Factor VIII structure and function

Factor VIII Structure and Function

P. A. Foster, T. S. Zimmerman SUMMA R Y. The relatively recent ability to obtain highly purilled factor VIII (FVIII) preparations from plasma products, the cloning of the FVIII gene, and the expression of recombinant FVIII have provided the basis for significant advancements in the understanding of the structure-function relationships of FVIII. Evaluation of the molecular structure of FVIII has revealed the presence of domains of significant internal amino acid sequence homology as well as homology with similar structural domains of factor V. Specific proteolytic cleavage sites have been identified in the molecule and the use of site directed mutagenesis has identified those proteolytic cleavage sites required for the activation of FVIII. Deletion and substitution variants of FVIII as well as the precise epitope mapping of FVIII antibodies which inhibit the procoagulant function of the protein or its binding to von Willebrand factor have provided insight into the identification of regions of FVIII which are required for normal function.

Factor VIII (FVIII) circulates in plasma with von Willebrand factor (vWF) as a noncovalently linked protein complex and plays an important role in the intrinsic pathway of coagulation. The clinical importance of FVIII procoagulant activity in the maintenance of normal hemostasis is evident in the severe and often fatal hemorrhagic disorder which accompanies its functional deficiency. The production of a FVIII molecule lacking procoagulant activity or the total lack of production of the FVIII protein, results in hemophilia A, an X-linked recessive bleeding disorder with a frequency of approximately 1 per 10000 in the general population.’ Much of the early observations of the properties of FVIII were obtained from the clinical study of hemophilia in an attempt to understand the basic pathophysiology of the disease. Division of Experimental Hemostasis, Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California. This work was supported in part by Grants HL 15491, HL 31950 and DK 07022 from the National Institutes of Health. This is publication number 553%BCR from the Research institute of Scripps Clinic. Correspondence: T. S. Zimmerman, MD, Scripps Clinic and Research Foundation, BCR 8, 10666 North Torrey Pines Road, La Jolla, California 92037, USA. B/ooJ Reviews (1989) 3. 180-191 Q 1989 Longman Group UK Ltd

Although descriptions of familial bleeding disorders affecting male members of certain families were recognized as early as the 2nd century, as documented in the writings of the Talmud,,* our understanding of the pathogenesis of hemophilia is relatively recent. Addis, in 1911, is generally credited with the first postulation that hemophilia was caused by the absence of a plasma factor which was later termed antihemophilic factor.3 It was not until 1936 that Patek and Stetson demonstrated the ability of normal plasma to shorten the prolonged clotting time of hemophilic blood,4 although the transfusion of whole blood was shown to successfully treat a bleeding episode in a hemophiliac as early as 1840. 5 A great deal of confusion was generated in the study of FVIII when early purified preparations of antihemophilic factor were demonstrated to not only correct the clotting time of hemophilic plasma but also to restore platelet adhesion to glass beads and to restore platelet aggregation, in the presence of the antibiotic ristocetin, in the plasma of patients with von Willebrand disease. Since von Willebrand disease was clearly a different disorder than hemophilia A by 0268-960X/89/ooO3-0l80

SlO.00

BLOOD REVIEWS

both genetic and clinical criteria, a controversy raged as to whether the above described properties of antihemophilic factor resided on different parts of the same protein or whether they resided on two separate proteins which co-purified. It has only been within the last decade that it was generally accepted that FVIII and vWF are two separate proteins which form a complex in plasma. The successful purification of FVIII from plasma and FVIII concentrates, the recent cloning of the genes for both FVIII and vWF, and the mapping of the epitopes of antibodies to FVIII and vWF which inhibit their functions, have resulted in much new information about the structure and function of both molecules. This review will focus on the structurefunction relationship of FVIII. Nomenclature Confusion concerning the molecular nature of FVIII is apparent in the literature because most reports characterizing the properties of FVIII prior to the mid to late 1970s are in fact describing the properties of the FVIII/vWF complex. This confusion is compounded by terminology which is somewhat imprecise given our current understanding of these two proteins. Along with our new understanding of the molecular nature of FVIII and vWF has come new recommendations for the nomenclature of each protein. In 1985, the subcommittee on factor VIII and von Willebrand factor of the International Committee on Thrombosis and Haemostasis formulated new nomenclature guidelines for FVIII and vWF.~ The symbol for factor VIII protein is now VIII instead of VIII:C. The symbol for factor VIII antigen is VIII: Ag and is to be used in the context of immunologic measurement of FVIII. Finally, the symbol for factor VIII activity is VIII:C and is to be used in the context of the functional measurement of cofactor activity. Likewise, the symbol for von Willebrand factor is vWF, and for the antigen is, vWF:Ag. No abbreviations are recommended for any of the various functional activities associated with vWF since no in vitro test completely reflects vWF activity. Cofactor Function Activated FVIII (FVIIIa) is a critical participant in the intrinsic pathway of coagulation (Fig. 1). The serine protease factor IXa (FIXa), in the presence of FVIIIa, negatively charged phospholipids, and calcium ions (tenase complex), cleaves an approximately 11 kilodalton (kDa) amino-terminal activation peptide from factor X (FX) to produce FXa.’ FXa is also an active serine protease. FXa, in the presence of activated factor V (FVa), phospholipid, and calcium ions (prothrombinase complex), is then able to convert prothrombin to its active enzymatic form thrombin.’ Neither FVIIIa nor FVa have been

181

Intrinsic Pathway of Coagulation Prekaiiikrein

4

X,,

Kallikrein HMK *“la XILXla IX

Tenase Complex

-L

q

IXa Villa ~Vlll Ca++

I{

XL

Prothromblnase Complex +V

Prothrombin &Thrombin Fibrinogen &Fibrin Fig. 1 Schematic representation of the intrinsic pathway of coagulation illustrating the components of the tenase complex and the prothrombinase complex. Coagulation factors are represented by their Roman numerals. Activated coagulation factors are represented by their Roman numerals followed by a small case a. Other abbreviations: high molecular weight kininogen, HMK; calcium ions, Ca++; phosphoiipid, PL.

demonstrated to contain any enzymatic activity and thus have been defined as cofactors. Although the association of the cofactor with the active enzyme is believed to increase the catalytic activity of the enzyme, the exact nature of their cofactor activity remains incompletely defined. Bovine FIXa has been demonstrated to directly activate FX in the absence of other cofactors, but only to a very small extent.’ In this study, the addition of phospholipid vesicles did not produce an effect on the catalytic activity of FIXa, but rather greatly decreased the apparent K, of FX. It is currently thought that the binding of FIXa and FX to the phospholipid surface increases the reactant’s relative concentration and proximity, and thus produces an apparent increase in the affinity of the enzyme for the substrate. The addition of FVIIIa to FIXa, FX, phospholipid, and calcium, leads to a marked acceleration in the rate of formation of factor Xa.9*‘0 This increased catalytic activity of FIXa is believed to be due to a direct association of FVIIIa with FIXa on the phospholipid surface. In a system using human FVIII, Hultin demonstrated that, in contrast to bovine FVIII, human FVIIIa produced both an increase in the rate of formation of FXa and also a decrease in the K of FX.’ ’ This suggests that FVIIIa may also play amdirect role in the modulation of FX binding to phospholipid and/or modulation of its interaction with FIXa. FVIII shares considerable structural as well as functional similarity with FVi2,13 (for recent review see 14). The association of FVa with FXa on a phospholipid membrane has been reported to allosterically induce a conformational change in the active site of FXa and to position the active site at a proper distance above the membrane for prothrombinase activity. 15 Because of the striking structural and functional homology between these two proteins, it is likely that FVIIIa plays the same role in the FVIIIa-

182

FACTOR

VIII STRUCTURE

AND

FUNCTION

FIXa-FX complex as FVa plays in the FVa-FXaprothrombin complex. Molecular Structure Two recent advances in the investigation of FVIII have resulted in a large amount of detailed information concerning the molecular structure of FVIII. The first was the successful purification of FVIII from bovine,i6 porcine,i7”* and human’9-25 sources. The second advance was the cloning of the gene for human FVII126*27 and the expression of recombinant cDNA produced FVIII.28*29

gation of the synthesis and secretion of recombinant FVIII by mammalian cells in tissue culture, it would appear that the predominant form of FVIII in plasma is the two chain form. Kaufman et al.j4 have reported detailed studies of the synthesis, processing and secretion of recombinant human FVIII by Chinese hamster ovary cells. In this system, single chain FVIII is synthesized and then cleaved to a 200 kDa heavy chain and a 80 kDa light chain form in the Golgi. Only small amounts (less than 10% of the total secreted FVIII) of unproteolized single chain FVIII is secreted into the culture media.34 The intracellular enzyme responsible for this cleavage has not been identified.

PuriJed Factor VIII

Human FVIII has been purified to a very high degree by several groups. 19-25*30 These preparations have been purified from commercial FVIII concentrates 19*21*23-25*30 cryoprecipitate,20 and plasma.22 The reported specific activity of these preparations range from 2294 U/mg” to 8000 U/mg2’ and represent an approximately 164 000 to 500 OOO-foldpurification compared to plasma. The majority of these groups have used immunoaffinity chromatography as a step in the purification of FVIII.‘9P20*22-25 FVIII purified from FVIII concentrates or cryoprecipiate consists of multiple polypeptides of approximately 80-210 kDa.*‘9,20,23-25,30 Western blot analysis, tryptic peptide mapping, and amino acid analysis of these polypeptides have shown that the 80 kDa peptide represents the carboxyl-terminal light chain of FVIII. 24,26,31 The 92-210 kDa peptides are all derived from the aminoterminal portion of the parent molecule. All of these latter peptides have the identical aminoterminal amino acid sequence and limited thrombin proteolysis of these fragments produces the 92 kDa aminoterminal heavy chain.24g31S32The enzyme which cleaves at Arg 1648 to generate the 80 kDa light chain and the enzyme(s) and sites which generate the loo-210 kDa heavy chain peptides are not known. This purified form of FVIII is felt to consist of heterodimers of a 80 kDa light chain in association with a heavy chain segment of 92-210 kDa.23,24,30P32 The two chains are presumed to be associated via a calcium linkage.l8,23,24.29,30,33 Two groups, however, have reported the recovery of a single chain, unproteolized form of FVIII from cryoprecipitate2’ and heparinized whole blood.22 It is not totally clear how much of the proteolysis of FVIII seen in the purified FVIII preparations recovered by most investigators is a result of proteolysis occurring in vitro during the purification process and how much occurs in vivo. Based on information from the investi*The specific peptides of purified FVIII with or without subsequent proteolysis have been described by several investigators with slight variations in reported molecular weight. For the purpose of discussion in this review, the molecular weights of FVIII peptides will be as reported by Fulcher et al.1g~32~42

Recombinant Factor VIII

Recently, the cloning of the human FVIII gene26,27 and the expression of active recombinant FVJI12*,29 have been reported. The amino acid sequence deduced from the FVIII gene and analysis of proteolytic cleavage products of FVIII have provided much information regarding the structure of the FVIII molecule.24~31*33 The cDNA codes for a mature single chain FVIII molecule of 2332 amino acid residues with a calculated molecular weight of 264 763 Da prior to glycosylation.31 This corresponds to a circulating glycosylated form of approximately 330 kDa.20 Structurally, FVIII can be divided into three distinct domains based on amino acid sequence homology (Fig. 2). The triplicated A domains (amino acid residues l-328, 380-711, and 1694-2019)33 contain approximately 30% internal amino acid residue sequence homology. 28,31 These domains also contain approximately 30% homology with the triplicated domains of the copper containing plasma protein ceruloplasmin’ 2,31 and 40% homology with the triplicated A domains of FV. ’ 3

FVlIl

372 1

’ Al

1669

711

709

FV

1

111 =A2

0

1016

0

1545

1YI

Ceruloplasmin ]Al] Fig. 2 Comparison of the structural domains of FVIII, N, and ceruloplasmin. The structural domains of each molecule are denoted by the letter within the boxed areas. The first and last amino acid residue of each domain is noted for FVll133 and FV.13 The hatched in areas of FVlll represent the two acidic regions. The arrows above Wll139 and FV13 indicate the thrombin cleavege sites necessary for activation. In the case of FV however, complete activation occurs with a single cleavage by Russels’ viper venom at residue 1545.“’

BLOOD REVIEWS

Between the second and third A domains lies the B domain (amino acid residues 712-1648)33 which is encoded entirely by exon 14 containing 3106 nucleotides (Fig. 2).‘* This domain has only 14% homology with the B domain of FV. l3 Ceruloplasmin does not contain a B domain between its second and third A domain. It is suspected that the B domain of FVIII represents a processed gene that has inserted itself into an ancestral gene resembling ceruloplasmin.27 It also has no known functional role other than possible protection of FVIII from proteolysis.35*36,37 The C domains of FVIII (amino acid residues 2020-2172 and 2173-2332)33 are at the carboxylterminus of the molecule and contain approximately 40% internal homology28,31 and 40% homology with the C domains of FV (Fig. 2).13 The C domain also contains approximately 20% amino acid sequence homology with the first 150 amino acid residues of the discoidin lectins of Dictyostelium.31 In addition to the structural domains based on amino acid homology as described above, the FVIII molecule also contains two regions which contain an unusually large number of acidic amino acid residues. The acidic region of the heavy chain extends from amino acid residue 33 l-372 and contains 15 aspartic acid and glutamic acid residues with only 4 lysine and arginine residues out of 42 amino acids.24*3’*33 The region also functions as a spacer region between the first (residues l-328) and second (residues 380-711) A domains.33 As will be discussed, this region appears to play an important role in the expression of FVIII procoagulant activity. An analagous acidic region occurs in the light chain of FVIII involving amino acid residues 1649-1689. This region also contains 15 aspartic acid and glutamic acid residues and only 4 lysine and arginine residues out of 41 amino acids.24*31,33 This region also appears to be important in the expression of FVIII procoagulant activity and contains a binding domain for vWF. There are no analogous acidic regions in FV, suggesting that these regions may be responsible for functions which are unique to FVIII. This supposition is supported by the localization of a binding domain for vWF to within the acidic region of FVIII light chain3’ as will be discussed later. Proteolytic Processing of Factor VIII Specific thrombin, activated protein C, and FXa cleavage sites have been identified within the FVIII heavy and light chain. Thrombin Thrombin digestion of the 80 kDa light chain of FVIII results in cleavage at Arg 1689 to form a carboxyl-terminal 72 kDa peptideZ0*24*31*32(Fig. 3). Thrombin digestion of the 92 kDa to 210 kDa polypeptides results in cleavage at Arg 740 with formation of the 92 kd aminoterminal heavy chain26 and subse-

1 w

TII APC Th Th X4 Xa

APC

? 336 372 444

:

92-kDa

183

Th APC Xa APC Th 740 & I

Th 7 xa xa 7 1330 164816691721 +,,+ 4 4 4 \\ 80-kD4 1690

2332 coon

2332

I

I’; 54-kDa

t 373

44-kDa

72-kDm

1 740

2332

1722 ‘L--Y 45-kDa

I

I 67-kDa

Fig. 3 Line diagram of FVlll indicating proteolytic cleavage sites. The known or proposed proteolytic cleavage sites of FVIII are denoted by the arrows above the line diagram of PVIII. The second and third lines indicate the FVIII fragments produced by thrombin (Th), activated protein C (APC), and factor Xa (FXa) cleavage. The numbers below the fragments indicate their molecular weight.

quently cleavage at Arg 372 with formation of a 54 kDa aminoterminal subunit and a 44 kDa carb(Fig. 3). Further oxyl-terminal subunit 20*23*24*31*32 thrombin digestion cleaves the 54 kDa peptide into a 30 kDa and a 20 kDa peptide. In addition to the above sites of cleavage, thrombin has been reported to produce cleavage at Arg 1330 and after prolonged incubation at Arg 336 in recombinant FVIII.39 Cleavage at any given site does not appear to be dependent on previous cleavage at any other site(s).40

Activated Protein C Proteolysis of FVIII by activated protein C has been demonstrated to occur by cleavage of the lOO210 kDa heavy chain species to produce the 92 kDa heavy chain and a 53 kDa species presumably from the aminoterminal region of the B domain. The 92 kDa heavy chain is further cleaved to form a 68 kDa aminoterminal intermediate peptidep41 This peptide is further proteolyzed to produce an approximately 45 kDa aminoterminal peptide and a 23 kDa peptide.24*41*42 The 45 kDa peptide has the same aminoterminal amino acid sequence as intact FVIII heavy chain. 24 Cleavage of the 92 kDa heavy chain or its 54 kDa thrombin cleavage fragment to form the 45 kDa fragment is proposed to occur at Arg 336 (Fig. 3).24 The residues at which the other cleavages occur have not yet been identified. No activated protein C cleavage sites have been demonstrated in FVIII light chain. 24 This is in contrast to the case for FV in which activated protein C has been demonstrated to cleave both the FV heavy and light chain, although it is the heavy chain cleavage which is inactivating.43 Factor X FXa cleaves the heavy chain of FVIII by proteolysis at the thrombin cleavage site at Arg 372 generating the aminoterminal 54 kDa and carboxyl-terminal

184 FACTORVIIISTRUCTUREANDFUNCTION 44 kDa subunit peptides (Fig. 3).24 FXa cleavage also produces a 45 kDa aminoterminal peptide. The site of this cleavage has not yet been identified but it is believed to occur at the proposed activated protein C cleavage site at Arg 336. 24 FXa is also believed to cleave at the thrombin cleavage site Arg 740.24 FXa cleaves the light chain of FVIII at the thrombin cleavage site at Arg 1689 generating the 72 kDa subunit and also at Arg 1721 with generation of a 67 kDa peptide. Factor VIII Activation The activation of FVIII requires specific proteolytic processing of the parent molecule. Thrombin is considered to play the most important role in the activation of FVIII in vivo and has been the most broadly studied in vitro. The exact subunit makeup of the active form (or forms) of FVIII has not been totally resolved. It is clear that elements from both the heavy and light chains are required for procoagulant function. This has been shown by the lack of procoagulant activity associated with the isolated FVIII heavy or light chain and by the development of procoagulant activity when the two chains are reconstituted.2g*37’44 It is also clear that the large central B domain of FVIII plays no apparent role in procoagulant activity in that recombinant FVIII molecules in which the entire B domain has been deleted retain normal procoagulant activity. 35,36,37,45 Several combinations of FVIII peptides have been proposed as the active form of FVIII based on the subunits present on polyacrylamide gel electrophoresis of thrombin activated FVIII. These include a 92 kDa/80 kDa heterodimer,20 a 92 kDa/72 kDa heterodimer 1g,32~42~46*47 and a 72 kDa/54 kDa/ 44 kDa heterotrimer.24*2g*30j33 M ore recently, Fay was able to demonstrate a thrombin activated FVIII preparation in which the 72 kDa and 54 kDa subunits eluted together after rapid gel filtration and contained procoagulant activity. 48 The 44 kDa subunit eluted separately and contained no procoagulant activity. This strongly suggests that the 72 kDa/54 kDa heterodimer is at least one form of active FVIII. The production of recombinant cDNA FVIII molecules with site-specific deletions and substitutions have helped further identify critical thrombin cleavage sites necessary for activation of FVIII. These studies have shown that modification or deletion of the thrombin cleavage sites at Arg 372 or Arg 1689 result in loss of or marked reduction of FVIII coNo loss of cofactor activity was factor activity. 3g*40*4g noted with modification of the arginine residue at positions 740, 1648, or 1721.40 It has also been demonstrated that alteration in the proposed activated protein C and FXa cleavage site at Arg 336 results in a FVIII molecule with higher specific activity than normal, possibly due to resistance to proteolytic inactivation4’

Factor VIII Inactivation The molecular events leading to inactivation of FVIII in plasma have not been totally resolved. In some instances, proteolysis is clearly implicated as the inactivating event. The inactivation of FVIII by activated protein C has been demonstrated to occur by cleavage of the 54 kDa peptide at the proposed cleavage site Arg 336 with generation of a 45 kDa fragment.24*2g*42 FX a also inactivates FVIII by proteolysis at the same proposed cleavage site at Arg 336. FXa also cleaves the 72 kDa light chain at Arg 1721 with the generation of a 67 kDa fragment.24* 2g Whether cleavage at Arg 1721 alone is sufficient for the inactivation of FVIII has not yet been determined.24 The inactivation of FVIII during prolonged incubation with thrombin is not as well understood. Several investigators have reported the loss of FVIII activity without further proteolysis of the active form24*30,48*50*51while others suggest proteolysis is involved.20~46~47The most suggestive evidence that the loss of activity of thrombin activated FVIII involves a conformational change in the molecule and not additional proteolysis was reported by Fay.4” It was demonstrated that FVIIIa and procoagulant inactive FVIIIa were both composed of the 72 kDa/ 54 kDa heterodimer. The active and inactive forms of this heterodimer could be differentially eluted from an anion exchange HPLC column, thus suggesting a difference in conformation had occurred which resulted in differing abilities of the two forms to bind to the anion exchange resin of the column. It was also noted that the inactivated form was still in the heterodimeric form thus inactivation did not occur as a result of dissociation of heavy chain from light chain. It was further demonstrated that a bifunctional crosslinking agent, which only formed intrachain crosslinks in FVIII, was able to stabilize FVIIIa from inactivation, suggesting that intrachain conformational changes may play a role in inactivation. The supposition that FVIII can be protected from inactivation by stabilization of conformation is indirectly supported by the observation that thrombin activated FVIIIa is stabilized by the association with FIXa and phospholipid5’ and by observations that FVIII in association with FIXa is protected from proteolysis by activated protein C.52*53 The primary mode of FVIII inactivation in vivo is unknown.

Procoagulant Activity Though the preceding studies have provided compelling evidence as to the importance of specific cleavage sites for activation and/or inactivation of FVIII, little is known about the specific regions of FVIII which mediate interaction with other components of the hemostatic system.

BLOOD REVIEWS

Inhibitory Antibody Epitopes In an attempt to identify areas of the FVIII molecule which are important to the expression of FVIII procoagulant activity, this laboratory has mapped the binding regions of FVIII inhibitory antibodies. Monoclonal antibodies were raised to purified human FVIII and polyclonal antibodies were raised against synthetic peptides based on the FVIII amino acid sequence. The binding areas of these antibodies were then mapped by testing the reactivities of these antibodies with synthetic FVIII peptides. The binding region of a monoclonal FVIII inhibitory antibody to the heavy chain of FVIII has been mapped to amino acid residues 351-365 by this method.54 This binding region resides within the acidic region of the heavy chain. It also lies between the activated protein C cleavage site at Arg 336 which inactivates FVIII, and the thrombin cleavage site at Arg 372 which is critical to FVIII activation. The mechanism of inhibition of FVIII by this antibody is unknown. Binding of the antibody may interfere with thrombin cleavage at Arg 372 either sterically or through an induced conformational change. It is also possible that it does not inhibit FVIII activation but prevents FVIIIa association with other components of the tenase complex. Again this could occur through steric effects or induction of conformational changes. Since FVIII is inactivated by activated protein C cleavage at Arg 336 which would result in cleavage of this acidic region from the 54 kDa subunit, it is likely that this region is involved with binding to one of the other components of the tenase complex. As will be discussed later, the acidic region of the light chain is important in protein-protein interactions as evident by the localization of a vWF binding domain within this region. Two polyclonal FVIII peptide antibodies with FVIII inhibitory activity have been mapped to residues 1663- 1669 and 1655- 1664, while a monoclonal FVIII antibody with no FVIII inhibitory activity was mapped to residues 1665- 1669.55 This suggests that residues 1663 and 1664 may be particularly important in the modulation of FVIII procoagulant activity, at least as determined by the epitopes of these two polyclonal antibodies. The binding areas of these two polyclonal antibodies resides within the acidic region of the light chain of FVIII. This acidic region, like that of the heavy chain, immediately preceeds a thrombin cleavage site (Arg 1689) which is critical to the activation of FVIII. The mechanism of inhibition of these antibodies is not known. Localization of human FVIII inhibitors may also implicate certain regions of the FVIII molecule as structurally important for procoagulant activity. Fulcher et al56v57 have demonstrated that the majority of FVIII alloantibodies arising in hemophilia and FVIII autoantibodies arising in nonhemophiliacs react with the 72 kDa FVIII light chain and/or the 44 kDa carboxyl-terminal subunit of the heavy chain.

Ref.

372

1689

1

1

185

Residues

59

338-362

I

54

351-365

I

50

379-538

107

701-766

55

1655-1669

58

2178-2332

I I m

Fig. 4 Binding areas of FVlll inhibitory antibodies. The schematic representation of the structural domains of FVIII is as described in Figure 2. The lines below indicate the approximate binding areas of the described FVIII inhibitory antibodies relative to the FVIII schematic above. The residue numbers of the binding area and the reference number for each are noted to the left.

These binding regions have been further localized to amino acid residues 379-538 and 2178-2332.” Two patients with antibodies with epitopes on the 54 kDa heavy chain subunit were also reported. Ware et als9 have also reported a human inhibitor epitope to be located within amino acid residues 338-362. Figure 4 represents a composite of the reported binding regions of monoclonal, polyclonal, and human FVIII inhibitory antibodies. The multiple binding regions for inhibitory antibodies implies heterogenous mechanisms of FVIII neutralization. Many of the currently defined binding areas of human inhibitors were mapped by demonstrating reactivity of the antibodies with bacterially expressed FVIII fragments of residues 379-538 and 2178-2332.58 As smaller FVIII fragments are used for testing, it will be interesting to see how small of an area of FVIII contains these inhibitory epitopes. It is also interesting to note that a large number of human FVIII inhibitors bind to the 44 kDa subunit of FVIII heavy chain whereas the current evidence suggests that the active form of FVIII is the 72 kDa/ 54 kDa heterodimer. This would suggest that areas of the FVIII molecule not directly responsible for procoagulant activity may play important roles in the modulation of the expression of that activity. Recombinant Factor VIII Studies Studies with recombinant FVIII molecules have also provided information concerning the importance of various parts of the FVIII molecule. The deletion of all or a large portion of the B domain has been reported by several groups to have no effect on procoagulant activity or vWF binding.35,36,39,45 A plasma derived form of FVIII consisting of only the 92 kDa/80 kDa heterodimer has been demonstrated to combine with vWF and restore FVIII activity in hemophilic dogs. 6o Although no loss of procoagulant activity has been demonstrated by these truncated recombinant molecules, it has been suggested that truncated FVIII molecules may contain new immunogenic determinants. One such new epitope has been

186

FACTOR VIII STRUCTURE AND FUNCTION

reported for a recombinant FVIII molecule with a deletion of residues 797-l 562.‘j1 The importance of the acidic region of the heavy chain for FVIII procoagulant activity has been demonstrated. A recombinant FVIII with an 880 amino acid deletion of the B domain has normal FVIII activity as discussed above.36 When the acidic region of the heavy chain of this recombinant FVIII is also deleted, leaving intact the 54 kDa and 44 kDa subunits, the molecule has a marked reduction in procoagulant activity (Pittman and Kaufman, personal communication). The importance of the acidic region for procoagulant activity is consistent with the localization of the binding region of a monoclonal FVIII inhibitor to residues 351-365 as discussed previously. 54 Deletion of the acidic region of the light chain however does not appear to result in a loss of procoagulant activity but does interfere with association with vWF.~’ The localization of polyclonal FVIII inhibitory antibodies to this acidic region5’ would suggest that they interfere with activation of FVIII and not with association of FVIIIa with other components of the tenase complex since this region is removed from FVIII by activation. The importance of this acidic region in vWF binding has also been suggested by antibody studies which will be described below. Von Willebrand Factor Interaction The best characterized interaction of FVIII with other components of the hemostatic system is its association with vWF. vWF is a multimeric glycoprotein which is hemostatically important in the mediation of platelet-vessel wall interactions at the site of vascular injury. 63*64vWF also functions as a carrier protein for FVIII which, because of its apparent stabilizing effect on FVIII, is also a hemostatically important function. In plasma, the circulating levels of FVIII are dependent upon FVIII complex formation with vWF. This is most clearly evident in severe von Willebrand disease in which patients with complete deletion of the vWF gene have markedly reduced FVIII levels.65 The importance of vWF in the stabilization of FVIII has long been suggested by the prolonged rise in FVIII levels seen after infusion with cryoprecipitate in patients with von Willebrand disease.66 The FVIII levels reached could not be accounted for by the amount of FVIII infused. The effect of vWF on the stabilization of FVIII activity in plasma has been demonstrated in vitro.67 Infusion studies of partially purified FVIII in humans68 and highly purified FVIII in dog@’ have demonstrated a marked reduction in the circulating half-life of infused FVIII in the absence of endogenous vWF. The effective expression of rcombinant FVIII is enhanced by the presence of vWF in the cell culture media or the coexpression of vWF by the transfected cells.34 The nature of this stabilization of FVIII by vWF

has not been totally established. vWF has been reported to protect FVIII from proteolysis by activated protein C6’ and by low concentrations of FXa7’ in purified systems. vWF does not however, protect FVIII from thrombin proteolysis.71 vWF has also been reported to protect recombinant FVIII heavy chain from proteolysis in cell culture media,34 possibly by preventing cleavage by activated protein C or similar protease. We72 and others73 have shown that a major binding site of vWF for FVIII lies within the first 272 amino acids of the vWF subunit. Our studies also indicated that intrachain disulfide bonds are necessary for vWF to retain FVIII binding function, but that interchain disulfide bonds are not. Each subunit of vWF contains one FVIII binding site. Whether each FVIII binding site in a multimer is actually capable of binding a FVIII molecule is not clear. Based on the relative concentrations of vWF and FVIII in human plasma, it is evident that not all sites are occupied. In an in vitro porcine system, it has been shown that each subunit of vWF can bind one FVIII molecule under saturating conditions.74 Using a different technique in an in vitro human system, Hamer et a175 estimated one FVIII molecule was bound per four vWF subunits under saturating conditions. A vWF binding domain of FVIII has already been localized by the authors to amino acid residues 1670-1684 of the FVIII light chain. This was accomplished by the mapping of the binding domain of a monoclonal FVIII antibody which inhibited FVIII binding to vWF to these residues.38 A second monoclonal FVIII antibody which also inhibits FVIII binding to vWF has been localized to residues 1673- 1689 by Leyte and Verbeet (personal communication). The localization of a vWF binding domain to the acidic region of the light chain of FVIII preceding the thrombin cleavage site at Arg 1689, which is critical for the activation of FVIII, is consistent with several previous observations. Fass et all8 suggested that FVIII light chain was involved in vWF binding based on the observation of a porcine FVIII antibody which reacted with and inhibited purified FVIII but reacted only weakly with FVIII in plasma. This antibody had reactivity only with light chain. It was suggested that the binding region of this antibody was not accessible when FVIII was bound to vWF, indicating that FVIII light chain may be involved with vWF binding. Direct evidence for the binding of FVIII light chain to vWF has now been reported by several groups. Hammer et a175 demonstrated the binding of radiolabeled purified FVIII to immobilized vWF. They were able to completely block this binding with a monoclonal FVIII antibody with light chain specificity. They also were able to demonstrate the binding of isolated FVIII light chain to vWF. Similarly, Sewerin et al, 76 Ezban and Nordfang,77 and Lollar et a178 were all able to demonstrate isolated FVIII light

BLOOD

chain binding to vWF. Isolated FVIII heavy chain could not be demonstrated to bind to vWF by any of these four groups or by Eaton and Vehar.33 These observations firmly establish the location of a vWF binding domain on FVIII light chain. We have previously referred to observations which support the localization of a vWF binding domain specifically to the acidic region which precedes the thrombin cleavage site at Arg 1689. Thrombin activation is believed to cause release of vWF from FVIII. This was initially suggested by the observation that vWF and FVIII: Ag are associated in plasma but dissociated in serum.79 It is also suggested by the loss of the ability of thrombin treated FVII178*80and thrombin treated FVIII light chain71v75,78 to bind to vWF. These observations are consistent with a vWF binding domain located on the acidic region of the 80 kDa light chain which is cleaved from the active form of the light chain (72 kDa) by activating cleavage at Arg 1689. Direct evidence for release of FVIII from vWF by thrombin proteolysis is provided by the evaluation of the release of FVIII fragments from FVIII/vWF complexes produced by thrombin. Three groups of investigators have demonstrated the release of the 72 kDa activated light chain by thrombin proteolysis of FVIII bound to immobilized vWF.‘~*‘~*‘* Also as previously discussed, a recombinant FVIII molecule in which the acidic region of the light chain was deleted, was unable to associate with vWF.‘j2 Only one group of investigators has reported the binding of 72 kDa light chain to vWF.~~ The preponderance of the evidence, therefore, is consistent with the localization of a major vWF binding domain to the acidic region of FVIII light chain. The mapping of a monoclonal FVIII antibody which blocks FVIII binding to vWF to residues 1670-1684 suggests these residues may be specifically involved. The question of whether a secondary vWF binding domain exists on FVIII heavy chain remains controversial. Thrombin digestion of FVIII/vWF complexes resulted in the release of all FVIII fragments as reported by Lollar et al” and Sewerin et a1.76 However, Hamer et al reported that the 54 kDa aminoterminal heavy chain subunit was released by thrombin proteolysis but the 44 kDa carboxyl-terminal subunit remained bound, suggesting a vWF binding domain on this fragment. 75 The same investigators, however, have reported release of both the 54 kDa and the 44 kDa fragments by FXa treatment of FVIII/vWF complexes.” Since both thrombin and FXa are believed to cleave FVIII at the same site (Arg 372) to produce these fragments, it is difficult to explain the apparent discrepancy in vWF binding of the 44 kDa heavy chain subunit. The majority of the evidence, therefore, suggests that the major vWF binding domain does reside within the acidic region of the light chain. The possibility of a secondary interaction between FVIII heavy chain and vWF exists but there currently is no conclusive direct evidence that such a site is present.

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187

Phospholipid Interaction

As discussed previously, negatively charged phospholipids are required for the effective interaction of FVIIIa, FIXa and FX for the production of FXa.9*81 If the analogy with the prothrombinase complex is valid, then the phospholipid-FVIIIa and phospholipid-FIXa interactions play a critical role in the subsequent tenase complex formation.82 The direct interaction of phospholipid with FVIII has been suggested by the observation that phospholipid preparations are able to adsorb FVIII from solution.83*84 This adsorption did not require calcium ions but was inhibited by increasing calcium concentration.83,84 In the same studies, the adsorption of FIXa and FXa were demonstrated to be dependent on the presence of calcium. Yoshioka et al demonstrated that the incubation of phospholipid with FVIII resulted in no change in FVIII activity but produced a marked decrease in FVIII : Ag detected by ELISA assay, thus suggesting FVIII-phospholipid complex formation. 85 Part of the loss of FVIII: Ag could be recovered by treatment of the complex with phopholipase C. The same results were found when platelet lysate was used instead of phospholipid. Since these experiments were carried out with FVIII/vWF complex and not purified FVIII, it is not certain that the interaction occurred with FVIII directly or through a phospholipid-vWF interaction. Other observations however suggest that there is a direct association of FVIII with phospholipid. The interaction of negatively charged phospholipid vesicles with FVIII/vWF complex has been demonstrated to result in displacement of vWF from FVIII activity and in the association of FVIII activity with the phospholipid vesicles, when evaluated by sucrose density ultracentrifugation.86~87 It has also been demonstrated that thrombin-activated FVIII migrates with the phospholipid layer with this technique.*’ Since thrombin-activated FVIII is unable to associate with vWF71,75,78.80 but is able to associate with phospholipid, it is unlikely that the vWF and phospholipid binding domains are identical. However, because of the apparent dissociation of FVIII from vWF by phospholipid, it may be that their respective binding domains are in close proximity. The phospholipid binding domain has been localized to the light chain of FVIII by both direct and indirect methods. A human FVIII inhibitory antibody has been described which binds only to purified FVIII and not to FVIII which had been incubated with phospholipid, thus presumably identifying a phospholipid binding site on FVIII. This antibody was demonstrated to react with a sample containing heavy chain and light chain peptides by direct immunoradiometric assay but not to preparations of isolated heavy chain, thus implicating a light chain binding site.*’ The association of purified FVIII to phospholipids has been shown in a more direct fashion by Bloom.go

188 FACTOR VIII STRUCTURE AND FUNCTION In an ELISA assay he was able to demonstrate the binding of an intact recombinant FVIII molecule to phosphatidylserine. A recombinant FVIII with a truncated B domain (deletion of residues 797-1562) also bound to phospholipid with the same affinity as that of intact recombinant FVIII. This indicated that the B domain was not required for phospholipid binding. In addition it was demonstrated that isolated FVIII light chain bound to phospholipid but isolated heavy chain did not. There has been no further localization of the phospholipid binding domain within a specific area of the light chain. Since FVIIIa must bind to phospholipid to be effective in the tenase complex, the phospholipid binding domain must reside on the carboxyl-terminal side of residue 1689, the cleavage site producing FVIII activation. A possible location is within the two C domains of FVIII (residues 2020-2 172 and 2 173-2332) which contain an approximately 20% amino acid sequence homology with the discoidin lectins of Dictyoselium.31 These discoidin lectins have been demonstrated to bind to negatively charged phospholipid. 91 The phospholipid binding domain of FV however, which also contains homology with the discoidin lectins in its C domains, is believed to be located in an area (A3 domain) within the aminoterminal region of the activated light chain but outside of the C domains.92q93 In addition to its presumed role in localizing FVIII and FIXa, phospholipid may also play a role in modulating a stable conformation of FVIIIa. Phospholipid appears to have a stabilizing effect on thrombin-activated FVIII in vitro.86 Phospholipid incubation with FVIII has also been reported to protect FVIII from inactivation by human FVIII inhibitory antibodies in an in vitro system94 It is interesting to recall that a large number of patients with FVIII inhibitors have antibodies which recognize the carboxyl-terminal region of FVIII light chain (residues 2178-2332).58 If the FVIII neutralizing effect of these inhibitors could be blocked by the incubation of FVIII with phospholipid, this would suggest that the C2 domain may be involved with phospholipid binding. It is apparent that both FVIII and FVIIIa bind to phospholipid. It is not clear, during in vivo clotting, whether FVIII is predominantly activated in fluid phase or after binding to the platelet membrane. Rick and Krizek reported that thrombin activation of FVIII was enhanced by the presence of activated pIatelets. g5 Their findings suggest that the association of FVIII with membrane phospholipids found predominantly on stimulated platelets results in more efficient activation of FVIII by thrombin. Hultin similarly found an enhancement of FVIII activation by stimulated platelets. g6 It has also been reported, however, that activation of FVIII by thrombin increases its binding to unstimulated platelets and that stimulation of the platelets did not result in any further increase in FVIIIa binding.”

Calcium Interaction Although there is no direct evidence demonstrating that calcium is required for FVIII cofactor activity within the tenase complex, calcium (or divalent metal ion) is necessary for the maintenance of the association of FVIII heavy and light chain. The calcium ion requirement for assembly of the tenase complex appears to be required for the effective binding of FIXa and FX to the phospholipid membrane.83,84,g8 Calcium is not required for FVIII binding to phospholipid and the association is inhibited with increasing concentrations of calcium.83*84 The removal of calcium from FVIII preparations has been demonstrated to result in loss of procoagulant activity. 99s100 The chelation of calcium with EDTA results in the separation of FVIII heavy chain from light chain. 18*23*2gq30The reconstitution of FVIII activity from isolated heavy chain and light chain requires the presence of divalent cations with Mn2+ having the strongest effect and then followed by Ca2+ and CO~+.~**** The domains of the FVIII subunits involved in calcium interaction have not yet been identified. Vehar et a131 noted that FVIII contains the four amino acid side chains proposed as the ligands for type I copper atoms of ceruloplasmin in the first (His 267, Cys 3 11, His 315, Met 320) and third A domains. It is possible that these residues participate in calcium binding. The fact that they are located in the first and third domains is consistent with an active form of FVIII consisting of the 72 kDa/54 kDa heterodimer. However there is no direct experimental evidence to implicate any area of FVIII as a calcium binding domain. Likewise, no area of FV has been identified as containing a calcium binding domain. Factor IX Interaction It has been reported that FVIII can be activated directly by FIXa. lo1 This occurred, however, only at extremely high concentrations of FIXa relative to thrombin concentrations required for FVIII activation. Other investigators were unable to detect any FVIII activation by FIXa. “*‘O FIXa has been demonstrated to protect FVIII from inactivation by activated protein C.41*52v53Since only FVIII heavy chain is cleaved by activated protein C, it is possible that a binding domain of FXIa resides on FVIII heavy chain. It is known that in the prothrombinase complex that the cofactor (FVa) mediates binding to the enzyme (FXa) via the light chain102~‘03*104 and also possibly via the heavy chain.‘05,‘06 Further evaluation is necessary to determine the FVIII binding domain(s) for FIXa. It is also not known whether FIX activation, FVIII activation or both are required for their interaction.

Factor X Interaction There is direct evidence for the binding of FVIII to FX. Vehar and Davie demonstrated the binding of

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FVIII to FX and used this as a final step in the purification of bovine FVIII.16 FX has also been demonstrated to enhance the association of FVIII heavy and light chain, presumably through an interaction with one or both chains.44 There have been no studies which have localized the site of interaction. In the prothrombinase complex, the substrate (prothrombin) binds to FV heavy chain”’ and thus it might be expected that FX binds to FVIII heavy chain. Further studies will be necessary to determine the binding domain of FX on FVIII. In conclusion, we have accepted to provide an update on the structure-function relationships of the FVIII molecule. As the production of FVIII be recombinant DNA technology becomes a reality, this knowledge becomes increasingly relevant. It is now possible to produce variant molecules which have advantages over the wild type product. Such may be the case in engineering molecules which lack antigenic sites for inhibitors but retain procoagulant activity.

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53. Rick M E, Kriezek D M, Esmon N L 1988 Factor IXa modifies the degradation of factor VIII by activated protein C. Clinical Research 36: 417A 54. Foster P A, Fulcher C A, Houghten R A, de Graaf Mahoney S, Zimmerman T S 1988 Localization of the binding regions of a murine monoclonal anti-factor VIII antibody and a human anti-factor VIII alloantibody, both of which inhibit factor VIII procoagulant activity, to amino acid residues Threonine 351-Serine 365 of the factor VIII heavy chain. Journal of Clinical Investigation 82: 123-128 55. Shima M, Fulcher C A, de Graaf Mahoney S, Houghten R A, Zimmerman T S 1988 Localization of the binding site for a factor VIII activity neutralizing antibody to amino acid residues Asp 1663-Ser166g.Journal of Biological Chemistry 263: 10198-10203 56. Fulcher C A, de Graaf Mahoney S, Roberts J R, Kasper C K, Zimmerman T S 1985 Localization of human factor VIII inhibitor epitopes to two polypeptide fragments. Proceedings of the National Academy of Sciences USA 82: 7728-1732 57. Fulcher C A, de Graaf Mahoney S, Zimmerman T S 1987 FVIII inhibitor IgG subclass and FVIII polypeptide specificity determined by immunoblotting. Blood 69: 1475-1480. 58. Scandella D, de Graaf Mahoney S, Mattingly M, Roeder D. Timmons L. Fulcher C A 1988 EDitoDe . . mannine .._ of human factor VIII inhibitor antibodies by deletion analysis of factor VIII fragments expressed in Escherichiu coli. Proceedings of the National Academy of Sciences USA 85: 6152-6156 59. Ware J, Lubahn B C, Reisner H M, Stafford D W 1987 An epitope of FVIII reactive with a human hemophilic inhibitor. Blood 70: 396a 60. Brinkhouse K M, Sandberg H, Garris J B, Mattsson C, Palm M, Griggs T, Read M S 1985 Purified human factor VIII procoagulant protein: comparative hemostatic response after infusion into hemophilic and von Willebrand disease dogs. Proceedings of the National Academy of Sciences USA 82: 8752-8756 61. Esmon P C, Mitra I, Foumel M A 1988 Recombinant factor VIII immunogenicity studies. FASEB Journal 2: Al 161 62. Pittman D D, Kaufman R J 1987 Internal deletions of factor VIII identify potentially important peptide sequences for binding to von Willebrand factor. Blood 70: 392a 63. Weiss H J, Turitto V T, Baumgartner H R 1978 Effect of shear rate on platelet interaction with subendothelium in titrated and native blood. I. Shear rate-dependent decrease of adhesion in von Willebrand’s disease and the BernardSoulier syndrome. Journal of Laboratory and Clinical Medicine 92: 75@764 64. Baumgartner H R, Tschopp T B, Meyer D 1980 Shear rate dependent inhibition of platelet adhesion and aggregation on collagenous surfaces by antibodies to human factor VIII/van Willebrand factor. British Journal of Haematology 44: 127-139 65. Ngo K Y, Trifard Glotz V, Koziol J A, Lynch D G, Gitschier J, Ranieri P, Ciavarella N, Ruggeri Z M, Zimmerman T S 1988 Honozygous and heterozygous deletions of the von Willebrand factor gene in patients and carriers of severe von Willebrand disease. Proceedings of the National Academy of Sciences USA 85: 2753-2757 66. Bennett B, Ratnoff 0 D, Levin J 1972 Immunologic studies in von Willebrand’s disease. Evidence that the antihemophilic factor (AHF) produced after transfusions lacks an antigen associated with normal AHF and the inactive material produced by patients with classic hemophilia. Journal of Clinical Investigation 51: 2597-2601 67. Weiss H J, Sussman I I, Hoyer L W 1977 Stabilization of factor VIII in plasma by the von Willebrand factor. Studies on posttransfusion and dissociated factor VIII and in patients with von Willebrand’s disease. Journal of Clinical Investigation 60: 390-404 68. Tuddenham E G D, Lane R S, Rotblat F, Johnson A J, Snape T J, Middleton S, Kernoff P B A 1982 Response to infusions of polyelectrolyte fractionated human factor VIII concentrate in human haemophilia A and von Willebrand’s disease. British Journal of Haematology 52: 259-267 69. Koedam J A, Meijers J C M, Sixma J J, Bouma B N 1987 Von Willebrand factor protects factor VIII from

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