinogen prepared from the plasma of these animals exhibiting an average of 7,715 counts per minute per mg of fibrinogen. The range of counts was 7,000–8,120. Two animals similarly prepared received 200 μc. ⁷⁵Se methionine each. The specific activity of the fibrinogen prepared from the plasma of these animals was 13,220 and 20,762 counts per minute per mg fibrinogen. The authors also established that identical procedures in non-nephrectomized animals resulted in considerably lower specific activity of the fibrinogen prepared from their plasma; the average activity of the fibrinogen being 4,691 counts per minute per mg.

Results obtained with flash labelling were compared with those obtained with the continuous infusion of the labelled amino acids. When 100 μc of ⁷⁵Se methionine was administered as a single intraperitoneal injection to two nephrectomized defibrinated rats the activity of the fibrinogen prepared from the plasma of these animals was 5,929 and 6,103 counts per mg fibrinogen.

In all experiments where thrombin defibrination and nephrectomy had been carried out the ratio of fibrinogen specific activity over total plasma protein specific activity was high; the average value being around 2.0 indicating selective incorporation of labelled seleno methionine into fibrinogen under these experimental conditions.

Comments

The technique as outlined provides an important research tool for the study of the turnover rates of fibrinogen and fibrin in a variety of physiologic and pathologic states. The theoretical advantages of producing a less altered, more natural fibrinogen through this technique are obvious. However, the procedures as outlined are cumbersome and the specific activity of the fibrinogen, although improved considerably by nephrectomy and thrombin defibrination in the animal, are still far below the specific activities readily achieved with in vitro labelling procedures. The use of in vivo labelling technique can therefore not be recommended for the labelling of fibrinogen to be used in vitro test systems.

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CORONARY HEART DISEASE | Hemostatic Factors

W. Gilmore , in Encyclopedia of Human Nutrition (Second Edition), 2005

Hemostatic Factors and Risk of Coronary Heart Disease

Elevated plasma coagulation factor levels are risk factors for coronary heart disease. Fibrinogen synthesis in the liver is stimulated by the proinflammatory cytokine interleukin-6 and, therefore, elevated levels are found during the acute phase response. It has been argued that it may be difficult to assign elevated plasma fibrinogen as a definitive risk factor since the pathology of CHD involves inflammation and the acute phase response, which will lead to increased fibrinogen anyway. The same argument has been used in the case of elevated white cell counts, which is also a risk factor for coronary events. However, it has been demonstrated that if fibrinogen and/or white blood cell count remain high after a vascular event then there is greater risk of subsequent events. Therefore, increased plasma fibrinogen and elevated white cell count are now considered a major risk factor for CHD.

Further studies have indicated that other blood clotting factors may act as risk factors for CHD. For example, a prospective study, The Northwick Park Heart Study, identified factor VII as a risk factor for CHD and showed that plasma levels of factor VII were predictive of CHD in a dose-dependent manner. Another study has shown that factor VIII may be a risk factor for cardiovascular disease. Increased levels of PAI-1 and decreased plasma levels of plasminogen activators have also been identified as risk factors for coronary heart disease.

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BLOOD BANKING

Leon L. Su , Lennart E. Lögdberg , in Blood Banking and Transfusion Medicine (Second Edition), 2007

Quantitative and Qualitative Fibrinogen Deficiency

The common coagulation pathway culminates in the formation of the fibrin clot through the action of thrombin on fibrinogen. Thus congenital abnormalities in fibrinogen synthesis, either hypofibrinogenemia/afibrinogenemia or dysfibrinogenemia, may result in spontaneous or posttraumatic hemorrhage of variable degree. Various clinical entities including hepatocellular disease, disseminated intravascular coagulopathy, and L-asparaginase therapy may be associated with clinically significant hypofibrinogenemia or dysfibrinogenemia. In general, fibrinogen levels greater than 100 mg/dL are considered to be adequate for hemostasis whereas fibrinogen levels below 100 mg/dL frequently are associated with severe bleeding. ³⁷ Although fibrinogen levels lower than 100 mg/dL result in the prolongation of the prothrombin and activated partial thromboplastin time, correction of the fibrinogen deficit is typically required only during episodes of active bleeding or before surgical procedures. ^38–40

In a review, Humphries ⁴¹ reported that the most common use of cryoprecipitate as fibrinogen replacement is in acquired fibrinogen deficiency. Dose and frequency of administration depend on the rate of consumption or destruction of fibrinogen, and this can be assessed by monitoring the fibrinogen level. In certain coagulopathies, repletion of other coagulation proteins in addition to fibrinogen may necessitate the use of FFP. ^{16, 42–47}

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Thrombin Time and Fibrinogen Evaluation

Mikhail Roshal MD, PhD , Morayma Reyes Gil MD, PhD , in Transfusion Medicine and Hemostasis (Third Edition), 2019

Physiologic Role of Fibrinogen

Fibrinogen is a plasma glycoprotein with a multitude of activities in the hemostasis system. The protein is a product of three genes FGA, FGB, and FGG and is primarily synthesized by hepatocytes, although extrahepatic fibrinogen synthesis has been observed in lung, kidney, and other tissues. Fully assembled fibrinogen is a hexamer of three dimers Aα, Bβ, and γ chains. The nomenclature refers to the small polypeptides A and B that are cleaved from Aα and Bβ chains by thrombin during conversion of fibrinogen to fibrin (see Chapter 115).

In addition to the plasma fibrinogen pool, the protein is also stored in the platelet alpha granules. The platelet fibrinogen pool provides a localized boost in fibrinogen concentration at the site of platelet activation. Fibrinogen serves as a scaffold for platelet aggregation via the activated form of integrin αIIbβ3 (also known as glycoprotein IIb/IIIa). Platelet aggregation via fibrinogen cross-linking provides an initial hemostatic barrier following blood vessel injury as part of the rapid primary hemostatic response. Subsequently, thrombin activation on the platelet surface leads to conversion of fibrinogen to fibrin and the formation of a more durable hemostatic barrier consisting of platelets and fibrin. During the conversion of fibrinogen to fibrin, thrombin cleaves fibrinopeptides A and B from fibrinogen Aα and Bβ chains, respectively, forming so-called fibrin monomers. Fibrin monomers polymerize into a noncovalently linked, staggered, linear network that stabilizes the initial platelet plug. Fibrin fibers are subsequently covalently cross-linked by activated factor XIII, a step that prevents premature dissolution of the fibrin clot. Subsequent degradation of fibrin requires action of plasmin. Paradoxically, fibrinogen also possesses antithrombotic activity, as it sequesters thrombin at nonsubstrate sites. Thus lack of fibrinogen in afibrinogenemia can lead to hemorrhagic and thrombotic complications. Dysfibrinogenemia can also present as either hemorrhagic and/or thrombotic complications, as abnormal fibrin results not only in defective clots but also may be resistance to plasmin cleavage and lysis.

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Coagulation Factor Autoantibodies

Massimo Cugno , Alberto Tedeschi , in Autoantibodies (Third Edition), 2014

Fibrinogen

Fibrinogen is a 340-kDa glycoprotein that is synthesized primarily in the hepatic parenchymal cells and participates in the final stages of the blood coagulation process. It consists of two copies of three different polypeptide chains, including two α chains (67.6 kDa), two β chains (52.3 kDa), and two γ chains (48.9 kDa). Fibrinogen synthesis is markedly increased during acute-phase states induced by tissue damage or inflammation. Epidemiologic data indicate that high levels of fibrinogen are associated with an increased risk of myocardial infarction and stroke. Thrombin binds to the fibrinogen central domain and liberates two fibrinopeptides named A and B, resulting in fibrin monomers that interact with each other, forming polymers that are then stabilized by activated factor XIII with the formation of a crosslinked fibrin network [1]. Antifibrinogen antibodies are extremely rare, with only a few cases reported in the current scientific literature. These antibodies are often associated with clinical bleeding, but reports of nonclinically relevant antibodies exist. Autoantibodies may alter fibrinogen by inhibiting the fibrinopeptide release and fibrin monomer polymerization [8,9].

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ATHEROSCLEROSIS AND THROMBOSIS IN DIABETES MELLITUS: NEW ASPECTS OF PATHOGENESIS

JOHN A. COLWELL , ... RUDOLF J. JOKL , in Levin and O'Neal's The Diabetic Foot (Seventh Edition), 2008

Coagulation

Activation of the coagulation system leads to fibrin formation by thrombin. Experimental and clinical data suggest that primary fibrin deposits and mural thrombi lead to the initial endothelial lesion and may contribute to the development of macrovascular and microvascular disease. In diabetes, there may be a general activation of blood coagulation, and fibrin lesions can be found in several organs of diabetic subjects. Most of the individual factors of both the intrinsic and the extrinsic coagulation pathway, as well as the inhibitors of coagulation, may be altered in diabetes. There are multiple data to support a pathogenetic rather than consequential role of hypercoagulation in the development of vascular disease in diabetes.

Attention has been directed at fibrinogen levels and dynamics in diabetes for a variety of reasons. It is now clear that the plasma level of fibrinogen is an independent risk factor for thrombotic and major cardiovascular events in population-based studies. ²⁷⁷ There have been many studies of fibrinogen levels and dynamics in diabetes mellitus. Generally, plasma fibrinogen levels are found to be elevated in diabetic individuals, particularly in those with previous hyperglycemia. Insulin deficiency results in an increase in fibrinogen synthesis in type 1 diabetes, and an insulin infusion will decrease the fibrinogen synthetic rate. Fibrinogen survival has been reported to be decreased in diabetes, and this abnormality is quickly reversed when euglycemia is achieved with insulin. Decreased fibrinogen survival in diabetes is also reversed by heparin, suggesting that intravascular fibrin formation may be taking place. Fibrinogen is glycated in diabetic subjects, and cross-linking of the α-chains of fibrinogen is impaired. Exercise conditioning will lower plasma fibrinogen levels in type 2 diabetic individuals. In the DCCT/EDIC cohort, fibrinogen was identified as a marker of nephropathy and peripheral vascular disease in type 1 diabetics. ²⁷⁸ These findings suggest that there might be increased fibrin formation in vivo in individuals with diabetes.

Because fibrinogen to fibrin formation is catalyzed by thrombin, investigations have centered on the regulation of thrombin activity in diabetes and on an in vivo index of thrombin activity, fibrinopeptide A (FPA). ²⁷⁹ FPA is cleaved from the α-chain of fibrinogen by the action of thrombin. This forms the first step in the conversion of fibrinogen to fibrin. FPA levels tend to be elevated in diabetes, especially when control is poor or vascular problems exist. Furthermore, recent studies have indicated that elevated FPA levels might be seen in diabetic individuals before vascular complications are present. A relation between plasma and urinary FPA and hyperglycemia in diabetes has been reported.

Prothrombin activation fragment F1+2 has been identified as a sensitive marker of coagulation in vivo. F1+2 is released from prothrombin when it is converted to thrombin by activated factor X. (Intima-media thickness, an index of subclinical atherosclerosis, is strongly associated with plasma F1+2 in the general population. ²⁸⁰ ) F1+2 generation proved to be very sensitive to even a short increase of glycemia. ²⁸¹ Positive correlation between F1+2 and glycated hemoglobin in diabetic patients has been found. ²⁸²

The most important inhibitor of the coagulation system is antithrombin III (AT III). AT III activity may be modulated by glucose both in vitro and in vivo. Hyperglycemia will cause a decrease in AT III activity in nondiabetic subjects, and activity returns to normal after a glucose infusion is stopped. Depressed levels of AT III activity are found in adult type 1 diabetic subjects, and infusion of insulin to produce normoglycemia will return AT III activity to normal. Nonenzymatic glycosylation of the AT III molecule might be the cause of its impaired functional activity. ²⁸³

Activated protein C is a vitamin K–dependent plasma protein that is another potent anticoagulant. It acts at the level of factors V and VIII in the intrinsic coagulation scheme. Several investigators have reported decreased protein C antigen and activity levels in type 1 diabetes, and such changes could theoretically promote coagulation. Glucose-induced hyperglycemia will lead to a fall in protein C levels and activity in normal and diabetic individuals. Depressed plasma levels and activity of protein C will rise with insulin-induced normoglycemia in type 1 diabetes.

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Other Congenital Coagulopathies

J. Petkova , K.D. Friedman , in Reference Module in Biomedical Sciences, 2014

Fibrinogen (Factor I) Deficiency

Fibrinogen is a soluble dimeric plasma glycoprotein. Its conversion into insoluble fibrin by the action of thrombin is the final step in clot formation. It also interacts with adhesion molecules, platelets, and the endothelium in the process of formation of the hemostatic plug.

The fibrinogen molecule is a dimer. Each monomer is composed of three polypeptide chains, designated A-alpha, B-beta, and G-gamma. The genes (FGA, FGB, FGG) for the three chains of fibrinogen are located on chromosome 4q31.3. The liver is the only major site of fibrinogen synthesis in humans ( Tennent et al., 2007). The plasma half-life of fibrinogen is 3–5 days. Synthesis of fibrinogen is upregulated by acute phase reactants. Fibrinogen is also endocytosed in and stored in the alpha granules of platelets (Rox et al., 2005).

Conversion of fibrinogen into the insoluble fibrin clot can be considered to take place in three stages. Initially, thrombin cleaves off fibrinopeptides A and B. This is followed by assembly of insoluble fibrin monomers into protofibrils and fibers. Finally, factor XIII cross-links these fibrin fibers by joining D regions in an end-to-end or longitudinal fashion (Spraggon et al., 1997).

Fibrin formation causes conformational changes that expose binding sites for tissue-type plasminogen activator (t-PA) and plasminogen. During healing and clot dissolution, activated plasmin then cleaves fibrin. However, thrombin generated by the coagulation process activates thrombin activatable fibrinolysis inhibitor (TAFI), which selectively removes these lysine residues, thus limiting fibrinolysis (Medved and Nieuwenhuizen, 2003).

Fibrinogen also interacts with platelet integrins PG IIb/IIIa to support platelet aggregation and it plays a role in adherence and migration of monocytes and neutrophils through the endothelium (Languino et al., 1995).

Complete deficiency of fibrinogen (afibrinogenemia) is an autosomal recessive disorder. The estimated frequency in Europe is 1 in 10⁶. Obligate heterozygotes have plasma fibrinogen levels of approximately half the normal level and are usually asymptomatic. The majority of mutations are in the FGA gene.

The clinical presentation of patients with afibrinogenemia is less severe than those with hemophilia (Lak et al., 1999). A characteristic finding is umbilical cord bleeding (85% of cases) (Lak et al., 1999). Muscle bleeds and hemarthroses are less frequent and rarely result in disability. Unlike hemophilia, epistaxis is also common (72%). Menorrhagia is common (70%). Gastrointestinal and urinary tract bleeding are less common (10–12%). Spontaneous thrombosis (venous as well as arterial) has been reported in patients with afibrinogenemia (Chafa et al., 1995).

Patients with incomplete deficiency of fibrinogen (hypofibrinogenemia) have milder phenotype, or may be asymptomatic (Peyvandi et al., 2006).

Dysfibrinogenemia generally arises from missense mutations in any of the three FG genes. It is usually inherited in an autosomal dominant fashion. The majority of patients are found incidentally and have no associated phenotype (55%), whereas 20% are associated with thrombosis and 25% with hemorrhage (Laffan, 2010).

Normal plasma concentration of fibrinogen is approximately 1.7–4.0 gl^{− 1} when measured by clotting assays. Patients with deficiency or abnormality of fibrinogen have prolongation of clotting times (aPTT, PT, thrombin time (TT)). In afibrinogenemia, all clotting times are prolonged, while in patients with milder deficiencies or dysfibrinogenemia, only the TT may be abnormal. TT is also prolonged in acquired dysfibrinogenemia.

The basis of therapy is replacement of fibrinogen. The principal source is cryoprecipitate, which contains approximately 1.5 g of fibrinogen per unit. Plasma-derived, virally inactivated concentrates are not readily available in the United States; however, they are the preferred method for replacement in Europe. A level of 1 gl^{− 1} is considered sufficient to provide hemostasis. If the severity of bleeding problems warrants prophylactic treatment, the long half-life of fibrinogen allows prophylactic infusions of fibrinogen concentrate or cryoprecipitate to be given weekly to maintain a trough level of 0.5 gl^{− 1} (Parameswaran et al., 2000). Patients with afibrinogenemia rarely develop antibodies, but these have been reported (Ra'anani et al., 1991). Menorrhagia may be controlled by use of antifibrinolytics and oral contraception.

In women who desire pregnancy, fibrinogen replacement should begin before 5 weeks of gestation to prevent abortion to maintain fibrinogen level >1.0 gl^{− 1}. Fibrinogen consumption increases during pregnancy, and infusions should be increased accordingly. A level of >1.5 gl^{− 1} is recommended for delivery (Kobayashi et al., 2000).

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Physiologic Changes Associated with Bed Rest and Major Body Injury

ROBERT JOHN DOWNEY , ... CHARLES WEISSMAN , in The Physiological Basis of Rehabilitation Medicine (Second Edition), 1994

The Immune System Connection: Cytokines

An explosion of knowledge has occurred about nonendocrine factors that may figure prominently in the response to stress. Many insights have been gained from increased understanding of the immune system. Watters and colleagues observed after etiocholanone injections an increase in the plasma activity of interleukin 1 (IL 1), ²¹³ a substance released by activated human monocytes and macrophages in response to various antigenic stimuli. Also called endogenous pyrogen or leukocyte endogenous factor, it modulates many of the tissue responses to inflammation. It induces hepatocytes to synthesize and release acute-phase reactants (e.g., macroglobulin, complement, immunoglobulins), ²¹⁵ makes endothelium adhesive for monocytes, ²¹⁵ promotes fibroblast growth, ²¹⁶ causes fever, ²¹⁷ and may be involved in muscle breakdown (Figure 17-2). ²¹⁸ Baracos and colleagues ²¹⁹ reported that a biologic extract rich in IL 1 simulated skeletal muscle proteolysis in vitro via protaglandin E₂ (PgE₂) formation. Clowes and coworkers ²²⁰ identified a polypeptide from the serum of septic patients that also caused muscle proteolysis in vitro.

Whether IL 1 or a related substance is involved in proteolysis is still unclear (see Protein Metabolism). IL 1 also activates the expression of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), and B cell-stimulating factor 2 (BSF-2, also called IL 6) in endothelial cells, helper T cells, bone marrow stroma cells, and fibroblasts. ^221–223 These factors in turn activate marrow progenitor cells, and leukocytosis results. ²²⁴ Luger and colleagues ²²⁵ observed decreased IL 1 activity in patients with fatal sepsis, but in those who survived the levels were normal. One explanation is that the elevated plasma concentrations of catecholamines suppress monocytic IL 1 production. Another monokine, hepatocyte-stimulating factor (also called BSF-2 and IL 6), has been shown to induce fibrinogen synthesis in hepatocytes. ^{226, 227} It is also produced by human endothelial cells in response to IL 1 tumor necrosis factor (TNF), and to bacterial liposaccharide stimulation. ²²⁸

Cachectin, or TNF, ²²⁹ is another important cytokine that appears to have metabolic effects. This protein is secreted by macrophages in response to exposure to endotoxin ²³⁰ and Candida albicans organisms. ²³¹ Administration of TNF to animals results in most of the manifestations of septic shock: hypotension, metabolic acidosis, hemoconcentration, hyperglycemia, hyperkalemia, hemorrhagic lesions of the gastrointestinal tract, and acute tubular necrosis. ²³² Waage and colleagues ²³³ noted a correlation between TNF levels, degree of septic shock, and subsequent death in patients with meningiococcemia. In addition, TNF causes fever by direct action on the hypothalamus and by inducing IL 1 secretion. ^{234, 235} The latter substance then mediates many of the changes described above. In a recent study, Michie and colleagues ²³⁶ infused normal subjects with endotoxin and found that serum levels of TNF peaked after 90 to 180 minutes. Associated with this peak were increases in plasma ACTH and epinephrine concentrations, body temperature, and heart rate. Pretreatment with ibuprofen did not affect the increase in TNF level but did suppress the increase in body temperature and ACTH. It has also been shown that TNF can dramatically decrease the synthesis and activity of lipogenic enzymes in cultured adipocytes, thus the name cachectin. ²³⁷ This mirrors the decreased lipogenesis observed with whole body measurements in septic and injured patients. Lymphotoxin, also called TNF-β, is a product of activated T cells and has biologic activity similar to that of cachectin. ^238–240

IL 2 (T-cell growth factor), another cytokine that may participate in the metabolic response to stress, is secreted by T cells in response to stimuli such as IL 1 and causes generation and proliferation of antigen-specific cytotoxic and helper T cells required for cell-mediated immunity. IL 2 production is reduced in injured patients, and the volume of production is inversely correlated with the severity of injury. ²⁴¹ This decreased IL-2 synthesis is likely due to excessive PgE₂ output by inhibitory monocytes. ²⁴² PgE₂ is associated with reduced lymphokine production, ²⁴³ inhibited lymphocyte-mediated cytolysis, and inhibition of lymphocyte mitogenesis. ²⁴³ Partial restoration of IL 2 synthesis can occur by blocking the cyclooxygenase pathway with indomethacin. ²⁴² Burn patients exhibit not only decreased IL 2 synthesis but also inability to effect expression of high-affinity functional IL 2 receptors. ²⁴⁴ The duration of reduced IL 2 production may be as long as 60 days after the burn injury, and it correlates with the severity of the burn. ²⁴² Also, septic burn patients produce less IL 2 than nonseptic ones. These alterations in IL 2 homeostasis may be one mechanism of postinjury depression of cell-mediated immunity.

One of the main interests in IL 2 is its use in cancer immunotherapy. ²⁴⁵ The complications of such therapy (when given in large doses) constitute a response similar to that seen after injury and sepsis: weight gain due to fluid retention, noncardiogenic pulmonary edema, hyperpyrexia, nephrotoxicity, and hepatotoxicity. ^{246, 247} It is still unclear whether this response is due to a pharmacologic effect or a physiologic one; and whether it is due to stimulation of other mediators or is just an effect of IL 2 itself. In patients receiving IL 2 infusions, the levels of ACTH, cortisol, GH, and interferon gamma (IFN-γ) in the blood have been shown to be elevated. ^248–251

IFN-γ, a glycoprotein released by stimulated T lymphocytes, is another mediator of the immune stress response. ²⁵² It activates macrophages to release IL 1, TNF, ²⁵³ , G-CSF, M-CSF, and BSF-2, ²²³ increases IL 2 receptors on monocytes, and reduces release of PgE₂ ²⁵⁴ and urokinase-type plasminogen activator. ²⁵³ It reduces immune suppressor activity by inhibiting PgE₂ release as well as inhibiting viral replication. Elevated serum levels of IFN-γ have been observed in patients with pelvic inflammatory disease. ²⁵⁵ Platelet-activating factor, a phospholipid product of activated macrophages, may also be active, ²⁵⁶ especially in the response to endotoxin.

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Common Genetic Determinants of Coagulation and Fibrinolysis

Angela M. Carter , ... Peter J. Grant , in Emery and Rimoin's Principles and Practice of Medical Genetics (Sixth Edition), 2013

54.2.5 Fibrinogen

Fibrinogen (or Factor I) is a soluble glycoprotein (GP) with a central role in blood clotting both as the substrate for fibrin formation and as the ligand for the platelet α_IIbβ_III receptor, which promotes platelet aggregation. Thrombin cleaves fibrinogen to fibrin monomers, which spontaneously polymerize to form half-staggered protofibrils. These aggregate to fibers and result in a three-dimensional fibrin network stabilized by activated FXIII, which serves as a scaffold for the emerging blood clot. Fibrinogen is a large and complex protein comprising pairs of three nonidentical polypeptides linked by a central domain. The subunits are referred to as Aα, Bβ and γ chains (roman capital letters denoting intact chains prior to proteolytic cleavage by thrombin), which are encoded by separate genes (FGA, FGB, FGG) located in the fibrinogen module on the short arm of chromosome 4 (44,45). Individual chains are produced and assembled in hepatocytes. In addition to its function as a coagulation factor, fibrinogen is also an acute phase protein, and raised levels have been consistently associated with CVD (18,46,47). Both environmental and genetic factors influence fibrinogen levels, and in heritability studies, the contribution of genetic factors to plasma levels of fibrinogen have been estimated to be in the region of 50% (5,6,48) . There are a number of polymorphisms in all three chains, but in vitro studies suggest that β-chain synthesis is rate limiting in the synthesis of the fibrinogen molecule and research has focused on this area. The substitution of G to A in fibrinogen β-455 is consistently associated with increased fibrinogen levels, in addition to promoter polymorphisms of the fibrinogen β gene (-1420G/A, -993C/T and -148 C/T), which are in complete linkage disequilibrium with β-455 G/A. All three fibrinogen genes have interleukin (IL)-6 responsive sequences, but only SNPs in the β-chain have been shown to modulate fibrinogen synthesis in response to IL-6 (49).This may explain why some environmental factors, such as smoking, result in higher plasma fibrinogen levels only in carriers of these SNPs, and underlines the confounding effect of genetic and environmental factors on plasma fibrinogen. More recent work identified common SNPs and haplotypes of the FGA and FGB genes as a major determinant for the variability of fibrinogen levels, concluding that genetic background represents a major determinant of fibrinogen, which modulates the response to proinflammatory stimuli (50). Two coding polymorphisms have been identified to date (Arg448Lys in the β-chain, and Thr312Ala in the alpha chain). Arg448Lys is in strong linkage disequilibrium with β-chain promoter polymorphisms and is associated with variation in plasma fibrinogen levels, functional properties of the fibrinogen molecule (51) and fibrin phenotype. Clots formed from recombinant as well as plasma-purified Lys448 fibrin have a more compact structure with thinner fibers and smaller pores and increased stiffness, even before cross-linking by FXIII (51). In addition, clots made from fibrinogen-depleted plasma substituted with recombinant Lys448 clots are more difficult to lyse (51). Such phenotypic properties have been associated with increased risk for CVD (52–55). In clinical studies, the Lys448 allele was associated with severity of CAD (56) and with stroke in female patients (57). In Caucasians, the polymorphism is estimated to occur at a frequency of 15–20%.

Thr312Ala occurs in a region of α-fibrinogen important in a number of FXIII-dependent processes, including α-fibrin/α-fibrin and α-fibrin/α2-antiplasmin cross-linking (amino acid residues Aα328 and 303, respectively (58,59)). The region of α-fibrinogen encompassing this polymorphism (residues Aα 242–424) is important in promoting the dissociation of the FXIII A and B subunit dimers thereby enhancing activation of FXIII itself (60,61). We found that fibrin clots formed from plasma-purified Ala312 fibrinogen have increased α-chain cross-linking, fibrin fiber diameter and clot stiffness compared with clots from Thr312 fibrinogen (62). In clinical studies we reported associations of the Ala312 allele with poststroke mortality in subjects with atrial fibrillation (63) and with pulmonary embolism (64) supporting a role for Ala312 in clot stability. Ala312 is in linkage disequilibrium with a haplotype on FGG that is associated with decreased plasma levels of the gamma chain variant γ'. This modified version of the gamma chain arises from alternative processing of γ-fibrinogen mRNA resulting in the inclusion of intron 9 and polyadenylation of the transcript at an alternative polyadenylation site in intron 9 (65). This leads to replacement of the last 4 amino acids by 20 alternative amino acids, which significantly alters the properties of the γ chain (see also review by Uitte de Willige (66)). Between 7 and 15% of plasma fibrinogen molecules contain γA/γ', which has been shown to bind FXIII zymogen, and it may represent a mechanism whereby the local concentration of FXIII is increased during clot formation (67). Fibrinogen γ' has a decreased platelet aggregation potential. Interaction of fibrinogen with the platelet α_IIbβ₃ receptor is decreased in recombinant, homodimeric (AαBβγ')₂, indicating that the binding of platelets to the fibrinogen γ chain is mediated by the four C-terminal residues of the γA chain (68). Clots made from γA/γ' fibrinogen in the presence of FXIII are more highly cross-linked and lyse more slowly than clots from γA/γA fibrinogen (69), and γA/γ' fibrinogen has also been shown to enhance the activation of FXIII (70). Furthermore, clots formed with γA/γ fibrinogen have a thinner average fiber diameter and a higher proportion of branch points than clots formed from γA/γA fibrinogen (71). A functional role for γA/γ fibrinogen in thrombosis is supported by findings by Lovely et al. (72) who found that γA/γ' fibrinogen levels are increased in patients with CAD compared with healthy controls. Similar findings were reported by Mannila et al., (73) who found significantly increased γA/γ' levels in patients with myocardial infarction (MI) compared to age- and sex-matched controls. In contrast, Uitte de Willige et al. (74) report decreased γA/γ' levels in patients suffering from deep venous thrombosis (DVT) compared to healthy controls in the Leiden Thrombophilia Study. This could suggest that the relationship between the γ' splice variant and thrombosis is dependent on the type of CVD, and exerts a pro-thrombotic effect in arterial disease, but is associated with protection against thrombotic events in venous disease. The putative mechanisms behind these observations—potentially mediated by differences in fibrin structure, platelet binding and FXIII activation—require further investigation.

Owing to the consistent association of increased plasma fibrinogen with CVD and heritability of ~50%, a number of studies have searched for loci associated with variance in plasma fibrinogen and two recent studies have reported GWAS analysis for plasma fibrinogen (75,76). In the study by Dehghan et al., (75) which involved a meta-analysis of >20,000 individuals from six independent studies, 73 SNPs localized to four chromosomal regions were identified with GWAS significant associations with fibrinogen. The most significant SNP was rs1800789 at 4q31.3 within the FGB gene; this SNP is in linkage disequilibrium with the -148 C/T and -455 G/A polymorphisms, which have been consistently associated with plasma fibrinogen (see earlier). Rs2522056 and rs1539019 were the most significant SNPs at 5q23.3 (3′ to the gene encoding interferon regulatory factor 1 [IRF1]) and 1q44 (NLR family, pyrin domain containing 3 isoforms [NLRD3], respectively, both of which are involved in the regulation of inflammatory processes (75)). Rs511154 at 3q22.3 occurs in intron 1 of the gene encoding propionyl coenzyme A carboxylase β-polypeptide [PCCB]. Together these polymorphisms accounted for <2% of the variance in fibrinogen. In an independent study of >17,000 healthy participants in the Women's Genome Health Study, Danick et al. (76) identified 19 SNPs meeting GWAS significance, localized to five distinct chromosomal regions. The strongest associations were with SNPs in 4q32.1 in the vicinity of the fibrinogen structural genes (rs6056 and rs1800788 were the most informative SNPs in this region), supporting the findings of Dehghan et al. The most significant SNP at 1q21.3 was rs8192784 (76), a nonsynonymous Asp358Ala polymorphism in the gene encoding the IL-6 receptor (IL6R), which has been shown to influence plasma levels of soluble IL6R and IL-6 (77). The other significant SNPs were rs7422339, a nonsynonymous Asp1405Thr polymorphism at 2q34 (carbamoyl phosphate synthetase 1 [CPS1]), rs1016988 and rs10479002 at 5q31.1 (solute carrier family 22, members 4 and 5 [SLC22A4, SLC22A5]) and rs10512597 at 17q25.1 (in the vicinity of a cluster of immunoglobulin superfamily members including CD300LF). Together these SNPs accounted for <2% of the variance in fibrinogen. Interestingly, rs8192284 and rs10512597 were also associated with plasma C-reactive protein (76), highlighting the role of inflammatory factors in the regulation of fibrinogen.

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Heterogeneity of congenital afibrinogenemia, from epidemiology to clinical consequences and management

Alessandro Casini , ... Philippe de Moerloose , in Blood Reviews, 2021

2 Fibrinogen biosynthesis

Fibrinogen is a hexameric glycoprotein made of two sets of three homologous polypeptide chains: Aα, Bβ and γ [7]. The three fibrinogen chains are encoded by three genes FGB, FGA and FGG, ordered from centromere to telomere on the long arm of human chromosome 4, that are co-regulated to generate mRNA at balanced levels to sustain fibrinogen secretion from the liver [8]. Alternatively spliced mRNAs produce two different isoforms for FGA: Aα and AαE, and FGG γ and γ' [7 ]. Epigenetic and post-transcriptional regulation by miRNAs also contribute to the overall rate of fibrinogen synthesis [ 9,10]. Each gene is separately transcribed and translated to produce a nascent polypeptide including a signal peptide, which is cleaved from each chain during translocation of the single chains into the lumen of the endoplasmic reticulum (ER) [11]. The three component chains are secreted as an assembled hexamer (AαBβγ)₂ primarily from hepatocytes [11]. In addition, a separate small fibrinogen pool with structural differences due to absence of the γ' isoform is also endocytosed in platelet alpha-granules [12]. Fibrinogen chain assembly is a complex machinery proceeding in the lumen of ER in a stepwise manner under the control of chaperones and glycosylation enzymes that efficiently support the correct assembly and folding of the protein [13–15] Misfolded, misassembled and surplus proteins are retained in the ER and, in the large majority of cases, ultimately degraded by lysosomes and proteasomes [16]. Hexamers containing mutant chains which escape this quality control can lead to Fibrinogen Storage Disease [17,18].

As shown in Fig. 1, the fibrinogen structure comprises two terminal D regions containing the γ-nodule and the β nodule formed by the COOH-terminal portions of the Bβ and γ chains, respectively as well as adjacent portions of the coiled coils. One central E region contains the central globular nodule formed by the NH2-terminal portions of all six chains and two adjacent portions of the coiled coil [19,20]. Fibrin polymerisation is initiated by the thrombin-mediated cleavage of fibrinopeptides A (FpA) and B with subsequent exposure of binding sites 'A' and 'B' in the E region complementary to constitutive sites 'a' and 'b' in the D regions [20]. Fibrin monomer molecules produced by the release of FpA interact with each other in a half-staggered manner via the knob-hole interactions resulting in larger fibrin oligomers growing in length to protofibrils [21]. The lateral aggregation of protofibrils supports their packing into fibrin fibers with a 22.5-nm periodic cross-striation [22]. The elongation and the thickening of fibrin fibers are accompanied by branching which leads to formation of a 3-dimensional fibrin clot network finally cross-linked by factor XIIIa which, together with platelets and red blood cells, provides structural integrity to the growing thrombus [23].

Fig. 1. Fibrinogen structure, fibrin formation and polymerization. The fibrinogen crystal structure is presented in the upper panel with its schematic representation displayed below. Fibrin formation starts with the cleavage of fibrinopeptides A (FpA) by thrombin to expose knobs 'A' which interact with holes 'a' in the C-terminal regions of the γ chains (indicated by dotted circle) to induce fibrin polymerization. Subsequent cleavage of fibrinopeptides B (FpB) by thrombin exposes knobs 'B' which can interact with holes 'b' in the C-terminal regions of β chains (indicated by dotted circle). These are thought to contribute to the lateral aggregation of protofibrils together with 'αC- αC' interactions (indicated by dotted circle). This image was prepared using BioRender.com with the fibrinogen crystal structure originally produced using PDB entry 3GHG [153], the Swiss-PdbViewer 4.1.0 and POV-Ray 3.7 software. (A)α, (B)β and γ chains are colored in grey, green and blue, respectively.

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