Blood online
Home About Blood Authors Subscriptions Permission Advertising Public Access contact us
 

 
Advanced
Current Issue
First Edition
Future Articles
Archives
Submit to Blood
Search
American Society of Hematology
Meeting Abstracts
Email Alerts
This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Right arrow Rights and Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lenting, P. J.
Right arrow Articles by Mertens, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lenting, P. J.
Right arrow Articles by Mertens, K.
Related Collections
Right arrow Review Articles
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Next Article next article arrow

Blood, Vol. 92 No. 11 (December 1), 1998: pp. 3983-3996

REVIEW ARTICLE

The Life Cycle of Coagulation Factor VIII in View of Its Structure and Function

By Peter J. Lenting, Jan A. van Mourik, and Koen Mertens

From the Departments of Plasma Protein Technology and Blood Coagulation, CLB, Sanquin Blood Supply Foundation, Amsterdam, The Netherlands.

    INTRODUCTION
Top
Introduction
References

THE PAST TWO DECADES have brought remarkable progress in our understanding of the molecular basis of hemophilia A. This disease, which has already been documented as a familial bleeding tendency in the fifth century,1 still persists as the most common hemorrhagic disorder, affecting 1 in approximately 5,000 males.2 Hemophilia has been associated with deficiency of a plasma component since 1937, when Patek and Taylor3 showed that the clotting defect of hemophilic plasma could be corrected by plasma of a normal individual. This component was called "antihemophilic factor," or "factor VIII" according to the more recent nomenclature. Subsequent studies using preparations enriched in factor VIII activity have established factor VIII as being the cofactor of activated factor IX in the factor X-activating complex of the intrinsic coagulation pathway.4 However, the molecular entity of factor VIII has remained unidentified until the early 1980s, when the protein was purified to complete homogeneity, and its cDNA was cloned.5-8 This breakthrough has triggered numerous studies on the genetic and molecular basis of hemophilia A, and consequently our knowledge on the structure and function of the factor VIII protein has been rapidly expanding since then.

The present review focuses on the "life cycle" of factor VIII, which comprises the sequence of events between biosynthesis and clearance of the protein (see Fig 1). These processes are discussed in view of our current knowledge on factor VIII structure and function, with particular reference to the proteolytic modulation of factor VIII, and its assembly into the factor X activating complex.


View larger version (20K):
[in this window]
[in a new window]
  		Fig 1. The lifespan of factor VIII. Factor VIII is synthesized by various tissues, including liver, kidney, and spleen, as an inactive single-chain protein. After extensive posttranslational processing, factor VIII is released into the circulation as a set of heterodimeric proteins. This heterogenous population of factor VIII molecules readily interacts with vWF, which is produced and secreted by vascular endothelial cells. Upon triggering of the coagulation cascade and subsequent generation of serine proteases, factor VIII is subject to multiple proteolytic cleavages. These cleavages are associated with dramatic changes of the molecular properties of factor VIII, including dissociation of vWF and development of biological activity. After conversion into its active conformation, and participation in the factor X activating complex, activated factor VIII rapidly looses its activity. This process is governed by both enzymatic degradation and subunit dissociation.

    BIOSYNTHESIS AND SECRETION OF FACTOR VIII

Factor VIII gene.   The gene of factor VIII is located at the tip of the long arm of the X chromosome.5 It spans over 180 kb, and as such is one of the largest genes known. Its transcription may require several hours assuming a transcription rate of 10 nucleotides per second, and yields a 9-kb mRNA product.5 The factor VIII gene comprises 26 exons, which encode a polypeptide chain of 2351 amino acids.6-8 This includes a signal peptide of 19 and a mature protein of 2332 amino acids. Analysis of the deduced primary structure determined from the cloned factor VIII cDNA showed the presence of a discrete domain structure: A1-a1-A2-a2-B-a3-A3-C1-C26-8 (Fig 2). The A domains display approximately 30% homology to each other. These domains further display a similar extent of homology to the copper-binding protein ceruloplasmin and to factor V, the cofactor in the prothrombinase complex.9 The A domains are bordered by short spacers (a1, a2, and a3) that contain clusters of Asp and Glu residues, the so-called acidic regions. The C domains are structurally related to the C domains of factor V. In addition, the lipid-binding lectin discoidin I, human and murine milk fat globule proteins, and a putative neuronal cell adhesion molecule from Xenopus laevis share amino acid sequence similarity to the factor VIII C domains.10-12 The B domain is unique in that it exhibits no significant homology with any other known protein.

Biosynthesis of factor VIII.   Several tissues have the potential of expressing the factor VIII gene. Factor VIII mRNA has been demonstrated in a variety of tissues, including spleen, lymph nodes, liver, and kidney.13-15 Transplantation studies in hemophilic animals showed that organs such as lung and spleen indeed contribute to the presence of circulating factor VIII.16,17 However, the liver most likely provides the primary source of factor VIII. This view is supported by liver perfusion18,19 and by liver transplantation studies in both animals and humans.20-22 A number of cases concerning hemophilic patients have been reported in which transplantation resulted in sustained, normalized levels of factor VIII.21,22

Several lines of evidence indicate that, within the liver, hepatocytes are the major factor VIII-producing cells. First, factor VIII mRNA is present in hepatocytes but not in sinusoidal cells.13 Second, the promotor region of the factor VIII gene comprises responsive elements that are characteristic for hepatocyte-specific expression.23 Finally, in immuno-ultrastructural studies factor VIII protein was detected in the rough endoplasmatic reticulum and the Golgi apparatus of hepatocytes.24 It should be mentioned that other reports showed the presence of factor VIII in hepatic endothelium using histochemical techniques.25-27 This is unexpected because these cells appear to lack factor VIII mRNA. It is possible that the latter observation reflects surface binding of factor VIII or internalization rather than factor VIII biosynthesis.

Secretion of factor VIII.   Studies on factor VIII biosynthesis and secretion have been limited by the lack of human cell lines that properly express significant amounts of factor VIII. Analysis of the factor VIII secretion process has therefore been restricted to autologous gene expression.28 These studies showed that, in general, factor VIII is poorly expressed. Low expression is associated with a low level of steady-state mRNA29 and inefficient secretion.30 The initial stage of secretion involves the translocation of the mature 2332 amino acid polypeptide into the lumen of the endoplasmatic reticulum (ER), where N-linked glycosylation occurs. Within the ER, factor VIII appears to interact with a number of chaperone proteins, including calreticulin, calnexin, and the Ig-binding protein (BiP).31-34 Due to the interaction with these chaperone proteins, a significant proportion of the factor VIII molecules is retained within the ER, thereby limiting the transport of factor VIII to the Golgi apparatus. The mechanism reponsible for the transport from the ER to the Golgi apparatus is not elucidated yet. However, recent studies indicate that this step involves an intracellular membrane lectin: endoplasmatic reticulum-Golgi intermediate compartment-53 (ERGIC-53).35

Within the Golgi apparatus, factor VIII is subject to further processing, including modification of the N-linked oligosaccharides to complex-type structures, O-linked glycosylation, and sulfation of specific Tyr-residues (Fig 2). In addition, factor VIII is among the many proteins that undergoes intracelullar proteolysis.36-39 The middle part and the carboxyterminal region of the B domain comprise a motif (Arg-X-X-Arg), which is similar to the motif that is recognized by intracellular proteases of the subtilisin-like family.38,39 The responsible endoprotease mediating intracellular factor VIII proteolysis, however, has not been identified. The Arg-X-X-Arg motif allows proteolysis at Arg1313 and at Arg1648. The latter event disrupts the covalent linkage of the factor VIII heavy chain (A1-a1-A2-a2-B) and light chain (a3-A3-C1-C2), giving rise to the heterodimeric molecule that circulates in plasma.

The factor VIII heavy and light chain remain noncovalently associated through the A1 and A3 domain in a metal-ion-dependent manner.7,40-42 Considering the structural homology of factor VIII to the copper-binding protein ceruloplasmin, it is not surprising that copper ions have been found in factor VIII as well.43,44 One molecule of copper is present per molecule of factor VIII. Most likely, the copper-ion binding site is composed of residues His265, Cys310, His315, and Met320 within the A1 domain.44 Binding of copper ions to this site may allow the A1 domain to adopt an A3-domain binding conformation. Alternatively, copper ions may directly bridge the A1 and A3 domain by interacting with both domains simultaneously. Whether only copper ions are involved in the association between heavy and light chain is unclear. Whereas in the absence of other metal ions copper ions are ineffective in promoting reassembly of dissociated heavy and light chain, calcium or manganese ions are considerably more efficient in this respect.45-47a However, specific activity of factor VIII dimers that were reassociated in the presence of calcium ions, is markedly enhanced upon the addition of copper ions.47a Apparently, copper ions serve an auxiliary role to enhance cofactor function of factor VIII. These observations suggest that multiple sites may be involved in the association between heavy and light chain. Irrespective of the precise mechanism, it is clear that metal ions serve an important role in maintaining the heterodimeric structure of secreted factor VIII.

Factor VIII secretion and hemophilia A.   Aberrant biosynthesis or secretion may result from several defects. Obviously, gross deletions or rearrangements may result in impaired transcription, RNA processing, or translation. No data are reported on the secretion of such gene products or on the stability within the circulation provided that these gene products are actually secreted. Defective secretion may further be caused by apparently minor gene defects like single missense mutations. Frequently known missense mutations associated with low levels of factor VIII are located in codon 2307, resulting in replacement of Arg2307 by Gln or Leu.48,49 Both mutations have been analyzed using recombinant factor VIII mutants expressed in mouse fibroblasts50 and COS-1 monkey kidney cells.51 Both proteins appear to be functionally normal, but are poorly secreted. The majority of the retained mutants are targeted into an ER-associated degradation pathway. The mechanism responsible for intracellular retention of these mutated factor VIII molecules is unknown.

Reduced levels of factor VIII protein may also result from defects located outside the factor VIII gene. One striking example concerns patients having combined factor V and VIII deficiency. The gene responsible for combined factors V and VIII deficiency has been mapped to the long arm of chromosome 18, between markers D18S849 and D18S1103,52,53 whereas the genes for factors V and VIII are located at chromosomes 1 and X, respectively. Recently, the unknown gene has been idenfied to encode the intracellular membrane lectin ERGIC-53, a resident protein of the ER-Golgi intermediate compartment.35 Indeed, affected individuals displayed mutations in this gene, in association with a complete lack of expression of ERGIC-53.35 Apparently, ERGIC-53 contributes to the secretion process of factors V and VIII, presumably by acting as a chaperone protein. Identification of the underlying mechanism should provide more insight in the intracellular routing and secretion of both cofactors.

    ASSEMBLY OF THE FACTOR VIII-VON WILLEBRAND FACTOR (vWF) COMPLEX

Binding sites for vWF.   Immediately after its release into the circulation, the factor VIII heterodimer interacts with its carrier protein vWF to form a tight, noncovalent complex. Each monomer of the multimeric vWF protein is able to bind one factor VIII molecule with high affinity (kd < 0.5 nmol/L).54-57 In vivo, however, the stoichiometry is limited by the number of factor VIII molecules present, resulting in approximately a 1:50 ratio.

Two peptide regions of factor VIII are implicated to be involved in binding vWF: one at the aminoterminal end of intact factor VIII light chain,55-59 and one at the carboxyterminal end (residues 2303-2332).57,60-62 Using proteolytically derived fragments of factor VIII light chain, it was shown that both these individual regions are capable of binding vWF.57 However, the affinity of these fragments for vWF is markedly lower compared with the intact factor VIII heterodimer (two and three orders of magnitude for the aminoterminal and carboxyterminal end, respectively).57 Apparently, both ends of factor VIII light chain act synergistically in the binding of vWF.

With respect to vWF binding to the aminoterminal region of factor VIII light chain, it appears that residues 1649 to 1671, thus including sulfated residue Tyr1664, are dispensable for vWF binding.57,63,64 In addition, the Arg1689-Ser1690 cleavage site has to be intact,57 suggesting that residues carboxyterminal of this thrombin cleavage site contribute to binding as well. Recombinant factor VIII synthesized in the presence of an inhibitor of sulfation displays reduced binding to vWF,63,65 suggesting a role for sulfated Tyr1680 in the interaction with the carier protein. Indeed, replacement of Tyr1680 by Phe results in loss of high-affinity binding to vWF, allowing only a low-affinity interaction.63,65,66 The presence of Tyr1680 in its sulfated form thus contributes to optimal binding of factor VIII to vWF.

Factor VIII interactions modulated by vWF.   One functional aspect of factor VIII-vWF complex formation may be to prevent premature binding of factor VIII to components of the factor X-activating complex. For instance, binding of factor VIII light chain to factor IXa is inhibited by vWF.67 Because the affinity of factor VIII for vWF exceeds that for factor IXa by approximately 100-fold,56,67 factor VIII highly favors vWF binding over factor IXa binding. The mechanism by which vWF inhibits factor IXa binding is not yet elucidated. Because binding of both proteins by factor VIII light chain seems to involve distinct parts of the molecule, it is unlikely that vWF competes with factor IXa for binding to the same site. Mechanisms that could contribute to inhibition include sterical hindrance and alteration of the factor VIII conformation so that factor IXa cannot bind.

Whereas factor IXa and vWF bind at different sites, vWF and phospholipids both bind to the C2-domain region 2303-2332.62,68 Using a human anti-factor VIII antibody, it has been shown that these sites, although in very close proximity, do not completely overlap.69 Nevertheless, the close proximity of these sites may explain the observations that binding of factor VIII to vWF is incompatible with factor VIII binding to membrane surfaces.70-73 It should be noted that, in comparison with noncomplexed factor VIII, factor VIII in complex with vWF is less susceptible to proteolytic attack by various lipid-binding proteases.74-77 These vitamin K-dependent serine proteases, which include activated protein C (APC) and factor Xa, require to assemble with factor VIII at a membrane surface for efficient proteolysis of factor VIII.76,78 In contrast, thrombin displays proteolytic activity independent of a membrane surface. Indeed, vWF does not protect factor VIII against cleavage by thrombin.79-81 Cleavage of factor VIII by thrombin results in loss of vWF binding and conversion of factor VIII into its active conformation.

vWF and hemophilia A.   The association with vWF serves an important role in factor VIII physiology, as vWF functions as a stabilizer of the heterodimeric structure of factor VIII.45,82-84 The physiological relevance of complex formation is particularly apparent in patients with von Willebrand disease (vWD) (type 3), who have no detectable vWF protein. Not only do these patients have a secondary deficiency of factor VIII, but they also have a considerably reduced half-life of intravenously administred factor VIII.83,85-88 This phenotype is also observed in patients with vWD with the so-called Normandy variant, which is defined as type 2N. Despite normal levels of circulating vWF, factor VIII levels are severely reduced.89,90 These patients harbor a mutation in the factor VIII-binding domain of vWF, which results in defective binding to vWF.

With regard to factor VIII, two distinct basepair substitutions have been reported that are associated with impaired complex assembly.49,91,92 Both mutations result in the replacement of one single amino acid, Tyr1680, and are associated with hemophilia A. So far, no mutations located within the C2 domain region have been reported that are associated with reduced affinity for vWF.

    ACTIVATION OF FACTOR VIII

Cleavage sites associated with factor VIII activation.   Within the factor X-activating complex, the proteolytic activity of factor IXa is markedly enhanced by factor VIII. It has been well established that proteolysis of factor VIII is required for the generation of its cofactor activity.79,93-97 The uncleaved factor VIII procofactor lacks the ability to enhance factor IXa activity.47,98 Enzymes that are able to endow factor VIII with its cofactor activity include thrombin and factor Xa.79,93,98-102 Thrombin cleaves factor VIII at one specific site within the light chain, Arg1689, and at two sites in the heavy chain: Arg372 and Arg740 (Fig 3).79 Proteolysis of factor VIII heavy chain by factor Xa involves three sites: Arg336, and the two thrombin-cleavage sites Arg372 and Arg740 (Fig 3).79 With regard to factor VIII light chain, factor Xa is able to cleave at Arg1689, a site that is shared with thrombin, but also at Arg1721, a site that is specific for factor Xa.79 It should be noted that this site is cleaved in human factor VIII, but not in porcine factor VIII.103 It has been unclear whether cleavage at Arg1721 contributes to factor VIII activation or inactivation, because prolonged incubation with factor Xa results in loss of factor VIII activity in parallel with cleavages at positions 336 and 1721.79 This has been resolved by studying reassociated dimers of intact factor VIII heavy chain with either thrombin- or factor Xa-cleaved factor VIII light chain.47 The resulting factor VIII dimers were functionally indistinguishable, demonstrating that factor Xa-cleavage of the light chain is not associated with inactivation. It has been reported that factor Xa-activated factor VIII displays less activity than thrombin-activated factor VIII.79,98,102 Because cleavage of the light chain cannot be responsible for this phenomenon, this is most likely due to additional factor Xa-cleavage at Arg336 in the factor VIII heavy chain.


View larger version (12K):
[in this window]
[in a new window]
  		Fig 2. The factor VIII protein. Mature factor VIII consists of 2332 amino acids, which are arranged in a discrete domain structure: A1 (residues 1-336), A2 (373-710), B (741-1648), A3 (1690-2019), C1 (2020-2172), and C2 (2173-2332). The A domains are bordered by acidic regions a1 (337-372), a2 (711-740), and a3 (1649-1689). Disulfide Bridges: Using B-domainless factor VIII, seven disulfide bonds have been identified: residues 153 and 179, 248 and 329 (A1 domain), 528 and 554, 630 and 711 (A2 domain), 1832 and 1858, 1899 and 1903 (A3 domain), and 2021 and 2169 (C1 domain).196 Within the C2 domain, residues 2174 and 2326 most likely also form a disulfide bridge. Free cysteine-residues have been identified at positions 310, 692, and 2000.196 Cys528 and Cys1858 may be present as free cysteines, because these residues are reactive toward a sulfhydryl-specific fluorphor.197 With regard to the Cys-residues in the B-domain it is unknown whether they are free or linked. N-Linked Glycosylation: Factor VIII contains 25 consensus sequences (Asn-Xxx-Thr/Ser) that allow N-linked glycosylation. Using either full-length or B-domainless factor VIII, the majority of these sites have been shown to be glycosylated: residues 42 and 239 (A1 domain), residues 757, 784, 828, 900, 963, 1001, 1005, 1055, 1066, 1185, 1255, 1259, 1282, 1300, 1412, and 1442 (B domain), residue 1810 (A3 domain), and residue 2118 (C2 domain).198-200 Nonglycosylated residues are present at positions 943 and 1384 (B domain) and at position 1685 (a3 acidic region). Residue 582 (A2 domain) has been reported to be nonglycosylated in two studies,199,200 whereas one study reported this residue to be partially glycosylated.198 Finally, it remains to be investigated whether residue 1512 (B domain) is glycosylated. Tyrosine Sulfation: The acidic regions contain consensus sequences that allow sulfation of Tyr-residues at positions 346 (a1 region), 718, 719, 723 (a2 region), 1664, and 1680 (a3 region). Analysis using recombinant proteins established that all sites indeed can be sulfated.


View larger version (5K):
[in this window]
[in a new window]
  		Fig 3. Limited proteolysis of factor VIII. The major part of factor VIII circulates as a set of heterogenous dimers, consisting of a light (a3-A3-C1-C2) and heavy chain (A1-a1-A2-a2-B). The heavy chain is variably sized due to limited proteolysis within the B domain. Some of these cleavages may occur intracellularly at positions 1313 and 1648 (open downward arrows). Factor VIII can be converted into its active form by proteolysis in both the heavy and light chain by various serine proteases (closed downward arrows), including thrombin and factor Xa. Because proteolysis by factor Xa but not thrombin is inhibited by vWF, thrombin is probably the physiological activator of factor VIII. Proteolytic degradation of factor VIIIa proceeds through cleavages within the A1 and A2 domains by various serine proteases (upward arrows), and results in release of the a1 acidic region and bisecting of the A2 domain. In contrast to what has previously been assumed, cleavages within the light chain by factor IXa or factor Xa do not result in inactivation of factor VIII, but contribute to the development of factor VIII cofactor activity.

Because activation of factor VIII involves proteolysis of both its heavy and light chain, it is of interest to compare the relative contribution of each cleavage to the development of factor VIII cofactor function. The contribution of cleavage at Arg740 to factor VIII activation is limited, because mutations at this position do not interfere with factor VIII activation or function.97 Selective cleavage of factor VIII at Arg372 by a snake venom-derived protease generates a factor VIII molecule that displays 60% of the activity of fully activated factor VIII.80 Factor VIII dimers cleaved within the light chain only have 25% to 30% of the activity displayed by fully activated factor VIII.47,104 These findings allow the conclusion that both cleavage at Arg372 and Arg1689 are required to exert full cofactor activity. This view is supported by the observation that recombinant factor VIII mutants containing replacements at either Arg372 or Arg1689 are unable to correct the clotting time of factor VIII-deficient plasma.97

Acidic regions.   With regard to the cleavage sites at positions 372, 740, and 1689, it is noteworthy that these residues are located at the carboxyterminal end of an acidic sequence interconnecting adjacent domains. These acidic regions (a1, a2, and a3, see Fig 2) contain several Tyr residues in a sequence that meets the consensus features for tyrosine sulfation.105,106 Sulfated Tyr residues may serve a role in various processes, including protein-protein interactions.106 Because thrombin is known to interact with a variety of acidic, sulfated sequences, it has been proposed that acidic regions within factor VIII serve a role in thrombin activation of factor VIII.107 Replacement of Tyr346 (a1 region) or Tyr1664 (a2 region) indeed results in factor VIII mutants that are activated by thrombin less efficiently.66 It should be mentioned that deletion of Tyr1664 leaves the activation kinetics of factor VIII by thrombin unaffected.108,109

Replacement of Tyr-residues by Phe in the a2 acidic region (residues 718, 719, and 723) did not affect thrombin activation, but rather resulted in mutants that displayed reduced ability to stimulate factor IXa enzymatic activity.66 In contrast, recombinant factor VIII variants (both full-length and B-domainless) containing nonsulfated Tyr at these positions are activated normally and display full cofactor activity.110,111 Although both approaches reveal apparently contrasting findings with regard to the effect of Tyr-sulfation in the a2 region on factor VIII cofactor function, they allow the conclusion that sulfation of these Tyr-residues does not contribute to activation of factor VIII. However, the acidic nature of this a2 sequence appears to be of importance for activation of factor VIII. First, a monoclonal antibody directed against a2 inhibits thrombin activation of factor VIII, but does not interfere with factor VIII cofactor activity.112,113 Secondly, deletion of a2 or part thereof in B-domainless factor VIII results in molecules that require higher thrombin concentrations than normal factor VIII for efficient activation, but display normal factor IXa cofactor activity.110,112 Interestingly, in both cases it was observed that reduced thrombin activation was caused by a reduced cleavage efficiency at Arg372 and Arg1689, suggesting that the a2 acidic region influences cleavage in remote regions in the factor VIII molecule. This hypothesis is supported by observations using a factor VIII chimera with a replacement in the a2 region. In this chimeric molecule, residues 716-736 of a2 have been replaced by a sequence that is known to have a high affinity for thrombin, ie, the amino acid sequence 51-80 of heparin cofactor II.114 This chimeric protein proved more potent than normal factor VIII in correcting the clotting time of factor VIII-deficient plasma. The increased intrinsic activity is caused by an increased rate of thrombin cleavage at Arg372 and Arg1689 compared with normal factor VIII. Thus, these findings suggest that the a2 region promotes proteolytic activation of factor VIII.

Apart from their role in thrombin activation, the acidic regions a1 and a3 contribute to factor VIII function also in an additional manner. The a1 region has been described to be involved in maintaining the stability of the factor VIII heterotrimer115 and in binding of factor X116,117 (see Factor VIII Inactivation section). The a3 region is important for high affinity binding of vWF (see Assembly of the Factor VIII-vWF Complex section). Further, factor VIII heterodimers consisting of uncleaved heavy and light chain do not posses any cofactor activity.47,48 In contrast, factor VIII exclusively cleaved at position 1689, thus lacking the a3 region, displays significant cofactor activity (approximately 25% of fully activated factor VIII).47,104 Apparently, the acidic region a3 functions as an activation peptide that needs to be cleaved off for exposure of cofactor activity.

Defective activation and hemophilia A.   Mutations resulting in replacement of amino acids at the factor VIII activation sites should predispose to hemorraghic diathesis. Indeed, missense mutations at Arg372, Ser373, and Arg1689 are associated with hemophilia A.49,118-124 With regard to the Arg1689 mutation, biochemical data predict residual activity to be at least half of that of normal factor VIII, as cleavage at Arg372 accounts for approximately 60% of total activity.80 However, residual activity appears to range between <1% and approx 12%, thus lower than expected.49 Several possibilities may be considered to explain this apparent discrepancy. First, due to substitution of Arg1689, the vWF binding site including sulfated Tyr1680 is not cleaved from the light chain. Therefore, the factor VIII-vWF complex may fail to dissociate upon thrombin treatment. As a consequence, factor VIII is unable to interact with factor IXa and phospholipids, and thus cannot assemble into the factor X-activating complex (see below). Secondly, in most of the documented patients with a mutation at Arg1689, this residue is replaced by a Cys.49 This Cys-residue has the potential to form an extra disulfide bridge within the factor VIII light chain.125,126 It seems conceivable that this results in misfolding within the factor VIII light chain, which precludes biological activity. Finally, residual activity may be affected by the type of amino acid substitution. This view is supported by the notion that a similar discrepancy exist for substitutions at Arg372. For instance, Arg372 to Pro substitutions exclusively result in severe hemophilia A, whereas Arg372 to His substitutions result in a mild to moderate phenotype.

    ASSEMBLY OF THE FACTOR X-ACTIVATING COMPLEX

Regulation of complex assembly.   To activate factor X, factor IXa and factor VIIIa assemble into a membrane-bound complex. To maintain the hemostatic balance, this complex should only assemble after initiation of the coagulation cascade, implying that participation of factor VIII in this complex is subject to a delicate regulatory mechanism. In this regard, vWF plays a central role. Although factor IXa displays similar affinity for nonactivated and activated factor VIII,127 the factor IXa binding site within nonactivated factor VIII is unlikely to be accesible when factor VIII is in complex with vWF.67 Furthermore, binding of factor VIII to the membrane surface is inhibited by vWF,70-73 suggesting that only activated factor VIII that is dissociated from vWF is able to bind to the membrane surface. However, it should be mentioned that the affinity of factor VIII for the membrane surface is dependent on the membrane composition, as affinities have been reported that differ 10- to 100-fold (10-9 to 10-11 mol/L).72,128-131 Thus, under particular conditions the affinity of vWF and the membrane surface for factor VIII is similar. Apparently, a delicate balance may exist between factor VIII being in complex with vWF or the membrane surface. Because in direct binding studies vWF prevents binding of factor VIII to the membrane surface,70-73 a minor part of the factor VIII population probably is in complex with the membrane surface. This situation changes dramatically upon cleavage of factor VIII light chain, which results in a 1,600-fold decrease in affinity for vWF.57 Because of this event, the balance will readily shift toward factor VIIIa binding to the membrane surface. This subsequently favors binding of factor IXa to factor VIIIa, which is no longer associated with vWF. Ultimately, this leads to the assembly of the membrane-bound factor VIIIa-factor IXa complex that activates factor X.

The role of the membrane surface in complex assembly.   The notion that in the absence of a membrane surface the generation of factor Xa by the factor VIIIa-factor IXa complex is negligible132 underscores the essential role of the membrane surface in the factor X-activating complex. The membrane surface may act in two distinct ways: first by positioning the enzyme-cofactor complex into an active conformation or, alternatively, by locating the enzyme and cofactor at the same site. At present, data have been reported that are in support of both mechanisms. On the one hand, it has been shown that the affinity of factor IXa for factor VIIIa is increased 2,000-fold in the presence of phospholipids,133 which suggests that the second mechanism is dominant. On the other hand, the affinity of (activated) factor VIII for factor IXa is reported to be similar in the presence (kd = 10-8 to 10-9 mol/L)104,127,132,134,135 and absence (kd = 10-8 mol/L)67,132 of a phospholipid surface. Gilbert and Arena132 showed that in the presence of phospholipids the catalytic activity of the enzyme-cofactor complex is increased 1,500-fold. These data favor the view that the membrane surface positions the enzyme and cofactor in a conformation that allows efficient substrate cleavage.

Location of factor IXa interactive sites.   The interaction between factor VIIIa and factor IXa has been investigated in several elegant studies using factor IXa molecules that carry a fluorescent-label in the active site.127,134-139 It became evident that in the presence of phospholipids, factor VIIIa induces a conformational change in the factor IXa protease domain. In addition, maximal changes in the factor IXa protease domain require the presence of the factor VIIIa A2-domain,134 suggesting that the A2 domain contains a factor IXa interactive site. Indeed, studies using a series of synthetic peptides showed that factor IXa binding can be attributed to the A2-domain sequence 558 to 565.137 Furthermore, a region within the carboxyterminal part of the A2 domain (residues 698 to 710) also has been proposed to comprise a factor IXa-binding site.140,141

Besides factor VIII heavy chain, the light chain also contributes to factor IXa binding. In equilibrium binding studies, isolated factor VIII light chain proved to bind factor IXa with high affinity.67 Moreover, factor VIII light chain and the intact factor VIII heterodimer are indistinguishable in terms of affinity for factor IXa, indicating that high-affinity binding to factor IXa is mediated by the factor VIII light chain. Binding of factor VIII light chain to factor IXa was found to be inhibited by the A3-domain directed monoclonal antibody CLB-CAg A, a strong inhibitor of factor VIII activity.67,113,142 By using synthetic peptides, the A3-domain sequence 1811 to 1818 has been identified as a site that binds factor IXa.142 Thus, interaction with factor IXa involves at least three sites on factor VIII: residues 558-565, 698-710, and 1811-1818.

Three-dimensional model of factor VIII and factor IXa.   Three-dimensional representations of the enzyme and cofactor have been published, based on factor IXa crystallography143 and factor VIII homology modeling144,145 (Fig 4). So far, it is unknown which residues in the factor IXa molecule are involved in binding factor VIII. However, the location of these sites should fit with the location of their counterparts on the factor VIII molecule. Although the amino acid numbering suggests that the factor IXa-binding regions are located in completely different parts of the factor VIII protein, the three-dimensional model indicates that the factor IXa-binding sites are in close vicinity, and are exposed at the same side of the molecule (Fig 4). Matching of the factor VIII and factor IXa models suggests that factor IXa comprises distinct sites involved in factor VIIIa binding, interacting with the A2 or A3 domain. Factor VIIIa binding appears to be mediated by both the factor IXa light chain and protease domain. The involvement of the protease domain is in agreement with the observation by Bajaj et al146,147 that the stimulation of factor IXa proteolytic activity by factor VIIIa is inhibited by a monoclonal antibody directed against the protease domain residues 231-265. This suggests that this protease domain region comprises a factor VIII-binding site. However, recombinant factor IXa molecules in which residues in the antibody-binding epitope have been mutated combine a strongly reduced affinity for the antibody with normal biological activity.148 Because normal activity is associated with normal factor VIII binding, these findings leave the exact location of the factor VIII binding site in the factor IXa heavy chain unidentified. Irrespective of its precise location, this site on the factor IXa heavy chain presumably interacts with the factor VIII A2 domain, because this domain induces the largest change in the conformation of the factor IXa active site.134


View larger version (94K):
[in this window]
[in a new window]
  		Fig 4. Model of the factor VIII and factor IXa molecules. Shown are representiations of porcine factor IXa (Protein Data Bank accession code ) and the triplicated A-domains of human factor VIII (Hemophilia A web site, http://europium.mrc.rpms.ac.uk), which are derived from crystallography and homology modeling, respectively. Factor IXa binding region in the factor VIII A3 domain (residues 1811-1818) is shown in white, whereas the binding regions in the A2 domain (residues 558-565 and 698-710) are shown in dark and light blue, respectively (space-filling representations). These sites are in close vicinity, and are exposed at the same side of the molecule. The factor VIII A2 domain is required to induce significant changes within the factor IXa protease domain, indicating that it binds to the factor IXa protease domain. The A3 domain of factor VIII has been proposed to interact with the factor IXa light chain. Within the factor IXa light chain, residues 12, 64, 69, 78, 92, and 94 (see refs 150 to 155) are indicated (red, space-filling representation). These residues have been reported to be associated with an abnormal response to factor VIIIa in factor X activation.

Assuming an interaction between the factor IXa protease domain and the factor VIII A2 domain, it seems conceivable that the A3 domain interacts with a region within the factor IXa light chain. This is in line with recent observations that the light chain of factor VIII binds to the light chain of factor IXa.138,149 Furthermore, mutations within the factor IXa light chain have been described that are associated with an abnormal response to factor VIIIa in factor X activation.150-154 These mutations are dispersed over the factor IXa light chain, indicating that multiple sites may contribute to binding of the factor VIII A3 domain. Alternatively, some mutations may destabilize the factor VIII binding site by affecting the conformation of the factor IXa light chain. This latter possibility has been reported for two distinct factor IX mutations.154,155

Collectively, by combining biochemical data with the three-dimensional models of factor IXa and factor VIII, it appears that the factor VIII A2 domain binds to the factor IXa heavy chain, and the factor VIII A3 domain to the factor IXa light chain (Fig 4). It is of importance to realize that for factor VIII as well as for factor IXa the current models not fully represent the biologically active molecules. The factor IXa structure has been determined in the absence of calcium ions,143 which are obligatory for optimal exposure of the factor VIII light chain binding site and of the catalytic centre.133,155 The factor VIII model provided by Pemberton et al145 comprises the A domains only and lacks the B and C domains and the acidic domain spacers. It cannot be excluded that these domains affect the structure of the A domains. In addition, this factor VIII model does not distinguish between the inactive procofactor or activated factor VIII. It is obvious that both factor VIII species will have different structural properties, because only factor VIII, which is cleaved at specific positions, is able to stimulate factor IXa activity. Despite these restrictions, both the factor VIII and factor IXa model provide an important basis for proper selection of residues that may be investigated for their contribution in the assembly of the factor VIII-factor IX complex.

Factor IXa binding and hemophilia A.   Inspection of the hemophilia A database shows that several mutations have been reported that are in or close to the factor IXa binding sites: codons 558, 565, and 566, codons 698, 701, and 704 in the heavy chain, and codons 1789, 1796, 1823, and 1825 in the factor VIII light chain.49 It seems reasonable to assume that the bleeding tendency which is associated with these mutations is caused by suboptimal assembly of the factor IXa-factor VIIIa complex. With regard to the 558 and 566 mutation, this view has been supported by studies using recombinant factor VIII.156 Interestingly, a mutation outside the factor IXa-binding regions, ie, at codon Arg527, also has been reported to be associated with inefficient stimulation of factor IXa proteolytic activity.157,158 Examination of the three-dimensional model shows that Arg527 is located in the immediate vicinity of the factor IXa binding sequence 558 to 565.145 The exposure of this factor IXa binding site may be affected by substitution of Arg527. Alternatively, Arg527 may be part of an extensive factor IXa-binding interface, involving multiple sites of the A2 domain.

    INACTIVATION OF FACTOR VIII

Mechanisms of inactivation.   Downregulation of the factor X-activating complex may involve inactivation or inhibition of either the enzyme factor IXa or the cofactor, factor VIIIa. Inactivation of the cofactor comprises two distinct pathways: proteolytic degradation and spontaneous dissociation. Once activated, factor VIII cofactor activity is rapidly lost.135,159-162 Compared with activated factor VIII, the procofactor is markedly more stabile, which is illustrated by its dissociation rate being 100-fold lower (kdiss approx 4 to 6 × 10-4 s-1 and 4 × 10-6 s-1 for factor VIIIa and factor VIII, respectively).47,163 The intrinsic instability of factor VIIIa can be attributed to the weak interaction between the A2 domain and the metal ion-linked A1/A3-C1-C2 dimer.164-166 The kd for this interaction is approximately 0.2 µmol/L.42 Because this value exceeds the factor VIII concentration in plasma 100- to 1,000-fold, equilibrium is in favor of the inactive, dissociated state of factor VIIIa.

Proteolytic degradation of factor VIIIa involves cleavages in the heavy chain at positions 336 and 562 by various enzymes, such as factor IXa, factor Xa, and APC.74-79,167-170 Cleavage at position 336 in factor VIIIa releases a1, the acidic sequence that interconnects the A1 and A2 domain. Because of this release, the A2 domain dissociates more rapidly from the factor VIIIa heterotrimer.115 This acidic spacer has been proposed to comprise a binding site for the substrate factor X,116,117 indicating that release of this site results in impaired substrate binding. Thus, cleavage at Arg336 affects both intramolecular (A2 domain dissociation) and intermolecular (factor VIII-factor X) interactions. Arg562, which is part of the A2 domain sequence that comprises a factor IXa interactive site, is exclusively cleaved by APC.168 It seems conceivable that loss of cofactor activity due to cleavage at this site reflects the loss of the ability to interact with factor IXa.

One intriguing question is whether proteolytic degradation or spontaneous dissociation dominates the inactivation of factor VIII in vivo. At present the relative contribution of each mechanism to factor VIII inactivation is not fully understood, although some reports indicate that spontaneous dissociation is dominant.162,171,172 This view is underscored by the observation that dissociation of the factor VIIIa heterotrimer may be accelerated by binding of the A2 domain to the low-density lipoprotein receptor-related protein.173 To describe the process of factor VIIIa inactivation, it should further be considered that factor IXa plays a dual role. It stabilizes factor VIIIa by linking the A2 domain to the A3 domain,137,142,159 and protects factor VIII against inactivation by APC.136,167,174 On the other hand, under certain conditions factor IXa may inactivate factor VIIIa by cleavage at position 336,169,170 a site that is shared with factor Xa and APC. The fact that factor IXa is involved both in stabilization and in inactivation of factor VIII complicates a final assessment of the regulatory role of factor IXa in intrinsic factor Xa formation.

Defects in factor VIII inactivation.   Impaired inactivation of factor VIIIa or its homologue factor Va may be associated with a disturbed balance between procoagulant and anticoagulant systems. With respect to factor Va this view is supported by the notion that mutation at Arg506, a site that is cleaved by APC, predisposes to venous thromboembolism.175-178 It has been investigated whether patients displaying venous thromboembolism carry analogous mutations at the APC cleavage sites in factor VIII (ie, Arg336 and Arg562).179,180 However, this association has not been observed, which suggests that mutations at these positions are rare. Alternatively, such mutations may not predispose to thrombotic disorders, indicating that proteolytic inactivation of factor VIIIa is less important than inactivation of factor Va with regard to the hemostatic balance. This would be in agreement with the fact that murine factor VIII lacks the inactivation site at position 336.15 In addition, in vitro data using genetically engineered factor VIII with mutations in the APC cleavage sites showed that these mutations were not associated with reduced clotting times in APC-resistance assays.181

Although APC-resistant factor VIII molecules have not been identified in patients, the possibility remains open that APC-resistance may modulate factor VIII inactivation in an indirect manner. Inactivation of factor VIII by APC is enhanced in the presence of the APC-cofactor protein S.74,167 Several investigators have reported that the factor V procofactor enhances the cofactor effect of protein S in factor VIII inactivation.162,182,183 This link between factor V and factor VIII inactivation becomes even more apparent by the finding of Váradi et al,183 who reported that factor V, which carries the Arg506 to Gln mutation, has impaired cofactor activity in APC- and protein S-dependent factor VIII inactivation. The physiological significance of this observation remains unclear. However, the possibility that APC-resistance also affects factor VIII inactivation is challenging and deserves further study.

Another intriguing finding is that APC-resistant factor V has the potential to bypass the absence of factor VIII activity to some extent in in vitro thrombin generation studies.184 Therefore, it cannot be excluded that hemophilic patients which carry the factor V Arg506 to Gln mutation display a less severe phenotype than expected. This indeed has been shown for some hemophilic patients as described by Nichols et al.185 However, Arbini et al186 did not find an association between the presence of APC-resistant factor V and the severity of the bleeding tendency in a population of 295 hemophilic patients. The ability of APC-resistant factor V to bypass factor VIII deficiency may be restricted to specific, thus far unrecognized conditions.

    CLEARANCE OF FACTOR VIII

At present, little is known about the mechanism by which factor VIII is cleared from the circulation. Obviously, vWF serves an important role, because in patients with severe vWD the factor VIII half-life is considerably decreased.83,86-88 As vWF protects factor VIII against proteolytic degradation in vitro (see Assembly of the Factor VIII-vWF Complex section), it cannot be excluded that the decreased half-life in the absence of vWF factor is associated with proteolytic degradation of factor VIII. However, experimental data in support of this possibility are lacking thus far. Another explanation for the rapid clearance could be binding of noncomplexed factor VIII to the surface of cells. In this respect it is of interest to mention that hepatic endothelial cells have been reported to contain factor VIII protein, but not its mRNA (see Biosynthesis and Secretion of Factor VIII section). It may be noteworthy that the copper-binding protein ceruloplasmin, which is structurally related to the factor VIII A domains, is internalized in the hepatic endothelial cells through a receptor-mediated process.187 It seems possible that factor VIII is taken up by hepatic endothelium by a similar mechanism.

Because the effect of factor V proteolysis on its survival has been investigated in a nonhuman primate model,188 it is of interest to compare factor VIII survival with that of factor V. Whereas the half-life of the factor V procofactor is approximately 14 hours, the half-life of its thrombin-activated derivative is dramatically different. The heavy and light chain are cleared very rapidly (t1/2 < 20 minutes). The half-life for factor V light chain is remarkably close to the half-life of 10 minutes reported for the isolated light chain of factor VIII after infusion into hemophilic dogs.189 Perhaps, clearance of both proteins involves a similar mechanism. Clearance of the factor V activation peptide, ie, the B domain, is considerably slower (t1/2 > 30 hours),188 suggesting a previously unrecognized role of this domain in preventing premature clearance of factor V. Whether this is also true for the factor VIII B domain is not clear. The half-lifes of normal factor VIII and B-domain deleted factor VIII are similar in hemophilic patients190 as well in hemophilic dogs,87,191,192 indicating that the B domain does not contribute to factor VIII survival. However, it is important to note that these data reflect factor VIII survival in the presence of vWF. Therefore, it would be of interest to investigate survival of B-domainless factor VIII in patients with vWD type 3.

    FUTURE DIRECTIONS

Structure-function studies have contributed significantly to our current understanding of factor VIII biology and the molecular background of hemophilia A. As such this has provided the basis for the development of second generation recombinant factor VIII molecules that may find a therapeutical application. Pertinent to this point are the B-domain-deleted factor VIII variants, which are subject to clinical (factor VIII-SQ)190 or preclinical108,191,192 testing. Furthermore, various factor VIII variants have been designed which in the future may be particular useful in the treatment of hemophilia A. Variants have been described which are less prone to inhibitor neutralization.193,194 Other examples include variants with enhanced hemostatic potency114 or stability.195

Despite the rapid accumulation of information regarding the structure and function of factor VIII, a number of questions remain to be answered. For instance, it is still unclear why factor VIII becomes a potent cofactor of factor IXa once it is cleaved within the heavy and light chain. What is the structural basis for such a dramatic increase in biological activity? How does factor VIIIa, together with the membrane surface, push factor IXa into an extremely potent configuration? It seems obvious that studies on three-dimensional modeling techniques based on crystal structures of factor VIII or factor VIII fragments complexed with their ligands (eg, factor IX) should provide a solid basis for a better understanding of the molecular aspects of factor VIII function and dysfunction. Because both factor VIII and factor IX are now potentially available in substantial quantities, determination of the crystal structure of complexes comprising wild-type or mutant proteins should be feasible and undoubtedly will lead to significant advances in this field. This approach will also facilitate the design of antagonistic inhibitors of the factor VIII system, which could provide novel anticoagulants for the treatment of thrombotic disorders.

The question of why exposure to factor VIII concentrates is associated with allo-immune reponses in some of the hemophilia A patients remains unanswered. What is the mechanism that causes these adverse reactions? Why and how is the immune system challenged under these conditions? Why do certain specific polypeptide regions of the factor VIII molecule play a prominent role in these immune reactions? Finally, little is known about the origin and expression of factor VIII at the cellular level. Which mechanisms trigger the specific cellular responses that govern elevations of the factor VIII plasma level under a variety of clinical conditions? Similarly, the mechanism of factor VIII clearance is an unexplored subject. Finding the answers to these questions is not only of fundamental, merely scientific appeal, but also has the potential of further improving our current strategies for the treatment of hemophilia A.

    ACKNOWLEDGMENT

We express our gratitude to Drs W.G. van Aken, J. Voorberg, O.D. Christophe, and K. Fijnvandraat for helpful discussions and critical reading of the manuscript. We also thank Dr G. Kemball-Cook for providing the coordinates of the factor VIII.

    FOOTNOTES

   Submitted June 5, 1997; accepted July 17, 1998.

Address reprint requests to Peter J. Lenting, PhD, Department of Plasma Protein Technology, CLB, Sanquin Blood Supply Foundation, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands; e-mail: P_Lenting{at}clb.nl.

    REFERENCES
Top
Introduction
References

1. Rosner F: Hemophilia in the talmud and rabbinic writings. Ann Intern Med 70:833, 1969

2. Sadler JE, Davie EW: Hemophilia A, Hemophilia B, and von Willebrand's disease, in Stamatoyannopoulos G, Nienhuis A, Leder P, Majerus P (eds): The Molecular Basis of Blood Diseases. Philadelphia, PA, Saunders, 1987, p 576.

3. Patek AJ, Taylor FHL: Hemophilia. II. Some properties of a substance obtained from normal plasma effective in accelerating the clotting of hemophilic blood. J Clin Invest 16:113, 1937

4. Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy S: Surface dependent reactions of the vitamin K-dependent enzyme complexes. Blood 76:1, 1990[Abstract/Free Full Text]

5. Gitschier J, Wood WI, Goralka TM, Wion KL, Chen EY, Eaton DH, Vehar GA, Capon DJ, Lawn RM: Characterization of the human factor VIII gene. Nature 312:326, 1984[Medline] [Order article via Infotrieve]

6. Wood WI, Capon DJ, Simonson CC, Eaton DL, Gitschier J, Keyt B, Seeburg PH, Smith DH, Hollingshead P, Wion KL, Delwart E, Tuddenham EGD, Vehar GA, Lawn RM: Expression of active human factor VIII from recombinant DNA clones. Nature 312:330, 1984[Medline] [Order article via Infotrieve]

7. Vehar GA, Keyt B, Eaton D, Rodriguez H, O'Brien DP, Rotblat F, Oppermann H, Keck R, Wood WI, Harkins RN, Tuddenham EGD, Lawn RM, Capon DJ: Structure of human factor VIII. Nature 312:337, 1984[Medline] [Order article via Infotrieve]

8. Toole JJ, Knopf JL, Wozney JM, Sultzman LA, Bueker JL, Pittman DD, Kaufman RD, Brown E, Shoemaker C, Orr EC, Amphlett GW, Foster WB, Coe ML, Knutson GJ, Fass DN, Hewick RM: Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 312:342, 1984[Medline] [Order article via Infotrieve]

9. Church WR, Jernigan RL, Toole J, Hewick RM, Knopf J, Knutson GJ, Nesheim ME, Mann KG, Fass DN: Coagulation factors V and VIII and ceruloplasmin constitute a family of structurally related proteins. Proc Natl Acad Sci USA 81:6934, 1984[Abstract/Free Full Text]

10. Stubbs JD, Lekutis C, Singer KL, Bui A, Yuzuki D, Srinivasan U, Parry G: cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences. Proc Natl Acad Sci USA 87:8417, 1990[Abstract/Free Full Text]

11. Larocca D, Peterson JA, Urrea R, Kuniyoshi J, Bistrain AM, Ceriani RL: A Mr 46,000 human milk fat globule protein that is highly expressed in human breast tumors contains factor VIII-like domains. Cancer Res 51:4994, 1991[Abstract/Free Full Text]

12. Takagi S, Hirata T, Agata K, Mochii M, Eguchi G, Fujisawa H: The A5 antigen, a candidate for the neuron recognition molecule, has homologies to complement proteins and coagulation factors. Neuron 7:295, 1991[Medline] [Order article via Infotrieve]

13. Wion KL, Kelly D, Summerfield JA, Tuddenham EGD, Lawn RM: Distribution of factor VIII mRNA and antigen in human liver and other tissues. Nature 317:726, 1985[Medline] [Order article via Infotrieve]

14. Levinson B, Kenwrick S, Gamel P, Fisher K, Gitschier J: Evidence for a third transcript from the human factor VIII gene. Genomics 14:585, 1992[Medline] [Order article via Infotrieve]

15. Elder B, Lakich D, Gitschier J: Sequence of the murine factor VIII cDNA. Genomics 16:374, 1993[Medline] [Order article via Infotrieve]

16. Groth CG, Hathaway WE, Gustafsson A, Geis WP, Putnam CW, Bjorken C, Porter KA, Starzl TE: Correction of coagulation in the hemophilic dog by transplantaion of lymphatic tissue. Surgery 75:725, 1974[Medline] [Order article via Infotrieve]

17. Veltkamp JJ, Asfaou E, van der Torren E, van der Does JA, van Tilburg NH, Pauwels EKJ: Extrahepatic factor VIII synthesis. Lung transplants in hemophilic dogs. Transplantation 18:56, 1974[Medline] [Order article via Infotrieve]

18. Shaw E, Giddings JC, Peake IR, Bloom AL: Synthesis of procoagulant factor VIII, factor VIII-related antigen, and other coagulation factors by the isolated perfused rat liver. Br J Haematol 41:585, 1979[Medline] [Order article via Infotrieve]

19. Owen CA, Bowie EJW, Fass DN: Generation of factor VIII coagulant activity by isolated, perfused neonatal pig livers and adult rat livers. Br J Haematol 43:307, 1979[Medline] [Order article via Infotrieve]

20. Marchioro TL, Hougie C, Ragde H, Epstein RB, Thomas ED: Hemophilia: Role of organ homografts. Science 163:188, 1969[Abstract/Free Full Text]

21. Lewis JH, Bontempo FA, Spero JA, Gorenc TJ, Ragni MV, Starzl TE: Liver transplantation in a hemophiliac. N Engl J Med 312:1189, 1985[Medline] [Order article via Infotrieve]

22. Bontempo FA, Lewis JH, Gorenc TJ, Spero JA, Ragni MV, Scott JP, Starzl TE: Liver transplantation in hemophilia A. Blood 69:1721, 1987[Abstract/Free Full Text]

23. Figueiredo MS, Brownlee GG: cis-Acting elements and transcription factors involved in the promotor activity of the human factor VIII gene. J Biol Chem 270:11828, 1995[Abstract/Free Full Text]

24. Zelechowska MG, van Mourik JA, Brodniewicz-Proba T: Ultrastructural localization of factor VIII procoagulant antigen in liver hepatocytes. Nature 317:729, 1985[Medline] [Order article via Infotrieve]

25. Stel HV, van der Kwast TH, Veerman ECI: Detection of factor VIII/coagulant antigen in human liver tissue. Nature 303:530, 1983[Medline] [Order article via Infotrieve]

26. van der Kwast TH, Stel HV, Cristen E, Bertina RM, Veerman ECI: Localization of factor VIII-procoagulant antigen: An immunohistological survey of the human body using monoclonal antibodies. Blood 67:222, 1986[Abstract/Free Full Text]

27. Kadhom N, Wolfrom C, Gautier M, Allain JP, Frommel D: Factor VIII procoagulant antigen in human tissues. Thromb Haemost 59:289, 1988[Medline] [Order article via Infotrieve]

28. Kaufman RJ, Pipe SW, Tagliavacca L, Swaroop M, Moussalli M: Biosynthesis, assembly and secretion of coagulation factor VIII. Blood Coagul Fibrinol 8:S3, 1997 (suppl 2)

29. Kaufman RJ, Wasley LC, Davies MV, Wise RJ, Israel DI, Dorner AJ: Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells. Mol Cell Biol 9:1233, 1989[Abstract/Free Full Text]

30. Dorner AJ, Bole DG, Kaufman RJ: The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins. J Cell Biol 105:2665, 1987[Abstract/Free Full Text]

31. Dorner AJ, Wasley LC, Kaufman RJ: Increased synthesis of secreted proteins induces expression of glucose regulated proteins in butyrate treated CHO cells. J Biol Chem 264:20602, 1989[Abstract/Free Full Text]

32. Marquette KA, Pittman DD, Kaufman RJ: A 110 amino acid region within the A1 domain of coagulation factor VIII inhibits secretion from mammalian cells. J Biol Chem 270:10297, 1995[Abstract/Free Full Text]

33. Swaroop M, Moussalli M, Pipe SW, Kaufman RJ: Mutagenesis of a potential immunoglobulin-binding-protein-binding site enhances secretion of coagulation factor VIII. J Biol Chem 272:24121, 1997[Abstract/Free Full Text]

34. Pipe SW, Morris JA, Shah J, Kaufman RJ: Differential interaction of coagulation factor VIII and factor V with protein chaperones calnexin and calreticulin. J Biol Chem 273:8537, 1998[Abstract/Free Full Text]

35. Nichols WC, Seligsohn U, Zivelin A, Terry VH, Hertel CE, Wheatly MA, Moussalli MJ, Hauri HP, Ciavarella N, Kaufman RJ, Ginsburg D: Mutations in the ER-Golgi intermediate compartment protein ERGIC-53 cause combined deficiency of coagulation factors V and VIII. Cell 93:61, 1998[Medline] [Order article via Infotrieve]

36. Kaufman RJ, Wasley LC, Dorner AJ: Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem 263:6352, 1988[Abstract/Free Full Text]

37. van de Ven WJM, Voorberg J, Fontijn R, Pannekoek H, van den Ouweland AMW, van Duijnhoven HLP, Roebroek AJM, Siezen RJ: Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes. Mol Biol Rep 14:265, 1990[Medline] [Order article via Infotrieve]

38. Hutton JC: Subtilisin-like proteinases involved in the activation of proproteins of the eukaryotic secretory pathway. Curr Opin Cell Biol 2:1131, 1990[Medline] [Order article via Infotrieve]

39. Barr PJ: Mammalian subtilisins: The long-sought dibasic processing endoproteases. Cell 66:1, 1991[Medline] [Order article via Infotrieve]

40. Fass DN, Knutson GJ, Katzmann JA: Monoclonal antibodies to porcine factor VIII procoagulant and their use in the isolation of active coagulant protein. Blood 59:594, 1982[Abstract/Free Full Text]

41. Fay PJ, Anderson MT, Chavin SI, Marder VJ: The heterodimeric structure of human factor VIII and changes produced by thrombin interaction. Biochim Biophys Acta 871:268, 1986[Medline] [Order article via Infotrieve]

42. Fay PJ, Smudzin TM: Characterization of the interaction between the A2 subunit and A1/A3-C1-C2 dimer in human factor VIIIa. J Biol Chem 267:13246, 1992[Abstract/Free Full Text]

43. Bihoreau N, Pin S, de Kersabiec AM, Vidot F, Fontaine-Aupart MP: Copper-ion identification in the active and inactive forms of plasma-derived FVIII and recombinant FVIII-triangle II. Eur J Biochem 220:41, 1994

44. Tagliavacca L, Moon N, Dunham WR, Kaufman RJ: Identification and functional requirement of Cu(I) and its ligands within coagulation factor VIII. J Biol Chem 272:27428, 1997[Abstract/Free Full Text]

45. Fay PJ: Reconstitution of human factor VIII from isolated subunits. Arch Biochem Biophys 262:525, 1988[Medline] [Order article via Infotrieve]

46. Nordfang O, Ezban M: Generation of active coagulation factor VIII from isolated subunits. J Biol Chem 263:1115, 1988[Abstract/Free Full Text]

47. Donath MJSH, Lenting PJ, van Mourik JA, Mertens K: The role of cleavages at positions Arg1689 and Arg1721 in subunit interaction and activation of human factor VIII. J Biol Chem 270:3648, 1995[Abstract/Free Full Text]

47a. Sudkahar K, Fay PJ: Effects of copper on the structure and function of factor VIII subunits: Evidence for an auxiliary role for copper ions in cofactor activity. Biochemistry 37:6874, 1998[Medline] [Order article via Infotrieve]

48. Gitschier J, Wood WI, Shuman MA, Lawn RM: Identification of a missense mutation in the factor VIII gene of a mild hemophiliac. Science 232:1415, 1986[Abstract/Free Full Text]

49. Kemball-Cook G, Tuddenham EGD: The factor VIII mutation database on the world wide web: the haemophilia A mutation, search, test and resource site. Hamsters update (version 3.0, http://europium.mrc.rpms.ac.uk). Nucleic Acids Res 25:128, 1997

50. Voorberg J, de Laaf RTM, Koster PM, van Mourik JA: Intracellular retention of a factor VIII protein with an Arg2307right-arrowGln mutation as a cause of haemophilia A. Biochem J 318:931, 1996

51. Pipe SW, Kaufman RJ: Factor VIII C2 domain missense mutations exhibit defective trafficking of biological functional proteins. J Biol Chem 271:25671, 1996[Abstract/Free Full Text]

52. Nichols WC, Seligsohn U, Zivelin A, Terry V, Arnold ND, Siemieniak DR, Kaufman RJ, Ginsburg D: Linkage of combined factors V and VIII deficiency to chromosome 18q by homozygosity mapping. J Clin Invest 99:596, 1997[Medline] [Order article via Infotrieve]

53. Neerman-Arbez M, Antonarakis SE, Blouin JL, Zeinali S, Akhtari M, Afshar Y, Tuddenham EG: The locus for combined factor V-factor VIII deficiency (F5F8D) maps to 18q21, between D18S849 and D18S1103. Am J Hum Genet 61:143, 1997[Medline] [Order article via Infotrieve]

54. Lollar P, Hill-Eubanks DC, Parker CG: Association of the factor VIII light chain with von Willebrand factor. J Biol Chem 263:10451, 1988[Abstract/Free Full Text]

55. Vlot AJ, Koppelman SJ, van den Berg MH, Bouma BN, Sixma JJ: The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor. Blood 85:3150, 1995[Abstract/Free Full Text]

56. Leyte A, Verbeet MP, Brodniewicz-Proba T, van Mourik JA, Mertens K: The interaction between human blood coagulation factor VIII and von Willebrand factor. Biochem J 257:679, 1989[Medline] [Order article via Infotrieve]

57. Saenko EL, Scandella D: The acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von Willebrand factor. J Biol Chem 272:18007, 1997[Abstract/Free Full Text]

58. Foster PA, Fulcher CA, Houghten RA, Zimmerman TS: An immunogenic region within residues Val1670-Glu1684 of the factor VIII light chain induces antibodies which inhibit binding of factor VIII to von Willebrand factor. J Biol Chem 263:5230, 1988[Abstract/Free Full Text]

59. Precub JW, Kline BC, Fass DN: A monoclonal antibody to factor VIII inhibits von Willebrand factor binding and thrombin cleavage. Blood 77:1929, 1991[Abstract/Free Full Text]

60. Shima M, Scandella D, Yoshioka A, Nakai H, Tanaka I, Kamisau S, Terada S, Fukui H: A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine. Thromb Haemost 69:240, 1993[Medline] [Order article via Infotrieve]

61. Shima M, Nakai H, Scandella D, Tanaka I, Sawamoto Y, Kamisue S, Morichika S, Murakami T, Yoshioka A: Common inhibitory effects of human anti-C2 domain inhibitor alloantibodies on factor VIII binding to von Willebrand factor. Br J Haematol 91:714, 1995[Medline] [Order article via Infotrieve]

62. Saenko EV, Shima M, Rajalakshmi KJ, Scandella D: A role for the C2 domain of factor VIII in binding to von Willebrand factor. J Biol Chem 269:11601, 1994[Abstract/Free Full Text]

63. Leyte A, van Schijndel HB, Niehrs C, Huttner WB, Verbeet MP, Mertens K, van Mourik JA: Sulphation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor. J Biol Chem 266:740, 1991[Abstract/Free Full Text]

64. Meulien P, Faure T, Mischler F, Harrer H, Ulrich P, Bouderbala B, Dott K, Marie MS, Mazurier C, Wiesel ML, van der Pol H, Cazenave JP, Courtney M, Pavirani A: A new recombinant procoagulant protein derived from the cDNA encoding human factor VIII. Protein Eng 2:301, 1988[Abstract/Free Full Text]

65. Pittman DD, Wang JH, Kaufman RJ: Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII. Biochemistry 31:3315, 1992[Medline] [Order article via Infotrieve]

66. Michnick DA, Pittman DD, Wise RJ, Kaufman RJ: Identification of individual tyrosine sulfation sites within factor VIII required for optimal activity and efficient thrombin cleavage. J Biol Chem 269:20095, 1994[Abstract/Free Full Text]

67. Lenting PJ, Donath MJSH, van Mourik JA, Mertens K: Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII. J Biol Chem 269:7150, 1994[Abstract/Free Full Text]

68. Foster PA, Fulcher CA, Houghten RA, Zimmerman TS: Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidyl serine. Blood 75:1999, 1990[Abstract/Free Full Text]

69. Saenko EL, Shima M, Gilbert GE, Scandella D: Slowed release of thrombin-cleaved factor VIII from von Willebrand factor by a monoclonal and a human antibody is a novel mechanism for factor VIII inhibition. J Biol Chem 271:27424, 1996[Abstract/Free Full Text]

70. Andersson LO, Brown JE: Interaction of factor VIII-von Willebrand factor with phospholipid vescicles. Biochem J 200:161, 1981[Medline] [Order article via Infotrieve]

71. Nesheim M, Pittman DD, Giles AR, Fass DN, Wang JH, Slonosky D, Kaufman RJ: The effect of plasma von Willebrand factor on the binding of human factor VIII to thrombin-activated platelets. J Biol Chem 266:17815, 1991[Abstract/Free Full Text]

72. Gilbert GE, Drinkwater D, Barter S, Clouse SB: Specificity of phosphatidylserine-containing membrane binding sites for factor VIII. J Biol Chem 267:15861, 1992[Abstract/Free Full Text]

73. Saenko EL, Scandella D: A mechanism for inhibition of factor VIII binding to phospholipid by von Willebrand factor. J Biol Chem 270:13826, 1995[Abstract/Free Full Text]

74. Koedam J, Meijers J, Sixma J, Bouma B: Inactivation of human factor VIII by activated protein C: Cofactor activity of protein S and protective effect of von Willebrand factor. J Clin Invest 82:1236, 1988

75. Rick ME, Esmon N, Krizek D: Factor IXa and von Willebrand factor modify the inactivation of factor VIII by activated protein C. J Lab Clin Med 115:415, 1990[Medline] [Order article via Infotrieve]

76. Koedam JA, Hamer RJ, Beeser-Visser NH, Bouma BN, Sixma JJ: The effect of von Willebrand factor on activation of factor VIII by factor Xa. Eur J Biochem 189:229, 1990[Medline] [Order article via Infotrieve]

77. Fay PJ, Coumans J-V, Walker FJ: von Willebrand factor mediates protection of factor VIII from activated protein C-catalysed inactivation. J Biol Chem 266:2172, 1991[Abstract/Free Full Text]

78. Fay PJ, Walker FJ: Inactivation of human factor VIII by activated protein C: Evidence hat the factor VIII light chain contains the activated protein C binding site. Biochim Biophys Acta 994:142, 1989[Medline] [Order article via Infotrieve]

79. Eaton D, Rodriguez H, Vehar GA: Proteolytic processing of human factor VIII. Biochemistry 25:505, 1986[Medline] [Order article via Infotrieve]

80. Hill-Eubanks DC, Parker CG, Lollar P: Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin. Proc Natl Acad Sci USA 86:6508, 1989[Abstract/Free Full Text]

81. Esmon CT, Lollar P: Involvement of thrombin anion-binding exosites 1 and 2 in the activation of factor V and factor VIII. J Biol Chem 271:13882, 1996[Abstract/Free Full Text]

82. Weiss HJ, Sussman II, Hoyer LW: Stabilization of factor VIII in plasma by the von Willebrand factor. J Clin Invest 60:390, 1977

83. Tuddenham EGD, Lane RS, Rotblat F, Johnson AJ, Snape TJ, Middleton S, Kernoff PBA: Response to infusions of polyelectrolyte fractionated human factor VIII concentrate in human haemophilia A and von Willebrand's disease. Br J Haematol 52:259, 1982[Medline] [Order article via Infotrieve]

84. Wise RJ, Dorner AJ, Krane M, Pittman DD, Kaufman RJ: The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII. J Biol Chem 266:21948, 1991[Abstract/Free Full Text]

85. Mannucci PM, Tenconi PM, Castaman G, Rodeghiero F: Comparison of four virus-inactivated plasma concentrates for treatment of severe von Willebrand disease: A cross-over randomized trial. Blood 79:3130, 1992[Abstract/Free Full Text]

86. Over J, Sixma JJ, Bouma BN, Vlooswijk RAA, Beeser-Visser NH: Survival of factor VIII in von Willebrand's disease. J Lab Clin Med 97:332, 1981[Medline] [Order article via Infotrieve]

87. Brinkhous KM, Sandberg H, Garris JB, Mattson C, Palm M, Griggs T, Read MS: Purified human factor VIII procoagulant protein: Comparative hemostatic response after infusions into hemophilic and von Willebrand disease dogs. Proc Natl Acad Sci USA 82:8752, 1985[Abstract/Free Full Text]

88. Morfini M, Mannucci PM, Tenconi PM, Longo G, Mazzucconi MG, Rodeghiero F, Ciavarella N, De Rosa V, Arter A: Pharmacokinetics of monoclonally-purified and recombinant factor VIII in patients with severe von Willebrand disease. Thromb Haemost 70:270, 1993[Medline] [Order article via Infotrieve]

89. Nishino M, Girma JP, Rothschild C, Fressinaud E, Meyer D: New variant of von Willebrand disease with defective binding to factor VIII. Blood 74:1591, 1989[Abstract/Free Full Text]

90. Mazurier C, Diveval J, Jorieux S, Delobel J, Goudemand M: A new von Willebrand factor defect in a patient with factor VIII deficiency but with normal levels and multimeric patterns of both plasma and platelet von Willebrand factor. Blood 75:20, 1990[Abstract/Free Full Text]

91. Higuchi M, Wong C, Kochhan L, Aronis S, Kasper CK, Kazazian HH, Antonarakis SE: Mutations in the factor VIII gene by direct sequencing of amplified genomic DNA. Genomics 6:65, 1990[Medline] [Order article via Infotrieve]

92. Traystmann MD, Higuchi M, Kasper CK, Antonarakis SE, Kazazian HH: Use of denaturating gradient gel electrophoresis to detect point mutations in the factor VIII gene. Genomics 6:293, 1990[Medline] [Order article via Infotrieve]

93. Vehar G, Davie EW: Preparation and properties of bovine FVIII (anti-hemophilic factor). Biochemistry 19:401, 1980[Medline] [Order article via Infotrieve]

94. Fass DN, Knutson GJ, Katzmann J: Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein. Blood 59:594, 1982

95. Fulcher C, Zimmerman T: Characterization of the human FVIII procoagulant protein with a heterologous precipitating antibody. Proc Natl Acad Sci USA 79:1648, 1982[Abstract/Free Full Text]

96. Rotblat F, O'Brien SDP, O'Brien FJ, Goodall AH, Tuddenham EGD: Purification of factor VIII:C and its characterization by western blotting using monoclonal antibodies. Biochemistry 24:4294, 1985[Medline] [Order article via Infotrieve]

97. Pittman DD, Kaufman RJ: Proteolytic requirements for thrombin activation of anti-haemophilic factor (FVIII). Proc Natl Acad Sci USA 85:2429, 1988[Abstract/Free Full Text]

98. Lollar P, Knutson GJ, Fass DN: Activation of porcine FVIII:C by thrombin and factor Xa. Biochemistry 24:8056, 1985[Medline] [Order article via Infotrieve]

99. Hoyer LW, Trabold NC: The effect of thrombin on human factor VIII. J Lab Clin Med 97:50, 1981[Medline] [Order article via Infotrieve]

100. Mertens K, Bertina RM: Activation of human blood coagulation factor VIII by activated factor X, the common product of the intrinsic and the extrinsic pathway of blood coagulation. Thromb Haemost 47:96, 1982[Medline] [Order article via Infotrieve]

101. Griffith MJ, Reisner H, Lundblad R, Roberts HR: Measurement of human factor IXa activity in an isolated factor X activating system. Thromb Res 27:289, 1982[Medline] [Order article via Infotrieve]

102. Neuenschwander PF, Jesty J: Thrombin-activated and factor Xa-activated factor VIII: Differences in cofactor activity and decay rate. Arch Biochem Biophys 296:426, 1992[Medline] [Order article via Infotrieve]

103. Parker ET, Pohl J, Blackburn MN, Lollar P: Subunit structure and function of porcine factor Xa-activated factor VIII. Biochemistry 36:9365, 1997[Medline] [Order article via Infotrieve]

104. Regan LM, Fay PJ: Cleavage of factor VIII light chain is required for maximal generation of factor VIIIa activity. J Biol Chem 270:8546, 1995[Abstract/Free Full Text]

105. Hortin G, Folz R, Gordon JI, Strauss AW: Characterization of sites of tyrosine sulfation in proteins and criteria for predicting their occurence. Biochem Biophys Res Commun 141:326, 1986[Medline] [Order article via Infotrieve]

106. Huttner WB: Tyrosine sulfation and the secretory pathway. Annu Rev Physiol 50:363, 1988[Medline] [Order article via Infotrieve]

107. Hortin GL, Benutto BM: Inhibition of thrombin clotting activity by synthetic peptide segments of its inhibitors and substrates. Biochem Biophys Res Commun 169:437, 1990[Medline] [Order article via Infotrieve]

108. Donath MJSH, de Laaf RTM, Biessels PTM, Lenting PJ, van de Loo JW, van Mourik JA, Voorberg J, Mertens K: Characterization of des-(741-1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa. Biochem J 312:49, 1995

109. Bihoreau N, Paolantonacci P, Bardelle C, Fontaine-Aupart MP, Krishnan S, Yon J, Romet-Lemonne JL: Structural and functional characterization of factor VIII-triangle II, a new recombinant factor VIII lacking most of the B-domain. Biochem J 277:23, 1991

110. Kjalke M, Heding A, Talbo G, Persson E, Thomsen J, Ezban M: Amino acid residues 721-729 are required for full factor VIII activity. Eur J Biochem 234:773, 1995[Medline] [Order article via Infotrieve]

111. Mikkelsen J, Thomsen J, Ezban M: Heterogeneity in tyrosine sulfation of chinese hamster ovary cell produced recombinant factor VIII. Biochemistry 30:1533, 1991[Medline] [Order article via Infotrieve]

112. Donath MJSH, Lenting PJ, van Mourik JA, Mertens K: Kinetics of factor VIII light-chain cleavage by thrombin and factor Xa. Eur J Biochem 240:365, 1996[Medline] [Order article via Infotrieve]

113. Leyte A, Mertens K, Distel B, Evers RF, De Keyzer-Nellen MJM, Groenen-van Dooren MMCL, De Bruin J, Pannekoek H, van Mourik JA, Verbeet MP: Inhibition of human blood coagulation factor VIII by monoclonal antibodies. Biochem J 263:187, 1989[Medline] [Order article via Infotrieve]

114. Voorberg J, van Stempvoort G, Klaasse Bos JM, Mertens K, van Mourik JA, Donath MJSH: Enhanced thrombin sensitivity of a factor VIII-heparin cofactor II hybrid. J Biol Chem 271:20985, 1996[Abstract/Free Full Text]

115. Fay PJ, Haidaris PJ, Huggins CF: Role of the COOH-terminal acidic region of A1 subunit in A2 subunit retention in human factor VIIIa. J Biol Chem 268:17861, 1993[Abstract/Free Full Text]

116. Regan LM, O'Brien LM, Beattie TL, Sudhakar K, Walker FJ, Fay PJ: Activated protein C-catalyzed proteolysis of factor VIIIa alters its interactions with factor Xase. J Biol Chem 271:3982, 1996[Abstract/Free Full Text]

117. Lapan KA, Fay PJ: Localization of a factor X interactive site in the A1 subunit of factor VIIIa. J Biol Chem 272:2082, 1997[Abstract/Free Full Text]

118. Gitschier J, Kogan S, Levinson B, Tuddenham EGD: Mutations of factor VIII cleavage sites in hemophilia A. Blood 72:1022, 1988[Abstract/Free Full Text]

119. O'Brien DP, Tuddenham EGD: Purification and characterization of factor VIII 1,689-Cys: A nonfunctional cofactor occurring in a patient with severe hemophilia A. Blood 73:2117, 1989[Abstract/Free Full Text]

120. Shima M, Ware J, Yoshioka A, Fukui H, Fulcher CA: An arginine to cysteine amino acid substitution at a critical thrombin cleavage site in a dysfunctional factor VIII molecule. Blood 74:1612, 1989[Abstract/Free Full Text]

121. Arai M, Higuchi M, Antonarakis SE, Kazazian HH, Philips JA, Janco RL, Hoyer LW: Characterization of a thrombin cleavage site mutation (Arg 1689 to Cys) in the factor VIII gene of two unrelated patients with cross-reacting material-positive hemophilia A. Blood 75:384, 1990[Abstract/Free Full Text]

122. Pattinson JK, McVey JH, Boon M, Ajani A, Tuddenham EGD: CRM+ haemophilia A due to a missense mutation (372right-arrowCys) at the internal heavy chain thrombin cleavage site. Br J Haematol 75:73, 1990[Medline] [Order article via Infotrieve]

123. Acquila M, Caprino D, Pecorara M, Baudo F, Morfini M, Mori PG: Two novel mutations at 373 codon of FVIII gene detected by DGGE. Thromb Haemost 69:392, 1993[Medline] [Order article via Infotrieve]

124. Johnson DJ, Pemberton S, Acquila M, Mori PG, Tuddenham EG, O'Brien DP: FVIII S373L: Mutation at P1' site confers thrombin cleavage resistance, causing mild haemophilia A. Thromb Haemost 71:428, 1994[Medline] [Order article via Infotrieve]

125. Aly AM, Arai M, Hoyer LW: Cysteamine enhances the procoagulant activity of factor VIII-East Hartford, a dysfunctional protein due to a light chain thrombin cleavage site mutation (arginine-1689 to cysteine). J Clin Invest 89:1375, 1992

126. Aly AM, Hoyer LW: Factor VIII-East Hartford (arginine 1689 to cysteine) has procoagulant activity when separated from von Willebrand factor. J Clin Invest 89:1382, 1992

127. Duffy EJ, Parker ET, Mutucumarana VP, Johnson AE, Lollar P: Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles. J Biol Chem 267:17006, 1992[Abstract/Free Full Text]

128. Bloom JW: The interaction of rDNA factor VIII, factor VIII-des-(797-1562), and factor VIII-des-(797-1562)-derived peptides with phospholipid. Thromb Res 48:439, 1987[Medline] [Order article via Infotrieve]

129. Gilbert GE, Furie BC, Furie B: Binding of human factor VIII to phospholipids. J Biol Chem 265:815, 1990[Abstract/Free Full Text]

130. Gilbert GE, Drinkwater D: Specific membrane binding of factor VIII is mediated by O-phospho-L-serine, a moiety of phosphatidylserine. Biochemistry 32:9577, 1993[Medline] [Order article via Infotrieve]

131. Spaargaren J, Giessen PLA, Janssen MP, Voorberg J, Willems GM, van Mourik JA: Binding of blood coagulation factor VIII and its light chain to phosphatidylserine/phosphatidylcholine bilayers as measured by ellipsometry. Biochem J 310:539, 1995

132. Gilbert GE, Arena AA: Activation of the factor VIIIa-factor IXa enzyme complex of blood coagulation by membranes containing phosphatidyl-L-serine. J Biol Chem 271:11120, 1996[Abstract/Free Full Text]

133. Mathur A, Zhong D, Sabharwal AK, Smith KJ, Bajaj SP: Interaction of factor IXa with factor VIIIa. J Biol Chem 272:23418, 1997[Abstract/Free Full Text]

134. Lamphear BJ, Fay PJ: Factor IXa enhances reconstitution of factor VIIIa from isolated A2 subunit and A1/A3-C1-C2 dimer. J Biol Chem 267:3725, 1992[Abstract/Free Full Text]

135. Curtis JE, Helgerson SL, Parker ET, Lollar P: Isolation and characterization of thrombin-activated human factor VIII. J Biol Chem 269:6246, 1994[Abstract/Free Full Text]

136. Regan LM, Lamphear BJ, Huggins CF, Walker FJ, Fay PJ: Factor IXa protects factor VIIIa from activated protein C. J Biol Chem 269:9445, 1994[Abstract/Free Full Text]

137. Fay PJ, Beattie T, Huggins CF, Regan LM: Factor VIIIa A2 subunit residues 558-565 represent a factor IXa interactive site. J Biol Chem 269:20522, 1994[Abstract/Free Full Text]

138. O'Brien LM, Medved LV, Fay PJ: Localization of factor IXa and factor VIIIa interactive sites. J Biol Chem 270:27087, 1995[Abstract/Free Full Text]

139. Mutucumarana VP, Duffy EJ, Lollar P, Johnson AE: The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa. J Biol Chem 267:17012, 1992[Abstract/Free Full Text]

140. Jorquera JI, McClintock RA, Roberts JR, MacDonald MJ, Houghten RA, Fulcher CA: Synthetic peptides derived from residues 698 to 710 of factor VIII inhibit factor IXa activity. Circulation 86:685, 1992 (abstr)

141. Liles DK, Monroe DM, Roberts HR: The factor VIII peptide consisting of amino acids 698 to 712 enhances factor IXa cleavage of factor X. Blood 90:463a, 1997 (abstr, suppl 1)

142. Lenting PJ, van de Loo JWHP, Donath MJSH, van Mourik JA, Mertens K: The sequence Glu1811-Lys1818 of human blood coagulation factor VIII comprises a binding site for activated factor IX. J Biol Chem 271:1935, 1996[Abstract/Free Full Text]

143. Brandstetter H, Bauer M, Huber R, Lollar P, Bode W: X-ray structure of clotting factor IXa: Active site and module structure related to Xase activity and hemophilia B. Proc Natl Acad Sci USA 92:9796, 1995[Abstract/Free Full Text]

144. Pan Y, DeFay T, Gitschier J, Cohen FE: Proposed structure of the A domains of factor VIII by homology modelling. Nature Struct Biol 2:740, 1995[Medline] [Order article via Infotrieve]

145. Pemberton S, Lindley P, Zaitsev V, Card G, Tuddenham EGD, Kemball-Cook G: A molecular model for the triplicated A domains of human factor VIII based on the crystal structure of human caeruloplasmin. Blood 89:2413, 1997[Abstract/Free Full Text]

146. Bajaj SP, Rapaport SI, Maki SL: A monoclonal antibody to factor IX that inhibits the factor VIII:Ca potentiation of factor X activation. J Biol Chem 260:11574, 1985[Abstract/Free Full Text]

147. Bajaj SP, Sabharwal AK, Gorka J, Birktoft JJ: Antibody-probed conformational transitions in the protease domain of human factor IX upon calcium binding and zymogen activation: Putative high affinity Ca2+ binding site in the protease domain. Proc Natl Acad Sci USA 89:152, 1992[Abstract/Free Full Text]

148. Hamaguchi N, Bajaj SP, Smith KJ, Stafford DW: The role of amino-terminal residues of the heavy chain of factor IXa in the binding of its cofactor, factor VIIIa. Blood 84:1837, 1994[Abstract/Free Full Text]

149. Lenting PJ, ter Maat H, Clijsters PPFM, Donath MJSH, van Mourik JA, Mertens K: Cleavage at arginine 145 in human blood coagulation factor IX converts the zymogen into a factor VIII binding enzyme. J Biol Chem 270:14884, 1995[Abstract/Free Full Text]

150. Rees DJG, Jones IM, Handford PA, Walter SJ, Esnouf MP, Smith KJ, Brownlee GG: The role of beta -hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX. EMBO J 7:2053, 1988[Medline] [Order article via Infotrieve]

151. Hughes PE, Morgan G, Rooney EK, Brownlee GG, Handford PA: Tyrosine 69 of the first epidermal growth factor-like domain of human factor IX is essential for clotting activity. J Biol Chem 268:17727, 1993[Abstract/Free Full Text]

152. Larson PJ, Stannfield-Oakly S, van Dusen WJ, Kasper SK, Smith KJ, Monroe DM, High K: Structural integrity of the gamma-carboxyglutamic acid domain of human blood coagulation factor IXa is required for its binding to cofactor VIIIa. J Biol Chem 271:3869, 1996[Abstract/Free Full Text]

153. Nishimura H, Takeya H, Miyata T, Suehiro K, Okamura T, Niho Y, Iwanaga S: Factor IX Fukuoka. J Biol Chem 268:24041, 1993[Abstract/Free Full Text]

154. Christophe OD, Lenting PJ, Kolkman JA, Rees DJG, Mertens K: Blood coagulation factor IX residues Glu78 and Arg94 provide a link between both epidermal growth factor-like domains that is crucial in the interaction with factor VIII light chain. J Biol Chem 273:222, 1998[Abstract/Free Full Text]

155. Lenting PJ, Christophe OD, ter Maat H, Rees DJG, Mertens K: Ca2+ binding to the first epidermal growth factor-like domain of human blood coagulation factor IX promotes enzyme activity and factor VIII light chain binding. J Biol Chem 271:25332, 1996[Abstract/Free Full Text]

156. Amano K, Sarkar R, Pemberton S, Kemball-Cook G, Kazazian HH, Kaufman RJ: The molecular basis for cross-reacting material-positive hemophilia A due to missense mutations within the A2-domain of factor VIII. Blood 91:538, 1998[Abstract/Free Full Text]

157. Mertens K, van Wijngaarden A, Bertina RM, Veltkamp JJ: The functional defect of factor VIII Leiden, a genetic variant of coagulation factor VIII. Thromb Haemost 54:650, 1985[Medline] [Order article via Infotrieve]

158. Celie PHN, Donath MJSH, Jorieux S, van Mourik JA, Mazurier C, Mertens K: Characterization of Arg527 and Arg531 substitution variants of factor VIII associated with mild haemophilia. Thromb Haemost 77:570, 1997 (abstr, suppl)

159. Lollar P, Knutson GJ, Fass DN: Stabilisation of thrombin activated porcine factor VIII:C by factor IXa and phospholipid. Blood 63:1303, 1984[Abstract/Free Full Text]

160. Lollar P, Parker ET, Fay PJ: Coagulant properties of hybrid human/porcine factor VIII molecules. J Biol Chem 267:23652, 1992[Abstract/Free Full Text]

161. Neuenschwander P, Jesty J: A comparison of phospholipid and platelets in the activation of human factor VIII by thrombin and factor Xa, and in the activation of factor X. Blood 72:1761, 1988[Abstract/Free Full Text]

162. Lu D, Kalafatis M, Mann KG, Long GL: Comparison of activated protein C/protein S-mediated inactivation of human factor VIII and factor V. Blood 87:4708, 1996[Abstract/Free Full Text]

163. Persson E, Ezban M, Shymko RM: Kinetics of the interaction between the human factor VIIIa subunits: Effects of pH, ionic strength, Ca2+ concentration, heparin, and activated protein C-catalyzed proteolysis. Biochemistry 34:12775, 1995[Medline] [Order article via Infotrieve]

164. Lollar P, Parker CG: Subunit structure of thrombin-activated porcine factor VIII. Biochemistry 28:666, 1989[Medline] [Order article via Infotrieve]

165. Lollar P, Parker ET: Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog. J Biol Chem 266:12481, 1991[Abstract/Free Full Text]

166. Fay PJ, Haidaris PJ, Smudzin TM: Human factor VIIIa subunit structure. J Biol Chem 266:8957, 1991[Abstract/Free Full Text]

167. Walker FJ, Chavin S, Fay PJ: Inactivation of FVIII by activated protein C and protein S. Arch Biochem Biophys 252:322, 1987[Medline] [Order article via Infotrieve]

168. Fay PJ, Smudzin TM, Walker FJ: Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa. J Biol Chem 266:20139, 1991[Abstract/Free Full Text]

169. O'Brien DP, Johnson D, Byfield P, Tuddenham EGD: Inactivation of factor VIII by factor IXa. Biochemistry 31:2805, 1992[Medline] [Order article via Infotrieve]

170. Lamphear BJ, Fay PJ: Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa. Blood 80:3120, 1992[Abstract/Free Full Text]

171. Fay PJ, Beattie TL, Regan LM, O'Brien LM, Kaufman RJ: Model for the factor VIIIa-dependent decay of the intrinsic factor Xase. J Biol Chem 271:6027, 1996[Abstract/Free Full Text]

172. van't Veer C, Golden NJ, Kalafatis M, Mann KG: Inhibitory mechanism of the protein C pathway on tissue factor-induced thrombin generation. J Biol Chem 272:7983, 1997[Abstract/Free Full Text]

173. Yakhyaev A, Mikhailenko I, Strickland D, Saenko E: Cellular uptake and degradation of thrombin activated factor VIII fragments. Blood 90:31a, 1997 (abstr, suppl)

174. Bertina RM, Cupers R, van Wijngaarden A: Factor IXa protects activated factor VIII against inactivation by activated protein C. Biochem Biophys Res Commun 125:177, 1984[Medline] [Order article via Infotrieve]

175. Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma PH: Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 369:64, 1994[Medline] [Order article via Infotrieve]

176. Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B: Activated protein C resistance caused by Arg506Gln mutation in factor Va. Lancet 343:1361, 1994[Medline] [Order article via Infotrieve]

177. Voorberg J, Roelse J, Koopman R, Büller H, Berends F, ten Cate JW, Mertens K, van Mourik JA: Association of idiopathic venous thromboembolism with a single point mutation at Arg506 of factor V. Lancet 343:1535, 1994[Medline] [Order article via Infotrieve]

178. Zöller B, Dählback B: Linkage between inherited resistance to activated protein C and factor V mutation in venous thrombosis. Lancet 343:1536, 1994[Medline] [Order article via Infotrieve]

179. Roelse JC, Koopman MMW, Büller HR, ten Cate JW, Montaruli B, van Mourik JA, Voorberg J: Absence of mutations at the activated protein C cleavage sites of factor VIII in 125 patiens with venous thrombosis. Br J Haematol 92:740, 1996[Medline] [Order article via Infotrieve]

180. Bokarewa MI, Falk G, Bremme K, Blomback M, Wiman B: Search for mutations in the genes for coagulation factors V and VIII with a possible predisposition to activated protein C resistance. Eur J Clin Invest 27:340, 1997[Medline] [Order article via Infotrieve]

181. Amano K, Michnick DA, Moussalli M, Kaufman RJ: Mutation at either Arg336 or Arg562 in factor VIII is insufficient for complete restistance to activated protein C (APC)-mediated inactivation: Implications for the APC resistance test. Thromb Haemost 79:557, 1998[Medline] [Order article via Infotrieve]

182. Shen L, Dahlbäck B: Factor V and protein S as synergistic cofactors to activated protein C in degradation of factor VIIIa. J Biol Chem 269:18735, 1994[Abstract/Free Full Text]

183. Váradi K, Rosing J, Tans G, Pabinger I, Kail B, Schwarz HP: Factor V enhances the cofactor function of protein S in the APC-mediated inactivation of factor VIII: Influence of the factor VR506Q mutation. Thromb Haemost 76:208, 1996[Medline] [Order article via Infotrieve]

184. van `t Veer C, Golden NJ, Kalafatis M, Simioni P, Bertina RM, Mann KG: An in vitro analysis of the combination of hemophilia A and factor V-Leiden. Blood 90:3067, 1997[Abstract/Free Full Text]

185. Nichols WC, Amano K, Cacheris PM, Figueiredo MS, Michaelides K, Schwaab R, Hoyer L, Kaufman RJ, Ginsburg D: Moderation of Hemophilia A phenotype by the factor V R506Q mutation. Blood 88:1183, 1996[Abstract/Free Full Text]

186. Arbini AA, Mannucci PM, Bauer KA: Low prevalence of the factor V Leiden mutation among "severe" hemophiliacs with a "milder" bleeding diathesis. Thromb Haemost 74:1255, 1995[Medline] [Order article via Infotrieve]

187. Tavassoli M, Kishimoto T, Kataoka M: Liver endothelium mediates the hepatocytes uptake of ceruloplasmin. J Cell Biol 102:1298, 1986[Abstract/Free Full Text]

188. Rand MD, Hanson SR, Mann KG: Factor V turnover in a primate model. Blood 86:2616, 1995[Abstract/Free Full Text]

189. Mertens K, Leyte A, Lenting PJ, van Mourik JA: Factor VIII-von Willebrand factor interaction and in vivo survival of factor VIII light chain. Thromb Haemost 65:658, 1991 (abstr)

190. Fijnvandraat K, Berntop E, ten Cate JW, Johnsson H, Peters M, Savidge G, Tengborn L, Spira J, Stahl C: Recombinant, B-domain deleted factor VIII (r-VIII-SQ): Pharmacokinetics and initial safety aspects in hemophilia A patients. Thromb Haemost 77:298, 1997[Medline] [Order article via Infotrieve]

191. Pittman DD, Alderman EM, Tomkinson KN, Wang JH, Giles AR, Kaufman RJ: Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII. Blood 81:2925, 1993[Abstract/Free Full Text]

192. Mertens K, Donath MJSH, van Leen RW, de Keyzer-Nellen MJM, Verbeet MP, Klaasse Bos JM, Leyte A, van Mourik JA: Biological activity of recombinant factor VIII variants lacking the central B-domain and the heavy chain sequence Lys713-Arg740: Discordant in vitro and in vivo activity. Br J Haematol 85:133, 1993[Medline] [Order article via Infotrieve]

193. Lubin IM, Healey JF, Scandella D, Runge MS, Lollar P: Elimenation of a major inhibitor epitope in factor VIII. J Biol Chem 269:8639, 1994[Abstract/Free Full Text]

194. Healey JF, Lubin IM, Nakai H, Saenko EL, Hoyer LW, Scandella D, Lollar P: Residues 484-508 contain a major determinant of the inhibitory epitope in the A2 domain of human factor VIII. J Biol Chem 270:14505, 1995[Abstract/Free Full Text]

195. Pipe SW, Kaufman RJ: Characterization of a genetically engineered inactivation-resistant factor VIIIa. Proc Natl Acad Sci USA 94:11851, 1997[Abstract/Free Full Text]

196. McMullen BA, Fujikawa K, Davie EW, Hedner U, Ezban M: Locations of disulfide bridges and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A). Protein Sci 4:740, 1995[Medline] [Order article via Infotrieve]

197. Fay PJ, Smudzin TM: Inter-subunit fluorescence energy transfer in human factor VIII. J Biol Chem 264:14005, 1989[Abstract/Free Full Text]

198. Sandberg H, Brandt J, Ålin P, Strömberg S, Gray E, Bartfai J, Castro V: Glycosylation pattern of a B-domain-deleted factor VIII molecule (r-VIII-SQ). Thromb Haemost 73:1214, 1995 (abstr)

199. Bihoreau N, Veillon JF, Ramon C, Scohyers JM, Schmitter JM: Characterization of a recombinant antihaemophilia A factor (factor VIII-triangle II) by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 9:1584, 1995[Medline] [Order article via Infotrieve]

200. Medzihradszky KF, Besman MJ, Burlingame AL: Structural characterization of site specific N-glycosylation of recombinant human factor VIII by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry. Anal Chem 69:3968, 1997

© 1998 by The American Society of Hematology.
 
0006-4971/98/9211-0154$3.00/0

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 haematolHome page
M. van den Biggelaar, E. A.M. Bouwens, N. A. Kootstra, R. P. Hebbel, J. Voorberg, and K. Mertens
Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells
Haematologica, May 1, 2009; 94(5): 670 - 678.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. van den Biggelaar, A. B. Meijer, J. Voorberg, and K. Mertens
Intracellular cotrafficking of factor VIII and von Willebrand factor type 2N variants to storage organelles
Blood, March 26, 2009; 113(13): 3102 - 3109.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. J. van Schooten, S. Shahbazi, E. Groot, B. D. Oortwijn, H. M. van den Berg, C. V. Denis, and P. J. Lenting
Macrophages contribute to the cellular uptake of von Willebrand factor and factor VIII in vivo
Blood, September 1, 2008; 112(5): 1704 - 1712.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Lacroix-Desmazes, A.-M. Navarrete, S. Andre, J. Bayry, S. V. Kaveri, and S. Dasgupta
Dynamics of factor VIII interactions determine its immunologic fate in hemophilia A
Blood, July 15, 2008; 112(2): 240 - 249.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. J. Gale, T. J. Cramer, D. Rozenshteyn, and J. R. Cruz
Detailed Mechanisms of the Inactivation of Factor VIIIa by Activated Protein C in the Presence of Its Cofactors, Protein S and Factor V
J. Biol. Chem., June 13, 2008; 283(24): 16355 - 16362.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
W. Cao, S. Krishnaswamy, R. M. Camire, P. J. Lenting, and X. L. Zheng
Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13
PNAS, May 27, 2008; 105(21): 7416 - 7421.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B. W. Shen, P. C. Spiegel, C.-H. Chang, J.-W. Huh, J.-S. Lee, J. Kim, Y.-H. Kim, and B. L. Stoddard
The tertiary structure and domain organization of coagulation factor VIII
Blood, February 1, 2008; 111(3): 1240 - 1247.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Dasgupta, A.-M. Navarrete, J. Bayry, S. Delignat, B. Wootla, S. Andre, O. Christophe, M. Nascimbeni, M. Jacquemin, L. Martinez-Pomares, et al.
A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes
PNAS, May 22, 2007; 104(21): 8965 - 8970.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Lacroix-Desmazes, B. Wootla, S. Dasgupta, S. Delignat, J. Bayry, J. Reinbolt, J. Hoebeke, E. Saenko, M. D. Kazatchkine, A. Friboulet, et al.
Catalytic IgG from Patients with Hemophilia A Inactivate Therapeutic Factor VIII
J. Immunol., July 15, 2006; 177(2): 1355 - 1363.
[Abstract] [Full Text] [PDF]

Home page
 Mayo Clin Proc.Home page
R. K. Pruthi
Hemophilia: A Practical Approach to Genetic Testing
Mayo Clin. Proc., November 1, 2005; 80(11): 1485 - 1499.
[Abstract] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
C. V. Denis, S. J. Roberts, T. M. Hackeng, and P. J. Lenting
In Vivo Clearance of Human Protein S in a Mouse Model: Influence of C4b-Binding Protein and the Heerlen Polymorphism
Arterioscler Thromb Vasc Biol, October 1, 2005; 25(10): 2209 - 2215.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
N. Bovenschen, K. Mertens, L. Hu, L. M. Havekes, and B. J. M. van Vlijmen
LDL receptor cooperates with LDL receptor-related protein in regulating plasma levels of coagulation factor VIII in vivo
Blood, August 1, 2005; 106(3): 906 - 912.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
B. Dahlback and B. O. Villoutreix
Regulation of Blood Coagulation by the Protein C Anticoagulant Pathway: Novel Insights Into Structure-Function Relationships and Molecular Recognition
Arterioscler Thromb Vasc Biol, July 1, 2005; 25(7): 1311 - 1320.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. C. Spiegel, P. Murphy, and B. L. Stoddard
Surface-exposed Hemophilic Mutations across the Factor VIII C2 Domain Have Variable Effects on Stability and Binding Activities
J. Biol. Chem., December 17, 2004; 279(51): 53691 - 53698.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. D. Blostein, B. C. Furie, I. Rajotte, and B. Furie
The Gla Domain of Factor IXa Binds to Factor VIIIa in the Tenase Complex
J. Biol. Chem., August 15, 2003; 278(33): 31297 - 31302.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Villard, S. Lacroix-Desmazes, T. Kieber-Emmons, D. Piquer, S. Grailly, A. Benhida, S. V. Kaveri, J.-M. Saint-Remy, and C. Granier
Peptide decoys selected by phage display block in vitro and in vivo activity of a human anti-FVIII inhibitor
Blood, August 1, 2003; 102(3): 949 - 952.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. Manithody, P. J. Fay, and A. R. Rezaie
Exosite-dependent regulation of factor VIIIa by activated protein C
Blood, June 15, 2003; 101(12): 4802 - 4807.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
N. Bovenschen, J. Herz, J. M. Grimbergen, P. J. Lenting, L. M. Havekes, K. Mertens, and B. J. M. van Vlijmen
Elevated plasma factor VIII in a mouse model of low-density lipoprotein receptor-related protein deficiency
Blood, May 15, 2003; 101(10): 3933 - 3939.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J. Tost, P. Schatz, M. Schuster, K. Berlin, and I. G. Gut
Analysis and accurate quantification of CpG methylation by MALDI mass spectrometry
Nucleic Acids Res., May 1, 2003; 31(9): e50 - e50.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Bovenschen, R. C. Boertjes, G. van Stempvoort, J. Voorberg, P. J. Lenting, A. B. Meijer, and K. Mertens
Low Density Lipoprotein Receptor-related Protein and Factor IXa Share Structural Requirements for Binding to the A3 Domain of Coagulation Factor VIII
J. Biol. Chem., March 7, 2003; 278(11): 9370 - 9377.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
P. V. Jenkins, J. Freas, K. M. Schmidt, Q. Zhou, and P. J. Fay
Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex
Blood, June 28, 2002; 100(2): 501 - 508.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. B. Doering, C. D. Josephson, H. N. Craddock, and P. Lollar
Factor VIII expression in azoxymethane-induced murine fulminant hepatic failure
Blood, June 17, 2002; 100(1): 143 - 147.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pathol.Home page
D J Bowen
Haemophilia A and haemophilia B: molecular insights
Mol. Pathol., April 1, 2002; 55(2): 127 - 144.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. E. K. Rosenblum, K. Schmidt, J. Freas, M. Mastri, and P. J. Fay
Cofactor Activities of Factor VIIIa and A2 Subunit following Cleavage of A1 Subunit at Arg336
J. Biol. Chem., March 29, 2002; 277(14): 11664 - 11669.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Stoilova-McPhie, B. O. Villoutreix, K. Mertens, G. Kemball-Cook, and A. Holzenburg
3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography
Blood, February 15, 2002; 99(4): 1215 - 1223.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pathol.Home page
D J Bowen
Haemophilia A and haemophilia B: molecular insights
Mol. Pathol., February 1, 2002; 55(1): 1 - 18.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
P. C. Spiegel Jr, M. Jacquemin, J.-M. R. Saint-Remy, B. L. Stoddard, and K. P. Pratt
Structure of a factor VIII C2 domain-immunoglobulin G4{kappa} Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII
Blood, July 1, 2001; 98(1): 13 - 19.
[Abstract] [Full Text] [PDF]

Home page
 NEJMHome page
P. M. Mannucci and E. G.D. Tuddenham
The Hemophilias -- From Royal Genes to Gene Therapy
N. Engl. J. Med., June 7, 2001; 344(23): 1773 - 1779.
[Full Text] [PDF]

Home page
 BloodHome page
E. N. van den Brink, E. A. M. Turenhout, N. Bovenschen, B. G. A. D. H. Heijnen, K. Mertens, M. Peters, and J. Voorberg
Multiple VH genes are used to assemble human antibodies directed toward the A3-C1 domains of factor VIII
Blood, February 15, 2001; 97(4): 966 - 972.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
E. N. van den Brink, E. A. M. Turenhout, C. M. C. Bank, K. Fijnvandraat, M. Peters, and J. Voorberg
Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain
Blood, July 15, 2000; 96(2): 540 - 545.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. S. Ahmad, J. M. Scandura, and P. N. Walsh
Structural and Functional Characterization of Platelet Receptor-mediated Factor VIII Binding
J. Biol. Chem., April 21, 2000; 275(17): 13071 - 13081.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
H. P. Schwarz, P. J. Lenting, B. Binder, J. Mihaly, C. Denis, F. Dorner, and P. L. Turecek
Involvement of low-density lipoprotein receptor-related protein (LRP) in the clearance of factor VIII in von Willebrand factor-deficient mice
Blood, March 1, 2000; 95(5): 1703 - 1708.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. H. N. Celie, P. J. Lenting, and K. Mertens
Hydrophobic Contact between the Two Epidermal Growth Factor-like Domains of Blood Coagulation Factor IX Contributes to Enzymatic Activity
J. Biol. Chem., January 7, 2000; 275(1): 229 - 234.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
W Li and G Keller
VEGF nuclear accumulation correlates with phenotypical changes in endothelial cells
J. Cell Sci., January 5, 2000; 113(9): 1525 - 1534.
[Abstract] [PDF]

Home page
 J. Biol. Chem.Home page
S. S. Stoylova, P. J. Lenting, G. Kemball-Cook, and A. Holzenburg
Electron Crystallography of Human Blood Coagulation Factor VIII Bound to Phospholipid Monolayers
J. Biol. Chem., December 17, 1999; 274(51): 36573 - 36578.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. J. Lenting, J. G. Neels, B. M. M. van den Berg, P. P. F. M. Clijsters, D. W. E. Meijerman, H. Pannekoek, J. A. van Mourik, K. Mertens, and A.-J. van Zonneveld
The Light Chain of Factor VIII Comprises a Binding Site for Low Density Lipoprotein Receptor-related Protein
J. Biol. Chem., August 20, 1999; 274(34): 23734 - 23739.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Do, J. F. Healey, E. K. Waller, and P. Lollar
Expression of Factor VIII by Murine Liver Sinusoidal Endothelial Cells
J. Biol. Chem., July 9, 1999; 274(28): 19587 - 19592.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Right arrow Rights and Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lenting, P. J.
Right arrow Articles by Mertens, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lenting, P. J.
Right arrow Articles by Mertens, K.
Related Collections
Right arrow Review Articles
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

click for free articles
home about blood authors subscriptions permissions advertising public access contact us
  Copyright © 1998 by American Society of Hematology         Online ISSN: 1528-0020