Deletion analysis of recombinant human factor V. Evidence for a phosphatidylserine binding site in the second C-type domain.

Human coagulation factor V is an integral component of the prothrombinase complex. Rapid activation of prothrombin is dependent on the interactions of this nonenzymatic cofactor with factor Xa and prothrombin in the presence of calcium ions and a phospholipid or platelet surface. Factor V is similar structurally and functionally to the homologous cofactor, factor VIII, which interacts with factor IXa to accelerate factor X activation in the presence of calcium and phospholipids. Both of these cofactors, when activated, possess homologous heavy and light chains. Binding to anionic phospholipids is mediated by the light chains of these two cofactors. In bovine factor Va, a phosphatidylserine-specific binding site has been localized to the amino-terminal A3 domain of the light chain. In human factor VIII, on the other hand, a region within the carboxyl-terminal C2 domain of the light chain has been shown to interact with anionic phospholipids. We have constructed a series of recombinant deletion mutants lacking domain-size fragments of the light chain of human factor V (rHFV). These mutants are expressed and secreted as single-chain proteins by COS cells. Thrombin and the factor V activator from Russell's viper venom process these deletion mutants as expected. The light chain deletion mutants possess essentially no procoagulant activity, nor are they activated by treatment with factor V activator from Russell's viper venom. Deletion of the second C-type domain results in essentially complete loss of phosphatidylserine-specific binding whereas the presence of the C2 domain alone (rHFV des-A3C1, which lacks the A3 and C1 domains of the light chain) results in significant phosphatidylserine-specific binding. The presence of the A3 domain alone (rHFV des-C1C2) does not mediate binding to immobilized phosphatidylserine. Increasing calcium ion concentrations result in decreased binding of recombinant human factor V and the mutant rHFV des-A3C1 to phosphatidylserine, similar to previous studies with purified plasma factor V and phospholipid vesicles. These results indicate that human factor V, similar to human factor VIII, possesses a phosphatidylserine-specific binding site within the C2 domain of the light chain.     

Thrombin cleavage analysis of a novel antihaemophilic factor variant, factor VIII delta II

Factor VIII delta II is a genetically engineered deletion variant of factor VIII expressed by recombinant Chinese hamster ovary cells, in which a major portion of the central (B) domain and a part of the light chain (Pro771-Asp1666) are missing. After immunoaffinity purification, the kinetics of thrombin cleavage of the novel molecule was analysed by SDS/PAGE, Western blotting and N-terminal amino acid sequencing. Thrombin first cleaves factor VIII delta II at Arg740-Ser741 to generate the 90-kDa heavy chain and an 80-kDa fusion polypeptide consisting of the remaining portion of the B domain and the 73-kDa light chain. The 90-kDa fragment is further cleaved, giving rise to 50-kDa and 40-kDa fragments while the 80-kDa fragment generates a 71/73-kDa doublet. The 71/73-kDa doublet, 50-kDa and 40-kDa fragments were further analysed by N-terminal amino acid sequencing and found to correspond to the predicted amino acid sequences. Our study shows that, in spite of the 900 amino acid deletion present in factor VIII delta II, the essential structural elements required for thrombin activation are conserved.     

Factor VIII C2 domain contains the thrombin-binding site responsible for thrombin-catalyzed cleavage at Arg1689

Thrombin-catalyzed factor VIII activation is an essential positive feedback mechanism regulating intrinsic blood coagulation. A factor VIII human antibody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation and cleavage at Arg(1689) by thrombin. The results suggested that A-FF prevented the interaction of thrombin with factor VIII and that the C2 domain was involved in the interaction with thrombin. We performed direct binding assays using anhydro-thrombin, a catalytically inactive derivative of thrombin in which the active-site serine is converted to dehydroalanine. Intact factor VIII, 80-kDa light chain, 72-kDa light chain, and heavy chain fragments bound dose-dependently to anhydro-thrombin, and the K(d) values were 48, 150, 106, and 180 nm, respectively. The C2 and A2 domains also dose-dependently bound to anhydro-thrombin, and the K(d) values were 440 and 488 nm, respectively. The A1 domain did not bind to anhydro-thrombin. A-FF completely inhibited C2 domain binding to anhydro-thrombin (IC(50), 18 nm), whereas it did not inhibit A2 domain binding. Furthermore, C2-specific affinity purified F(ab)'(2) of A-FF, and the recombinant C2 domain inhibited thrombin cleavage at Arg(1689). Our results indicate that the C2 domain contains the thrombin-binding site responsible for the cleavage at Arg(1689).     

Identification of residues contributing to A2 domain-dependent structural stability in factor VIII and factor VIIIa

Factor VIII circulates as a heterodimer composed of heavy (A1A2B domains) and light (A3C1C2 domains) chains, whereas the contiguous A1A2 domains are separate subunits in the active cofactor, factor VIIIa. Whereas the A1 subunit maintains a stable interaction with the A3C1C2 subunit, the A2 subunit is weakly associated in factor VIIIa and its dissociation accounts for the labile activity of the cofactor. In examining the ceruloplasmin-based factor VIII A domain model, potential hydrogen bonding based upon spatial separations of <2.8A were found between side chains of 14 A2 domain residues and 7 and 9 residues in the A1 and A3 domains, respectively. These residues were individually replaced with Ala, except Tyr residues were replaced with Phe, and proteins stably expressed to examine the contribution of each residue to protein stability. Factor VIII stability at 55 degrees C and factor VIIIa activity were monitored using factor Xa generation assays. Fourteen of 30 factor VIII mutants showed >2-fold increases in either or both decay rates compared with wild type; whereas, 7 mutants showed >2-fold increased rates in factor VIIIa decay compared with factor VIII decay. These results suggested that multiple residues at the A1-A2 and A2-A3 domain interfaces contribute to stabilizing the protein. Furthermore, these data discriminate residues that stabilize interactions in the procofactor from those in the cofactor, where hydrogen bonding in the latter appears to contribute more significantly to stability. This observation is consistent with an altered conformation involving new inter-subunit interactions involving A2 domain following procofactor activation.     

Lower inhibitor development in hemophilia A mice following administration of recombinant factor VIII-O-phospho-L-serine complex

Factor VIII is a multidomain protein composed of A1, A2, B, A3, C1, and C2 domains. Deficiency or dysfunction of factor VIII causes hemophilia A, a bleeding disorder. Administration of exogenous recombinant factor VIII as a replacement leads to development of inhibitory antibodies against factor VIII in 15-30% of hemophilia A patients. Hence, less immunogenic preparations of factor VIII are highly desirable. Inhibitory antibodies against factor VIII are mainly directed against immunodominant epitopes in C2, A3, and A2 domains. Further, several universal epitopes for CD4+ T-cells have been identified within the C2 domain. The C2 domain is also known to interact specifically with phosphatidylserine-rich lipid vesicles. Here, we have investigated the hypothesis that complexation of O-phospho-l-serine, the head group of phosphatidylserine, with the C2 domain can reduce the overall immunogenicity of factor VIII. The biophysical (circular dichroism and fluorescence) and biochemical studies (ELISA and size exclusion chromatography) showed that O-phospho-l-serine binds to the phospholipid-binding region in the C2 domain, and this interaction causes subtle changes in the tertiary structure of the protein. O-Phospho-l-serine also prevented aggregation of the protein under thermal stress. The immunogenicity of the factor VIII-O-phospho-l-serine complex was evaluated in hemophilia A mice. The total and inhibitory antibody titers were lower for factor VIII-O-phospho-l-serine complex compared with factor VIII alone. Moreover, factor VIII administered as a complex with O-phospho-l-serine retained in vivo activity in hemophilia A mice. Our results suggest that factor VIII-O-phospho-l-serine complex may be beneficial to increase the physical stability and reduce immunogenicity of recombinant factor VIII preparations.     

Proteolysis of blood coagulation factor VIII by the factor VIIa-tissue factor complex: generation of an inactive factor VIII cofactor.

Activation of factor VIII by thrombin occurs via limited proteolysis at R372, R740, and R1689. The resultant active factor VIIIa molecule consists of three noncovalently associated subunits: A1-a1, A2-a2, and A3-C1-C2 (50, 45, and 73 kDa respectively). Further proteolysis of factor VIIIa at R336 and R562 by activated protein C subsequently inactivates this cofactor. We now find that the factor VIIa-tissue factor complex (VIIa-TF/PL), the trigger of blood coagulation with restricted substrate specificity, can also catalyze limited proteolysis of factor VIII. Proteolysis of factor VIII was observed at 10 sites, producing 2 major fragments (47 and 45 kDa) recognized by an anti-factor VIII A2 domain antibody. Time courses indicated the slow conversion of the large fragment to 45 kDa, followed by further degradation into at least two smaller fragments. N-Terminal sequencing along with time courses of proteolysis indicated that VIIa-TF/PL cleaved factor VIII first at R740, followed by concomitant cleavage at R336 and R372. Although cleavage of the light chain at R1689 was observed, the majority remained uncleaved after 17 h. Consistent with this, only a transient 2-fold increase in factor VIII clotting activity was observed. Thus, heavy chain cleavage of factor VIII by VIIa-TF/PL produces an inactive factor VIII cofactor no longer capable of activation by thrombin. In addition, VIIa-TF/PL was found to inactivate thrombin-activated factor VIII. We hypothesize that these proteolyses may constitute an alternative pathway to regulate coagulation under certain conditions. In addition, the ability of VIIa-TF/PL to cleave factor VIII at 10 sites greatly expands the known protein substrate sequences recognized by this enzyme-cofactor complex.     

Inactivation of factor VIII by activated protein C and protein S

Factor VIII was inactivated by activated protein C in the presence of calcium and phospholipids. Analysis of the activated protein C-catalyzed cleavage products of factor VIII indicated that inactivation resulted from the cleavage of the heavy chains. The heavy chains appeared to be converted into 93- and 53-kDa peptides. Inactivation of factor VIII that was only composed of the 93-kDa heavy chain and 83-kDa light chain indicated that the 93-kDa polypeptide could be degraded into a 68-kDa peptide that could be subsequently cleaved into 48- and 23-kDa polypeptides. Thus, activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. The addition of protein S accelerated the rate of inactivation and the rate of all of the cleavages. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products, including the 93-, 68-, and 53-kDa polypeptides. The addition of factor IX to the factor VIII-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.     

Production of Recombinant Human Factor VIII in Different Cell Lines and the Effect of Human XBP1 Co-Expression

Recombinant factor VIII is one of the most complex mammalian proteins and a biotechnology venture required for the treatment of hemophilia A. The complexity of the protein, post-translational modifications and limitations of expression elements make the production of active recombinant FVIII a challenge. Here we report the production of biologically active Factor VIII in two different cell lines, CHO and HepG2, by transient transfection. Two expression vectors based on the CMV promoter were used: one harboring CMV Intron A (InA) and the other without it. To bypass difficulties in secretion, we also studied the influence of co-expression of the human splice isoform of the XBP1 gene. We report the production of recombinant FVIII possessing bioengineered FVIII heavy and light chains, linked by a minimal B domain. In our study, HepG2, a human hepatocyte cell line, expressed Factor VIII ten-fold more than a CHO cell line, and in HepG2 cells, the expression of XBP1 improved Factor VIII activity. For CHO cells, expression was improved by the presence of InA, but no further improvement was noted with XBP1 co-expression. These data suggest that the minimal B domain rFVIII preserves Factor VIII biological activity and that different expression elements can be used to improve its production.     

Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A.

Factor VIII-specific affibodies were selected from phage displayed libraries constructed by combinatorial mutagenesis of an alpha helical bacterial receptor domain derived from staphylococcal protein A. Bead-immobilized recombinant human factor VIII (rVIII) (80 and 90 kDa chains) protein was used during competitive biopannings in the presence of free 80-kDa chain protein, resulting in the selection of several binders that showed dissociation constants (Kd) in the range 100-200 nM as determined by biosensor analyses. One variant (Z[rVIII:3], 90-kDa chain specific) was further characterized in small-scale affinity chromatography experiments, and showed efficient and selective recovery of biologically active rVIII from Chinese hamster ovary cell supernatant-derived feed stocks. The purity of the enriched rVIII was comparable with rVIII material purified by immunoaffinity chromatography using a 90-kDa chain-specific monoclonal antibody. Interestingly, epitope mapping showed that the monoclonal antibody and the affibody ligand competed for the same or at least overlapping epitopes on rVIII. In addition, the Z[rVIII:3] variant was produced by peptide synthesis with a C-terminal cysteine to enable directed coupling to solid supports. This 59-residue protein was analyzed by circular dichroism and showed a secondary structure content similar to that of the parental Z domain used as scaffold. In biosensor studies, the synthetic affibody was immobilized recruiting the C-terminal cysteine residue, and demonstrated to bind both recombinantly produced and plasma-derived factor VIII. From a secondary library, constructed by re-randomization of relevant positions identified after alignment of the first-generation variants, a panel of affinity-improved second-generation affibodies were selected of which one clone showed a dissociation constant (Kd) for rVIII of 5 nM. Several of these variants also showed higher apparent binding efficiencies towards rVIII when analyzed as immobilized ligands in biosensor experiments. Taken together, the results suggest that affibody ligands produced by bacterial or synthetic routes could be of interest as an alternative to monoclonal antibodies in purification processes or as diagnostic or monitoring tools.     

Role of factor VIII C2 domain in factor VIII binding to factor Xa.

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248-2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K(d)) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nM, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248-2285) demonstrated that a peptide designated EP-2 (residues 2253-2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.     

Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex

Factor VIII is a procofactor that plays a critical role in blood coagulation, and is missing or defective in hemophilia A. We determined the X-ray crystal structure of B domain-deleted human factor VIII. This protein is composed of five globular domains and contains one Ca(2+) and two Cu(2+) ions. The three homologous A domains form a triangular heterotrimer where the A1 and A3 domains serve as the base and interact with the C2 and C1 domains, respectively. The structurally homologous C1 and C2 domains reveal membrane binding features. Based on biochemical studies, a model of the factor IXa-factor VIIIa complex was constructed by in silico docking. Factor IXa wraps across the side of factor VIII, and an extended interface spans the factor VIII heavy and light chains. This model provides insight into the activation of factor VIII and the interaction of factor VIIIa with factor IXa on the membrane surface.     

The 1.7 ? X-Ray Crystal Structure of the Porcine Factor VIII C2 Domain and Binding Analysis to Anti-Human C2 Domain Antibodies and Phospholipid Surfaces

The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the “non-classical” antibody epitopes. Furthermore, antibody-binding results illustrate that the “classical” 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU)/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.     

Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule

The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.     

Trp2313-His2315 of Factor VIII C2 Domain Is Involved in Membrane Binding: STRUCTURE OF A COMPLEX BETWEEN THE C2 DOMAIN AND AN INHIBITOR OF MEMBRANE BINDING*

Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 Å) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed β-strand of the C2 domain, Trp²³¹³-His²³¹⁵. This result indicates that the Trp²³¹³-His²³¹⁵ segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.     

The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII

von Willebrand factor (vWF) is a multimeric glycoprotein that promotes platelet aggregation and stabilizes coagulation factor VIII in the plasma. vWF is also required for the stable accumulation of recombinant factor VIII secreted from cells in a heterologous expression system. In this report, we show that vWF can promote the in vitro reconstitution of factor VIII activity from dissociated heavy and light chains of factor VIII, suggesting that vWF may act to promote stable assembly of factor VIII subunits at the site of secretion. The structural requirements for vWF propeptide cleavage and for vWF multimerization in its binding and stabilization of factor VIII was examined using specifically altered recombinant vWF. The mutant vWF molecules were also assayed for their function in ristocetin-induced platelet agglutination mediated through the platelet receptor GPIb. Deletion of the vWF propeptide produced a dimeric vWF molecule that failed to mediate platelet agglutination, suggesting that multimerization is required for vWF to attain functional GPIb binding. This mature dimeric form of vWF, however, was fully capable of binding to and supporting stable secretion of factor VIII. A vWF mutant with an altered propeptide cleavage site formed large multimers of uncleaved pro-vWF that functioned in platelet agglutination. However, this noncleavage mutant neither bound to or supported stable accumulation of factor VIII. Analysis of the vWF propeptide, expressed independently, demonstrated that it could not bind factor VIII or stabilize its secretion. These results show that the dimeric mature vWF subunit is sufficient to bind and stabilize factor VIII and that the presence of uncleaved vWF propeptide inhibits both factor VIII binding and stabilization.     

Inactivation of human factor VIII by activated protein C: evidence that the factor VIII light chain contains the activated protein C binding site

Factor VIII is represented as a series of heterodimers composed of an 83(81) kDa light chain noncovalently bound to a variable size (93 to 210 kDa) heavy chain. Activated protein C inactivates factor VIII causing several cleavages of the factor VIII heavy chain(s). When factor VIII subunits were dissociated and component heavy and light chains isolated, the heavy chains were no longer a substrate for proteolysis by activated protein C. However, when factor VIII heavy chains were recombined with light chain, the reconstituted factor VIII activity was inactivated by activated protein C. The rate of factor VIII inactivation catalyzed by activated protein C was reduced by the presence of free light chain. The extent of this inhibition was dependent upon the concentration of light chain. Control experiments indicated that this protective effect of free light chain was not the result of inhibition of the activated protein C - lipid interaction. Fluorescence analysis demonstrated binding between the factor VIII light chain, chemically modified with eosin maleimide, and activated protein C, modified at its active site by dansyl-Glu-Gly-Arg chloromethyl ketone. Similar to proteolysis of factor VIII by activated protein C, this binding was dependent upon a lipid surface. Based upon the degree of fluorescence quenching, a spatial distance of 26 A was calculated separating the two fluorophores. These results demonstrate direct binding of activated protein C to the factor VIII light chain and suggest that this binding is an obligate step for activated protein C-catalyzed inactivation of factor VIII.     

Factor VIII activity and factor VIII-associated antigen in healthy newborns and in newborns with stressing perinatal factors

Factor VIII coagulation activity (VIII:C) and factor VIII associated antigen (VIII:AGN) were determined in healthy newborns and in children with charging perinatal factors ("risk children"). VIII:C values of healthy newborns may be compared with those of grown-ups with normal coagulation. Risk children have somewhat higher values than newborns, the difference, however, being statistically not significant. The concentration of VIII:AGN is clearly increased in both groups on the first day of life. Moreover, VIII:AGN is being eliminated more slowly in risk children. The increased VIII:AGN concentrations are considered as a sequel of stress conditions caused by birth, whereas the discrepancy between VIII:C and VIII:AGN is due to a thrombin effect.     

Thrombin-activated and factor Xa-activated human factor VIII: differences in cofactor activity and decay rate.

The decay of human coagulation factor VIIIa has been studied by kinetic methods that ensure no interference through proteolytic feedback. The rate of decay of factor VIIIa activity was found to vary with the activator used to activate factor VIII. Thrombin-activated factor VIII-von Willebrand factor complex (fVIII-vWf) decayed at a rate of 0.31 min-1, whereas factor Xa-activated fVIII-vWf decayed at 0.11 min-1 under the same conditions. Factor VIII free of von Willebrand factor (factor VIII: C), although decaying at a generally slower rate after activation, still showed a dependence of decay rate on activator: thrombin-activated factor VIII:C decaying at a rate of 0.06 min-1, and factor Xa-activated factor VIII: C at 0.01 min-1. Readdition of von Willebrand factor (18 micrograms/ml) to factor VIII:C did not alter the observed activity or decay rate. The decay of the two species of factor VIIIa was studied, using the fVIIIa-vWf complex, in the presence of varying levels of factor IXa. Plots of reciprocal decay rates vs factor IXa concentration were linear, and nearly parallel for the two factor VIIIa species, with a mean slope of 0.56 min.nM-1. In addition to these studies, we have confirmed previous studies showing that the two forms of factor VIIIa differ in cofactor activity, but they do so in the same ratio as in their decay rates. We suggest that this difference and that observed in decay rate have a common cause, and incorporate this into a potential kinetic model of factor VIII activation and decay.     

Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion

Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine factor VIII. Novel hybrid human/porcine factor VIII molecules were constructed to identify porcine factor VIII domains that confer high level expression. Hybrid human/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human factor VIII. Additionally, hybrid murine/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency.     

Reconstitution of human factor VIII from isolated subunits

Human factor VII heterodimers were fractionated into component heavy and light chains using an anti-light chain specific monoclonal antibody immunosorbant. Neither the light chain nor the heavy chain alone possessed activity. Factor VII activity was reconstituted by recombining the subunits in the presence of Mn2+ or Ca2+. Reconstitution of activity also showed ionic strength dependence suggesting the importance of hydrophobic and electrostatic interactions. All factor VIII heavy chains (93 to 210 kDa) recombined with the 83 kDa light chain as judged by retention of all reconstituted heterodimeric forms by the monoclonal immunosorbant. Maximum specific activity (3 units/micrograms) was obtained at a 1:1 molar ratio of light chain:heavy chain. The presence of von Willebrand factor enhanced the rate of factor VIII reconstitution as much as 5-fold. This effect was both ionic strength-dependent and dose-dependent up to a 25-fold weight excess of von Willebrand factor over factor VIII.