The protein structure and effect of factor VIII

Thrombosis Research (2007) 119, 1 — 13

intl.elsevierhealth.com/journals/thre

REVIEW ARTICLE

The protein structure and effect of factor VIII Hong Fang *, Lemin Wang, Hongbao Wang Department of Cardiology, Tongji Hospital Affiliated to Tongji University, Shanghai (200065), China Received 14 June 2005; received in revised form 6 November 2005; accepted 26 December 2005 Available online 17 February 2006

KEYWORDS Factor VIII; Molecular structure; Mutation; Bleeding disorders

Abstract Factor VIII (FVIII) is a key component of the fluid phase of the blood coagulation system. The proteases efficiently cleave FVIII at three sites, two within the heavy and one within the light chain resulting in alteration of its covalent structure and conformation and yielding the active cofactor, FVIIIa. FVIIIa is a trimer composed of A1, A2 and A3—C1—C2 subunits. The role of FVIIIa is to markedly increase the catalytic efficiency of factor IXa in the activation of factor X. Variants of these factors frequently also lead to severe bleeding disorders. D 2006 Elsevier Ltd. All rights reserved.

Contents The protein structure of factor VIII. . . . . . . . . . . . . . . . . . . . . . . A domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of FVIII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of A1 subunit and A2 subunit . . . . . . . . . . . . . . . . . . The relation between A3—C1—C2 subunits and the activity of FVIII . . . Factor VIII and metal ions . . . . . . . . . . . . . . . . . . . . . . . . . . To activate FVIII and the role of FVIIIa . . . . . . . . . . . . . . . . . . . The hemophilia and mutation of protein structure in factor VIII . . . . . . The relation between mutations and hemophilia A . . . . . . . . . . . . Inhibitors and hemophilia . . . . . . . . . . . . . . . . . . . . . . . . . . Recombinant FVIII protein and therapeutics for patients with hemophilia . The effect of recombinant FVIII protein . . . . . . . . . . . . . . . . . . FVIII bioengineered for improved secretion . . . . . . . . . . . . . . . . Prevent FVIII inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. E-mail address: [email protected] (H. Fang). 0049-3848/$ - see front matter D 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2005.12.015

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2 Factor VIII (FVIII), an essential blood coagulation protein, is a key component of the blood coagulation system. The glycoprotein FVIII in its activated form, factor VIIIa (FVIIIa) functions as a cofactor for prothrombinase and tenase complexes. FVIIIa acts as a cofactor to the serine protease FIXa in the conversion of the zymogen FX to the active enzyme (FXa) [1]. The role of FVIIIa is to markedly increase the catalytic efficiency of factor IXa (FIXa) in the activation of factor X (FX). Activation of FVIII results from limited proteolysis catalyzed by thrombin or FXa, which binds the FVIII substrate over extended interactive surfaces. Natural variants of FVIII may lead frequently to severe bleeding disorders. FVIII also plays an important role at sites of vascular injury, for the amplification of the clotting cascade. Recent evidence suggests a direct relationship between high plasma levels of FVIII and an increased risk for arterial and venous thrombosis [2—5]. In this review, we discussed the protein structure and effect of FVIII.

The protein structure of factor VIII The primary structure of human FVIII was deduced from its cDNA sequence [6]. It was consistent with partial sequence analysis of tryptic peptides [7]. FVIII is synthesized as a 300 kDa precursor protein, which is a 2332-residue single-chain glycoprotein, composed of three distinct domain types in the arrangement, namely (NH2) A1—A2—B—A3—C1—C2 (COOH) [8] (see Fig. 1). Among species, amino acids are highly homologous except for their B domains. The FVIII protein is secreted as a heterodimer, a series of divalent metal ion-dependent heterodimers following at least two intracellular cleavages within the B domain, including variable lengths of heavy (A1—A2—B domains) and light chains (A3—C1—C2). Both chains contribute to high-affinity interaction with lipoprotein receptorrelated protein (LRP) [9]. The A1 subunit in the heavy chain and the light chain (A3—C1—C2) retain the stable divalent metal ion-dependent linkage, whereas the A2 subunit of heavy chain is weakly associated with the dimer through electrostatic interactions [1]. We summarize the accumulated knowledge on domains of FVIII below.

H. Fang et al.

A domain The A domains are homologous with FV and ceruloplasmin. There are three A domains in FVIII, i.e. A1, A2, A3 domains. Two highly conserved ß-barrel core structures, which were tilted somewhat from each other, were found in every A domain and they were arranged concentrically. The second of each subdomain interacts with the first of the next domain. The core disulfide bonds are along the bbottomQ surface of the structure [10]. Each was conserved in all three proteins. FVIII binds a single atom of copper in the A domain and it is in the colorless cuprous ion form [11]. The FVIII core structures are likely to be very close to the modeled structure but surface loop structures can only be approximated. One dominant A2 epitope for alloantibodies or autoantibodies is in an additional surface loop, residues 484—508 [12]. Another one on A2 includes a sequence, residues 558—565 [13,14], which interacts with FIXa and is likely the major catalytic interaction between the active cofactor and the enzyme [15]. The A2 domain is the most readily dissociated in FVIIIa. The surface peptide sequence near the amino terminus of A3, which was residues 1810—1818, appears to provide a secondary binding site for FIXa [16]. In the absence of phospholipids, FX binding includes a site within the carboxy-terminal portion of the A1 domain’s acidic connecting peptide, ba1Q, or residues 337—372 [17,18]. Activated protein C binding involves a site between His2007 and Val2016 within the carboxy-terminal half of the A3 domain [19]. Within residues 227—336, near the carboxy-terminus of the A1 domain, there are several candidate sequences for intracellular binding to Bip, and mutagenesis within this regionaffected secretion [20]. phe309 appears to be an important residue in Bip binding [21].

B domain The B domain of FVIII contains most of the protein’s Asn-linked carbohydrate chains, and there is wide variability among FVIIIs from different species. The large, central B domain is highly glycosylated but has a variable sequence. The activity of FVIII was not controlled by B domain. Most of the large, central B domain (N700 aa) can be deleted without losing activity [22,23].

Figure 1 The protein structure of factor VIII. There are 2332 residues in factor VIII, composed of 3 distinct domain types in the arrangement (NH2) A1—A2—B—A3—C1—C2 (COOH).

The protein structure and effect of factor VIII The B domain largely functions in intracellular processing while providing sites for cleavage both prior to secretion and during activation by thrombin to an active cofactor. Asparagine-linked oligosaccharide structures of the FVIII B domain recognize the carbohydrate recognition domains of asialoglycoprotein receptor and that the asialo-orosomucoidsensitive mechanism, most likely asialoglycoprotein receptor, contributes to the catabolism of coagulation FVIII in vivo [24].

C domain Domain C1 consists of 153 amino acids with 42% identity, and domain C2 contains 160 amino acids. The crystal structure of the factor VIII C2 domain consists of a ß-sandwich core from which ß-hairpins and loops extend to form a hydrophobic surface [25]. The hydrophobic surface includes M2199 and F2200 at the tip of the 1st ß-hairpin. Two pairs of hydrophobic residues, which extend from adjacent loops, may be central to a major lipid-binding surface. Domain C1 residues align closely to those of C2, except for lacking ß-strands-3, -4 and the proposed lipid-binding site [26]. The putative lipid binding surface in domain C2 is opposite from the amino- and carboxy-terminal sequences and single disulfide bond. It binds a human monoclonal antibody [27] which blocks phospholipid binding. Sitespecific mutagenesis of selected hydrophobic or positively charged residues present on this surface reduces binding of domain C2 to some fractions of anti-FVIII inhibitors [28]. Mutagenesis studies support the prediction of the C2 domain’s crystallographic structure that lipid binding involves two pairs of adjacent, solvent-exposed hydrophobic residues, Met—Trp2200 and Leu—Leu2252 [29,30]. FVIII C2 domain also contains VWF-, thrombin-, and factor Xa-binding sites [31—33]. There are binding sites in isolated domain C2 for both thrombin and FXa, although comparison with potential zymogen binding was not made. Although domain C1 has not been expressed by itself, baculoviral peptides with C1—C2 sequence suggest that domain C1 considerably enhances binding of von Willebrand factor (vWF) to domain C2. C1—C2 also bind FIX or FX zymogens, or their active forms, perhaps representing a less-specific, oriented interaction between FVIII with the enzyme and substrate in intrinsic system clotting to facilitate FX activation [34].

The effect of FVIII FVIII is a plasma glycoprotein that functions as an essential cofactor in blood coagulation [35]. FVIII

3 binds to and circulates with vWF as an inactive precursor. It is activated by thrombin or FXa, which cleaves FVIII at Arg372, between the A1 and A2 domains; at Arg740, between the A2 and B domains; and at Arg1689, which releases the A2 peptide from the light chain [36]. In vitro, trace amounts of thrombin can markedly enhance apparent FVIII clotting activity [37] and the apparent specific activity can increase 50-fold. Once thrombin activates FVIII, it dissociates from vWF and is concentrated by binding to a phospholipid surface [38,39]. There it orients to interact with FIXa and FX. Binding to FIXa involves sequences in A2, residues Ser558 through Gln565, and sequences in A3, residues Glu1811 through Lys1818 domains [40], and may also involve an orienting interaction with the FVIII C domains. In addition, thrombin and FXa appear to bind to a site on the surface of C2. On a lipid surface, FVIIIa enhances catalysis of FX by FIXa N 105 [41,42]. Similar involvement of C2 is seen in binding to phospholipids from activated platelets [43]. There may be additional, lower-affinity lipidbinding sites within the A3—C1—C2 light chain of FVIIIa. Lipid and vWF—FVIII C domain-binding sites overlap [44] and are blocked by antibodies whose epitopes include these hydrophobic residues. When thrombin cleaves the acidic a3 peptide at the amino terminus of the A3 domain, FVIII loses one of its high-affinity binding sites to vWF. This presumably allows a hemostatically active lipid surface to compete with vWF to displace it from the FVIIIa C2 domain [45]. FVIII is converted to its active form, FVIIIa, upon proteolytic cleavage by thrombin and is a heterotrimer composed of the A1, A2 and A3—C1—C2 subunits. A1 subunits of FVIIIa terminates with 36-residue segment (Met337— Arg372) rich in acidic residues. This segment is removed after cleaving at Arg336 by activated protein C, which results in the inactivation of the cofactor [46].

The effect of A1 subunit and A2 subunit Three regions in the A2 subunit (373—395, 418—428, and 518—533) interact with the A1/A3—C1—C2 dimer [47]. The isolated A2 subunit possesses limited cofactor activity in stimulating FIXa-catalyzed activation of FX. This activity is markedly enhanced by the A1 subunit. The C-terminal region of A1 subunit (residues 337—372) is thought to represent an A2interactive site. This region appears critical to FVIIIa. The region of A1 appears necessary to properly orient A2 subunit relative to FIXa in the cofactor rather than directly stimulate A2. Truncated A1 (A1(336)) showed similar affinity for A2 subunit and stimulated its cofactor activity. However,

4 A1(336) was unable to reconstitute FVIIIa activity in the presence of A2 and A3—C1—C2 subunits [48]. The residues 337—372, comprising the acidic Cterminal region in A1 subunit, interact with FXa during the proteolytic inactivation of FVIIIa. Peptides 337—372 are important for FXa-catalyzed activation of FVIII. Residues 337—372 directly interact with FXa. They markedly inhibited cofactor activation, consistent with a delay in the rate of cleavage at the A1—A2 junction. Studies using the isolated FVIII heavy chain indicated that the peptide completely blocked cleavage at the A1—A2 junction and partially blocked cleavage at the A2—B junction. Mutant FVIII molecules in which clustered acidic residues in the 337—372 segment were converted to alanine were evaluated for activation by FXa. Of the mutants tested, only FXa-catalyzed activation of the D361A/D362A/D363A mutant was inhibited with peak activity of approximately 50% and an activation rate constant of approximately 30% of the wild-type values. The 337—372 acidic region separating A1 and A2 domains and, in particular, the cluster of acidic residues at position 361—363 contribute to a unique FXa-interactive site within the FVIII heavy chain which promotes FXa docking during cofactor activation [49]. Both A1 termini are necessary for functional interaction of A1 with A2. Furthermore, the C terminus of A1 contributes to the K(m) for FX binding to FXase, and this parameter is critical for the activity assessed in plasma-based assays [50]. Nogami et al. suggest the acidic region comprising residues 389—394 in FVIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg(740) during procofactor activation [51]. The release of the A2 domains is necessary for FVIII to dissociate from vWF. This is a requisite step in blood coagulation because the binding of FVIII to phospholipid membranes and to vWF is mutually exclusive. vWF promotes release of FVIII into the circulation and increases its circulatory lifetime.

The relation between A3—C1—C2 subunits and the activity of FVIII A3—C1—C2 was an effective competitor of FVIIIa binding to FIXa. A significant component of the interprotein affinity is contributed by FVIIIa subunits other than A3—C1—C2 in the membranedependent complex. The isolated A2 subunit of FVIIIa interacts weakly with FIXa, and recent modeling studies have implicated a number of residues that potentially contact the FIXa protease domain. Both A2 and A3—C1—C2 subunits contribute to the affinity of FVIIIa for FIXa in the membranedependent FXase [52]. Inactivating cleavages cat-

H. Fang et al. alyzed by FXa at Lys(36) and Arg(336) are regulated in part by the A3—C1—C2 subunit. Furthermore, cleavage at Lys(36) appears to be selectively modulated by the C-terminal acidic region of A1, a region that may interact with FXa via its heparinbinding exosite [53]. Multiple sites in FVIII contribute to high-affinity LRP binding, one of which is the FVIII A3 domain region Glu(1811)—Lys(1818). This suggests that LRP binding to the FVIII A3 domain involves the same structural elements that also contribute to the assembly of FVIII with FIXa. The model of the phospholipid-binding site was determined by the crystal structure of the C2 domain of FVIII [54]. Within loop 4, the solvent-exposed amino acid, Trp(2313), is believed to contribute to FVIIIphospholipid binding. The crystal structures of the FVIII light chain C2 domains have been determined and they show hydrophobic and electrostatic interactions important for anchoring to the phospholipid membrane. Several studies have shown that the FVIII C2 domain also contains VWF-, thrombin-, and FXa-binding sites [55]. The C2 domain of FVIII is not only important for FVIII—phospholipid interaction but for FVIII—VWF as well. The C2 domain surfaceexposed residue, Trp(2313), is critical for the secretion of protein [56]. The C2 domain, and possibly C1, contributes to vWF binding [57,58] that is essential for the stabilization of FVIII in circulation. Its carboxyl-terminal bC2Q domain is responsible for binding to both activated platelet surfaces and vWF. When thrombin activates FVIII, FVIIIa dissociates from vWF and is concentrated by binding to a phospholipid surface, where it interacts with FIXa. In circulation, FVIII is stabilized by binding to vWF with a plasma half-life of about 10 h. The half-life of FVIII is much shorter than that of vWF. About 95% of FVIII is bound at any time such that an equilibrium exists in vivo. After specific thrombin cleaving that removes the remainder of the B domain and one of the high-affinity vWF binding sites, FVIII becomes heterotrimeric FVIIIa (see Fig. 2). This is consistent with the 1% to 4% FVIII clotting activities in plasma of patients with severe type 3 vWD and with the observation that plasma from patients with severe hemophilia A infused into those with severe vWD was associated with a delayed but sustained endogenous FVIII response [59]. Infusion of isolated porcine vWF into a pig model with severe deficiency caused a 3-fold rise in FVIII clotting activity without quantitative increase of the liver’s mRNA for FVIII [60]. Thus, increased synthesis does not occur, such that the rise is due to increased stability of FVIII bound to the transfused vWF [61]. Lewis found that the F2200A mutant had slightly lower cofactor activity at subsaturating phospholip-

The protein structure and effect of factor VIII

5

Figure 2 The relation between FVIII and vWF. After thrombin cleaving that removes the remainder of the B domain and one of the high-affinity vWF binding sites, FVIII becomes heterotrimeric FVIIIa.

id concentrations. However, single mutations at N2198 and M2199 had relatively little impact on cofactor activity, or on phospholipid and vWF binding [62]. Spiegel characterized the effect of 20 hemophilia-associated missense mutations across this domain (i.e. all occur in patients in vivo) on its stability and its binding activities. At least six of these mutations were severely destabilizing, and another four caused moderate destabilization and corresponding reductions in both binding functions. One mutant (A2201P) displayed a significant reduction in its membrane binding activity but normal vWF binding, while two others (P2300S and R2304H) caused the opposite effect. Several mutations (including L2210P, V2223M, M2238V, and R2304C) displayed near wild-type stabilities and binding activities and may instead affect mRNA splicing or alternative properties or functions of the protein. This study demonstrated that vWF and membrane binding activities can be uncoupled and uniquely disrupted by different mutations and that either effect can lead to similar reductions in clotting activity [35].

Factor VIII and metal ions Generation of factor VIII cofactor activity requires divalent metal ions such as Ca2+ or Mn2+. Evaluation of cofactor reconstitution from isolated factor VIIIa subunits revealed the presence of a functional Ca2+ binding site within the A1 subunit. Isothermal titration calorimetry demonstrated at least two Ca2+ binding sites of similar affinity within the A1 subunit. Ca2+ binding affinity was greatly reduced (or lost) in several mutants including E110A, E110D,

D116A, E122A, D125A, and D126A. Alternatively, E113A, D115A, and E124A showed wild type-like activity with little or no reduction in Ca2+ affinity [63]. Although not necessary for thrombin cleavages, Ca2+ binding is essential for clotting activity [64]. However, Mn2+ affinity was minimally altered except for mutant D125A (and D116A). Region 110— 126 serves a critical role for Ca2+ coordination with selected residues capable of contributing to a partially overlapping site for Mn2+, and that occupancy of either site is required for maximal cofactor activity [63].

To activate FVIII and the role of FVIIIa FVIIIa is formed by thrombin cleavage at residues 372 (A1—A2 junction) and 740 (A2—B junction) in the heavy chain, and residue 1689 in the light chain of FVIII. FVIIIa is a trimer composed of A1, A2, and A3— C1—C2 subunits: A1, residues 1—372; A2, residues 373—740; and A3—C1—C2, residues 1690—2332 [1] (see Fig. 3). It is a metal-linked heterotrimer composed of the A1/A2/A3—C1—C2 domains and lacking the B domain. It functions in the intrinsic pathway of blood coagulation by binding to factor IXa, phospholipid, and possibly FX, and by promoting the activation of FX by FIXa. When thrombin activates FVIII, FVIIIa dissociates from vWF and is concentrated by binding to a phospholipid surface, where it interacts with FIXa. FVIIIa enhances the rate of activation of FX by FIXa by more than 105-fold [41]. Activation of plasma FVIII/VWF requires cleavages at Arg372 and Arg1689 for cofactor activity, whereas cleavage at Arg740 appears not to be rate limiting. Cleavage at Arg1689 releases an acidic 14-

6 kDa fragment that leads to dissociation of VWF, allowing the association of FVIIIa to the phospholipid membrane [65]. In the absence of phospholipid, the k(cat) for FVIIIa-dependent, FIXa-catalyzed conversion of FX was markedly less than that observed in the presence of phospholipid and decreased as the ionic strength of the reaction increased. At low salt concentration, the k(cat) was approx. 8-fold greater than that observed at near physiologic ionic strength. However, this level of salt showed minimal effects on the intermolecular affinities of FVIIIa (or isolated A2 subunit) for FIXa or on the K(m) for FX. Alternatively, the association of A2 subunit with A1 subunit was sensitive to increases in salt and paralleled the reduction in k(cat) observed with FVIIIa. This instability was not observed in phospholipid-containing reactions. These results indicate that, in the presence of FIXa, the salt-dependent dissociation of FVIIIa subunits is significantly enhanced in the absence of phospholipid, promoting a reduced k(cat) for the cofactor-dependent generation of FXa [66]. Optimal rates of FX activation require occupancy of receptors for FIXa, FVIII, and FX on the activated platelet surface. The presence of FVIII and FX increases 5-fold the affinity of FIXa for the surface of activated platelets, and the presence of FVIII or FVIIIa generates a high affinity, low capacity specific FX-binding site on activated platelets. The A2 domain of FVIII significantly increases the affinity and stoichiometry of FVIIIa binding to platelets and contributes to the stability of the FXactivating complex. Both FVIII and FVIIIa binding were specific, saturable, and reversible. FVIII binds

H. Fang et al. to specific, high affinity receptors on activated platelets and FVIIIa interacts with an additional 300—500 sites per platelet with enhanced affinity. FVIIIa binding to activated platelets in the presence of FIXa and FX is closely coupled with rates of F—X activation. The presence of EGR—FIXa and FX increases both the number and the affinity of binding sites on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a threereceptor model in the assembly of the F—X-activating complex on the platelet surface [67]. FVIIIa cofactor activity is controlled, at least in part, by degradative cleavages at Arg336—Met and Arg562— Gly bonds by APC [68]. Unlike FV inactivation, in which a common mutation removing one of two APC cleavage sites, Arg508 to Gln, predisposes to thrombosis (FV Leiden), no parallel at either FVIII cleavage site has been identified. This may be because inactivation of FVIII requires cleavage of both bonds [69] or, especially at low concentrations that likely occur during clotting in vivo, that A2 domain dissociation is the predominant determinant of inactivation. Furthermore, binding to FIXa protects FVIIIa from APC cleavage. Inactivation of FVIIIa occurs by A2 dissociation or by specific cleavages within A1 and A2 by activated protein C. Contributions of FVIIIa subunits to cofactor association with FIXa were evaluated. Jenkins indicated that a significant component of the interprotein affinity was contributed by FVIIIa subunits other than A3—C1—C2 in the membrane-dependent complex. The isolated A2 subunit of FVIIIa interacts weakly with FIXa, and recent modeling studies have

Figure 3 To activate FVIII. FVIII circulates as a heterogeneous mixture of heterodimers formed by variable intracellular processing of the B domain. The diagram shows the expected size fragments because of thrombin cleavage at Arg372, Arg740, and Arg1689. FVIIIa is formed by thrombin cleavage at residues A1—A2 junction and A2—B junction in the heavy chain, and residue 1689 in the light chain of FVIII.

The protein structure and effect of factor VIII implicated a number of residues that potentially contact the FIXa protease domain [15]. Site-directed mutagenesis of candidate residues in the A2 domain was performed, and recombinant proteins were stably expressed and purified. Functional affinity determinations demonstrated that one mutant, FVIII (Asp712Ala) exhibited an 8-fold increase in K(d) (35 F 1.5 nM) relative to wild-type, suggesting a contribution of approximately 10% of the FVIIIa—FIXa binding energy. Thus, both A2 and A3—C1—C2 subunits contribute to the affinity of FVIIIa for FIXa in the membrane-dependent FXase [52].

The hemophilia and mutation of protein structure in factor VIII Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A [70]. Hemophilia A is a sex-linked disorder that results from a deficiency of functional FVIII. Deficiency of FVIII activity leads to hemophilia A, a congenital bleeding tendency of variable severity that is due to a large number of distinct FVIII gene mutations [71].

The relation between mutations and hemophilia A Some hemophilic mutations lead to circulating, dysfunctional proteins, whereas others affect expression, secretion or stability in circulation. Stabilization of FVIII is largely due to its interaction with vWF, through binding sites within an acidic peptide, ba3,Q at the carboxy-terminal of the FVIII B domain and, as more recently appreciated, within its C domains [72]. Mutations responsible for mild/moderate hemophilia A were extensively characterized over the last 15 years and more than 200 mutations have been identified. However, most of the molecular mechanisms responsible for the reduced FVIII levels in patients’ plasma were determined only recently. Recent progresses in the study of the FVIII molecule of three-dimensional structure provided a major insight for understanding molecular events leading to mild/moderate hemophilia A. This allowed prediction of mutations impairing FVIII folding and intracellular processing, which resulted in reduced FVIII secretion. Mutations potentially slowing down FVIII activation by thrombin were also identified. A number of mutations were also predicted to result in altered stability of activated FVIII. Biochemical analyses allowed identification of mutations reducing FVIII production. Mutations

7 impairing FVIII stability in plasma, by reducing FVIII binding to VWF were also characterized. Defects in FVIII activity, notably slow activation by thrombin, or abnormal interaction with FIXa, were also recently demonstrated. Biochemical analysis of FVIII variants provided information regarding the structure/function relationship of the FVIII molecule and validated predictions of the three-dimensional structure of the molecule. These observations also contributed to explain the discrepant activities recorded for some FVIII variants using different types of FVIII assays. Altogether, the study of the biochemical properties of FVIII variants and the evaluation of the effects of mutations in threedimensional models of FVIII identified molecular mechanisms potentially explaining reduced FVIII levels for a majority of patients with mild/moderate hemophilia A. It is expected that these studies will improve diagnosis and treatment of this disease [73]. The amino-terminal heavy chain (A1—A2—BV domains) is noncovalently bound to the light chain (A3—C1—C2) [74]. From mutagenesis of these Arg residues, the A2—BV cleavage is not essential for activity, whereas those between A1 and A2 (in a1) and a3 and A3 are necessary for detectable clotting activity in vitro. Hemophilic mutations of the latter two Arg residues markedly reduced to absent FVIII activity. Fluorescence anisotropy with FIXa labeled at its active site indicated that cleavage between A1 and A2 allows interaction between FVIIIa and FIXa. Hemophilic mutations predicted by the ceruloplasmin-based model to involve the A2 interface with either A1 or A3 lead to increased intracellular degradation. Employing FVIII derived from patient plasma and recombinant FVIII variants explored that substitution of Arg527 (YTrp) and Arg531 (YGly, His) in FVIII caused the functional defect. Mutation of these residues is associated with mild hemophilia A. For both FVIII—R527W and FVIII—R531H, activity was lower than antigen, indicating a functional defect for both variants. In contrast to FVIII— R527W, the amount of FVIII—R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. FX activation experiments employing recombinant FVIII—R531G revealed that the activated FVIII—R531G heterotrimer was less stable than normal FVIIIa, apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg531 is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer [75]. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. Patients with hemophilia A have been described whose plasmas display a discrepancy between their FVIII activities, where

8 the 1-stage activity assay displays greater activity than the 2-stage activity assay. The molecular basis for one of these mutations, (ARG)531(HIS), is an increased rate of A2 subunit dissociation. Examination of a homology model of the A domains of FVIII predicted (ARG)531 to lie at the interface of the A1 and A2 subunits and stabilize their interaction. Indeed, patients with mutations either directly contacting (ARG)531 ((ALA)284(GLU), (ALA)284(PRO)) or closely adjacent to the A1—A2 interface in the tightly packed hydrophobic core ((SER)289(LEU)) have the same phenotype of 1stage/2-stage discrepancy. A2 dissociation was 3fold faster for both FVIIIa mutants compared to FVIIIa wild-type. Therefore, these mutations within the A1 subunit of FVIIIa introduce a similar destabilization of the FVIIIa heterotrimer compared to the (ARG)531(HIS) mutation within the A2 subunit and support that these residues stabilize the A domain interface of FVIIIa [76].

Inhibitors and hemophilia Inhibitor formation occurs at a frequency of 20% to 30% in severe hemophilia A, and 3% in hemophilia B. Today, it represents the major complication in patient care and renders classical substitution therapy ineffective. Genetic factors, such as FVIII gene mutations and immune response genes, particularly the major histocompatibility complex, have been shown to constitute decisive risk factors for the development of inhibitors. In severe hemophilia A and B, those mutations that result in the absence or severe truncation of the FVIII/FIX proteins are associated with the highest risk for inhibitor formation, indicating that a major driving force in inhibitor development is the presentation of a novel antigen to the patient’s immune system. An alternative pathomechanism may underlie inhibitor development in patients with mild hemophilia A. Missense mutations, especially those in the C1/C2 domains, may alter the immunogenicity of the FVIII protein, eliciting an inhibitor response against the mutated epitope [77].

Recombinant FVIII protein and therapeutics for patients with hemophilia The effect of recombinant FVIII protein Greater understanding of the structure—function relationships in the FVIII gene and protein will continue to guide development of more effective recombinant therapeutics and of gene therapy for

H. Fang et al. patients with disease. The past 10 years of clinical experience have demonstrated the safety and efficacy of recombinant FVIII(rFVIII). Now, insights from our understanding of clotting factor structure and function, mechanisms, gene therapy advances and a worldwide demand for clotting factor concentrates leave us on the brink of embracing targeted bioengineering strategies to further improve therapeutics. The ability to bioengineer recombinant clotting factors with improved function holds promise to overcome some of the limitations in current treatment, the high costs of therapy and increased availability to a broader world hemophilia population. Most research has been directed at overcoming the inherent limitations of rFVIII expression and the inhibitor response. This includes techniques to improve rFVIII biosynthesis and secretion, functional activity, half-life and antigenicity/immunogenicity. Some of these proteins have already reached commercialization and have been utilized in gene therapy strategies, while others are being evaluated in pre-clinical studies. These novel proteins partnered with advances in gene transfer vector design and delivery may ultimately achieve persistent expression of FVIII, leading to an effective longterm treatment strategy for hemophilia A. In addition, these novel FVIII proteins could be partnered with new advances in alternative recombinant protein production in transgenic animals yielding an affordable, more abundant supply of rFVIII [78—80]. Hemophilia A is now treated by infusions of pure FVIII, but the activity of FVIII is limited because it is unstable following activation by thrombin. This instability of activated FVIII is the result of dissociation of the A2 subunit. The recombinant FVIII were used in the field. To obtain increased stability in FVIIIa, a disulfide bond between the A2 domain and the A3 domain, preventing A2 subunit dissociation, has been engineered. A FVIII mutant containing Cys664 and Cys1826 was produced and purified (C664—C1826 FVIII). Immunoblotting showed that a disulfide bond did form to link covalently the A2 and the A3 domains. Following activation of the recombinant C664—C1826 FVIII by thrombin, the mutant FVIIIa had increased stability and retained more than 90% of its clotting activity at a time at which wildtype FVIIIa lost more than 90% of its activity. This remarkably stable C664—C1826 FVIIIa provides a unique approach for studies of the cofactor activity of FVIIIa and also for new, improved therapy for hemophilia A [81]. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. Oh found that a recombinant mutant heavy chain of

The protein structure and effect of factor VIII FVIII (rFVIII—Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII—L) were expressed in Baculovirus—insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII—Hm and rFVIII—L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85  10 2 U/mg protein) that sustained the high level activity during in vitro incubation at 37 8C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus—insect cell system is inactivation-resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A [46]. B-domain-deleted recombinant factor VIII is safe and effective and has hemostatic activity similar to that of full-length FVIII concentrates. It was found to be redundant for maintaining hemostasis [82]. However, preservation of some of the B-domain glycosylation sites enhances secretion [83]. Distinction of the relative roles of hepatocyte as opposed to sinusoidal endothelial cell synthesis [84] and of native promoter and enhancer elements will allow design of efficient targeted expression constructs. A FVIII was made with the A2 and A3 domains covalently linked in order to prevent A2 dissociation [85]. This resulted in a species that was active and was effective in animal models, although it lacked a high-affinity binding site for vWF and had a survival about one-fourth that of native FVIII. Saenko et al. thought the strategy to prolong FVIII lifetime by disrupting FVIII interaction with its clearance receptors and demonstrate how construction of human— porcine FVIII hybrid molecules can reduce their reactivity towards inhibitory antibodies [86].

FVIII bioengineered for improved secretion Expression of FVIII in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. However, FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)—FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport, Miao engineered several asparagine-linked oligosac-

9 charides within a short B-domain spacer within BDD—FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals [87].

Prevent FVIII inhibitors FVIII replacement therapy remains the mainstay in hemophilia A care. The major complication of replacement therapy is formation of antibodies, which inhibits FVIII activity, thus dramatically reducing treatment efficiency. FVIII inhibitors most frequently target the A2, C2 and A3 domains of FVIII and interfere with important interactions of FVIII at various stages of its functional pathway; a class of FVIII inhibitors inactivates FVIII by proteolysis. FVIII administration elicits specific inhibitory antibodies (Abs) in about 25% of patients with hemophilia A, the majority of which are reactive with its C2 and A2 domains. The choice between options to treat hemophilia A patients with inhibitors should depend on the level of inhibitors and consideration of efficacy, safety, and availability of particular regimens. Advances of basic science open avenues for alternative targeted, specific and long-lasting treatments, such as the use of peptide decoys for blocking FVIII inhibitors, bypassing them with human/porcine FVIII hybrids, neutralizing FVIII-reactive CD4 T cells with anti-clonotypic antibodies, or inducing immune tolerance to FVIII with the use of universal CD4 epitopes or by genetic approaches [88—94]. Gilles found that mAbBO2C11 was a high-affinity human monoclonal antibody (mAb) directed toward the C2 domain, which is representative of a major class of human FVIII inhibitors. Anti-idiotypic Abs were raised to mAbBO2C11 to establish their neutralizing potential toward inhibitors. One mouse anti-idiotypic mAb, mAb14C12, specifically prevented mAbBO2C11 binding to FVIII C 2 domain and fully neutralized mAbBO2C11 functional inhibitory properties. Modeling of the 3-D conformation of mAb14C12 VH and alignment with the 3-D structure of the C2 domain showed putative 31 surfaceexposed amino acid residues either identical or homologous to the C2 domain. These included one C2 phospholipid-binding site, Leu2251—Leu2252, but not Met2199—Phe2200. Forty putative contact residues with mAbBO2C11 were identified. mAb14C12 dose-dependently neutralized mAbBO2C11 inhibitory activity in mice with hemophilia A reconstituted with human rFVIII, allowing full

10 expression of FVIII activity. It was also neutralized in an immunoprecipitation assay approximately 50% of polyclonal anti-C2 Abs obtained from 3 of 6 unrelated patients. mAb14C12 is the first example of an anti-idiotypic Ab that fully restores FVIII activity in vivo in the presence of an anti-C2 inhibitor. The present results establish the in vitro and in vivo proof of concept for idiotype-mediated neutralization of a major class of FVIII inhibitors [95]. Recently, Lei and Scott demonstrated that B-cell presentation of FVIII domains on an Ig backbone specifically prevents or decreases existing antibodies in hemophilia A mice [96]. A second approach is to make FVIII proteins that have reduced immunologic reactivity to inhibitors. One such strategy incorporates hybrid proteins with stretches of the porcine FVIII sequence to replace epitopes in regions where there is little to no cross-reactivity [97]. Mechanisms of immune responses to human FVIII are being elucidated in a murine model of hemophilia A [98—100]. Once differences in human responses to the human FVIII protein are identified, this may well lead to the design of peptides or peptide conjugates to induce tolerance or prevent alloimmunity in patients with hemophilia A. In general, the study of the biochemical properties of FVIII and the evaluation of the effects of mutations in three-dimensional models of FVIII identified molecular mechanisms potentially explaining the pathogenesis of bleeding and thrombosis disorders. It is expected that these studies will improve diagnosis and treatment of these diseases.

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