Version May 2024
INTRODUCTION — Hemostasis is the process of blood clot formation at the site of vessel injury. This process begins with the formation of the platelet plug, followed by activation of the clotting cascade and propagation and stabilization of the clot (figure 1).
One of the major multicomponent complexes in the coagulation cascade is the intrinsic X-ase (ten-ase) complex. This complex consists of activated factor IX (factor IXa) as the protease, activated factor VIII (factor VIIIa), calcium, and phospholipids as the cofactors, and factor X as the substrate. Factor IXa can be generated by either factor XIa, activation of the intrinsic pathway, or by the tissue factor/factor VIIa complex. (See "Overview of hemostasis", section on 'Multicomponent complexes'.)
The biology of factor VIII and factor IX is reviewed here. Deficiencies in either of these coagulation factors lead to hemophilia (hemophilia A and hemophilia B, respectively), which is discussed separately. (See "Clinical manifestations and diagnosis of hemophilia".)
FACTOR VIII — The F8 gene is located on the X chromosome. It is one of the largest known genes, divided into 26 exons that span 186,000 base pairs [1,2]. Factor VIII is synthesized as a single chain polypeptide of 2351 amino acids. A 19-amino acid signal peptide is cleaved by a protease shortly after synthesis so that circulating plasma factor VIII is a heterodimer.
Factor VIII circulates in plasma in a noncovalent complex with von Willebrand factor. Cleavage of plasma factor VIII by thrombin or factor Xa at Arg372 and Arg1689 is necessary to activate factor VIII and allow it to participate in the intrinsic pathway X-ase [3,4]. Activated factor VIII (factor VIIIa) is inactivated by activated protein C in conjunction with protein S; the site of proteolytic inactivation is Arg336 in the A1 subunit [3]. Factor VIIIa is also cleaved by activated protein C at Arg562 in the A2 subunit.
Structure — Factor VIII consists of a heavy chain with A1 and A2 domains (figure 2); a connecting region with a B domain; and a light chain with A3, C1, and C2 domains [5-8]. The functions of factor VIII reflect binding at specific sites within the molecule:
C2 domain — The C2 domain binds to the procoagulant phospholipid phosphatidylserine on activated platelets and endothelial cells [9,10] and to von Willebrand factor [10,11]. An overlap exists in the epitope for both binding functions [10,11]. A different site in the C2 domain appears to contain the binding site for factor Xa. In vitro, an antibody directed against this binding site prevents factor VIII activation by factor Xa [12]. A thrombin binding site also is present in the C2 domain that is responsible for factor VIII cleavage at Arg1689 [13].
The importance of the C2 domain is illustrated by observations in non-hemophilic patients with a bleeding tendency caused by acquired autoantibodies against factor VIII. In some of these patients, the antibodies are directed against the C2 domain and prevent interaction with phosphatidylserine [5,6]. (See "Acquired hemophilia A (and other acquired coagulation factor inhibitors)".)
A2 domain — The A2 domain is a site of factor IXa binding, the active enzyme in the X-ase pathway [14-18].
A1/A3-C1-C2 dimer — The A1/A3-C1-C2 dimer also contributes to factor IXa binding [17,18], a part of which occurs within the A3 domain [14,18].
B domain — The heavily glycosylated B domain displays no significant homology to any known protein. It serves as a connecting region that separates the second and the third A domains but is not required for clotting activity [19]. The B domain plays a role in intracellular quality control and factor VIII secretion and potentially regulatory roles in plasma during factor VIII activation, platelet binding, inactivation, and clearance [20].
Antibodies directed against these domains, occurring in patients with hemophilia or as an acquired disorder in patients without hemophilia, can lead to bleeding episodes or, in patients with hemophilia, bleeding that is more difficult to control. In some cases of acquired disease, for example, the antibodies are directed against the C2 domain and prevent interaction with phosphatidylserine [5,6]. (See "Inhibitors in hemophilia: Mechanisms, prevalence, diagnosis, and eradication" and "Acquired hemophilia A (and other acquired coagulation factor inhibitors)".)
Site of production — The site of factor VIII production has been controversial, but studies using gene targeting have identified endothelial cells as the primary (and perhaps exclusive) source of factor VIII synthesis and secretion [21,22]. Endothelial cell knockout of the F8 gene or the gene responsible for factor VIII secretion cause hemophilia in mice, whereas liver-specific knockout of either gene does not affect plasma factor VIII levels. (See "Tools for genetics and genomics: Specially bred and genetically engineered mice", section on 'Knockout mice'.)
Factor VIII production is thus different from all other coagulation factors, which are made by hepatocytes, and similar to von Willebrand factor, which is produced in endothelial cells. (See 'Binding to von Willebrand factor' below.)
Factor VIII was previously thought to be produced at least partially by the liver, based on observations from liver transplantation experiments in animals and clinical results in patients with hemophilia A who undergo liver transplantation for liver failure or hepatocellular carcinoma. Liver transplantation from an animal without hemophilia to a hemophilic animal partially corrected factor VIII deficiency in the recipient, and liver transplantation from a hemophilic animal into a non-hemophilic animal partially depleted factor VIII in the recipient. The presence of a significant vascular bed in the transplanted liver likely explains this finding.
Binding to von Willebrand factor — Factor VIII circulates in plasma in a noncovalent complex with von Willebrand factor (VWF) (figure 2). The interaction between factor VIII and VWF has two main effects:
●Binding to VWF protects factor VIII from proteolytic inactivation by activated protein C and its cofactor, protein S [23-25]. VWF slows the inactivation of factor VIII 10- to 20-fold [24,25]; this prolongs its half-life in the circulation fivefold [23]. It also modulates clearance of the VWF-factor VIII complex [26].
●Binding to VWF is thought to concentrate factor VIII at sites of vascular injury and active hemostasis through the attachment of VWF to subendothelial matrix proteins and adherent platelets. (See "Pathophysiology of von Willebrand disease", section on 'VWF functions'.)
However, a factor VIII fusion protein that prevents factor VIII binding to native VWF resulted in similar platelet accumulation as unmodified recombinant factor VIII in a hemophilia A mouse model [27]. (See "Hemophilia A and B: Routine management including prophylaxis", section on 'Efanesoctocog alfa (factor VIII-VWF fusion)'.)
The C2 domain of factor VIII [10,11] binds to the D1 and adjacent D3 domains of VWF [24]. VWF can bind factor VIII only when factor VIII has not been cleaved by thrombin [28]. After thrombin cleavage, activated factor VIII (factor VIIIa) is released from VWF and becomes a fully functional, active cofactor in the ongoing generation of thrombin; however, it is also now more susceptible to inactivation [25].
The clinical importance of VWF binding can be illustrated by the findings in type 2N von Willebrand disease (VWD) and in some F8 variants leading to decreased binding to VWF:
●Type 2N VWD, transmitted as an autosomal recessive trait, is characterized by pathogenic variants in the VWF gene within the factor VIII binding domain [29,30]. Affected patients present with low levels of factor VIII (usually <10 percent), because of unimpeded proteolytic cleavage of factor VIII, along with a clinical pattern of bleeding similar to that seen in hemophilia A, rather than the predominance of mucosal bleeding associated with classical VWD. Type 2N VWD, rather than hemophilia A, should be suspected in the following situations [31]:
•Families in which an autosomal (rather than X-linked) inheritance pattern for factor VIII deficiency is suggested
•Isolated factor VIII deficiency
•Individuals with a low FVIII level but no likely causative variant found on genetic testing
•Individuals who carry a diagnosis of mild hemophilia but have suboptimal response to factor VIII therapy despite absence of an inhibitor
(See "Pathophysiology of von Willebrand disease", section on 'Stabilization of factor VIII'.)
●Some patients with genetic variants in the C1 domain of the factor VIII molecule were found to have normal factor VIII specific activity (the ratio between factor VIII coagulant activity and factor VIII antigen) but reduced binding to VWF, resulting in mild to moderate hemophilia A [32].
Formation of factor VIIIa — On exposure to thrombin or factor Xa, a nonessential cleavage at Arg740 transiently converts the heavy chain into 92 kd fragments. Concurrently, cleavage at Arg372 converts the heavy chain into 54 kd and 44 kd fragments. At the same time, a small fragment is cleaved from the light chain to separate factor VIII from VWF [28]. These cleavages produce the activated factor VIII heterotrimer factor VIIIa, which is composed of the 54 kd and 44 kd heavy chain fragments and the 72 kd light chain fragment.
Intrinsic pathway X-ase — The physiologic role of factor VIII is to accelerate the cleavage of factor X by factor IXa; the binding sites for factor IXa are in the A2 and A3 domains [14-18]. As noted above, the intrinsic X-ase complex consists of factor IX; the protease; factor X; the substrate; and the cofactors, factor VIIIa, calcium, and phospholipids [4,33]. It has been proposed that factor VIIIa has a stabilizing action that forces the catalytic modules factor IXa and factor X together, markedly accelerating the velocity of the X-ase complex [34]. In addition, the enzyme and the substrate are concentrated on a phospholipid surface of activated platelets or endothelial cells, decreasing the Km by several orders of magnitude.
Factor VIII cannot become part of the X-ase complex until it is released from VWF, because interaction with VWF inhibits its binding to phospholipid surfaces. The separation from VWF requires the cleavage of the factor VIII light chain by thrombin or factor Xa.
Although the generation or exposure of tissue factor at the wound site is the primary physiologic event in initiating clotting via the extrinsic pathway, the intrinsic pathway X-ase also is required for normal hemostasis, as illustrated by the bleeding disorders hemophilia A and B. The importance of the intrinsic pathway reflects the limited amount of tissue factor generated in vivo and the presence of tissue factor pathway inhibitor which, when complexed with factor Xa, inhibits the tissue factor/factor VIIa complex [35]. Thus, sustained generation of thrombin depends upon the activation of both factor IX and factor VIII. This process is amplified because factor VIII is activated by both factor Xa and thrombin [3,4] and factor IX is activated by thrombin-induced activation of factor XI [36-38]; as a result, there is a progressive increase in activation of factors VIII and IX as factor Xa and thrombin are formed [35]. (See "Overview of hemostasis".)
Inactivation of factor VIIIa — Factor VIIIa is an unstable molecule that rapidly loses its cofactor function, in part because it is no longer bound to VWF [24,25]. The inactivation of factor VIIIa occurs by subunit dissociation in a process that is initiated by the formation of thrombin. The binding of thrombin to thrombomodulin induces a conformational change in thrombin such that it acquires the ability to activate protein C and no longer promotes platelet activation or the cleavage of fibrinogen (see "Overview of hemostasis", section on 'Activated protein C and protein S'). Activated protein C (APC) proteolytically inactivates factors VIIIa and Va, thereby inactivating the intrinsic X-ase and prothrombinase, respectively [39,40]. Factor VIIIa is first cleaved at Arg336 within the A1 subunit and then Arg562, bisecting the A2 subunit [40].
The rate of APC-mediated inactivation of factor VIIIa is increased several fold by protein S, in concert with APC-inactivated factor V [41], which facilitates cleavage of the A2 subunit; this latter effect is impaired by factor IXa [40]. APC has only a limited effect on non-activated factor VIII, which is protected from catabolism by binding to von Willebrand factor [23-25]. The A2 and A3 domains interact with a macrophage receptor low-density lipoprotein receptor-related protein, enabling these fragments to be removed from the circulation [42]. The sinusoidal endothelial receptor CLEC4M has also been implicated in VWF-independent factor VIII clearance [43].
Elevated levels of factor VIII — Elevated plasma factor VIII coagulant activity is now accepted as an independent marker of increased thrombotic risk. This subject is discussed separately. (See "Overview of the causes of venous thrombosis", section on 'Factor VIII'.)
FACTOR IX — The F9 gene is located near the terminus of the long arm of the X chromosome. The gene codes for a signal peptide, a propeptide (promoter region), and the mature 415 amino acid protein that circulates in the plasma (figure 3). Vitamin K-dependent gamma-carboxylation of specific glutamic acid residues occurs as a posttranslational process in the endoplasmic reticulum of the hepatocyte. The propeptide participates in defining a recognition site that designates an adjacent glutamic acid-rich domain for gamma-carboxylation [44]. The amino acids of this recognition site bind directly to the vitamin K-dependent carboxylase [45]; disruption of this site leads to deficient or absent gamma-carboxylation [44]. The processing steps, through which the signal peptide and the propeptide are removed, occur in the Golgi apparatus prior to secretion.
Structure and function — Factor IX is a two-chain, disulfide-linked molecule, composed of a light chain (MW 18,000) and a heavy chain (MW 28,000). The N-terminal light chain consists of a gamma-carboxyglutamic acid domain containing 12 gamma-carboxyglutamic acid (Gla) residues dependent upon the vitamin K carboxylase system, followed by a short hydrophobic stack domain that is not required for activation, two epidermal growth factor (EGF)-like domains, and the activation peptide and its flanking sequences [34,46-48].
Gla confers calcium-binding properties on the vitamin K-dependent proteins. The addition of calcium causes factor IX to undergo a structural transition that leads to exposure of a phospholipid binding site [49-51]. The calcium-stabilized form of factor IX binds to membranes, whereas the magnesium-stabilized form does not [51].
The EGF-like domains share homology with protein C and factor X, and they may function as binding sites for platelet flanking sequences. A calcium-binding region in the EGF-like domains appears to mediate protein-protein interactions [52].
The first EGF-like domain is required for activation by tissue factor/factor VIIa but is not essential for activation by factor XIa or for phospholipid binding [53]. The second EGF-like domain is required for the formation of the intrinsic pathway X-ase on the surface of activated platelets [54]. However, the residues responsible for binding to activated platelets are different from those that mediate formation of the X-ase [55].
The physiologic importance of the EGF-like domains in factor IX function is illustrated by the following observations in humans:
●Deletion of the first EGF-like domain leads to severe hemophilia B [56].
●Pathogenic variants in the second EGF-like domain also are associated with hemophilia B; one such variant is associated with a 50-fold reduction in activation by factor XIa (compared to wild-type factor IX), a 20-fold reduction in activation by the tissue factor/factor VIIa complex, and a 14-fold reduction in activation of factor X [57].
The N-terminal also contains the site for binding of factor IX to the endothelium, which appears to be mediated through collagen IV [58].
The C-terminal heavy chain comprises the catalytic domain of factor IXa. This domain possesses a trypsin-like serine protease fold consisting of two interconnected domains with the catalytic residues His221, Asp269, and Ser365 found at the interface of the two domains [34]. In addition, numerous surface loops exist, connecting the secondary structure elements.
The serine protease recognizes specific cleavage sites within factor X and is also a target of protease inhibitors, which play an important role in the inactivation of the enzyme. The variable surface loops bordering the substrate binding groove contribute to the specific interaction with both inhibitors and substrates [59-61]. Factor VIIIa has a stabilizing action that forces the catalytic modules factor IXa and factor X together, markedly accelerating the velocity of the X-ase complex [34] (see 'Intrinsic pathway X-ase' above).
Activation — Factor IX, like factor VIII, is a zymogen that must be activated to factor IXa to participate in coagulation (figure 3). Activation occurs in two ways:
●By the tissue factor/factor VIIa complex of the extrinsic pathway X-ase [62-64]
●By factor XIa [38,65], which is formed during activation of the intrinsic coagulation pathway (figure 1), or by thrombin [36,37]
Factor IX is activated by cleavage of peptide bonds Arg145-Ala146 and Arg180-Val 181, resulting in cleavage of the activation peptide [66]. As noted above, factor IXa is the active protease in the intrinsic pathway X-ase, with factor VIIIa, calcium, and phospholipids as cofactors [4,33].
Inactivation of factor IXa — Factor IXa is inactivated slowly by antithrombin, a serpin that forms a stable equimolar complex at the Asp359 substrate binding pocket [67]. AT also neutralizes thrombin and factors Xa, XIa, and XIIa.
NATURALLY-OCCURRING GAIN-OF-FUNCTION VARIANTS — Naturally-occurring variants in the F8 and F9 genes have been reported that increase circulation factor levels and/or activity.
These variants may cause inherited thrombophilia, although the prevalence and relative risk of thrombosis compared with other thrombophilias is unknown, and testing for these conditions is not performed routinely. (See "Overview of the causes of venous thrombosis", section on 'Elevated clotting factors and chemokines'.)
Factor VIII Padua — A case of inherited thrombophilia associated with a gain-of-function variant in the F8 gene (tandem duplication of the promoter, exon 1, and most of intron 1) was reported in 2021 [68]. The proband and related relatives had multiple deep vein thromboses and a factor VIII activity (and antigen) levels >400 percent. Screening of 50 unrelated VTE patients with very high factor VIII levels identified a second pedigree with the identical F8 variant, suggesting a founder effect.
Factor IX Padua — A case of inherited thrombophilia associated with a gain-of-function variant in the F9 gene (F9-R338L, factor IX Padua) was reported in 2009 [69]. The plasma level of the factor IX was normal, but the clotting activity was approximately 8 times normal due to a faster rate of factor X activation [70].
This variant is being used in F9 gene therapy constructs as a means of increasing the levels of circulating factor IX activity relative to wild-type F9 and the viral vector, potentially increasing efficacy and reducing vector-related toxicity. (See "Gene therapy and other investigational approaches for hemophilia".)
SUMMARY
●Role in coagulation cascade – Factors VIII and IX are major components in the generation of the intrinsic pathway X-ase (ten-ase) complex, which, in activated forms (ie, factors VIIIa and IXa) in conjunction with calcium and phospholipids, convert factor X to activated factor X (ie, factor Xa). Factors VIIIa and IXa are responsible for amplification of the coagulation cascade after initial thrombin generation through the extrinsic pathway (figure 1). (See 'Intrinsic pathway X-ase' above and "Overview of hemostasis", section on 'Clotting cascade and propagation of the clot'.)
●Factor VIII – The F8 gene is located on the X chromosome. It is one of the largest known genes, divided into 26 exons that span 186,000 base pairs. Pathogenic variants in the F8 gene are responsible for the sex-linked bleeding disorder hemophilia A. (See 'Factor VIII' above and "Genetics of hemophilia A and B", section on 'F8 gene (hemophilia A)'.)
•Factor VIII circulates in plasma in a noncovalent complex with von Willebrand factor (figure 2). (See 'Binding to von Willebrand factor' above.)
•Cleavage of plasma factor VIII by thrombin or factor Xa is necessary to activate factor VIII and allow it to participate in the intrinsic pathway X-ase complex. Activated factor VIII is inactivated by activated protein C in conjunction with protein S.
●Factor IX – The F9 gene is also located on the X chromosome. Pathogenic variants of this gene are responsible for the sex-linked bleeding disorder hemophilia B. (See 'Factor IX' above and "Genetics of hemophilia A and B", section on 'F9 gene (hemophilia B)'.)
•Factor IX contains 12 gamma-carboxyglutamic acid (Gla) residues dependent upon the vitamin K carboxylase system. Gla confers calcium-binding properties on the vitamin K-dependent proteins, which allows factor IX to bind to phospholipids. (See "Vitamin K-dependent clotting factors: Gamma carboxylation and functions of Gla".)
•Factor IX must be activated to factor IXa to participate in coagulation (figure 3). Activation occurs via the tissue factor/factor VIIa complex of the extrinsic X-ase pathway as well as by factor XIa. Factor IXa is inactivated slowly by antithrombin.
●Gain-of-function variants – Naturally-occurring gain-of-function variants in the genes for factors VIII and IX have been described. The F9 variant is being used in gene therapy constructs for hemophilia B. (See 'Naturally-occurring gain-of-function variants' above.)
ACKNOWLEDGMENTS — The editorial staff at UpToDate acknowledge W Keith Hoots, MD, and Amy D Shapiro, MD, who contributed to earlier versions of this topic review.
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