Authors: Mariarosaria Miranda1, Eleonora Nardini2, Puneet Tomar3 and Sarah Scatigna4
Affiliations: 1Stichting Sanquin Bloedvoorziening, 2Vumc, 3University of Milan, 4Goethe University
Factor VIII plays a key role in the intrinsic pathway of the blood coagulation cascade. In its active form, FVIIIa acts as a cofactor for the proteolytic activation of FX by the serine protease FIXa.
Biosynthesis and secretion of Factor VII
The FVIII encoding gene, located on the long arm of the X chromosome, encodes a polypeptide chain of 2351 amino acids. This includes a signal peptide of 19 and a mature protein of 2332 amino acids, composed of a domain structure: A1-a1-A2-a2-B-a3-A3-C1-C2. The A domains display homology to each other, to the copper-binding protein ceruloplasmin and to factor V. These domains are flanked by acidic regions (a1, a2 and a3), which contain clusters of Asp and Glu residues. The C domains are homologous to the C domains of FV. In contrast, the B domain is unique (1).
Although several tissues, such as spleen, lymph nodes and kidney, contribute to the presence of FVIII in circulation, the primary source is the liver (2). The mature polypeptide is secreted into the lumen of the endoplasmic reticulum (ER), where it undergoes N-linked glycosylation. Then, FVIII is transported and further processed into the Golgi apparatus. Some of the main modifications are the O-linked glycosylations and the sulfation of specific Tyr-residues. Prior to its secretion into the plasma, FVIII is subjected to intracellular proteolysis at the B-A3 junctions and within the B domain, which disrupts the covalent linkage of the FVIII heavy chain (A1-a1-A2-a2-B) and light chain (a3-A3-C1-C2). The outcome is a metal ion-linked heterodimers (3) (Fig.1). The main ions involved in the promotion of the reassembly of dissociated heavy and light chains and in the enhancement of cofactor function are calcium, manganese and copper ions (4).
Factor VIII-Von Willebrand factor (vWF) complex assembly and FVIII activation
In circulation, the heterodimeric FVIII interacts in a tight and noncovalent manner with its carrier proteins, the Von Willebrand factor. The binding to vWF involves the A3 domain and two C2-domain regions. The functional purpose of the FVIII-vWF complex is to prevent the premature formation of the Xase complex. Since the FVIII affinity for vWF exceeds that for FIXa, FVIII highly favors vWF binding. In addition, the close proximity between the phospholipids and vWF binding sites on FVIII makes the FVIII-vWF complex incompatible with FVIII binding to the membrane. As a consequence, FVIII-vWF complex is less susceptible to proteolytic attack by lipid-binding proteases. Indeed, these proteases, which include activated protein C (APC) and FXa, efficiently inactivate FVIII when it binds the membrane surface. In contrast, thrombin has a proteolytic activity independent of a membrane surface. For these reasons, vWF does not protect FVIII against thrombin cleavage (3).
Thrombin and FXa share some cleavage sites. Thrombin cleaves at one site in the light chain, Arg1689, and at two sites in the heavy chain, Arg372 (A1-A2 junction) and Arg740 (A2-B junction). The cleavage of FVIII by FXa involves three sites in the heavy chain, Arg336, Arg372 and Arg740, and two sites in the light chain, Arg1689 and Arg1721. Both the cleavages at Arg372 and Arg1689 are required to exert the cofactor activity, whereas cleavage at Arg740 is not rate limiting. Cleavage at Arg1689 releases an acidic fragment that leads to the dissociation of vWF. The final product is a metal-linked hetero-trimer lacking the B domain (4) (Fig. 2).
The active FVIII loses the binding with vWF and binds to FIXa forming a well-organized membrane-bound complex at the phosphatidylserine rich activated platelet membrane in the presence of Ca2+ ions (5). The role of the membrane surface could be explained in two different ways: the membrane contributes both to position enzyme-cofactor complex in an active conformation and to locate the enzyme and the cofactor in the same site (3).
The formation of FVIIIa-FIXa complex, also known as tenase complex, involves the binding both of the factor VIII A2 domain to the factor IXa heavy chain, and of the factor VIII A3 domain to the factor IXa light chain. FVIIIa induces a conformational change in the protease domain of FIXa, thus enhancing the rate of activation of FX by FIXa by more than 105 times (4).
Inactivation and clearance of FVIII
The factor VIII protein has a half-life of 12 hours in adults (shorter in children), when stabilized by the von Willebrand factor. No longer protected by vWF, activated FVIII is inactivated and quickly cleared from the bloodstream (6).
Inactivation of FVIII involves both proteolytic degradation and spontaneous dissociation. In contrast to inactive FVIII, FVIIIa is more unstable. Its proteolytic degradation is due to cleavages in positions 336 and 562 of the heavy chain by diverse enzymes, such as FIXa, FXa and APC, that affect both intramolecular and intermolecular interactions (7).
The clearance of FVIII from the bloodstream involves hepatic receptors, such as the low-density lipoprotein-related protein 1 (LRP1) and the low-density lipoprotein receptor (LDLR). LRP1 catabolizes FVIII and FVIII-vWF complex, whereas LDLR acts in concert with LRP1 in catabolizing free FVIII (8) (9).
Mutation and diseases
Mutations that affect expression, secretion and stability of FVIII lead to the onset of bleeding disorders.
Aberrant biosynthesis, secretion, activation and clearance of FVIII may be caused by gross deletions, rearrangements and missense mutations in FVIII encoding gene. The qualitative or quantitative deficiency of FVIII results in an X-linked recessive disease, known as Hemophilia A (10).
However, reduced levels of FVIII could also be related to mutations that affect other genes. This is the case of the combined deficiency of factor V and factor VIII, caused by mutations in the genes that encode ERGIC-53 and MCFD2, two cargo receptors that facilitate the transport of FV and FVIII from ER to the Golgi apparatus (11). Furthermore, altered levels of FVIII could be detected in patients affected by some variants of von Willebrand disease (vWD) (12).
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