mutating factor viii: lessons from structure to function

Blood Reviews (2005) 19, 15–27

www.elsevierhealth.com/journals/blre

REVIEW

Mutating factor VIII: lessons from structureto function

Philip J. Faya,*, P. Vincent Jenkinsb

a Departments of Biochemistry and Biophysics and Medicine, School of Medicine,University of Rochester, P.O. Box 712, 601 Elmwood Ave., Rochester, NY 14642, USAb Katharine Dormandy Haemophilia Centre and Haemostasis Unit, Royal Free Hospital,Rowland Hill Street, London NW3 2QG, UK

Summary Factor VIII, a metal ion-dependent heterodimer, circulates in complexwith von Willebrand factor. At sites of vessel wall damage, this procofactor isactivated to factor VIIIa by limited proteolysis and assembles onto an anionicphospholipid surface in complex with factor IXa to form the intrinsic factor Xase; anenzyme complex that efficiently converts factor X to factor Xa during thepropagation phase of coagulation. Factor Xase activity is down-regulated bymechanisms that include self-dampening by dissociation of a critical factor VIIIasubunit and proteolytic inactivation by the activated protein C pathway. Recentstudies identify putative metal ion coordination sites as well as ligands involved inthe catabolism of the activated and procofactor forms of the protein. Ourknowledge of these multiple intra- and inter-molecular interactions has beenfacilitated by the application of naturally occurring and site-directed mutations tostudy factor VIII structure and function. In this review, we document important andnovel contributions following this line of investigation.

�c 2004 Elsevier Ltd. All rights reserved.

KEYWORDSFactor VIII;Factor VIIIa;Factor IXa;Intrinsic factor Xase;Site-directedmutagenesis;CRMþ hemophilia

Introduction

Deficiency or defects in factor VIII result in hemo-philia A, the most common of the severe inheritedbleeding disorders. Information derived from thefactor VIII gene structure1–3 and evaluation of pa-tient samples has allowed for a comprehensivedatabase 4 that lists hundreds of mutations yieldingthe hemophilia phenotype. Of interest is that asignificant number of mutations include CRMþ

(cross reactive material positive) missense (point)

* Corresponding author. Tel.: +1-585-275-6576; fax: +1-585-473-4314.

E-mail address: [email protected] (P.J. Fay).

0268-960X/$ - see front matter �c 2004 Elsevier Ltd. All rights reserdoi:10.1016/j.blre.2004.02.003

mutations wherein a defect resides in a singleamino acid residue of the circulating protein. Thisinformation provides an excellent complement tohomology models and limited high-resolutionstructures available for factor VIII, which representa scaffold upon which interactive and functionallyimportant sites may be mapped. Conversely thesehigher order structures extend the utility of thedatabase as a predictor of important residues forfunction. Application of site-directed mutagenesisto recapitulate point mutations yielding a hemo-philic phenotype as well as the generation of novelmutations followed by expression of recombinantfactor VIII protein has generated important re-agents to probe critical intra- and inter-molecular

ved.

mail to: [email protected]

16 P.J. Fay, P.V. Jenkins

interactions. In this review, we summarize how theanalyses of mutant factor VIII proteins have yieldedvaluable insights to the intra- and inter-molecularinteractions important for the structure and func-tion of the circulating protein.

Intra-molecular interactions probed bymutagenesis

Factor VIII

Factor VIII circulates as a heterodimer, comprisedof a heavy (contiguous A1–A2–B domains) and lightchain (contiguous A3–C1–C2 domains), that asso-ciate by metal ion-dependent and -independentinteractions (Fig. 1). The latter interaction ac-counts for approximately 80% of the thermody-namic stability for the inter-chain interaction.5

Residues involved in this interaction are primarilylocalized within the A1 and A3 domains, which ischaracterized by both electrostatic and hydropho-bic binding interactions.6;7 Little direct informationis available on specific residues that participate inthese metal ion-independent interactions. Model-ing of the A domains of factor VIII suggests points ofcontact. Examination of the hemophilia A data-base4 indicates a number of point mutations atresidues that may indeed represent interactivesites, however, little direct experimental evidenceconfirming their participation is available.

Metal ion-dependent interactions appear vitalfor both factor VIII structure and function. Twodistinct interactions have been probed by recon-stitution of isolated subunits as well as limited

Figure 1 Factor VIII and factor VIIIa. The A and C domains aa2 and a3 are in green. The B domain in silver is not to scale.linked by a metal ion (Cu)-dependent bridge depicted by lightarrows. Approximate locations for macromolecular and metaElectrostatic association of A1 and A2 is denoted by the plum

studies using mutagenesis. These interactions in-clude the binding of copper ion and calcium/man-ganese ions. A single copper ion8 (Cuþ 9) has beenidentified in factor VIII and this ligand is lost upondissociation of the factor VIII chains. Biophysicalstudies examining the reconstitution of factor VIIIfrom isolated heavy and light chains revealed aprimary role for copper was in enhancing the inter-chain affinity by �100-fold.5 That study alsoshowed that copper, while alone is insufficient toregenerate the active conformation of factor VIII,in the presence of calcium (see below) does mar-ginally increase the specific activity of factor VIII.However, the site(s) involved in coordinating theCu ion remains controversial. Based upon the ho-mology of factor VIII A domains with ceruloplas-min,10;11 three potential copper sites have beenidentified in the cofactor. Type 1 sites exist in boththe A1 and A3 domains, while a type 2 site is pro-posed to span the A1 and A3 domains (Fig. 2).These sites are designated based upon character-istic spectral properties. From mutagenesis stud-ies, Tagliavacca et al.9 suggested that the type 1site in A1 contained a bound Cuþ. The basis for thisconclusion was a marked reduction in the specificactivity of a transiently expressed Cys310 to Serfactor VIII. These investigators also showed thatconversion of Cys2000 to Ser and His1957 to Ala didnot affect factor VIII specific activity, whereasHis99 to Ala yielded �25% the control value. Morerecent data evaluating mutagenesis at His99 andHis1957 (type 2 site residues) suggest that occu-pancy at this site does not contribute to enhancedspecific activity, whereas modification at Cys310and Cys2000 does not alter the inter-chain affinity(Wakabayashi et al.,12 unpublished observations).

re in pink and gold, respectively. Acidic rich segments a1,Heavy and light chains of the factor VIII heterodimer areblue circles. Activating cleavage sites are shown in greenl ion interactive sites are denoted by the blue lollipops.dashed line. Modified from Ref.18

Figure 2 Putative binding sites for Cu, Ca and Mn ions. Metal ion sites are localized within the 5-domainal model87

showing A1 (blue), A2 (cyan), A3 (copper) and C domains (gold), which is drawn bound to a surface (red). Lettersdenote the indicated site: (a) type 1 Cu site in A1, (b) type 2 Cu site bridging A1 and A3, (c) type 1 Cu site in A3, and (d)putative Ca2þ (upper) and Mn2þ (lower) sites in A1.

Mutating factor VIII: lessons from structure to function 17

Thus, these data support a model where copperion-occupancy of the type 2 site bridging the A1and A3 domains makes a direct contribution to theinter-chain binding affinity, whereas copper occu-pancy of both type 1 sites appears necessary formaximal specific activity of the cofactor.

The generation of cofactor activity following thereconstitution of factor VIII from its isolated lightand heavy chains requires divalent metal ions suchas Ca2þ (or Mn2þ). Recently, it was reported thatresidues 94–110 in the homologous protein, factorV, a region rich in acidic amino acids, formed aCa2þ binding site.13 While Ca2þ coordination infactor V functions to increase inter-chain affinity,14

the role of Ca2þ (or Mn2þ) in factor VIII appears tomodulate activity by altering protein conforma-tion.5;15 Site-directed mutagenesis of the homolo-gous region in factor VIII (residues 110–126) wasperformed to evaluate the role of this region inmetal ion coordination.16 Stimulation of cofactoractivity in response to added divalent metal ion(Ca2þ or Mn2þ) was used as an indicator to deter-mine affinity values. Ca2þ binding affinity wasgreatly reduced (or lost) following substitution atGlu110, Asp116, Glu122, Asp125, or Asp126 withalanine. Alternatively, Ala-substitution at Glu113,Asp115, or Glu124 showed wild-type-like activity

with little or no reduction in Ca2þ affinity. Exami-nation of Mn2þ affinity resulting from mutation atthese sites showed minimal effects except for themutations at Asp116 and Asp125. Assuming that theloss of a ligand for divalent metal ion coordinationresults in a reduction in metal ion binding affinity,it was proposed that Glu110, Asp116, Glu122,Asp125, and Asp126 likely coordinate Ca2þ,whereas Asp116 and Asp125 may contribute toMn2þ coordination (Fig. 2).

It was speculated in the above study that Ca2þ orMn2þ coordination would stabilize an elongatedregion defined by A1 residues 110–116 which jux-taposes the C1 domain (see Fig. 2). While A1 and A3domains appear to associate with a relatively ex-tended interface, the interface between A1 and C1is small. Thus stabilizing a segment in A1 near C1may add structure to a “hinge” region separatingthe A and C domains. This hypothesis is supportedby results obtained with Mn2þ, which is typicallycoordinated by acidic residues and/or His residues.There are two His residues in C1 (His2082 andHis2137) that are in close proximity to residues110–126 in A1. Thus these His residues may con-tribute to Mn2þ coordination with D125 (and pos-sibly D116) and this explanation is compatible withresults showing that Ca2þ and Mn2þ bind different


sites17 yet generate active factor VIII of similarspecific activity. Overall, the stabilization im-parted from metal ion binding near the A1–C1junction may be necessary to provide proper ori-entation of factor VIIIa subunits within the factorXase complex (see below).

Factor VIIIa

Proteolytic activation of factor VIII (described be-low) includes cleavage at the A1–A2 domainalboundary at Arg372, converting the factor VIIIheterodimer into a heterotrimer (see Fig. 1). Thus,while metal ion-dependent and -independent link-ages between A1 and A3 are preserved in factorVIIIa, the generation of a new subunit, A2, dictatesthe requirement for additional intra-molecular in-teractions. The association of A2 subunit with theA1/A3–C1–C2 dimer is a weak affinity interactionand dissociation of A2 represents the primarymechanism for the observed lability of factor VIIIaand the self-dampening of factor Xase activity (seeRef. 18 for review).

The importance of A2 subunit retention is borneout by a unique hemophilia A phenotype. It hasbeen long recognized that a subset of patientsdisplay discrepant assay values when factor VIII is

Figure 3 A domainal model illustrating one-stage/two-stageFactor VIII A domains11 are represented in ribbon format shdenote the a-carbon position for the indicated residues.

measured in a one-stage assay compared with atwo-stage assay.19 A significant difference in assayis that the former is a measure of procofactor(factor VIII) activity, while the latter, a measure ofcofactor (factor VIIIa) activity, requires prior acti-vation of factor VIII. Thus discrepancies when theone-stage yields a greater activity value than thetwo-stage is an indicator that the cofactor pos-sesses increased lability, likely a result of an in-creased dissociation rate for A2 subunit. A numberof missense mutations have been reported thatgive rise to this phenotype.20–24 As seen in Fig. 3,these mutations occur in A1, A2 and A3 domains offactor VIII and many lie at the inter-domainal in-terfaces with the A2 domain. This observationsuggests these residues participate, either directlyor indirectly, in interactions involving A2 subunitretention following cleavage at the A1–A2 bound-ary during cofactor activation. Alternatively, al-terations of these residues may lead to adetrimental interaction at the interface. Thesespeculations have been confirmed by several stud-ies (cited above) where specific point mutationswere recapitulated and recombinant proteins ex-pressed and subjected to physical analysis, such assurface plasmon resonance, to assess rates of A2subunit dissociation in the mutant factor VIIIaforms. The dissociation rate constants obtained

assay discrepancy mutations at the domainal interfaces.owing A1 (blue), A2 (cyan) and A3 (red). Green spheres


from these studies are consistent with enhancedrates of A2 subunit dissociation following activa-tion. Results from these analyses help to validatethe A domainal homology model as well as begin toprovide insights into potentially important inter-subunit interactions within the factor VIIIa het-erotrimer.

Inter-molecular interactions

Factor VIII circulates in complex with von Wille-brand factor (VWF), an association that stabilizesand protects the procofactor, as well as potentiallyserving to localize it to sites of vessel wall damage.Activation by proteinases such as thrombin con-verts the procofactor to active cofactor followinglimited proteolysis. Subsequent association of fac-tor VIIIa and factor IXa on an anionic phospholipidsurface yields factor Xase, which efficiently con-verts substrate factor X to factor Xa. Dampening offactor Xase is achieved by mechanisms includingsubunit dissociation and/or proteolytic inactivationvia the activated protein C pathway. Catabolism ofthe cofactor (as well as procofactor) proceeds bypathways involving interactions with heparan-likeglycosaminoglycans and the lipoprotein receptor-related protein (LRP). Thus a number of significantinter-molecular interactions occur over the circu-lating “life cycle” of factor VIII and several recentreviews address the biochemistry of these inter-actions.18;25;26

The factor VIII–VWF complex

The low levels of factor VIII in patients with severeVWF deficiency demonstrate the crucial role ofVWF in prolonging factor VIII half-life. Factor VIIIbinds via its light chain with high affinity to VWFand circulates in plasma as a non-covalently linkedcomplex. At least three regions of the light chain,the acidic a3 residues N-terminal to the A3 do-main,27 the C2 domain,28;29 and more recently theC1 domain,30 have been identified as containingVWF binding sites. The binding site within theacidic region has been localized within Lys1673 andArg1689.27 Mutation at the sulfated Tyr1680 resi-due has been shown to affect VWF binding, re-sulting in �5-fold decrease in affinity31 and yieldingmild hemophilia A.32

A series of hemophilia A-associated mutations inthe C1 and C2 domains have been identified thatresult in decreased binding of factor VIII to VWF.Examination of the residue locations in the C2 do-

main crystal structure33 and homology model of theC1 domain shows that most of the mutation sitesare located on the surface of the domains and ingeneral map within the same plane. Liu et al.34

have suggested that hemophilia-yielding mutationsites on the surface of the C1 and C2 domains occurin three distinct clusters. The cluster Thr2154,Gln2100 and Arg2150 in the C2 domain may directlyaffect C1–C2 positioning and/or VWF binding. Asecond cluster, Arg2307 and Trp2229, also in the C2domain likely interacts directly with VWF, while athird cluster of Ser2119, Arg2116 and Tyr2105 oc-curs at the N-terminus of the C1 domain. Based onthe proximity of the latter cluster to the A3 do-main, these investigators suggested that mutationsin this segment affect the positioning of the C1domain relative to the A3 domain. The observa-tions that mutations at distinct regions and inmultiple factor VIII domains alter the affinity offactor VIII and VWF indicate a complex inter-mo-lecular interaction that occurs over an extendedinterface.

Activation of the procofactor

Activation of factor VIII proceeds by limited pro-teolysis catalyzed by thrombin or factor Xa, withthe former likely representing the physiologic ac-tivator.35 Sites of cleavage occur at stretches ofacidic residues at domainal boundaries and includeP1 sites Arg372 (a1–A2 junction) and Arg740 (a2–Bjunction) in the factor VIII heavy chain and Arg1689(a3–A3 junction) in the light chain (Fig. 1). Currentinformation on the molecular mechanisms leadingto conversion of inactive procofactor to activecofactor has been addressed in a recent review.18

Briefly, cleavage of the factor VIII heavy chain atArg372 is essential to expose a factor IXa-interac-tive site,36 whereas cleavage at Arg740 removesthe B domain or its fragments from A2 subunit.Furthermore, cleavage of the light chain drives thedissociation of factor VIII from VWF.37 This cleav-age also contributes several-fold to the specificactivity of factor VIIIa.38;39

Examination of the hemophilia A database re-veals a number of point mutations at position 372and position 1689. Studies in the literature wherethe mutant factor VIII, either obtained from pa-tient plasma or recapitulated as a recombinantprotein, is assessed for cleavage by thrombin in-variably show the factor VIII chain is resistant tocleavage at the mutated site. These results areconsistent with the mutation inhibiting cleavage,and in so doing blocking the conversion to the


active cofactor molecule. Missense substitutionsidentified at these sites include Pro, Cys and His.4

Interestingly, while the Pro and Cys substitutionsinvariably yield a moderate to severe hemophiliaphenotype, the substitution of His for Arg appearsto produce a mild hemophilia. Thus the phenotypemay be dependent upon the given residue at the P1position. Interactions between the vitamin K-dependent serine proteinases and their substratesinvolve complex interactions that are not re-stricted to the binding of active site with scissilebond (see below), and limited rates of proteolysishave been observed following alterations at the P1residue (see for e.g. Ref.40) This and other similarstudies emphasize the importance of exositedocking relative to active site tethering. Consistentwith this observation, one may speculate thatthrombin-catalyzed cleavage at a P1 His residuemay be slow relative to Arg, but greater than thatobserved for a P1 Cys or Pro, and these marginaldifferences in cleavage rate may dictate the se-verity of the phenotype.

Specificity of the enzyme substrate interactionsis largely governed by exosites or regions in theenzyme removed from the active site that bindsubstrate molecules at regions distinct from sitesof cleavage. Earlier studies using the site-specificinhibitors heparin41 and hirugen42 suggested thatboth anion-binding exosites 1 and 2 in thrombinparticipated in the activation of factor VIII. Morerecently, Ala-scanning mutagenesis identified sev-eral basic residues in each exosite as contributingto the interaction with substrate factor VIII basedupon reduced rates of proteolysis following sub-stitution by alanine at these sites.43

Other mutations in factor VIII, removed from theP1–P10 site, have been observed that result in re-duced rates of thrombin activation and conse-quently a hemophilic phenotype. This class ofmutations has been characterized as having a dis-crepancy where the activity determined by a two-stage clotting assay is greater than that obtainedby the one-stage assay. Thus this phenotype rep-resents the reciprocal activity discrepancy to thatdescribed above as having enhanced rates of A2subunit dissociation. Mutations identified with this“activation deficient” characteristic includeGlu321Lys,44 Tyr346Cys44;45 and Glu720Lys.46

Tyr346 is sulfated in factor VIII47 thus contributingthe acidic character of the a1 region connectingthe A1 and A2 domains. While Glu321 is not con-tained within the a1 connecting region, this residuemay be juxtaposed to a1 in space. Finally, Glu720contributes to the acidic character of a2, whichconnects the A2 and B domains. Taken together,these residues share a common feature of con-

tributing to the acidic character of inter-domainalconnecting regions, and as such may provide im-portant contacts for anion-binding exosites in theactivating proteinases.

The factor Xase complex

The intrinsic factor Xase is the functional catalystfor conversion of factor X to Xa during the propa-gation phase of coagulation. Association of factorVIIIa with factor IXa occurs over an extended sur-face comprising both the A2 and A3 domains of thecofactor (Fig. 4). Complementary sites on factorIXa appear to localize primarily to the proteasedomain and factor IXa light chain (Gla–EGF1–EGF2domains), respectively (see Ref.48 for review). Gi-ven that the phospholipid binding sites are withinthe C2 and Gla domains of factors VIIIa and IXa,respectively, this orientation indicates factor VIIIacontributes a surface proximal interactive sitemediated by the A3 domain and a surface distalinteractive site mediated by the A2 subunit.

The high affinity (Kd � 15 nM) of the isolatedfactor VIII light chain for factor IXa49 is consistentwith the A3–C1–C2 subunit of factor VIIIa provid-ing the majority of the binding energy for this in-teraction. To date only one region within theA3–C1–C2 subunit has been identified as a factorIXa-interactive site. Inhibition studies using amonoclonal antibody localized this site within res-idues Gln1778–Asp1840.49 Subsequent peptide in-hibition studies mapped a minimum sequencerequired for this interaction to Glu1811–Lys1818,although more recent mutagenesis studies em-ploying a chimera where sequences of factor VIIIwere substituted for the homologous sequences infactor V have proposed the 1803–1810 residues asfactor IXa-interactive.50 A number of mutationsassociated with hemophilia A have been identifiedwithin the Gln1778–Asp1840 region, including Ar-g1781His, Ser1784Tyr, Leu1789Phe, Met1823Ile,Pro1825Ser, Thr1826Pro and Ala1834Val/Thr,4

supporting the essential role of this high affinityinteraction in contributing to cofactor function.However, no studies have been published to dateon the effects on these mutations and no delete-rious mutations have been identified within thesegment 1803–1818.

Based upon the capacity for factor IXa to spe-cifically block activated protein C-catalyzedcleavage at Arg562 in the A2 subunit51 and inhibi-tion of factor VIIIa stimulation of factor IXa by apeptide to the factor VIII residues 558–565,52 thisregion of A2 was identified to represent a factor

Figure 4 Factor Xase. Factor VIIIa (left) and factor IXa (right) are drawn in ribbon format based upon the 5-domainalmodel of factor VIII87 and the crystal structure of factor IXa,88 respectively. Factor IXa is shown in green with the 330helix in red. Spheres indicated the a-carbon positions of the active site residues. Factor VIII domains are coded as A1(blue), A2 (cyan) and A3 (red) and C domains (copper). Spheres indicate a-carbon positions for the indicated factor IXa-interactive sites.


IXa-interactive site. Interestingly, evaluation ofthe hemophilia A database supported this conten-tion based upon a number of missense mutationswithin this region yielding variable severities ofhemophilia. For example, substitution of Ser558with a bulky Phe residue results in a mild to mod-erate hemophilia phenotype.4 Other point muta-tions (plus indicated severity) include Val559Ala(mild), Asp560Ala (mild), Gln565Lys (moderate)and Asp569Pro (severe). Also contained within thisregion is a novel mutation that creates a new N-linked glycosylation site.53 Mutation of Ile566 toThr yields the sequence 564Asn–Gln–Thr, a con-sensus sequence for N-linked glycosylation at theasparagine residue. Indeed, glycosylation at thissite creates a severe hemophilia phenotype.Treatment of the partially purified, mutant factorVIII with N-glycanase to remove the sugar yielded amolecule with restored activity. These observa-tions were consistent with interference of a criticalinter-protein interactive site by either substitutionof a non-permissive amino acid side chain or addi-tion of a carbohydrate moiety.54

In a recent study, Jenkins et al.55 recapitulatedseveral of the CRMþ mutations in the 558–565 loopregion of A2, stably expressed and purified theproteins from BHK cells and kinetically evaluatedthe role of these residues in contributing to factor

Xase function. Results from that study suggestedthat, of the residues tested, Asp560 likely made aminor contribution to inter-protein binding. How-ever, this and several other residues within thisregion individually contributed to the overall cat-alytic efficiency (kcat). Thus this region appearsimportant in both binding factor IXa and generationof the cofactor effect. This segment of factor VIIIappears to be interactive with the 330 helix offactor IXa.56 Mutations in the helix result in a he-mophilia B phenotype characterized as not showingstimulation of activity by factor VIIIa.57 Based uponthe hypothesis that the 558 loop of A2 interactswith the 330 helix of factor IXa, Bajaj et al.56

suggested that this interface represented an ex-tended structure comprised of a number of po-tential interactions contributed by residues withinand adjacent to these structures (Fig. 5). Recentstudies have focused on further delineation of thisinterface, largely by use of mutagenesis tech-niques. For example, A2 residue Asp712, which inthe model forms a salt bridge with Lys294 of factorIXa, likely contributes to this ionic tethering sinceits substitution with Ala reduced the inter-proteinaffinity several-fold (equivalent to a stability con-tribution of � 1 kcal/mol).58 Other residues in A2,not accounted for by the Bajaj model, may alsoplay a role in modulating factor IXa activity by

Figure 5 The A2-factor IXa interface model. Themodeled interface represents only charged residuesproposed to participate in binding interactions. FactorIXa residues, shown in white, are numbered based uponthe factor IX sequence rather than the chymotrypsinnumbering system as originally illustrated by Bajajet al.56 A2 subunit residues are in yellow. Reproducedwith permission.


mechanisms that remain to be elucidated. Muta-tion at Arg527, which yields a mild phenotype andshows defective stimulation of factor IXa activ-ity,59 is located very close to the 558 loop in thefactor VIII A domainal model. Thus this residue mayindirectly contribute to function by stabilizing afactor IXa-interactive site.

Another region in A2, residues 484–509, repre-sents a major epitope for inhibitor antibodies,60

confirming an important role in factor VIII function.In a recent study to examine the contribution ofthis region to catalysis, Ala-scanning of chargedresidues (individually or in clusters) was performedand resultant factor VIII mutants assessed for ratesof factor Xa generation.61 One cluster mutant,where residues Arg489, Arg490 and Lys493 weresubstituted with alanine demonstrated reducedrates of catalysis, but exhibited little if any effectson affinity for factor IXa binding or the Km forfactor X. Interestingly, these reductions in reactionrate were not observed with the single site muta-tions, consistent with this region modulating kcatvia its basic electrostatic potential. The exactmechanism by which these charged residues con-tribute to catalysis remains to be determined.

Determination of a high-resolution structure forthe factor VIII C2 domain by X-ray crystallography33

has made a substantive contribution to our knowl-edge of the interaction of factor VIII with the an-ionic phospholipid surface. Based upon thisstructure, a binding model was proposed whereinboth hydrophobic and electrostatic interactionscontributed to the association of cofactor withsurface. The former interaction occurs via twohydrophobic spikes comprised of Met2199/Phe2200and Leu2251/Leu2252 that penetrate the lipid bi-layer and associate with the fatty acyl hydrocarbonchains. Electrostatic interactions are mediated bya ring of basic residues including Arg2215, Arg2220,Lys2227 and Lys2249, localized just above the hy-drophobic residues, that form salt linkages with theanionic polar head groups of phosphatidyl serine.While a number of point mutations resulting inhemophilia A map to the C2 domain, they tend tocluster at the protein core region rather than aputative membrane binding sites. One explanationproposed for this observation62 is that the bindingenergy reflects the additive nature of several sidechains at the membrane interface. Thus singlepoint mutations within this generally flexible re-gion may be well-tolerated. Conversely, mutationswithin the tightly packed protein core of the C2domain could result in major disruptions in foldingand/or secretion leading to loss of function.

In a recent study, the contributions of the hy-drophobic spikes to membrane binding were eval-uated following Ala-scanning mutagenesis ofindividual and clustered residues.63 Substitutingalanine for residues 2199/2200 and 2151/2152 re-sulted in �40% and �60% reductions in specificactivity and �20- and �30-fold reductions in af-finity for phospholipid vesicles, respectively.However, conversion of all four hydrophobic resi-dues to Ala in a single mutant yielded an �95%reduction in specific activity and >35-fold reduc-tion in affinity for the vesicles. These results sup-port the additive effects of the residues involved inthe interactions of factor VIII with phospholipidsurface.

Clearance of factor VIII and factor VIIIinhibitors

Both the procofactor and activated cofactor formsare likely removed from circulation by the hepaticclearance receptor, low-density LRP. 64–66 At leasttwo distinct sites, one localized to within residues1804–1834 of the A3 domain67 and the othermapped to residues 484–509 in the A2 domain,65


are involved in the interaction with LRP. Thus thepresence of two binding sites allows for uptake offactor VIII as well as factor VIIIa subunits A1/A3–C1–C2 and A2 that have undergone dissocia-tion. Interestingly, the affinity of the factorVIII–LRP interaction is not particularly strong (Kd

values of �60–120 nM,64;65) and this observationhas lead investigators to speculate on mechanismsthat would contribute to the concentration offactor VIII(a) on the cell surface. Indeed, cell-sur-face heparan sulfate proteoglycans (HSPG) showhigh affinity for factor VIII and a HSPG-binding sitewas localized to residues 558–565 within the A2domain.68 Limited information is available on fur-ther resolution of LPR- and HSPG-interactive sitesfollowing mutagenesis. A recent report describedconstruction of a chimeric factor V/factor VIIImolecule wherein factor VIII residues 1811–1818were replaced with the homologous residues infactor V.67 Although LRP binding was reducedmarkedly, cofactor activity was largely eliminated.

Binding of inhibitory antibodies to factor VIII,which may lead to increased rates of clearance,indeed reduce and/or eliminate the concentrationof functional factor VIII in circulation. Inhibitorsare largely restricted to severe hemophilic indi-viduals, affecting �25% of that population. A de-tailed description of these interactions can befound in several recent reviews.69–71 Initial studies

Figure 6 Localization of inhibitor epitopes. The 5-domainascribed in the legend to Fig. 4. Prominent inhibitor epitopes

from the Scandella laboratory72 employed re-combinant fragments of factor VIII expressed inEscherichia coli and mapped prominent epitopes ofthese inhibitors to the A2 and C2 domains (seeFig. 6). Consequently, a seminal study from theLollar laboratory showed that replacement of theA2 domain of human factor VIII with the homolo-gous porcine domain yielded a functional factor VIIIthat eliminated a major inhibitor epitope.73 Thatstudy set the stage for identification of residuesinvolved in forming the epitope for inhibitors, aswell as provided a proof-of-principle that human/porcine hybrids may represent a viable alternativefor reducing factor VIII antigenicity (see below).For example, a common inhibitor epitope withinthe factor VIII A2 domain was mapped to residues484–509.60 Antibodies to this region appear toblock a functional interaction of the A2 domainwith factor IXa74 by a mechanism likely related tointeraction of factor Xase with substrate factorX.61;75 Subsequent Ala-scanning mutagenesis of thisregion identified several residues within this seg-ment (Tyr487, Ser488, Arg489, Pro492, Val495,Phe501, and Ile508) that appeared critical for in-teraction with antibodies from several inhibitor-containing plasmas.76

A second major inhibitor epitope occurs withinthe C2 domain (residues 2173–2332). Binding ofantibody at this site disrupts interaction with VWF

l model of factor VIII87 is drawn in ribbon format as de-are illustrated in space-filling format.


as well as the phospholipid surface, thus attenu-ating the protective effects of VWF and blockingformation of factor Xase. Mapping studies applyingthe above strategy that porcine sequences showless reactivity with inhibitors than do human se-quences, identified a major factor VIII inhibitorepitope determinant that was bounded byGlu2181–Val2243.77 Inasmuch as this region con-tains the hydrophobic spikes, Met2199/Phe2200and Leu2251/Leu2252 implicated in factor VIIIbinding to phospholipid, a subsequent studyshowed the single mutations Met2199Ile,Phe2200Leu and Leu2252Phe all demonstrated re-duced antigenicity to most inhibitors tested,78

suggesting that most C2-directed inhibitors fre-quently target these spike structures. This specu-lation was confirmed following X-raycrystallographic analysis of an Fab fragment de-rived from an immortalized patient inhibitor IgG(BO2C11) complexed with a recombinant C2 do-main.79 That analysis showed that the all four hy-drophobic residues forming the spikes were buriedin the Fab complex. Furthermore, two residues(Arg 2215 and Arg2220), that contribute to thebasic ring proposed to interact with anionic polarhead groups, formed salt bridges with Asp residuesin the Fab. Thus functional studies assessed usingthe factor VIII mutants were confirmed in a high-resolution structure.

Superior factor VIII molecules bysite-directed mutagenesis

The continuing stream of information on factor VIIIstructure and function provides a resource fromwhich novel mutations may be envisioned thatpossess characteristics more desirable than thenative molecule in terms of a therapeutic.80–82

Modification of the factor VIII sequence to enhanceits circulating half-life, preserve the stability of theactivity cofactor and/or reduce immunogenicitywould constitute beneficial properties and theseareas are briefly addressed below.

Increasing the circulatory half-life of factor VIIIis dependent upon reducing its affinity for LRP and/or HSPGs. However, this approach is problematicsince sites that participate in these interactionsalso represent either factor IXa-interactive sites(558–565,52 1804–1818,50;83; see Fig. 1) or sitesthat enhance catalytic activity (the basic cluster in489–493,61) Gross modification of these sites,while attenuating the catabolic interaction, wouldlikely result in reduced factor VIII activity, asdemonstrated in the study by Bovenschen et al.67

described above. Thus a fine point dissection ap-proach will be required to preserve cofactor ac-tivity while minimizing these auxiliary interactions.It is of interest to note that recently reportedmutations in charged residues clustered within the484–509 region of factor VIII, and removed fromthe basic cluster at 489–493, retain wild-type-likeactivity.61 At the time of this writing, these factorVIII forms are under evaluation for interactionswith LRP.

Two novel, recombinant factor VIII reagentshave been described that possess enhanced co-factor stability by preventing dissociation of A2subunit in the factor VIIIa heterotrimer. In oneconstruct, designated IR8,84 a largely B-domainlessfactor VIII molecule retains a short length of Bdomain contiguous with the A3 domain but with thethrombin cleavage site at 1689 eliminated. Thuscleavage at Arg372, necessary to expose factor IXasites(s) within the A2 subunit, allows for separationof the A1 and A2 domains, but the latter remainscovalent with the (B segment)–A3–C1–C2 subunit.This molecule showed an apparent 5-fold higherspecific activity than wild-type factor VIII and re-tained �40% of its cofactor activity four hours afterthrombin activation. However, this moleculeshowed a 10-fold weaker affinity for VWF. A secondconstruct makes use of a novel disulfide bridge tocovalently bond A2 subunit within the factor VIIIamolecule.85 In this factor VIII mutant, Tyr664 in A2and Thr1826 in A3 were both changed to Cys. Basedupon the close spatial separation of these residuesas predicted by the A domainal homology model,11

these investigators speculated that the Cys re-placements would allow for formation of a disulfidebond. Indeed, this bond was formed in the re-combinant factor VIII. Activity assays revealed noloss in factor VIIIa during an extended reactiontime course.

A final consideration relates to immunologicalissues of reducing interaction with existing inhibi-tor antibodies (antigenicity) as well as attenu-ating the antibody response (immunogenicity). Asdescribed earlier, selective mutagenesis as carried-out by Lollar and colleagues86 following substitu-tion of porcine for human sequences, particularlywithin A2 and C2 domains, shows significant re-ductions in factor VIII antigenicity. Thus thesechimeric molecules would represent a novel ther-apeutic in the arsenal for treatment of inhibitorpatients. More daunting problems relate to issuesof immunogenicity. While issues related to toler-ization are beyond the scope of this review, theidentification and potential elimination of immu-nodominant sequences will certainly be advancedby a mutagenesis approach. It remains unclear why


a limited subset of exposed factor VIII sequencesdominates the inhibitor response. Alteration ofintrinsically immunogenic sequences with care topreserve cofactor function offers one solution toreducing immunogenicity.

Conclusions

Mutant factor VIII molecules have proven to rep-resent useful tools in the dissection of intra- andinter-molecular interactions involving the cofac-tor. Importantly, use of site-directed mutagenesiscoupled with valuable insights obtained from thehemophilia A database provide a synergy in ap-proach to assess basic questions of factor VIIIstructure and function, as well as potentially yieldtherapeutic reagents for the management of he-mophilia A.

Acknowledgements

Support is acknowledged from National Institutesof Health Grants HL30616, HL38199 and HL76213 toP.J.F., and the Haemophilia Society (UK) andKatharine Dormandy Trust to P.V.J. We thank Dr.H. Wakabayashi for the preparation of Fig. 2.

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mutating factor viii: lessons from structure to function

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