The human Factor VIII, also known as anti-hemophilic factor (AHF), is an essential blood-clotting protein [1]. It consists of 2332 residues [2], whose gene is located on the X chromosome [1][3].
Factor VIII is produced inside the liver (by the sinusoidal cells) and outside (by the endothelial cells) and acts in the intrinsic pathway of blood coagulation [1]. It is actually the lack or the deficiency of the factor VIII (which is a plasma glycoprotein) that causes a bleeding disorder: hemophilia A [2].
Factor VIII is much studied in order to find a cure for hemophilia A (also written as HEMA), for instance by designing mimicking factors [4].
History
1937: first use of the factor VIII (known at this period as “Antihemophilic Globulin”) to cure the blood coagulation disorder of hemophilia patients thanks to the discovery of F.H.L Patek and A.J Taylor [5].
1964: Usual utilisation of concentrated factor VIII to treat hemophilia [6].
1984: Factor VIII was first characterized by scientists at Genentech [7].
2017: Concentrated factor VIII with extended half-life [8].
Function
Factor VIII plays a central role in blood coagulation
“Factor VIII” is an inactive form. The Factor VIII circulates in the bloodstream in this inactive form, bound to another molecule called Von Willebrand Factor, until an injury that damages blood vessels occurs. Indeed, in plasma, factor VIII exists in two forms: free or as a complex with the von Willebrand factor. The complex is the predominant form and exists at a concentration of 0.1 µg/ml in the blood because factor VIII is stabilized by von Willebrand factor, while in its free state, it is rapidly cleaved by protease serines [1][7].
The coagulation process
In response to an injury, the coagulation factor VIII is separated from von Willebrand factor. The active form is called “Factor VIIIa” and is obtained by a proteolytic cleavage of the B-domain of Factor VIII by thrombin [1][2]. Then the two remaining chains are linked together thanks to a metal link (probably calcium ion) [2].
Thus the factor VIIIa is a non-covalent dimer [2].
It is the catalyst for the activation reaction of the factor X (to Factor Xa) by activated Factor IXa in the presence of calcium ion and phospholipids.
The factor X activation reaction by factor IXa is accelerated approximately 200,000 times when factor VIII interacts with factor IXa. [1][2][7]
Then, no longer protected by the von Willebrand factor, the factor VIIIa is proteolytically inactivated and quickly cleared from the blood stream, whereas, factor Xa becomes able (with the help of other factors) to stop the bleeding by forming a blood clot. [1][7]
Structure
Primary Structure
In humans, factor VIII is encoded by the F8 gene [2][9][10]. This gene maps on the most distant band of the long arm of the X-chromosome (region Xq28). It is 186 kb in size (0.1 % of the whole size of the chromosome) and contains 26 exons [3].
Secondary Structure
Factor VIII protein is composed of six globular domains: A1-A2-B-A3-C1-C2 and contains one Ca2+ and two Cu2+ ions. It has a molecular weight of 330 kDa [2][7][9].
The three A domains are homologous to the A domains of the copper-binding protein ceruloplasmin [1][7] . Together, they form a triangular heterotrimer where the A1 and A3 domains interact with the C2 and C1 domains, respectively [2].
The C domains belong to the phospholipid-binding discoidin domain family [1]. They are adjacent at the base of the triangular heterotrimer. Moreover, C1 and C2 domains are structurally homologous and they have the ability to bind the membrane. Indeed, both C domain protrude three β-hairpin loops with hydrophobic and basic residues in the same direction. Thanks to these loops the factor VIII might interact with the phospholipid bilayer. [2]
Factor VIIIa is obtained by cleavage and release of the B domain [1][2][11]. Although factor VIIIa can be formed from at least two cleavages involving Arg372 and Arg1689, fully factor VIIIa is obtained by a third cleavage at Arg740 [2].
The two chain that result are a heavy and a light chains [2][7][10].
The has a various size (90 or 120 kDa) [1][12]. It consists of the A1-A2 domains [1][7][12]. Both A1 and A2 domains are built up of two connected β barrels [2].
The has a molecular weight of 80 kDa and is composed of 684 amino acids [12]. It contains two domains: a unique A domain of 371 amino acids and a duplicated C domain of 153 amino acids and 160 amino acids, respectively [12]. These domains are ranked in the following order A3-C1-C2 [1][12]. It is composed of 42 % irregular structure, 36 % β-strands, and 22 % α-helices [12]. The C1 and C2 domains are defined by a distorted β barrel, while A3, as well as A1 and A2, is composed of two connected β barrels [2]. This chain also contains of the major binding site of von Willebrand Factor at its N-terminus [12].
Both chains are no covalently associated through to a calcium ion to form the active heterodimers [2][12]. This complex is the pro-coagulant factor VIIIa [1].
Such an association is essentialfor the functioning of the factor VIII [12].
Ligands
Alpha-D-mannose, calcium ion (Ca2+), copper ion (Cu2+) and N-acetl-D-glucosamine are the four ligands the factor VIII is able to bind to [10].
In factor VIII there are two copper ions and their binding sites are located internally within the and the domain. In the latter, there is another ligand, a , bound to its binding site [2].
Disease
Hemophilia is a genetic disorder characterized by a permanent tendency to hemorrhage because of a lack of blood coagulation [2].
There are different types of hemophilia: A or B, caused by a deficiency of two different factors.
Hemophilia A (HEMA), is four times as common as hemophilia B.
It is caused by a deficiency of factor VIII. [13]
This deficiency in factor VIII clotting activity results in prolonged oozing after injuries, tooth extractions, or surgery, and delayed or recurrent bleeding prior to complete wound healing [14].
Although hemophilia A is usually an inherited disease and therefore runs in families [13], about one-third of people with the disease are caused by a spontaneous mutation [13] such as misense or nonsense mutations, gene deletions or inversions [2].
Inheritance
Hemophilia A is inherited in an X-linked recessive manner:
Females inherit two X chromosomes, one from their mother and one from their father (XX). Males inherit an X chromosome from their mother and a Y chromosome from their father (XY). This means that if a son inherits an X chromosome carrying hemophilia from his mother, he will have hemophilia. By contrast, daughters have two X chromosomes, even if they inherit the hemophilia gene from their mother, they inherit a healthy X chromosome from their father and as a result they are only carrier but not affected.
Thus, because of the recessivity only men are affected by this disease and women are carriers that may pass the gene on to their children (50% chance of transmitting it in each pregnancy). [13]
The risk for boys to carry the disease therefore depends on the carrier status of the mother because affected males transmit the pathogenic variant to all of their daughters and none of their sons [14].
Hemophilia A can be mild, moderate, or severe, depending on the level of Factor VIII clotting activity:
• Severe hemophilia A: factor VIII’s proportion in the blood ≤ 1%
• Moderate hemophilia A: 1% ≤ factor VIII’s proportion in the blood ≤ 5%
• Mild hemophilia A: 6% ≤ factor VIII’s proportion in the blood ≤ 40% [14][15]
The major treatment of the bleeding disorder associated with hemophilia A is the infusion of factor VIII, which leads to the correction of hemostasis [13].
Relevance
Hemophilia occurs in approximately 1 in 5,000 live births but it is severe in approximately 60% of cases.
The main medication to treat hemophilia A is concentrated factor VIII protein, called “clotting factor”. Getting this “clotting factor” is therefore a major concern for hemophilia-affected people [13].
Nowadays, recombinant coagulation factor VIII products, which are developed in a lab through the use of DNA technology [7][11]. For instance, Toole and colleagues have created a biologically fully active factor with improved heterologous expression efficiency by deleting the B-domain from the native human factor VIII [11].
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