Who invented factor 8

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The total length of the coding sequence of this gene is 9 kbp [ 10 , 11 ] Fig. Transcribed are two products of alternative splicing. The number of different recorded mutations per bp of the coding sequence is shown. Numbers of histogram columns correspond to the exon numbers, names of protein domains are stated below the exon numbers. Length of exon 1 in mRNA is b; the only coding part of this exon including the signal peptide , b, was included in the calculations; length of exon 26 in mRNA is b, the only coding part of this exon — b — was used in the calculations.

Primary data taken from [ 3 ], extracted July 18, Three acidic subdomains, which are denoted as a1—a3 — A1 a1 —A2 a2 —B— a3 A3—C1—C2, localize at the boundaries of A domains and play a significant role in the interaction between FVIII and other proteins in particular, with thrombin. Mutations in these subdomains reduce the level of factor VIII activation by thrombin [ 19 , 20 ]. There currently is some controversy regarding the accurate definition of the boundaries of FVIII domains; the most common versions are listed in Table 1.

The region — of the A2 domain determines the binding of factor IXa and its conformational rearrangement within tenase [ 28 ] Fig. Core residues of N-linked glycans are shown in red, FVIII heavy chain is shown in blue, light chain is shown in green.

N-glycosylation sites are denoted by circles. Filled black circles — occupied sites, half-filled circle — partially occupied site, filled gray circle — presumably occupied site, red circles — unoccupied sites. Disulphide bonds are denoted by brackets, gray bracket — presumably existing disulphide bond.

Red vertical lines — reduced Cys residues, the actual state of Cys residues in the B domain is unknown. S inside a circle — sulfated Tyr residues.

SP — signal peptide and propeptide. These domains are capable of binding glycoconjugates and acidic phospholipids [ 33 ]. Numbers represent the homology level of amino acids for domain groups. Discoidin I was obtained from D. The B-domain encoded by a single long exon is partially removed from the mature protein. The B-domain contains 25 potential N-glycosylation sites, 16—19 of which are occupied and exhibit a significant level of microheterogeneity.

The homology of FVIII B-domains in humans and mice is low; however, these domains are highly glycosylated in both species, which can attest to the significance of this modification for post-translational processing of a protein [ 35 ].

Interestingly, the functional A and C domains of these proteins are conserved, while the similarity between the B-domains is limited to a high degree of glycosylation, which also attests to the functional significance of the high density of oligosaccharide groups in the FVIII B-domain [ 17 , 25 — 27 ]. The highly glycosylated B-domain can participate in the intracellular transport of the FVIII precursor and its processing.

However, abundant experimental data have demonstrated that deletion of the B-domain region enhances the secretion of functionally active FVIII [ 36 , 37 ]. The interaction between the FVIII polypeptide chain and metal ions determines the structural integrity of the mature protein and its cofactor function. The presence of copper ions within FVIII has been demonstrated by atomic adsorption spectrometry; dissociation of the FVIII chains results in complete dissociation of copper ions [ 38 ].

In turn, the reassociation of the split FVIII chains is possible only in the presence of copper salts [ 39 ]. The presence of two coordinated copper ions in direct contacts with the H, C, H and H, C, and H residues i.

Both copper ion binding pockets localize near the contact surface of the A1 and A3 domains; however, they do not directly participate in the formation of noncovalent bonds between the domains. Simultaneously, evidence of functional significance has been obtained only for the binding site of copper ions in the A1 domain both via point substitution of cysteine residues [ 40 ] and by direct monitoring of the coordination of copper ions by FRET [ 39 ]. The CF mutation in the FVIII gene [ 2 ] causing a severe form of hemophilia A is an additional argument in favor of the physiological significance of the copper-binding site in the A1 domain.

Both copper ions and calcium or manganese ions are required to recover the procoagulant activity of FVIII during chain dissociation—re-association [ 43 , 44 ]. Calcium or manganese ions do not affect chain dimerization, but they ensure that the heterodimer FVIII molecule has an active conformation [ 39 ] as they bind to the sites located on both protein chains [ 45 , 46 ]. FVIII is synthesized in the liver, which has been supported by the fact that liver transplantation can cure hemophilia A.

When isolating and purifying liver cell populations, it has been ascertained that secretion of significant amounts of FVIII 0. No successful attempts to immortalize cultured liver sinusoidal endothelial cells have been documented thus far; hence, all the experimental data on the features of FVIII biosynthesis have been obtained using heterologous expression systems that are usually characterized by artificially increased productivity [ 49 ].

Translocation of the growing FVIII polypeptide chain to the lumen of the endoplasmic reticulum ER , processing of the signal amino-acid-long peptide, and the primary events of disulfide bond formation and attachment of the high-mannose nuclei of N-linked oligosaccharides to the FVIII chain seem not to limit the total rate of its biosynthesis and have been thoroughly described in [ 47 ].

Meanwhile, the subsequent events of modifying oligosaccharide chains, disulfide isomerization, and folding of FVIII molecules may overload the corresponding enzyme groups in the cell and activate the systems responsible for retention of the incorrectly processed proteins in ER or the recycling systems for these proteins.

After the third glucose residue is eliminated, the protein is normally released from its complex with CN X and CRT and is transferred to the Golgi apparatus. Indeed, it has been demonstrated in pulse-chase experiments with proteasomes inactivated by lactacystin [ 50 ] that a significant portion of FVIII undergoes degradation via the ER AD pathway instead of being translocated to the Golgi apparatus; however, in those experiments proteasome inactivation increased the amount of intracellular FVIII but not its concentration in the culture medium.

Since the major fraction of Nlinked oligosaccharides in the FVIII molecule is localized in the B-domain, the deletion FVIII variants are less susceptible to retention in the ER during the calnexin cycle, which partially explains their increased level of secretion.

There are no conclusive data on the state of the cysteine residues in the B-domain. When conducting a series of substitutions of cysteine residues by serine or glycine residues, S. Pipe et al. It is rather possible that the removal of the only exposed disulfide bond results in suppression of FVIII retention in the ER occurring due to the translocation control by the disulfide isomerases [ 53 ]. However, the specific mechanism of such control with respect to FVIII and the participating proteins has hardly been studied.

BiP synthesis is typically induced when cells experience glucose starvation, during N-glycosylation inhibition, and in the presence of incorrectly folded proteins in the ER [ 55 ] in particular, during FVIII overexpression [ 56 ]. Since the F residue is adjacent to C, the key residue in the copper-coordination site in the A1 domain, one can assume that BiP interacts in the attachment of copper ions to FVIII as well.

The replacement of the region — of FVIII by the homologous region of FV reduces its degree of aggregation and affinity to BiP, as well as increases secretion [ 60 ]. This motif is supposed to contain a conformational epitope and a carbohydrate moiety to ensure that only the correctly folded and post-translationally modified proteins can be transported.

It is thought to be released due to the change in the local pH value and calcium concentration [ 71 ]. FVIII seems to be further transported inside vesicles of unknown composition or via transport containers associated with microtubules.

Six active sites of sulfation of tyrosine residues at positions , , , , , and have been detected in human FVIII; they predominantly localize near the acidic subdomains a1, a2, a3 and surround the points where FVIII is cleaved by thrombin. All the six sulfation sites are required to ensure the full activity of factor VIII; the inhibition of sulfation has resulted in a fivefold decrease in the functional activity of FVIII [ 74 ].

It has also been demonstrated that sulfation of the Y residue is required to ensure efficient interaction between FVIII and the von Willebrand factor. The natural mutation of YF manifests itself as moderate hemophilia A. In patients carrying this mutation, FVIII retains its normal activity level but is characterized by a decreased half-life value [ 75 ]. Kaufman et al. The necessity of sulfating residue so that the complex with the von Willebrand can form has also been confirmed in [ 76 ]. The final stage of FVIII processing in the trans -Golgi prior to the secretion involves limited proteolysis of the single-chain precursor at residues R and R, giving rise to a light and a heavy chain [ 22 ].

Mature natural FVIII, which can occur in one of several forms with a molecular weight of — kDa, is present in the blood plasma at a concentration of 0. Almost all the FVIII in plasma is complexed with a chaperone, the von Willebrand factor, which is secreted by vascular endothelial cells. The FVIII regions responsible for binding to this chaperone have been mapped in the light chain: in the acidic subdomain a3 [ 78 ], domains C2 [ 34 , 79 ] and C1 [ 80 ].

In a significant number of hemophilia A patients, inhibitors of injected exogenous FVIII emerge in the bloodstream, blocking its procoagulation activity [ 90 ]. The etiology of emergence of inhibitory antibodies has not been elucidated; some particular correlations between the emergence of inhibitory antibodies and the HLA haplotype [ 92 ] or the nature of the mutation of the factor VIII gene [ 93 ] were recently found.

IgG antibodies are the predominant class of inhibitory antibodies [ 94 ]. The area of direct interaction between FVIII and FX has been found in the acidic C-terminal subdomain of the A1 domain — [ 71 , 77 ]; however, this interaction most probably has no significant effect on the function of the tenase complex. The presence of phospholipids is required for FVIII to perform its cofactor function [ 7 , , ]. FVIII interacts in vivo with the phospholipids of activated platelets and damaged endothelial cells.

FVIII activation increases its affinity to phospholipids by 10 times [ ]. Tenase complex assembly on the cell membrane. Blood clotting factors are bound to the membrane surface, Factor III is the integral membrane protein. The thickness of the reaction arrows corresponds to the reaction rates.

When activating FVIII by thrombin, the breakdowns are introduced at positions R, R, and R [ ] and result in removal of the B domain, in cleavage of the heavy chain into the A1 and A2 domains that remain noncovalently bound, and in elimination of the short acidic region a3 preceding the A3 domain. In a number of publications, the region a3 is referred to as the FVIII activation peptide; however, efficient activation of FVIII cannot be reduced simply to elimination of region a3 from the molecule.

It involves the introduction of breakdowns at positions R and K36 [ 73 ], resulting in destabilization of the A1 domain and in the accelerated dissociation of the unbound A2 domain. The FVIII—vWF complex is mainly eliminated from the bloodstream by the specialized clearance receptor LRP low-density lipoprotein receptor-related protein that localizes on the hepatocyte membrane [ — ].

Today, there are three generations of pharmaceuticals based on recombinant blood-clotting factors [ 68 ]: the first-generation drugs contain human serum albumin and contact with animal-derived compounds during the production process; the excipients list of the second-generation drugs contains no albumin; in the third-generation drugs, contact with animal-derived compounds and components of the donated plasma is ruled out during the entire production process.

The minimization of the use of plasma components and animal- derived proteins can potentially reduce the risk of transmission of viral and prion infections [ ]. No confirmed evidence of transmission of infectious agents when using the first- and second-generation drugs based on recombinant FVIII has been documented thus far.

The recombinant FVIII drugs inevitably contain trace amounts of proteins from the producer cells and murine IgG; thus, the emergence of antibodies against these impurities in patients and the effect of these antibodies on the effectiveness of therapy have been studied during clinical trials. The antibodies formed in most patients; however, the relationship between the immune response to impurity proteins and the effectiveness of therapy has not been elucidated [ 68 ].

The natural FVIII circulating in the bloodstream contains multiple forms of the truncated B domain, which are formed by proteolysis of a full-length two-chain molecule.

The procoagulant properties of these FVIII variants have no significant differences [ ]; thus, variants of the recombinant FVIII with targeted deletion of the B domain have been obtained and characterized in a number of studies. The region encoding the amino acid residues — i.

A similar predominant accumulation of the single-chain FVIII form was also detected in the cases of deletion of regions —, —, and — [ 74 ]. The partial deletion of the amino acid residues to in the B domain variant gave rise to a fully active FVIII [ ]; however, this variant when injected into rabbits caused the emergence of specific antibodies against the protein linker region [ ], which could potentially have increased the frequency of formation of inhibitory antibodies when used in therapy.

Contamination of the product with the nonprocessed form of the light chain 90 kDa , which seems to carry the C-terminal fragment of the B domain, remains the only but unavoidable limitation of this FVIII expression system. When conducting a systematic, exhaustive search, P. Lind et al.

Meanwhile, no significant proteolytic chain cleavage at other sites has been observed. In this deletion variant, the linking point between the polypeptides of the heavy and light FVIII chains lay in the middle of the separating residue-long linker peptide.

The predominant cleavage of the precursor after residues R and S i. The lyophilized form of BDD SQ was stable for two years [ ] and was used in subsequent clinical trials, which demonstrated its pharmacological effectiveness and safety [ ]. The productivity of a similar cell line based on CHO cells, which had been obtained independently, was 0. The effectiveness and safety of FVIII BDD SQ drugs has been confirmed by clinical trials [ — ]; however, the subsequent meta-analysis of the data of numerous post-marketing studies has cast doubt.

Gruppo et al. The resistance to slight variations in the initial data robustness of the employed meta-analysis method has been discussed in a separate publication and has been confirmed for a wide range of coefficients for recalculating the number of cases of bleeding observed in different studies [ ]. Since the probability of emergence of inhibitors varies widely depending on the mutation type that caused hemophilia, certain HLA genotypes, and specific features of the substitution therapy, the data obtained in different medical centers may differ to a significant extent [ ].

However, the degrees of processing of the single-chain form for BDD SQ and human-cl variants turned out to be almost identical. The expression system of the deletion FVIII variant, which seemed to exhibit the highest efficiency at that moment, was obtained using special hybrid human cells HKB This is attributable to the improved transport of the precursor protein from the ER to the Golgi apparatus [ ] as compared with the full-length form and to a decrease in adsorption of the secreted FVIII onto the membrane surface of producer cells [ ] as compared with the regular deletion variants.

A significant decrease in the transcription level of hybrid genes containing an open reading frame ORF of FVIII were first described in studies devoted to the cultivation of retroviral vectors [ , ]. The observed effect depended on orientation and, to a significant extent, on the position. It was delocalized as the removal of different parts of the ORF fragment under study resulted in partial recovery of the transcription elongation level.

An orientationindependent bp-long transcriptional silencer was later detected in another region of FVIII ORF [ ]; its activity was suppressed by sodium butyrate [ ], which made it possible to enhance the level of FVIII secretion in the cell culture approximately sixfold.

Stress induced by processing of the FVIII precursor can be limited by suppression of FVIII gene transcription in the dividing culture, while only the dense nondividing cell culture is exposed to stress during subsequent induction of FVIII expression by adding sodium butyrate. The replacement of some codons by ones characterized by the highest frequency for H. Similar data were obtained for overexpression of the anti-apoptotic genes Aven and E1BK [ 72 ].

These data attest to the fact that this pathway of anti-apoptosis re-engineering of FVIII producers can be efficient only for a super-dense BHK cell culture. The addition of the antioxidant butylated hydroxyanisole to the culture medium simultaneously with sodium butyrate an agent inducing FVIII gene expression enabled a fourfold increase in the secretion level of the full-length FVIII.

FVIII predominantly binds to phospholipid membranes containing phosphatidylserine. An increase in FVIII adsorption on the membrane of apoptotic cells which also contains an elevated fraction of phosphatidylserine has been shown by flow cytometry.