Factor VIII – novel insights into form and function (2002)

Factor VIII (FVIII) is a key cofactor in blood coagulation, deficiency of which results in the serious bleeding disorder haemophilia A. The traditional treatment for haemophilia is by replacement therapy using plasma-derived or recombinant FVIII products. Recent developments in FVIII biochemistry have improved our understanding of how FVIII interacts with other macromolecules in the performance of its normal procoagulant function and how naturally occurring mutations result in the spectrum of clinical phenotypes found in haemophilia A. This review describes elements of recent studies on the structure of the FVIII molecule (as determined by X-ray crystallography, electron crystallography and modelling studies), its interaction with plasma von Willebrand factor and how this relates to the activation state of FVIII, interactions with procoagulant phospholipid surfaces and factor IXa in the formation of the ‘tenase’ complex and with the low-density lipoprotein receptor-related protein (LRP), a hepatic cell-surface receptor now known to be at least partly responsible for FVIII clearance from the circulation. Our improving understanding of the life cycle of the FVIII molecule carries with it the potential for better treatment for haemophilia, either by augmentation of its functional characteristics or by suppression of the natural clearance mechanisms. Normal human haemostasis involves two major constituents: the extrinsic, tissue factor (TF)/activated factor VII-dependent pathway and the intrinsic, factor VIII (FVIII)-dependent pathway. While initiation of blood coagulation is ascribed to the extrinsic pathway, in which small quantities of activated factors IX (FIXa) and X (FXa) are generated, the intrinsic pathway catalyses conversion of FX into FXa approximately 50-fold more efficiently, thus dramatically amplifying the coagulation events triggered by the TF-dependent pathway (Mann, 1999). The intrinsic pathway of blood coagulation is indispensable for maintaining haemostasis, as shown by the fact that deficiency or structural defects in FVIII lead to a coagulation disorder, haemophilia A. In the intrinsic pathway, activated FVIII (FVIIIa) functions as a cofactor for the serine protease FIXa, and their membrane-bound complex (Xase complex) activates FX to FXa (van Dieijen et al, 1981). In its turn, FXa participates in a similar complex for the conversion of prothrombin into thrombin, the key enzyme of the coagulation cascade. In normal circulation, the membrane surface required for the assembly of the Xase complex is classically provided by activated platelets (Nesheim et al, 1988). A major role of the platelet phospholipid (PL) surface is to concentrate components of the Xase complex, limit their interactions from three- to two-dimensional space and provide the optimal mutual orientation of the Xase components. This results in a dramatic acceleration of FXa generation due to the decrease in the Michaelis constant (Km) for FX (van Dieijen et al, 1981; Rawala-Sheikh et al, 1990) and increase in the catalytic constant of the reaction (kcat) (Gilbert & Arena, 1996). The FVIII molecule (∼300 kDa, 2332 amino acid residues) consists of three homologous A domains, two homologous C domains and the unique B domain, which are arranged in the order A1-A2-B-A3-C1-C2 (Fig 1; Toole et al, 1984; Wood et al, 1984). Prior to its secretion into plasma, FVIII is processed within the cell to a series of metal ion-linked heterodimers produced by cleavage at the B-A3 junction and by a number of additional cleavages within the B domain (Fay et al, 1986). These cleavages generate the heavy chain (HCh), consisting of the A1 (1–336), A2 (373–719) and B domains (741–1648), and the light chain (LCh), composed of the domains A3 (1690–2019), C1 (2020–2172) and C2 (2173–2332). The remaining intervening portions, C-terminal to the A1 domain (amino acids 337–372) and the A2 domain (amino acids 720–740) and N-terminal to A3 in LCh (amino acids 1649–1689), contain a high proportion of negatively charged residues and are called acidic regions, a1, a2 and a3 respectively. FVIII structure and regions involved in its major functional interactions. Non-activated FVIII is shown as a multidomain structure in which A1 and A3 subunits are non-covalently linked via a metal ion-mediated interaction (dotted line) and three A domains are flanked by acidic regions a1, a2 and a3. The regions of FVIII involved in binding of VWF, FX, FIXa, FXa and PL are shown in blue. The binding sites of FVIII identified for their interaction with LRP and heparan sulphate proteoglycans (HSPGs) are shown in red and green respectively. The basic residues within LRP and HSPGs binding sites, which are potentially involved in the corresponding FVIII interactions, are highlighted in red. Activation of FVIII by cleavages at positions 372 and 740 and 1689 leads to release of B domain and a3. Activated FVIII is a heterotrimer consisting of A1, A2 and A3-C1-C2 domains, in which A1 and A3 domains retain the metal ion-mediated interaction, and the stable A1/A3-C1-C2 dimer is weakly associated with the A2 subunit through electrostatic interactions. The LRP and HSPGs binding sites within the A2 subunit and the LRP site within the A3-C1-C2 subunits are shown. Immediately after release into the circulation, FVIII forms a tight non-covalent complex with von Willebrand factor (VWF), with a dissociation constant (Kd) of approximately 0·4 nmol/l (Saenko & Scandella, 1995, 1997), which is required for maintaining the normal FVIII level in plasma. The binding to VWF prevents premature assembly of the Xase complex prior to activation of FVIII and also protects FVIII from inactivation by activated protein C (APC), FIXa and FXa. VWF deficiency in both humans (Morfini et al, 1993) and animals (Schwarz et al, 2000) leads to a secondary deficiency of FVIII, which is most likely due to a lack of its protection from proteases and faster clearance from the circulation. There are at least two regions within the LCh of FVIII that are directly involved in binding to VWF (Fig 1): a3, residues 1649–1689, preceding the A3 domain (Leyte et al, 1991; Saenko & Scandella, 1997), and a region or regions within the C1 and C2 domains. Until recently, a C2 site within the sequence 2303–2332 was believed to be the primary second area modulating VWF and PL binding, based on synthetic peptide studies (Foster et al, 1990; Saenko & Scandella, 1997), and this was supported by the finding that in free solution the 2303–2324 peptide assumed an amphipathic helical conformation with affinity for PL membranes (Gilbert & Baleja, 1995). However, resolution of the crystal structure of the human C2 domain by X-ray crystallography has prompted re-interpretation of these studies as the 2303–2332 sequence was found to lie largely within the domain core (Pratt et al, 1999). Involvement of C1 in addition to C2 was first demonstrated by the fact that mutation of three C1 residues (Tyr2105, Arg2116 and Ser2119) results in haemophilia A with a direct or indirect effect on VWF binding (Jacquemin et al, 2000b) and that Arg 2150 is also closely associated with VWF interaction (Gilles et al, 1999; Peerlinck et al, 1999; Jacquemin et al 2000a). Secondly, another study suggests that the isolated C1-C2 fragment interacts with VWF much more tightly than does the isolated C2 domain (Liu & Thompson, 2000). Taken together, these recent studies suggest that VWF interaction may be mediated by contributions from the a3 peptide, C1 and C2, arranged closely in space to form a ‘patch’ for interaction either with VWF or with PL surface (Stoilova-McPhie et al, 2002). The HCh does not interact with VWF directly, but is required for the maximal affinity of the FVIII/VWF interaction as evidenced by a 10-fold higher affinity of the whole FVIII molecule for VWF in comparison with its isolated LCh (Saenko & Scandella, 1997). At the site of a coagulation event, FVIII is activated by two major physiological activators: thrombin and FXa. Both proteases cleave the FVIII molecule at Arg372 and Arg740 within HCh and at Arg1689 within LCh, producing A1, A2 and A3-C1-C2 fragments, which form heterotrimeric activated FVIII (FVIIIa) (Fig 1). In FVIIIa, the A1 and A3 domains retain the metal ion–mediated interaction, and the relatively stable A1/A3-C1-C2 dimer is more weakly associated with the A2 subunit through electrostatic interactions. Although the structural basis of FVIII activation by thrombin and FXa was shown well over a decade ago (Eaton et al, 1986), knowledge of the FVIII sites responsible for binding of these activators is not yet complete. An important FXa binding site was localized to C2 residues 2253–2270 (Nogami et al, 1999) based on inhibition of FXa/FVIII interaction by overlapping synthetic peptides covering this region (Fig 1). Although the thrombin binding sites have not been precisely mapped, the site responsible for thrombin-catalysed cleavage at Arg1689 in the LCh was recently assigned to the C2 domain of FVIII (Nogami et al, 2000). As the a3 region bears the VWF binding site, removal of a3 by cleavage at Arg1689 is a vital requirement for the release of FVIIIa from VWF and a substantial increase in FVIIIa affinity for platelets (Saenko et al, 1998). In addition, the cleavage at Arg1689 is required for generation of the maximal cofactor activity of FVIIIa as evidenced by a threefold lower activity of a FVIIIa derivative with intact LCh in comparison with FVIIIa cleaved at Arg1689 (Regan & Fay, 1995). Removal of a3 leads to a 10-fold decrease in the association rate constant and 160-fold increase in the dissociation rate constant of interaction of FVIIIa with VWF in comparison with non-activated FVIII (Saenko & Scandella, 1997). This results in a 1600-fold decrease in FVIIIa affinity for VWF, allowing FVIIIa to effectively bind to a PL surface and to FIXa during assembly of the Xase complex. The loss of a3 promotes FVIII dissociation from VWF, as a3 is also required for maintaining the C2 conformation optimal for VWF binding. A conformational change within the C2 domain upon removal of a3 was suggested from a reduced affinity of thrombin-cleaved LCh (compared with intact LCh) for a conformation-sensitive anti-C2 monoclonal antibody (mAb) NMC-VIII/5 (Saenko & Scandella, 1997). Additional evidence of this conformational change was obtained using another mAb, ESH8. This antibody has a discontinuous epitope composed of two portions of the C2 domain, residues 2227–2241 and 2259–2279 (Villard et al, 2001). The binding of ESH8 to these distinct, but spatially close, regions of C2 may account for the unique ability of this antibody to ‘lock’ the C2 conformation, thus preventing conformational changes within C2 induced by removal of a3. It was found that the affinity of FVIIIa for VWF in the presence of ESH8 (Kd 36 nmol/l) (Saenko et al, 1996) was 20-fold higher than in the absence of mAb (Kd 640 nmol/l) (Saenko & Scandella, 1997). This finding confirmed that upon FVIII activation the C2 domain undergoes a conformational change, which is required for complete release of FVIIIa from VWF. The cofactor activity of FVIIIa in the assembled intrinsic Xase complex is provided by three essential interactions of FVIIIa: with the PL membrane, with the enzyme FIXa and the substrate FX. Firstly, with regard to FVIII interaction with phospholipids of the cell membrane, FVIII LCh was shown to be entirely responsible for their high-affinity binding (Saenko et al, 1998). Thus, in contrast to the stabilizing role of the HCh in FVIII/VWF interaction, it does not appear to contribute to LCh binding to PL. Remarkably, the conformational changes within the C2 domain associated with FVIIIa release from VWF also result in a ∼10-fold increase in its affinity for PL membranes. This was concluded from the ability of the ESH8 antibody, which ‘locks’ the conformation of C2, to prevent the increase in FVIII affinity for PL upon activation (Saenko et al, 1998). Taking into account that the plasma concentration of FVIII is approximately 1 nmol/l, the decrease of KD value for FVIIIa/PL interactions to a comparable nanomolar level (0·4–1·1 nmol/l) (Saenko et al, 1998) provides conditions favourable for FVIIIa association with membranes at the site of coagulation. For some considerable time, it was believed that the major FVIII site involved in PL binding is located within the residues 2303–2332 of the C2 domain, based on the ability of synthetic peptides encompassing this region to inhibit FVIII/PL binding (Foster et al, 1990; Gilbert & Baleja, 1995). More recently, localization of the epitope of mAb NMC-VIII/5 and those of patient inhibitory antibodies (all of which prevent FVIII/PL interaction) to the C2 region 2181–2243 indicated that it should contain FVIII residues critical for interaction with PL (Healey et al, 1999). The 1·5 Å X-ray structure of the human FVIII C2 domain, which has recently become available, further implicated this latter FVIII region in PL binding. Analysis of the X-ray structure revealed the presence of three hydrophobic ‘feet’ formed by the side chains of Met2199/Phe2200, Val2223 and Leu 2251/Leu2252, which were suggested to penetrate the membrane bilayer (Pratt et al, 1999; Barrow et al, 2001). Additionally, four basic residues, Arg2215, Arg2220, Lys2227 and Lys2249, which lie to the have been to FVIII/PL binding by electrostatic interaction with negatively charged PL (Stoilova-McPhie et al, 2002). direct of binding obtained by electron crystallography has the resolution of functional domains of FVIII C1 and on a PL and the of these three with a in the region (Stoilova-McPhie et al, 2002). this does lie within the sequence 2303–2332 implicated in PL binding. FVIII domains and the PL surface as in the electron crystallography The based on the X-ray structure of C2 and electron crystallography studies of FVIII were confirmed by the that mutations of the residues the hydrophobic ‘feet’ and dramatically reduced the affinity of FVIII for PL (Gilbert et al, 2001). It is in with their effect on FVIII/PL interaction, these mutations also to a of FVIII affinity for VWF, that these two hydrophobic ‘feet’ are also important in FVIII interaction with VWF. Factor VIII to a PL The functional domains of FVIII arranged on a PL surface as determined in an electron crystallography study (Stoilova-McPhie et al, 2002). The domains, which are shown in are the as the domain hydrophobic in the four in the C2 domain implicated in PL binding are shown in green and of the PL membrane is as a The high-affinity interaction FVIIIa and FIXa is provided by the LCh of FVIII, which an important FIXa binding site (Kd nmol/l) formed by the A3 domain residues et al, 1996). of FVIIIa, which directly interacts with FIXa, is the A2 Although the affinity of isolated A2 for FIXa is (Kd this interaction the cofactor activity of FVIIIa, as the A2 domain the activity of FIXa by modulating its site located in the protease domain (Fay & 1998). The A2 domain three regions formed by amino acid residues (Fay et al, et al, et al, and (Fay & Scandella, which have been suggested to be involved in interaction with FIXa (Fig 1). the important A2 sites that FIXa activity appear to become upon cleavage at Arg372 within the HCh produced by either thrombin or FXa (Fay et al, 2001). Involvement of A2 and LCh sites in FVIIIa interaction with FIXa FVIIIa within the Xase complex, FIXa as a the A2 subunit and A1/A3-C1-C2 1999). This the rate of inactivation of FVIIIa heterotrimer due to a dissociation of the A2 subunit et al, et al, 1996). studies using FIXa et al, & Fay, et al, have the effect of the cleavages at Arg1689 and at Arg372 within the LCh and on the assembly of the functional Xase complex. The increase in by a FVIII molecule with cleaved LCh was shown to be higher than that for FVIII with intact LCh and comparable to that for FVIIIa (Regan & Fay, 1995). As the change in with the of of the FIXa site by FVIII, these suggest that removal of a3 upon cleavage at Arg1689 is required for optimal mutual orientation of FVIIIa and The latter provides a A2 and the of the FIXa protease domain in to its The also revealed that the maximal of the FIXa site cleavage A1 and A2 at 372 in addition to removal of a3 (Fay & et al, 2001). addition of FX to a further increase in that FX also to the orientation of the FIXa site in the assembled Xase complex (Fay & 1998). the FVIII involved in interaction with FX, has been localized to 337–372) based on the ability of the corresponding synthetic peptide to inhibit FX binding to the isolated A1 domain, and also on of this peptide with FX & Fay, 1997). This FX site was to residues & Fay, 1998). It is to the FVIII structure with PL determined in electron crystallography studies (Stoilova-McPhie et al, with the crystal structure of FIXa et al, to a which be as a for the functional Xase complex. a in which known sites on both FVIII and FIXa be in This a of and and in addition at the to an et al, 2001). The is the A2 sequence which implicated in binding to FIXa (Fay & Scandella, 1999) be at the of the two a finding also by et al Thus, it is that in the Xase complex, formed of FVIIIa with FIXa, some additional small conformational change has which involves this This may be supported by the fact that A2 which FIXa appear to become upon cleavage at Arg372 by either thrombin or FXa (Fay et al, 2001). Factor IXa complex FVIII (Stoilova-McPhie et al, is into a phospholipid membrane with the FVIII domains are the as the domain are shown as and the and C are shown as cleavage sites and are with FVIII regions implicated in FIXa binding and are highlighted as and in the the crystal structure for FIXa et al, is with the domain into the membrane and with the and serine protease domains are shown as and C of the FIXa light and heavy chains are shown as and the site as implicated in FVIII binding and FIXa are highlighted as red and in the A red also a FVIII interaction site at the The FVIII and FIXa were that the regions, were in the by approximately for of of by While the role of FVIII in the intrinsic Xase complex is well the responsible for FVIII have it has been found that FVIII is mediated by low-density lipoprotein receptor-related protein et al, 1999; Saenko et al, 1999; et al, a hepatic clearance receptor with a et al, 1998). A spectrum of LRP involved in blood coagulation and as FIXa et al, FXa et al, et al, a complex of thrombin with tissue factor pathway activators and their with et al, 1998). LRP is a of the low-density lipoprotein receptor of which in humans is by low-density and receptor et al, 1998). LRP is a non-covalently associated which a subunit responsible for binding and an subunit consisting of the domain the receptor in the plasma and the The binding of to LRP via A a of negatively charged amino which interact with charged residues of The of LRP in FVIII was based on the that FVIII was to bind to that it was and by cell and further that FVIII in the of a be by LRP using its a protein (Saenko et al, 1999; et al, 2000). FVIII binding sites are involved in interaction with within the C2 domain of the LCh et al, 1999) and the other within the A2 domain of the HCh (Saenko et al, 1999). The A2 domain LRP binding site has been to the region (Saenko et al, 1999). While the C2 site of FVIII has not been mapped, it most likely with the VWF binding site, as an anti-C2 domain mAb which FVIII binding to LRP also its binding to VWF et al, 1999; et al, 2001). Thus, in the circulation, FVIII is in a complex with VWF, the A2 LRP binding site be and FVIII This was supported by the finding that of the isolated A2 subunit and of FVIII from its complex with VWF by similar et al, 2001). of the C2 site in clearance was suggested from comparison of FVIII by in the presence and absence of VWF. In these it was found that isolated FVIII which the C2 site is was and approximately as as FVIII in complex with VWF (Saenko et al, 1999). An in the C2 site may be in with von Willebrand lack plasma VWF. in FVIII was shown to have an approximately three et al, et al, in comparison with normal et al, and haemophilia A have normal of VWF et al, 1997). The role of LRP in the clearance of FVIII in the absence of VWF is with a recent finding that in clearance of FVIII be by of et al, 1999; et al, et al, 2000). to of the C2 site is dissociation of FVIII from VWF upon activation to It is likely that of both the A2 and C2 LRP binding sites in heterotrimeric FVIIIa may be important for of FVIIIa at the site of et al, 2000a). In addition, FVIIIa is an molecule to its but dissociation into A2 and A1/A3-C1-C2 (Fay & The that both the isolated A2 domain et al, and A3-C1-C2 subunit of FVIII et al, be by may the of an pathway for clearance of the two of activated FVIII in via the A2 and C2 LRP binding As two other components of the Xase complex FXa et al, 1998) and FIXa et al, 2000b) are also LRP LRP may at least the Xase activity at the site of coagulation by the of FIXa, for which FVIIIa as a and the of of This be with of LRP on the surface of and et al, that become upon Although LRP and of FVIII, the relatively affinity of their interaction nmol/l et al, KD nmol/l (Saenko et al, suggests the of a for FVIII on the cell For LRP lipoprotein 1 complex and is by cell-surface heparan sulphate proteoglycans of the major components of the studies on FVIII have revealed that clearance of FVIII from its complex with VWF is also by HSPGs et al, 2001). of LRP and HSPGs in FVIII clearance was confirmed by the of these two which to a more of FVIII in than the of LRP The region of FVIII involved in binding to HSPGs was localized within the A2 domain residues et al, 2001). The knowledge FVIII is in the shown in The pathway of FVIII involves the binding of FVIII/VWF complex to which concentrate the complex on the cell surface and it to by of of FVIII binding of FVIII/VWF complex via interaction with by either of FVIII occurring via or by mediated directly by HSPGs As VWF does not FVIII in the pathway in cell studies (Saenko et al, that it from FVIII prior to FVIII into The is with from et al This in understanding the of FVIII a to the of FVIII in by LRP HSPGs binding sites in a recombinant FVIII of a molecule may be based on the of binding to LRP and This binding is mediated by interaction of charged residues of a with negatively charged residues of in LRP et al, 1998) or negatively charged sulphate and of chains in HSPGs (Mann, to the of the A2 domain of FVIII et al, 1997), the LRP binding site charged residues, three at positions and and three Arg at positions and which are tightly and on the A2 and Arg at positions and are also within the HSPGs binding site or in to These basic residues, which are potentially responsible for FVIII interaction with LRP and for of FVIII interaction with its LRP and HSPGs potentially FVIII in and thus increase the of FVIII replacement therapy of haemophilia A.