Activation of factor VIII and mechanisms of cofactor action

Blood Reviews (2004) 18, 1–15

www.elsevierhealth.com/journals/blre

Activation of factor VIII and mechanisms of cofactor action Philip J. Fay* Departments of Biochemistry and Biophysics and Medicine, PO Box 712, University of Rochester School of Medicine, Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA

KEYWORDS Factor VIII; Factor VIIIa; Factor IXa; Intrinsic factor Xase; Factor X; Catalytic rate constant; Thrombin; Factor Xa

Summary The factor VIII procofactor circulates as a metal ion-dependent heterodimer of a heavy chain and light chain. Activation of factor VIII results from limited proteolysis catalyzed by thrombin or factor Xa, which binds the factor VIII substrate over extended interactive surfaces. The proteases efficiently cleave factor VIII 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, factor VIIIa. The role of factor VIIIa is to markedly increase the catalytic efficiency of factor IXa in the activation of factor X. This effect is manifested in a dramatic increase in the catalytic rate constant, kcat , by mechanisms that remain poorly understood. c 2003 Elsevier Ltd. All rights reserved.


Introduction

Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein, factor VIII. Factor VIII circulates in an inactive, procofactor form in complex with von Willebrand factor (VWF), which stabilizes factor VIII and potentially helps to localize it to sites of vascular injury. Proteolytic activation of factor VIII releases the active cofactor, factor VIIIa, from the carrier VWF and enhances its affinity for anionic phospholipids, facilitating its association in the intrinsic factor Xase complex. This complex, consisting of the serine protease factor IXa and factor VIIIa assembled on a thrombogenic (anionic phospholipid) surface catalyzes the conversion of factor X to factor Xa, a reaction that is critical for the propagation phase of coagulation. The role of factor VIII in this complex is to increase the catalytic effi-

ciency of factor IXa toward factor X by several orders of magnitude. The primary kinetic effect of the cofactor is to increase the catalytic rate constant (kcat ) by modulating the active site region of factor IXa and interaction of enzyme and substrate. Although interest in factor VIII is commensurate with its biochemical and clinical importance, the intermolecular interactions involved in proteolytic conversion to the active cofactor form and molecular basis for its role in rate enhancement within the factor Xase complex remain poorly understood. This review addresses recent information relating to the activation of factor VIII, structural and conformational changes in the cofactor that accompany cleavage of scissile bonds, and how these alterations contribute to the cofactor effect.

Factor VIII structure and association with von Willebrand factor

*

Corresponding author. Tel.: +585-275-6576; Fax: +585-4734314. E-mail address: [email protected] (P.J. Fay).




Factor VIII is synthesized primarily by liver sinusoidal endothelial cells1 as a large (300 kDa; 2332

0268-960X/$ - see front matter c 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0268-960X(03)00025-0

2 residues) single chain protein.2;3 Internal sequence homology analysis identifies three “A’’ domains that are homologous to the A domains in factor V and ceruloplasmin, two “C’’ domains that are homologous to the lipid-binding region of a cellular agglutinin from Dictystelium discoideum, and a large “B’’ domain that is not homologous to other known structures.2;4 Short segments (30–40 amino acids; and designated by a lower case “a’’) possessing a high concentration of acidic residues directly follow the A1 (residues 337–372) and A2 domains (residues 711–740) and precede the A3 domain (residues 1649–1689). Thus the domainal structure of factor VIII is ordered as (NH2 ) A1–a1–A2–a2–B–a3–A3–C1–C2 (COOH) (Fig. 1). Several noteworthy sequence characteristics are attributed to factor VIII. Five Cys residues are contained in each A domain with four participating in intra-domainal disulfide bonds.5 Thus a free Cys residue is contained in the A1 (Cys310), A2 (Cys692) and A3 (Cys2000) domains. The reactive thiol groups of these residues offer unique sites for protein modification and have been employed for incorporation of fluorescent reporter groups in assaying intra- and intermolecular interactions. N-linked glycosylation, which heavily modifies the B domain as well as several sites in the A domains,4 appears dispensable for factor VIII activity based upon observations that enzymatic depletion of the carbohydrate does not affect cofactor function.6;7 Posttranslational modification includes sulfation of specific tyrosine residues in the acidic regions that

P.J. Fay

follow A1 (Tyr346), A2 (Tyr718, Tyr719 and Tyr723) and precede A3 (Tyr1664 and Tyr1680) domains.8 Select sulfated tyrosine residues in the a2 and a3 acidic segments contribute to cofactor function and VWF binding (see below), respectively; whereas the sulfation site in the a1 acidic segment can be mutated without loss of factor VIII function.8 Structural information on factor VIII is limited. Homology modeling of the A domains based upon the crystal structures of nitrite reductase9 and ceruloplasmin,10 the latter of which shows 34% identity with factor VIII A domains, has proved a useful tool in predicting interactive sites for macromolecules as well as mapping mutations and predicting structure-function correlates. The recent X-ray structure of the C2 domain11 provides important information on the mechanism for cofactor binding to surfaces. This binding is accomplished by both hydrophobic and electrostatic interactions. The former involves several residues, (Met2199, Phe2200, Val2223, Leu2251, and Leu2252) which penetrate the lipid bilayer and interact with the aliphatic hydrocarbon chains. The latter interaction is contributed by a ring of basic residues (Arg2215, Arg2220, Lys2227, and Lys2249) localized above the hydrophobic residues. These residues associate with the anionic polar head groups of phosphatidylserine moieties. Studies on 2-D crystals of factor VIII bound to phospholipid have yielded gross information (15  A resolution) on domainal orientation and have been employed to construct a 5-domainal model of factor VIII

Figure 1 Factor VIII domains. Residue numbers denoting domainal boundaries are shown below the schematic. A and C domains are in pink and gold, respectively. Acidic rich segments a1, a2 and a3 are in green. The B domain in silver is not to scale. Approximate locations of free Cys residues (SH), sulfated Tyr residues (SO4 ), and a predicted Ca2þ binding site are indicated.

Figure 2 Factor VIII activation to factor VIIIa and subsequent proteolytic inactivation. Domainal designations are as described in the legend to Fig. 1. Heavy and light chains are linked by a metal ion (Cu)-dependent bridge depicted by light blue circles. Activating and inactivating cleavage sites are shown in green and red arrows, respectively. Approximate locations for macromolecular interactive sites are denoted by the blue lollipops. Electrostatic association of A1 and A2 (fragments) is denoted by the plum dashed line.

Activation of factor VIII and mechanisms of cofactor action consisting of the three A and two C domains.12 This model may prove useful in providing insights into conformational changes associated with cofactor activation. Factor VIII circulates as a series of heterodimers,13–15 formed as a result of proteolysis at the B–A3 junction plus additional cleavage at sites within the B domain.4 Thus factor VIII contains a constant-sized light chain comprised of the A3–C1–C2 domains and a heavy chain, minimally represented by the A1–A2 domains but variable in size due to the presence of some or all of the contiguous B domain (Fig. 2). The function of the factor VIII B domain, which represents 40% of the mass of factor VIII, is poorly understood, but does not appear to contribute to procoagulant activity. Recombinant16 or naturally occurring14;15 heterodimers lacking this region possess normal coagulant activity, indicating that this region is dispensable for function. Binding of the two chains is noncovalent and requires a metal ion-dependent linkage with the residues responsible contained within the A1 and A3 domains.4;17 The role of this linkage, and identity of metal ion(s) and residues involved are not yet resolved. A single copper ion has been identified in factor VIII18;19 and its location is controversial. A type 1 copper-binding site has been proposed to exist in both the A1 and A3 domains.4;9;10 However, the absence of characteristic spectral properties typical of a type 1 copperbinding protein suggests these sites are unoccupied. A single type 2 copper site is postulated at the heavy chain (A1 domain)-light chain (A3 domain) interface.10 Recent evidence indicates copper marginally contributes to the specific activity of factor VIII,20 yet markedly enhances the interchain affinity.21 The latter studies, employing fluorescence energy transfer techniques, indicated the inter-chain affinity in the absence of copper (Kd 50 nM) is increased about 100-fold in the presence of copper (Kd  0.5 nM). Furthermore, since this analysis employed subunits that were modified with fluorophores at Cys310 (heavy chain) and Cys2000 (light chain), residues proposed to participate in the type 1 copper site, this observation provides indirect evidence in support of a functional role for the copper 2 site. Based upon reconstitution studies using isolated factor VIII chains, copper alone is insufficient to generate cofactor activity.20;22 However, recombining purified factor VIII chains in buffers containing Ca2þ or Mn2þ yields active factor VIII.22 These ions do not contribute to the inter-chain affinity but rather promote cofactor activity by modulating the conformation of the heterodimer.21

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Ca2þ23 and Mn2þ24 bind to both heavy and light chains of factor VIII, yet this binding appears to occur at non-identical sites. A putative site for Ca2þ binding is proposed in the A1 domain (residues 108–124),23 based upon to homology of this region to a site proposed for factor V.25 Interestingly, this site is directed away from the factor IXa-interactive sites on the A2 and A3 domains (see below), suggesting that the binding of Ca2þ must transduce a conformation change over a significant distance. Thus, evidence to date indicates that Cu2þ=þ promotes the inter-chain association and contributes to the specific activity of factor VIII, whereas Ca2þ or Mn2þ is required to promote the active cofactor conformation. Factor VIII circulates in noncovalent complex with von Willebrand factor (VWF), a large, multimeric protein comprised of identical 220 kDa subunits linked by disulfide-linked bonds in a headto-head and tail-to-tail configuration (see26 for review). Factor VIII binds VWF in a high affinity interaction (Kd  0:3 nM) mediated by residues in the factor VIII light chain,27 which provides 90% the binding energy for this interaction. Critical sites for this interaction have been localized to the N-terminal acidic region preceding the A3 domain (a3, residues 1649–1689).28;29 Interactive residues for binding VWF also appear to overlap with those required for the association of factor VIII with anionic phospholipids30 or activated platelets,31 thus blocking premature association with thrombogenic surfaces. While several studies have suggested a critical role for the C-terminal residues 2303–2332 of the C2 domain, these results are not fully compatible with the X-ray structure of C2, which suggests this region lies within the domainal core.11 Recent insights provided by the A1–A2–A3–C1–C2 domainal model suggests a3–C1–C2 form an extended surface for interaction with VWF.32 The association of factor VIII in this complex results in several positive attributes that include increased circulatory half-life, increased stability of the heavy chain-light chain interaction and protection from proteolysis (see33 for recent review). This latter attribute, largely a result of inhibition of the interaction of factor VIII with phospholipid surfaces as well as direct competition with protease binding sites,34 is particularly relevant in that it limits catalysis by surface-dependent activators and/or inactivators such as factor Xa and activated protein C. Cleavage of the factor VIII light chain (at Arg1689) during cofactor activation (see below) drives the dissociation of factor VIIIa from VWF. This conclusion is illustrated by point mutations at the light chain cleavage site (Arg1689) which

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yields a cleavage-resistant molecule possessing a hemophilia phenotype due to inability of the cofactor to dissociate from the VWF carrier protein.35 Removal of the acidic N-terminal region of the light chain results in a nearly 2000-fold reduction in the affinity of factor VIII for VWF.36 Thus, the competing interaction of the cleaved factor VIII for association with the anionic phospholipid surface now becomes the dominant interaction.

Activation of factor VIII Factor VIII is proteolytically activated by thrombin or factor Xa, and this process results in cleavages at sites within both the factor VIII heavy and light chains (Fig. 2). Thrombin attacks three bonds in factor VIII whereas factor Xa cleaves at these sites plus two additional ones. Common sites include cleavage in the heavy chain at Arg372 (A1–A2 junction) and at Arg740 (A2–B junction) while the light chain is cleaved near its NH2 terminus at Arg1689.37 This action converts the inactive procofactor form of factor VIII to the active cofactor. The molecular mechanisms as well as the significance of these cleavages with respect to expression of factor VIIIa activity are described below. Detailed mechanisms of protease action on factor VIII are not fully understood but appear to indicate the importance of several regions on the proteases as well as on factor VIII that are involved in forming the bimolecular complex. These intermolecular interactions are considered separately for the two proteases.

Activation by thrombin Earlier studies showed that heparin blocked activation of factor VIII by thrombin.38 Activation was inhibited by 50% at 0.1 U/ml heparin using a physiologic concentration of factor VIII, a result suggesting a novel anticoagulant effect of heparin. Mechanistically, this observation suggested a role for the thrombin anion binding exosite 2 in interacting with the factor VIII substrate. Mutagenesis studies confirmed a role for this region of the enzyme in cleaving the procofactor. Substitution of Arg residues 93, 97, and 101 with Ala yields a thrombin molecule possessing a triple mutation, designated thrombin RA, that did not bind heparin39 and was slow to activate factor VIII.40 Activity attributed to this mutation was 10% that of wild-type thrombin and yielded reduced rates of cleavage at all three sensitive bonds. Two other thrombin molecules possessing triple mutations in

P.J. Fay

this exosite (Arg89Ala/Arg93Ala/Glu94Ala and Arg 245Ala/Lys248Ala/Glu251Ala) each showed 60% wild-type activity in activating factor VIII,41 suggesting a marginal contribution of some or all of these residues to the interaction with substrate. However, results from that study also showed that a single point mutation in thrombin, Arg98Ala, possessed 40% the activity of wild-type thrombin. Interestingly, this mutation showed disparate activities relative to the scissile bonds. While similar rates of cleavage of light chain (Arg1689–Ser1690) were observed for the mutant and wild-type enzymes, the mutant protein showed only 30% of the wild-type activity directed towards cleaving factor VIII at the Arg372–Ser373 site. This result suggested that the relative contribution of distinct interactive sites may vary depending upon the location of the cleavage site. A role for thrombin exosite 1 in the activation of factor VIII was established based upon inhibition studies showing that hirugen, an exosite 1-directed dodecapeptide,42 effectively blocked cleavage at all sites in the procofactor. In a subsequent study, evaluation of an extensive collection of exosite 1 point mutations identified several critical residues involved in this interaction.41 Specifically, Alascanning mutagenesis at six sites (Lys21, His66, Lys65, Arg68, Arg70, and Tyr71) showed 6 50% wild-type like activity in activating the procofactor. Two of these mutations, Arg68Ala and Tyr71Ala showed approximately 10% or less activity and showed reduced activity at cleaving the thrombinsensitive bonds in the light chain and at the A1–A2 domainal junction. Mutagenesis studies have also implicated other structures in thrombin as contributing to the interaction with factor VIII.41 Alteration of residues participating in the Naþ -binding site, which contributes to the thrombin active site-cleft and the 50insertion loop, which contributes to the specificity of thrombin by defining the S2 subsite, were shown to diminish the reactivity of the mutant proteins in catalyzing cleavage of factor VIII. Similar to the mutagenesis results observed for exosite 1, these mutations showed defective cleavage in both the factor VIII light chain and the A1–A2 domainal junction. Taken together, these data suggest a number of residues contribute to thrombin-catalyzed proteolysis of the procofactor. However, it is important to generally qualify results of the mutagenesis work since the substitutions constructed may have a global affect on the conformation of the protease rather than a direct effect in modulating the binding of protease to factor VIII substrate. The above studies demonstrating extensive involvement of sites in thrombin removed from the

Activation of factor VIII and mechanisms of cofactor action

active site indicate that complementary interactive regions in the substrate factor VIII are not restricted to sites in-and-around the scissile bond. Limited information is currently available concerning the locations of these protease-interactive sites in factor VIII. In a recent study, Nogami et al.43 examined the binding of anhydrothrombin, an inactive derivative of the active enzyme in which the active site serine residue is chemically modified to dehydroalanine, to isolated factor VIII and VIIIa subunits/domains. Binding of anhydrothrombin was observed using factor VIII heavy chain, light chain, A3–C1–C2, A2, and C2 with affinity (Kd ) values between 100 and 500 nM. Of the subunits tested, only A1 failed to bind the reagent. That study also showed that an inhibitor antibody directed to the C2 domain, as well as the purified C2 domain itself, blocked thrombin cleavage of the light chain. These results indicated that a thrombin site is localized to C2 and occupancy of this site by thrombin allows for proteolysis of Arg1689. Post-translational modification of factor VIII by tyrosine sulfation appears to play a role in its activation by thrombin. Based upon analysis of point mutants where selected Tyr residues were converted to Phe,8 it was suggested that sulfation at Tyr346, near the scissile bond at the A1–A2 domainal junction, and at Tyr1664, near the cleavage site in the light chain, accelerate the rate of thrombin cleavage of factor VIII. Interestingly, a naturally occurring Tyr346Cys mutation results in CRMþ hemophilia A.44 This defect was characterized by a discrepancy in one-stage and two-stage clotting assays, with the latter (which measures cofactor activity of the pre-activated sample) yielding a greater activity value than the former (which measures procofactor activity). Thus, an apparent reduction in activation rate was the basis for the pathology. One may speculate that sulfation at these sites, which occur in highly acidic regions of the cofactor, contributes to the overall anionic nature of these sequences thereby facilitating interaction with anion-binding exosites on thrombin. The kinetics of factor VIII activation have been studied in purified systems. A high catalytic efficiency (kcat /Km 5106 M1 s1 ) was observed for the activation of porcine factor VIII.45 Analysis of proteolysis products using SDS–polyacrylamide gel electrophoresis has allowed for analysis for individual cleavage reactions. Cleavage of the heavy chain appears ordered with initial cleavage at the A2–B junction (Arg740) followed by cleavage at the A1–A2 junction (Arg372).15;37 Catalytic efficiency for cleavage at the initial site

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is 20-fold greater than the latter site.46 While association of factor VIII with VWF showed no effect on heavy chain cleavage, its presence resulted in an 8-fold increase in cleavage efficiency of the light chain, such that the rate limiting step became cleavage at Arg372.46 Thus VWF does not appear to affect the overall rate of cofactor activation. The mechanism by which VWF promotes light chain cleavage is not well understood, but appears to result from a conformation change in light chain enhancing its interaction (affinity) with thrombin since the observed increase in catalysis primarily reflected a reduction in Km .46 Overall, contributions to the thrombin-factor VIII interaction are derived from both anion-binding exosites 1 and 2 of the protease and the factor VIII C2 and A2 domains, plus the potential involvement of the acidic-rich connecting regions (a1, a2, and a3). Fine-point definition of these regions is varied, especially with respect to sites in factor VIII, and further work is required to identify critical residues. However, as indicated by Esmon and Lollar,40 this extensive complementarity between thrombin and factor VIII likely accounts for the observation that this protease is an extremely efficient activator of factor VIII.

Activation by factor Xa Factor Xa has been shown to attack factor VIII at identical sites as those cleaved by thrombin.37 However, this protease attacks additional sites in the factor VIII/VIIIa substrate. Cleavage within the A3 domain at Arg172137 is likely benign based upon factor VIIIa reconstitution studies using factor VIII light chain cleaved at an adjacent site (Arg1719) by factor IXa.47 Three additional cleavage sites have been mapped within the A1 domain/subunit of factor VIII/VIIIa. Porcine factor VIII is cleaved at Arg219 as a part of the activation process, yielding a factor VIIIa form possessing two subunits derived from the original A1 domain.48 This residue is Gln in human factor VIII molecule and thus is not attacked. Factor Xa also cleaves at Arg336,37 preceding the anionic Cterminal region and Lys36.49 Proteolysis at these two sites correlates with inactivation of cofactor function and these mechanisms are considered below. While the interaction of this enzyme-substrate pairing is less well-understood, several parallels can be drawn concerning the interactive sites of factor Xa and factor VIII to those identified for the interaction of thrombin with the cofactor.

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Protease exosite involvement appears to be crucial. A series of basic residues have been identified forming the heparin-binding exosite of factor Xa.50 Preliminary data that heparin directly blocks factor Xa- catalyzed proteolysis of factor VIII (Nogami and Fay, unpublished observation) suggests involvement of this region for the binding of enzyme and substrate. Several lines of evidence also suggest a role for the C2 domain in containing a factor Xa-interactive site including antibody inhibition and anhydro-factor Xa binding studies.51 Results from that study suggested the factor Xa-binding portion of C2 was not identical with the thrombinbinding portion. Some evidence also suggests the C-terminal region of A1 subunit interacts with the protease. Zero-length crosslinking showed a salt bridge existing between this region of the A1 and a region of the factor X protease domain exclusive of the activation peptide sequence.52 Furthermore, kinetic studies demonstrate that A1 residues 336—372 appear essential for factor Xa-catalyzed cleavage at Lys36, a reaction also selectively blocked by heparin (Nogami and Fay, unpublished observation). Conversely, mechanistic differences with thrombin exist. For example, the Tyr346Phe and Tyr1664Phe mutants are efficiently cleaved by factor Xa indicating that tyrosine sulfation does not influence binding or catalysis of this proteinase.8 Factor Xase comprised of factor Xa-activated factor VIIIa shows a several-fold lower catalytic efficiency than factor Xase comprised of the thrombin-activated cofactor.45 This reduced efficiency reflects both a lower kcat value and a higher Km value for the former factor Xase.48 Overall, the significance of factor VIII activation by factor Xa is unclear and this may represent a minor pathway to factor VIIIa. The basis for this contention is severalfold. First, factor Xa requires an anionic phospholipid surface for efficient cleavage of factor VIII.37 This situation is precluded by the association of factor VIII with VWF, which effectively blocks the cofactor-surface interaction.53 Furthermore, VWF may directly block the association of protease with substrate factor VIII by blocking an interactive site in C2, based upon analogy of the competitive inhibition of activated protein C binding to C2 by VWF.34 In addition, studies using thromboplastin-activated plasma in the presence of the thrombin-specific inhibitor, hirudin, demonstrated that thrombin, and not factor Xa, was responsible for factor VIII activation.54 Finally, mutations at critical sulfation sites, such as Tyr346Cys (see above), which result in a hemophilia phenotype44 suggest the importance of thrombin in the activation of the procofactor.

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Factor VIIIa and formation of the intrinsic factor Xase The proteolytic cleavages detailed above convert the procofactor, factor VIII, to the active cofactor, factor VIIIa. The molecular mechanisms by which this conversion occurs are not fully understood. Cleavage of the light chain functions to release factor VIII from VWF55 following removal of the a3 region which contains a critical interactive site for the carrier protein. Removal of a3 also appears to promote a change in the conformation within the C2 domain, facilitating dissociation from VWF36 and enhancing the affinity of factor VIIIa for anionic phospholipid surfaces including the activated platelet.56 This cleavage also appears to contribute to increased specific activity of the cofactor,57;58 although the mechanism for this effect is not known. Cleavage of the heavy chain at residue 740 yields an 90 kDa heavy chain form (contiguous A1–A2 subunit) that requires further cleavage (at residue 372) to express factor VIIIa activity.15 Cleavage at this site is essential to expose a functional factor IXa-interactive site(s) in the A2 domain that is cryptic in the unactivated molecule.59 Factor VIIIa is a heterotrimer composed of the heavy chain-derived A1 and A2 subunits and the A3–C1–C2 subunit derived from the light chain60;61 (Fig. 2). The A1 and A3–C1–C2 subunits retain the metal ion-dependent linkage.17;62 The association of A2 subunit with the A1/A3–C1–C2 dimer is metal ion-independent and is mediated by electrostatic interactions.61 Association of A2 with dimer is reversible (Kd  260 nM at physiologic pH).17;63 Approximately 90% of binding energy for A2 association is derived from its interaction with the A1 subunit based on fluorescence energy transfer.49 The highly acidic C-terminal region (a1, residues 337–372) of A1 subunit, originally believed to be critical for retention of A2,64 appears to contribute primarily to properly orient the A2 subunit relative to other factor VIIIa subunits in facilitating interaction with the active site of factor IXa.65 Recent studies have identified segments in A2 subunit rich in basic residues (373–385, 418–428) that may be interactive with the A1/A3–C1–C2 dimer.66 The significance of this weak affinity interaction and its contribution to the down regulation of factor Xase is considered below. Changes in conformation that are manifested upon conversion of the heterodimer to heterotrimer are critical to expression of cofactor function. Several studies employing chemical and physical techniques have suggested that these changes, in

Activation of factor VIII and mechanisms of cofactor action

large part, are subtle. For example, reaction of factor VIIIa with the zero- length cross-linker, ethyldimethyl carbodiimide (EDC), resulted in formation of a covalent linkage between A1 and A2 subunits,67 indicating the presence of a salt bridge at the site of cross-linking. However, treatment of factor VIII with EDC prior to cleavage by thrombin showed no linkage between the subunits, suggesting the inter-domainal salt bridge was not present in the unactivated form. In addition, examination of binding of the apolar probe, bis anilinonapthalsulfonic acid, to isolated factor VIII and factor VIIIa subunits revealed two exposed hydrophobic sites on the isolated heavy chain68 possessing affinities (Kd values) that were different from those for the single sites localized to the isolated A1 and A2 subunits, suggesting a change in conformation in and around these regions following thrombin cleavage. Furthermore, CD studies suggest an increase in b-sheet structure in factor VIIIa formed from factor VIII.69 While these studies support the importance of conformation changes resulting in altered function, direct correlation of a given alteration to gain of a specific function such as functional interaction with factor IXa or increased affinity for anionic phospholipid surfaces have not been determined. Assembly of the factor Xase complex is mediated by protein-lipid and protein- protein interactions (see70 for review). Factors VIIIa and IXa associate with anionic phospholipid surfaces with high affin-

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ity (Kd values 1 nM30 and 15 nM,71 respectively). In addition, studies using physiologic surfaces have provided evidence for coordinate binding interactions of the enzyme, cofactor and substrate to discrete surface structures (receptors). For example, the presence of both (active site-modified) factor IXa and factor X increased both the number and the affinity of binding sites on activated platelets for factor VIIIa.72 However, physical evidence indicating classical receptor proteins for the factor Xase constituents remains to be established. Inter-protein interactions between factor IXa and VIIIa, the subject of numerous studies, likely occur over an extended interface. Two domains of factor VIII (VIIIa) appear to be critical for this interaction (Fig. 3). The A3 domain of the light chain is proposed to contain a high affinity site (Kd 14 nM) based upon binding studies using isolated factor VIII chains.73 This affinity is similar to values observed for interaction with intact factor VIII (2 nM74 ), suggesting the majority of binding energy is derived from this interaction. Analyses employing antibodies (monoclonal as well as naturally occurring inhibitor antibodies) and inhibition studies with synthetic peptides have localized this region to residues 1811–1818 within the A3 domain.75 Similar approaches have been employed to identify interactive regions in the A2 subunit including residues 484–50976;77 and 558–565.78 Mutagenesis of residues within the 558–565 loop and subsequent characterization of the recombinant

Figure 3 Factor IXa-interactive sites in factor VIIIa. A1, A2 and A3 domains are shown as ribbon format in gray, blue and green, respectively. Sites implicated as interacting with factor IXa include A2 residues 484–509 (magenta), 511–530 (teal), 558–565 (red), 698–712 (orange) and A3 residues 1811–1818 (yellow).

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proteins have shown a specific role for this region in contributing to the kcat effect.79 Roles for additional factor VIII residues, such as Arg527, are based upon mutations yielding a hemophilic phenotype due to reduced affinity for factor IXa.80 Preliminary data also suggest a role for residues 708–713 in functional interaction with the enzyme81;82 and this notion is supported by a marked reduction in kcat for an Asp712Ala mutant factor VIII (Jenkins and Fay, unpublished observation). Studies examining the stimulation of factor IXa activity by the isolated A2 subunit (see below) indicate that the interaction of this subunit with the protease is of relatively weak affinity (Kd  300 nM83 ). Complementary regions in factor IXa have been proposed. Factor IXa, a member of the vitamin Kdependent serine protease family, possesses the modular structure of (N-terminus) Gla-EGF1-EGF2activation peptide-protease domains (C terminus). The active site of factor IXa is localized high (>70  A) above the phospholipid surface,84 and is modulated by the isolated A2 subunit83 as well as the A2 subunit of factor VIIIa.76;85 Thus, the interaction of A2 with the protease domain of factor IXa would represent a surface-distal interaction. Participation of a region referred to as the 330 helix of factor IXa (residues 330–338) in the interaction with factor VIIIa is indicated by point mutations in this region that yield a hemophilia B phenotype and result in defective interaction with factor VIIIa.86 Recently, a study assessing the functional interaction of isolated A2 subunit with native and 330 helix point mutations suggested a molecular model wherein the 558 loop of A2 and 330 helix of factor IXa, in addition to spatially adjacent residues, form an extended interface.87 That model, however, failed to include participation of residues 484–509 in the interactive surface. The reason for this omission is not clear, but one alternative may reflect docking of the individual, static structures, whereas a conformation change within A2, known to occur upon activation to expose a functional factor IXa-interactive site,59 may result in realignment such that the 484–509 region now juxtaposes the enzyme. Conversely, the association of the A3 domain of factor VIIIa with factor IXa likely represents a more surface-proximal interaction.85 While the exact complementary site in factor IXa is not well resolved, ligand-blotting studies appear to indicate the light chain of factor IXa (Gla-EGF1-EGF2) is involved.88 This interaction requires activation of the zymogen as well as integrity of the Ca2þ binding site in EGF189 and maintenance of a salt bridge connecting the EGF1 and EGF2 domains.90 How-

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ever, the direct contribution of this region to the high affinity binding site remains controversial. Other studies suggest a more indirect role of the EGF domains in the proper positioning of the protease domain above the surface for optimal interactions with the cofactor and substrate.86 In addition, it has been noted that factor IXa stabilizes the weak affinity association of A2 with the A1/A3–C1–C2 dimer by 10-fold.47 The mechanism for this effect has been attributed to factor IXa tethering interactive sites in the A2 and A3–C1–C2 subunits.85 A model for the factor Xase complex, based upon known and modeled structures, is presented in Fig. 4.

Mechanisms of cofactor activity contributing to rate enhancement The mechanism(s) by which factor VIIIa enhances catalytic efficiency (kcat =Km ) for factor Xa generation remain poorly understood. The primary kinetic component affected by factor VIIIa is kcat , which is increased by 104 –106 using either synthetic phospholipid vesicles91 or native surfaces such as activated platelets.92 Variability in this value results from differences in assay protocols and complications related to the lability of factor VIIIa subunit structure (see below). Table 1 lists kinetic parameters that are representative of values which appear in the literature. Recent studies employing isolated subunits of factor VIIIa have shown that while neither the A1 nor A3–C1–C2 affect the enzymatic activity of factor IXa in catalyzing the conversion of factor X to factor Xa, the isolated A2 subunit enhanced the kcat for this reaction by 100-fold.83 Thus elucidation of kinetic mechanisms for cofactor action may be facilitated by examination of functional, component interactions. Results from the X-ray structure studies of factor IXa reveal that the active site region of the enzyme is not fully formed and persists in a “zymogen-like’’ state.93;94 This results in large part from the 99-loop (chymotrypsin numbering system) blocking substrate binding (S2–S4) sites, a configuration that is locked in place by Tyr345 (Tyr177 in chymotrypsin numbering).94 Thus factor IXa shows poor capacity to catalyze conversion of natural as well as small molecule substrates. The marked kcat increase is brought about by a factor VIIIa-induced conformational change at or near the active site in factor IXa, which has been suggested to alleviate the inhibitory effect of the loop.95 Studies using fluorescence dyes incorporated in the active site of the enzyme have shown that factor VIIIa alters both

Activation of factor VIII and mechanisms of cofactor action

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Figure 4 Factor Xase. Factor VIIIa (left) and factor IXa (right) are shown associated with a phospholipid surface. Proteins are drawn in ribbon format based upon the 5-domainal model of factor VIII32 and the crystal structure of factor IXa.93 The yellow balls show residues of the catalytic triad. The 558 loop in the factor VIIIa A2 domain and the 330 helix in the factor IXa protease domain are shown in red and blue, respectively.

Table 1 Kinetic parameters for factor IXa-catalyzed activation of factor X. Conditiona

Km (lM)

kcat (min1 )

kcat /Km (lM1 min1 )

(x-fold)

Factor IXa +A2 +A2+A1 +FVIIIa

0.121 ± 0.027 0.101 ± 0.028 0.107 ± 0.027 0.033 ± 0.010

0.013 ± 0.002 0.98 ± 0.07 13.7 ± 1.6 202 ± 21

0.107 9.8 128 6121

(1) 92 1197 57,200

a

Reactions were run in the presence of 10 lM PS/PC/PE vesicles. Reactions run in the presence and absence of A2 subunit (600 nM) contained 5 nM factor IXa and those run in the presence of factor VIIIa (10 nM) contained 0.5 nM factor IXa. Results are from Fay et al.83;99

the fluorescence intensity of the dye as well as parameters such as anisotropy indicating increased constraints in the rotational freedom of the dye.84 Increases in fluorescence anisotropy were observed following cleavage of factor VIII light chain at Arg168957 and heavy chain at Arg37259;85 appear to correlate with the generation of cofactor activity and/or increases in specific activity. Interestingly, in the absence of cofactor, substrate factor X shows little effect on the active site of the enzyme as judged by parameters such as anisotropy,76 suggesting that formation of the enzyme–substrate complex is not reflective of a productive conformation. However, in the presence of factor VIIIa, substrate factor X imparts a substantial increase in anisotropy.76 Furthermore, recent evidence indi-

cates that factor X increases the affinity of factor IXa for factor VIIIa by 15-fold.96 Overall, results from these types of studies indicate that a binding site (or sites) in factor IXa for the cofactor is conformationally coupled to the active site region of the protease and that formation of this complex allows for productive interaction with substrate. Unequivocal conclusions related to cofactorinduced structural changes will require examination of crystallographic data for factor IXa obtained in presence of cofactor. Kinetic, mutational and modeling studies suggest that the factor IXa 330 helix, which links directly to residues in the extended substrate binding site, contributes to the modulation of the structure yielding altered activity.86 It is of interest to note that a homologous

10

region in factor VIIa contributes to its interaction with its cofactor, tissue factor.97 Recent mutational data employing a truncated factor IX lacking the Gla and first EGF domains showed that mutation at Tyr345 together with substitutions at other residues in the 99-loop resulted in an 7000-fold increase in amidolytic activity toward small molecule substrates.98 These authors suggest that factor VIIIa binding close to the Tyr345 segment releases the locked 99-loop conformation, with subsequent factor X binding rearranging the released 99-loop yielding access to the active site cleft. As described above, residues in and around the 330 helix of factor IXa form an interactive surface with the 558 loop and adjacent regions of A2 subunit. A functional consequence of interaction of isolated A2 with factor IXa is an 100-fold increase in kcat for factor Xa generation. This effect is further enhanced synergistically at least 10-fold in the presence of A1 subunit99 (Table 1). The role of A1 subunit appears to alter the conformation of A2, making it more “factor VIIIa-like’’ in facilitating reaction rates. The primary component of kcat is derived from cofactor–enzyme interactions. However, the phospholipid surface also affects this parameter. Studies on the role of surface in reaction rate parameters are problematic since the lipid makes a dramatic contribution to the inter-protein affinity of factor VIIIa and factor IXa, reducing the Kd by as much as 1000-fold,100 as well as markedly reducing Km for substrate factor X. The Km effect results from restricting the reactants to a twodimensional surface, thereby increasing their collisional frequency. Although reports suggest that the surface contribution to kcat exceeds 1000fold,101 this value is likely a significant over-estimate since, in the absence of surface factor VIIIa activity is impaired by weakened interaction with factor IXa and increased sensitivity to ionic strength of the reaction mix, leading to enhanced rates of A2 subunit dissociation.102 Thus the net contribution of surface to kcat appears to be an increase of 30-fold. Since this relative increase is observed using either factor VIIIa or the isolated A2 subunit, it suggests a role for lipid in orientating the complex of enzyme plus factor VIIIa (A2 subunit) relative to substrate. While enhancement of kcat represents the primary kinetic effect of the cofactor to catalytic efficiency of factor Xase, factor VIIIa also reduces the Km for substrate factor X about 4-fold (Table 1) and this effect may be significant at plasma concentrations of factor X. Information on the interaction of factor X with factor VIIIa is limited. Solid phase assays using isolated factor VIII chains and

P.J. Fay

factor VIIIa subunits revealed that the A1 subunit Cterminal region (residues 336–372) contains a binding site for factor X.103 Affinity determinations estimated a moderate affinity (Kd  1  3 lM), however, non-equilibrium methods were used in this approach. Studies using a zero length crosslinking reagent, EDC, showed that the association between factors VIII and X was primarily electrostatic and mediated by formation of a salt bridge(s).52 Thus it was suggested that acidic residues within the C-terminus of A1 bond basic residues in factor X. That study also restricted the factor X-interactive site to residues 349–372 within A1, and localized the interactive site in factor X to within the protease domain exclusive of the activation peptide. The latter result was based upon retention of the crosslink following cleavage with the factor Xa activator in Russell’s Viper venom. The importance of the contribution of factor VIIIa to reducing Km is suggested by experiments comparing activities of native factor VIIIa with factor VIIIa possessing a C-terminal truncated A1 subunit in a clotting assay versus a factor Xa generation assay using purified components. Factor VIIIa reconstituted from isolated A1, A3–C1–C2 and A2 subunits possesses native cofactor activity in either assay. However, when A1 subunit is cleaved at Arg336 by inactivating enzymes such as activated protein C (see below), the resulting subunit, A1336 yields disparate effects when used to reconstitute factor VIIIa. Cofactor activity measured in the clotting assay, where factor X is contributed by plasma and is diluted to typically 25% the plasma concentration or 40 nM, is markedly reduced to a level of <10% that observed for native factor VIIIa.65 This result reflects a measured Km for factor X of 200 nM using factor Xase in which factor VIIIa is cleaved at Arg336,49 in contrast to a Km of 30 nM for factor X using native factor Xase (Table 1). However, activity measured using the truncated factor VIIIa in a factor Xa generation assay, where factor X is present at Vmax concentration (>300 nM), is 60% the level of native factor VIIIa.49 These results suggest that loss of the factor X interactive site within A1 subunit reduces reaction rate in plasma, in part, as a result of increased Km , yielding a rate-limiting concentration of substrate factor X.

Dampening of FXase The down regulation of factor Xase is largely mediated by alterations in cofactor activity. While

Activation of factor VIII and mechanisms of cofactor action

this topic is of high importance in understanding the regulation of factor VIIIa, it falls somewhat outside the scope of this review. For this reason, the below section summarizes (potentially) significant mechanisms, including subunit dissociation and proteolytic inactivation, by which the cofactor activity of factor VIIIa is inhibited. The activity of factor VIIIa is unstable and decays spontaneously. This lability reflects the dissociation of A2 subunit.61;62 The observed “selfdampening’’ of factor Xase is largely attributed to this process. Studies showing the decreased coagulant activity of human factor VIIIa compared with porcine factor VIIIa attribute this effect to weaker association of the A2 subunit.104 Kinetic analysis demonstrated that association of human factor VIIIa in the factor Xase complex reduced the rate of A2 subunit dissociation by as much as 10-fold compared with uncomplexed factor VIIIa.47 This result was consistent with an earlier study showing the stabilizing effects of complex formation on factor VIIIa.105 The lability of this interaction contributes a regulatory role in coagulation. Indeed, mathematical simulations of clotting require inclusion of constraints associated with factor Xase lability due to factor VIIIa dissociation.106 The physiological importance of this weak intersubunit affinity is further reflected in a class of hemophilia A mutations having a one stage assay (measure of factor VIII procofactor activity) versus two stage assay (measure of factor VIIIa cofactor activity) discrepancy.107 While native factor VIII shows equivalence in either assay, several point mutations108–110 exhibit less relative activity in the latter assay compared with the former. This observation reflects a greater rate of A2 dissociation than observed in native factor VIIIa and is attributed to alterations in or around critical residues involved in A2 subunit retention. Activated protein C (APC) is a potent anticoagulant, selectively inactivating the cofactors factor Va and factor VIIIa. Cleavage of factor VIIIa occurs at Arg562, bisecting the A2 subunit, and Arg336, preceding the C-terminal acidic region of the A1 subunit.111 The latter site is also attacked by factors IXa and Xa (see below). Both cleavages affect the factor VIIIa-dependent modulation of the factor IXa active site, as judged by fluorescence anisotropy studies.112 Cleavage of Arg562 occurs within a critical factor IXa interactive site (558–565 loop78 ) and thus would directly alter the interaction of cofactor with factor IXa. Recent studies have provided addition details to inhibitory mechanisms attributed to proteolysis at the A1 site. Cleavage at Arg336 alters the interaction between the A1 and A2 subunits reflecting reduced

11

kcat values,65 as well as resulting in a several-fold increase in Km of factor Xase for substrate factor X.49 Factor IXa selectively protects factor VIIIa from cleavage at Arg562; however, this effect is abrogated by inclusion of protein S.113 In a purified system, comparison of rates of cofactor inactivation attributable to APC action suggest factor Va is the primary substrate, since the majority of factor VIIIa activity lost was not the result of proteolysis but rather spontaneous dissociation of A2 subunit.114 Furthermore, studies in a reconstituted coagulation model show greater degradation of factor Va compared with factor VIIIa by the APC pathway.115 That study also suggested little effect of APC action on factor Xa generated by the intrinsic factor Xase, although protein S, a cofactor for APC required to overcome the factor IXa-dependent protection of factor VIIIa, was not included in the reaction mixture. Collectively, these data suggest that while APC action is largely responsible for down-regulation of prothrombinase by attack of factor Va, cofactor inactivation via this pathway may be secondary to A2 subunit dissociation for dampening factor Xase activity. Two other proteases to consider are factor IXa and factor Xa. Both proteases attack the same bond in the A1 subunit, Arg336, preceding the anionic C-terminal region. Inasmuch as these factors represent the enzyme and product of the factor Xase complex, respectively, both have opportunity to catalyze the inactivation of the cofactor. Inhibitory mechanisms resulting from cleavage in the A1 site are described above. In the case of factor IXa, cleavage at the A1 site occurs over a prolonged time course.116;117 While a rigorous kinetic evaluation of this interaction is lacking, studies modeling the decay of factor Xase using native and cleavage resistant factor VIII molecules show a contribution of this event to activity loss, however, relatively high concentrations of factor IXa were required to observe this effect.47 While the significance of factor Xa as a factor VIII activator is unclear, the product of factor Xase could contribute to the down-regulation of the complex responsible for its generation. Factor Xa attacks two sites in the A1 subunit of factor VIIIa, Arg33637 and Lys36.49 Loss of activity resulting from cleavage of A1 at Lys36 is less well understood than cleavage than at Arg336, but it appears to alter A1 subunit conformation, markedly affecting the affinity of this subunit for A2 subunit.49 The physiological significance of factor Xa-catalyzed cleavage of A1 subunit resulting in inactivation of cofactor function remains to be determined. Although the catalytic rates for these inactivating cleavage reactions are slow, the concentrating

12

effect of the thrombogenic surface in restricting reactants to a two-dimensional space and the generation of high local concentrations of protease by the factor Xase complex support the hypothesis that factor Xa may contribute to the dampening of factor Xase.

Conclusions Recent studies of macromolecular interactions leading to proteolytic activation (and inactivation) of factor VIII, and the role of covalent and conformational alterations that convert the procofactor to the active form have yielded valuable insights into the generation and regulation factor VIIIa. However, important issues related to the structure of the active cofactor, and mechanisms by which the cofactor effect is transduced to the active site region of factor IXa remain to be determined. Continued evaluation of high resolution structures, coupled with rigorous biochemical evaluation employing novel reagents such as isolated subunits and recombinant proteins to map critical interactive sites will be required to resolve these fundamental questions.

Acknowledgements I wish to thank Dr. Vince Jenkins for assistance in drawing Figs. 3 and 4, and acknowledge support from National Institutes of Health Grants HL30616 and HL38199.

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