Factor IX Deficiency

Factor IX (FIX) deficiency or dysfunction, or hemophilia B, is an X-linked inherited bleeding disorder, usually manifested in males and transmitted by females who carry the causative mutation on the X chromosome. Hemophilia B results from a variety of defects in the FIX gene. FIX deficiency is 4-6 times less prevalent than factor VIII (FVIII) deficiency (hemophilia A).

The most significant breakthroughs in comprehending the mechanisms associated with coagulation first came from an understanding of the individual causes of the bleeding disorders. Hemophilia B was differentiated from hemophilia A in 1952, when it was found that mixing plasma from patients with the two conditions corrected the clotting time. The hemophilia B patient in that study had the surname Christmas, and hence the disorder became known as Christmas disease.

The existence of inherited bleeding disorders in males had long been recognized, however. The newspaper item below demonstrates what appears to be a late 19th-century record of hemophilia passed from mother to sons.

Obituary in the Salem Gazette (Massachusetts) of a 19-year-old man, March 22, 1796.

Hemophilia B may be classified as severe, moderate, or mild, based on the plasma levels of factor IX in affected individuals (<1%, 2-5%, 6-30%, respectively).^[1] A variety of of underlying mutations have been identified and linked with different levels of clinical severity.

Structure, production, and half-life

FIX, a vitamin K–dependent single-chain glycoprotein, is synthesized first by the hepatocyte as a precursor protein (protein in vitamin K absence). It then undergoes extensive posttranslational modification to become the fully gamma-carboxylated mature zymogen that is secreted into the blood.

The precursor protein has the following parts, starting with (1) a signal peptide at the amino (NH₂) terminal end (as marked in the diagram below), which directs the protein to the endoplasmic reticulum in the liver, and continuing with (2) the prepro leader sequence recognized by the gamma-glutamylcarboxylase, which is responsible for the posttranslational modification (carboxylation) of the glutamic acid residues (Gla) in the NH₂ -terminal portion of the molecule. These 2 parts of the molecule are removed before the protein is secreted into the circulation.

Major components of the factor IX structure.

Single-chain plasma FIX has the Gla domain (12 gamma-carboxyglutamic acid residues) at its amino terminal end; this is a characteristic feature of all vitamin K–dependent factors. The Gla domain is responsible for Ca²⁺ binding, which is necessary for the binding of FIX to phospholipid membranes. The Gla region is followed by (1) two epidermal growth factor regions, (2) the activation peptide, which is removed when the single-chain zymogen FIX is converted to activated factor IX (FIXa), ie, the 2-chain active enzyme, and (3) the catalytic domain, which contains the enzymatic activity.

Before secretion from the hepatocyte, the FIX protein undergoes extensive posttranslational modifications, which include gamma-carboxylation, beta-hydroxylation, and removal of the signal peptide and propeptides, addition of carbohydrates, sulfation, and phosphorylation. Gamma-carboxylation, as demonstrated in the diagram below, is a vitamin K–dependent process in which the enzyme gamma-glutamylcarboxylase binds to specific sites on the propeptide region of the precursor protein in the liver. The process of gamma-carboxylation of the glutamic acid residues forms gamma-carboxyglutamyl (Gla) residues in the mature protein and requires reduced vitamin K, oxygen, and carbon dioxide to perform its functions.

Vitamin K–dependent carboxylation of precursor factor IX to procoagulant factor IX. Carboxylation of glutamate (Glu) to gamma-carboxyglutamate (Gla) residues in the precursor protein of the vitamin K–dependent factors occurs in the endoplasmic reticulum of the hepatocyte. Reduced vitamin K is oxidized in this process. Warfarin prevents the reduction and recycling of oxidized vitamin K.

These Gla regions are the high affinity Ca²⁺ binding sites necessary for binding FIXa to lipid membranes so FIXa can express its full procoagulant activity. All of the vitamin K–dependent procoagulants and anticoagulants are biologically inactive unless the glutamic acid residues at the amino terminal end are carboxylated; the exact number of Gla regions varies with each protein.

Warfarin prevents the reduction and recycling of oxidized vitamin K (vitamin K epoxide) that is generated during this carboxylation reaction. As a result of the indirect inhibition of the carboxylation reaction resulting from a lack of available reduced vitamin K, hypocarboxylated and decarboxylated forms of the vitamin K–dependent factors are found in the circulation of patients ingesting warfarin. These abnormal forms have reduced or absent biological activity. Following these modifications, the carboxyterminal (C-terminal) region is recognized by the hepatic secretion process. Mutations that increase the charge of this region result in decreased hepatic secretion of all vitamin K–dependent proteins, including FIX, and lead to deficiencies of multiple vitamin K–dependent factors.

FIX is present in a concentration of 4-5 µg/mL with a half-life of approximately 18-24 hours. A 3-fold variation in the activity of FIX in plasma is normal. Since FIX is smaller than albumin, it distributes in both the extravascular and intravascular compartments. Following intravenous (IV) administration, recovery of FIX concentrates varies significantly, which has been ascribed to the development of nonneutralizing antibodies. In vivo binding of FIX to collagen IV has been proposed as another reason for reduced recovery of FIX following infusion of FIX concentrates in hemophilia B patients. FIX concentrates generally are replaced every 18-24 hours under steady state conditions. Lower recoveries are seen with recombinant factor IX (rFIX) compared to FIX concentrates.^[2]

Extensive homology is found between FIX and the other vitamin K–dependent proteins (procoagulants factor VII [FVII], factor X [FX], factor II [FII] and anticoagulant proteins C and S), especially in the prepro sequence and the Gla regions. Despite numerous similarities, each vitamin K–dependent protein performs a different function in the hemostatic pathway, which is diagrammed in the following image.

The hemostatic pathway: role of factor IX.

Activation

The gamma-carboxylated region of FIX is essential for calcium binding and is the site at which vitamin K–dependent coagulation proteins bind to cell surface phospholipids and efficient coagulation reactions take place. Ca²⁺ binding to the Gla region results in a conformational change leading to exposure of previously buried hydrophobic residues in the FIX molecule, which then can be inserted into the lipid bilayer.

Tissue factor (TF) is a glycosylated membrane protein present in cells surrounding blood vessels and in many organs. On the other hand, endothelial cells, tissue macrophages, and smooth muscle cells express TF only when stimulated by serine proteases, such as thrombin, and by inflammatory cytokines. In vivo, under physiologic conditions, only a trace amount of FVII is present in the activated form (activated factor VII [FVIIa] of approximately 1%). When TF becomes available, it complexes with FVII or FVIIa, and current concepts support the view that activation of FIX to FIXa is more rapid with the TF-FVII complex than with activated factor XI (FXIa).^[3] The activation peptide for FIX is detectable in the plasma of control subjects.^[4] The image below diagrams the activation of FIX.

Activation of factor IX and function of the intrinsic tenase complex. Activation of factor IX is followed by formation of the intrinsic tenase complex, which activates factor X to activated factor X, leading to a second and larger burst of thrombin production during activation of hemostasis.

Following activation, the single-chain FIX becomes a 2-chain molecule, in which the 2 chains are linked by a disulfide bond attaching the enzyme to the Gla domain. Activated factor VIII (FVIIIa) is the specific cofactor for the full expression of FIXa activity. Platelets not only provide the lipid surface on which solid-phase reactions occur, but they also possess a binding site for FIXa that promotes complex formation with FVIIIa and Ca²⁺. The complex of FIXa, FVIIIa, Ca²⁺, and activated platelet (phospholipid surface) reaches its maximum potential to activate FX to activated factor X (FXa). This activator complex, which contains FIXa, is termed the intrinsic tenase complex in contradistinction to the FVIIa-TF (extrinsic tenase) or FXa, activated factor V (FVa), Ca²⁺, and phospholipid (prothrombinase) complexes; all ultimately lead to thrombin generation.

In vivo, the active FVIIa-TF complex is responsible for the initial activation of FX to FXa, leading first to the generation of small amounts of thrombin. When the FIXa generated by the FVIIa-TF complex is part of the intrinsic tenase complex, it activates additional FX to FXa and leads to the second and explosive burst of thrombin generation with subsequent clot formation.

Many feedback loops exist in the coagulation pathway, and some evidence suggests that FIXa can activate FVII and FVIII in addition to FX. Support for the important role of FIX in producing FVIIa, essential for normal hemostasis in vivo, was provided by a sensitive highly specific FVIIa assay, which showed that healthy individuals had basal FVIIa levels of 4.34 ng/mL. Patients with severe FIX deficiency were found to have markedly reduced FVIIa levels of 0.33 ng/mL, whereas individuals with severe FVIII deficiency had FVIIa levels of 2.69 ng/mL, values higher than those seen in patients with severe hemophilia B.

Antithrombin is the most important physiologic inhibitor of FIXa. Clinically, hemophilias A and B are indistinguishable. Variability in bleeding manifestations in patients with similar reductions in FVIII, FIX, or factor XI (FXI) is a well-known fact to clinicians. Modulation of the hemorrhagic disorder induced by deficiencies of intrinsic coagulation factors by co-inheritance of thrombophilic mutations is another well-recognized determinant of the extent of disruption of hemostasis in patients with a bleeding diathesis.

Possible interactions between deficiencies of FIX and thrombin activatable fibrinolytic inhibitor

The demonstration that thrombi generated in plasmas obtained from patients with hemophilia A or B underwent premature lysis generated the hypothesis that bleeding in patients with hemophilia may be due not only to failure of adequate thrombin generation and clot formation, but also to a failure of adequate suppression of fibrinolysis leading to accelerated clot removal.

Proof of the concept of the latter has been provided for decades in patients with hemophilia, long before the role of thrombin activatable fibrinolytic inhibitor (TAFI) was even suspected, by the amply proven hemostatic adequacy of a single dose of replacement factor when combined with prolonged inhibition of fibrinolysis in patients with severe hemophilia undergoing dental or other mucocutaneous procedures. The demonstration in vitro of rapid clot lysis in hemophilic plasmas was followed by a demonstration of rapid clot lysis in plasmas deficient in FXI or factor XII (FXII), with prolongation of clot lysis by restitution of the missing factor.

Recently, a large amount of information has accrued regarding the pathophysiologic role of TAFI in thrombohemorrhagic disorders. TAFI, a single-chain carboxypeptidase B–like zymogen, is activated by thrombin to generate activated TAFI (TAFIa). Thrombin, plasmin, and trypsin all can activate TAFI, but thrombin bound to thrombomodulin has an approximate 1250-fold greater catalytic rate than thrombin alone; however, thrombin alone is sufficient to achieve significant TAFI activation.

The importance of TAFIa in influencing fibrinolysis is emphasized by the fact that conversion of only 1% of the zymogen to TAFIa is sufficient to suppress normal fibrinolysis by approximately 60%. TAFIa suppresses fibrinolysis by removing C-terminal lysine and arginine residues in a fibrin clot that has been partially degraded by plasmin. Removal of C-terminal lysine residues reduces the rate of plasminogen activation by a number of mechanisms, attenuating fibrinolysis. This effect is counterbalanced in normal plasma by the activation of protein C, which has profibrinolytic properties due to its ability to suppress thrombin generation by its major effect in degrading FVa and, to a lesser extent, FVIIIa.

In normal plasma, a balance exists between the effects of activated protein C on the one hand (profibrinolytic) and TAFIa on the other (antifibrinolytic). Thrombin secures survival of the thrombus created by its action on fibrinogen by activating TAFI, thereby inhibiting fibrinolysis. In this context, note that cross-linking of fibrin induced by activated factor XIII (FXIIIa, activated by thrombin) also renders the clot insoluble (for more information, see Factor XIII). Thus, thrombin uses multiple prongs to assure survival of its creation, fibrin, and affects the normal delicate balance between thrombus formation and thrombus resolution.

A reduction in the level of FIX via reduction of thrombin generation reduces TAFI activation and increases fibrinolysis, whereas persistence of FVa (as is the case with co-inheritance of factor V [FV] Leiden) leads to increased (persistent) thrombin production and TAFI activation, thereby inhibiting fibrinolysis.

These data, along with the known effects of epsilon-aminocaproic acid (EACA; Amicar) certainly raise the question of the efficacy of prolonged fibrinolytic inhibition in individuals with hemophilia as a possible mechanism with which not only to reduce the frequency of spontaneous bleeding but also to provide reduction in product usage in surgically induced bleeding in which fibrinolytic inhibitors currently are not used as adjuvant therapy. An expansion in the role of fibrinolytic inhibitors to control all types of bleeding in individuals with hemophilia could be explored in properly designed prospective clinical trials. Such trials could provide the first objective data on the true frequency of thromboembolic and other complications involved in the use of fibrinolytic inhibitors with replacement therapy.

Cell surface–directed hemostasis

The concept of coagulation as a waterfall or cascade, with a series of reactions each impacting the subsequent reaction, has been prevalent for a long time. The fact that fluid-phase reactions are inefficient and that platelets and other cell surfaces provide the anionic phospholipids needed for complex formation so that reactions can proceed efficiently also has been recognized. This model allowed the reader to conceptually visualize activated partial thromboplastin time (aPTT) and prothrombin time (PT) tests as the intrinsic and extrinsic pathways. One review proposed that coagulation is essentially a cell surface–based event in overlapping phases, suggesting the need for a paradigm shift from the old concept in which coagulation reactions were controlled by coagulation proteins to a new concept in which the "process is controlled by cellular elements."

In this model, diagrammed below, 3 phases are proposed including (1) initiation of coagulation on the surface of a TF-bearing cell, with formation of FXa, FIXa, and thrombin, (2) amplification of this reaction next on the platelet surface as platelets are activated, adhere, and accumulate factors/cofactors on their surfaces, and (3) the propagation phase in which the large second burst of thrombin occurs on the platelet surface resulting from the interaction of proteases with their cofactors, resulting in fibrin polymerization. Platelets are an early and essential feature of hemostasis, making them an ideal cell to regulate this process, and these authors provide a series of cogent reasons for switching to this new concept of hemostasis.^{[5, 6]}

Cell surfaced-directed hemostasis. Initially, a small amount of thrombin is generated on the surface of the tissue factor–bearing (TF-bearing) cell. Following amplification, the second burst generates a larger amount of thrombin, leading to fibrin (clot) formation. (Adapted from Hoffman and Monroe, Thromb Haemost 2001, 85(6): 958-65.)

Frequency

United States

Incidence of hemophilia B is approximately 1 case per 30,000 male births.

International

Frequency by ethnic background (countries) is currently not available. FIX deficiency has been found in many parts of the world.

Mortality/Morbidity

The consequences of the repeated bleeding experienced by individuals with hemophilia are serious and result from the repeated need for FIX replacement to control bleeding. Availability of replacement products has changed the lives of patients with FIX deficiency, although serious problems were incurred by the use of the only available, less pure, earlier products. Currently available concentrates and recombinant products have a better safety profile.^[7]

Persons with severe hemophilia have recurrent joint and muscle bleeds, which are spontaneous or follow minor trauma and cause severe acute pain and limitation of movement. The presence of blood in the joint leads to synovial hypertrophy, with a tendency to rebleed, which results in chronic synovitis, with destruction of synovium, cartilage, and bone leading to chronic pain, stiffness of the joints, and limitation of movement because of progressive severe joint damage.

Intramuscular hemorrhage, the second most common bleeding event, also produces acute pain, swelling, and limitation of movement. Other sites of bleeding and many other complications (discussed later) contribute to morbidity and mortality.

Current treatment methods have succeeded in reducing not only the morbidity but also the death rate, and for the first time, persons with hemophilia have been able to pursue economically viable careers. However, several problems remain.

Spontaneous or trauma-related hemarthroses and bleeding are controlled better using home care programs, which allow on-demand and prompt treatment of bleeds by the use of prophylactic and/or therapeutic infusions of FIX concentrates. This has led to a marked improvement in the quality of life for persons with hemophilia and allows them to participate in activities previously denied to them.

Highly purified FIX concentrates are not associated with thromboembolic complications and are associated with a reduced incidence of transmission of hepatitis and HIV. With currently available products, some individuals with hemophilia B can achieve a normal lifespan.

Death results from central nervous system (CNS) bleeding, progressive hepatitis with hepatic failure, anaphylaxis in children, development of inhibitors with severe bleeding, and AIDS.

Development of inhibitors (alloimmunization) in persons with hemophilia exposed to FIX-containing products or autoantibodies to FIX represents a serious complication, adding to morbidity and mortality.

Race

The disorder is found in all ethnic groups, and it does not have a specific ethnic or geographic distribution.

Ethnic differences in polymorphisms close to or in the FIX gene are important because they provide linkage data when identifying carriers, particularly when the mutation is unknown or for identification of de novo mutations.

A common G10430A mutation (Gly 60 Ser) in the factor IX gene was described in the moderate and mild hemophilia B in the majority of the Gujarati population.^[8]

Sex

The disorder is X-linked, with the FIX gene located on the long arm of the X chromosome. Consequently, males with hemophilia B usually are symptomatic, while females usually are silent carriers (no bleeding disorder).As demonstrated in the diagram below, all female offspring of a male with hemophilia B are obligatory carriers, while no male offspring are carriers. Chances are 50/50 that each female offspring of a carrier female is a carrier and 50/50 that each male offspring of a carrier has hemophilia.

Possible genetic outcomes in individuals carrying the hemophilic gene.

Carrier females usually are asymptomatic but can have bleeding (eg, easy bruising, menorrhagia, or excess bleeding after trauma) when they have significant reductions in FIX levels, which are caused by the greater (extreme) inactivation of the normal FIX gene, compared with the hemophilic FIX gene, during early embryogenesis. Other reasons a female may have clinical bleeding resulting from reduced levels of FIX include X-mosaicism, Turner syndrome, testicular feminization, or situations in which the father has hemophilia B and the mother is a carrier for the disorder. Carriers with basal levels of FIX of less than 30% can be expected to have a clinically evident bleeding disorder.

Age

Hemophilia B can be detected prenatally by measuring FIX activity in fetal blood samples obtained at 20 weeks of gestation by fetoscopy, but the presence of maternal FIX in amniotic fluid complicates the assessment. In addition, the procedure carries a high risk of complications, with a risk of fetal death of up to 6%. Detection of hemophilia B by linkage studies or gene mutation analysis (when the defect is known) can be performed by chorionic villous sampling at 12 weeks of gestation or by amniocentesis from 16-20 weeks, with complication rates of up to 2.0%.

Postnatal evaluation is triggered by a history of bleeding, which can start immediately after birth or, in mild hemophilia, can be delayed to a later age. Newborns without hemophilia have reduced levels of approximately 40%, with a gradual rise in the first year into the low-normal adult range. Prematurity is associated with lower levels due to the immaturity of the liver.

An age- and puberty-related (testosterone induced) rise in FIX levels, with an amelioration in bleeding symptoms, occurs in patients with FIX Leyden.

A review of written guidelines and practices of obstetricians, hematologists, and neonatologists in the United States for the treatment of pregnant carriers and newborns with hemophilia and intracranial hemorrhage (ICH) showed that more than 94% of the major facilities reviewed had no written guidelines. Survey findings led to the following recommendations^{[9, 10]} :

• Vacuum devices and fetal scalp monitors should not be used during vaginal delivery of known carriers of hemophilia
• All infants with intracranial hemorrhage should be evaluated for a bleeding disorder,
• Women with postpartum hemorrhage should have a coagulation workup
• A national registry should be created
• National guidelines should be developed, which should improve care for pregnant women with bleeding disorders