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R J Kaufman

Publications and source records attributed to R J Kaufman.

At least 73 records · Page 4Linked to original sources
PubMed1998

Can we improve on nature? "Super molecules" of factor VIII.

Treatment of haemophilia A requires frequent infusion of plasma- or recombinant-derived factor VIII. This regimen is limited due to the high cost and inconvenient access to peripheral veins. In addition, patients frequently develop inhibitory antibodies that limit available therapeutic regimens. Two major advances in factor VIII research over the past 15 years were the ability to isolate homogeneous preparations of factor VIII and the isolation of the factor VIII gene that provided for a detailed biochemical and structural characterization of the factor VIII molecule. With an increased understanding of the requirements for factor VIII function, studies have attempted to produce improved factor VIII molecules for replacement therapy. These findings have produced forms of factor VIII that are more efficiently produced, that are less immunogenic, and that have higher specific activity. The future will see the engineering of novel factor VIII molecules with increased therapeutic efficiency while minimizing inhibitor antibody development. In addition, there are now structural models of factor VIII available that should in the future direct development of novel peptidomimetics that may eventually overcome the requirement for replacement therapy with factor VIII protein.

Animals↗
PubMed1998

Combined factors V and VIII deficiency--the solution.

Combined deficiency of coagulation factor V and factor VIII is an autosomal recessive disorder which has been observed in a number of populations around the world. However, this disease appears to be most common in the Mediterranean basin, particularly in Jews of Sephardic and Middle Eastern origin living in Israel. We have taken a positional cloning approach toward identifying the gene responsible for this disorder. We initially studied 14 affected individuals from nine unrelated Jewish families using a panel of polymorphic genetic markers spaced throughout the human genome. The combined factors V and VIII deficiency gene was mapped to a locus on the long arm of chromosome 18 with a maximal LOD score of 13.22. A detailed genetic analysis identified two distinct haplotypes among these families, suggesting two independent founders or, alternatively, a single ancient founder with a more recent split of these subpopulations. Further work to identify and characterize the gene responsible for combined factors V and VIII deficiency should provide important insights into the biosynthesis of these homologous proteins.

Chromosome Mapping↗
PubMed1998

Protein serine/threonine phosphatase Ptc2p negatively regulates the unfolded-protein response by dephosphorylating Ire1p kinase.

Cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by increasing the transcription of the genes encoding ER-resident chaperone proteins. Ire1p is a transmembrane protein kinase that transmits the signal from unfolded proteins in the lumen of the ER by a mechanism that requires oligomerization and trans-autophosphorylation of its cytoplasmic-nucleoplasmic kinase domain. Activation of Ire1p induces a novel spliced form of HAC1 mRNA that produces Hac1p, a transcription factor that is required for activation of the transcription of genes under the control of the unfolded-protein response (UPR) element. Searching for proteins that interact with Ire1p in Saccharomyces cerevisiae, we isolated PTC2, which encodes a serine/threonine phosphatase of type 2C. The Ptc2p interaction with Ire1p is specific, direct, dependent on Ire1p phosphorylation, and mediated through a kinase interaction domain within Ptc2p. Ptc2p dephosphorylates Ire1p efficiently in an Mg2+-dependent manner in vitro. PTC2 is nonessential for growth and negatively regulates the UPR pathway. Strains carrying null alleles of PTC2 have a three- to fourfold-increased UPR and increased levels of spliced HAC1 mRNA. Overexpression of wild-type Ptc2p but not catalytically inactive Ptc2p reduces levels of spliced HAC1 mRNA and attenuates the UPR, demonstrating that the phosphatase activity of Ptc2p is required for regulation of the UPR. These results demonstrate that Ptc2p downregulates the UPR by dephosphorylating Ire1p and reveal a novel mechanism of regulation in the UPR pathway upstream of the HAC1 mRNA splicing event.

Amino Acid Sequence↗
PubMed1998

Mutation at either Arg336 or Arg562 in factor VIII is insufficient for complete resistance to activated protein C (APC)-mediated inactivation: implications for the APC resistance test.

Activated protein C (APC)-mediated inactivation of factor VIII (FVIII) correlates with cleavage at either Arg336 and/or Arg562. To elucidate the APC cleavage requirements for inactivation of FVIII, APC cleavage site mutants in FVIII (R336I, R562K and R336I/R562K) were made by site-directed mutagenesis. Analysis of these FVIII mutants expressed in COS-1 monkey cells demonstrated the thrombin-cleaved mutant R562K was resistant to APC cleavage at residue 562 but not at Arg336 and the thrombin cleaved mutant R3361 was mostly resistant to APC cleavage at residue 336, but was sensitive to APC cleavage at Arg562. The double mutant R336I/R562K was mostly resistant to cleavage at residue 336 and completely resistant to cleavage at residue 562. Thus, APC cleavage of FVIII does not require a specific order of cleavage at either residue. The functional inactivation by APC was studied using partially purified preparations of FVIII expressed in Chinese hamster ovary cells. Both single mutants were inactivated at similar rates but slower than wild-type FVIII, whereas the double mutant R336I/R562K was resistant to inactivation. The ability of a commercially available APC-resistance assay kit to detect APC resistant FVIII was tested by reconstituting FVIII deficient plasma with the APC resistant mutants. Only the R336I/R562K demonstrated a reduced APC-resistance ratio, indicating that this assay can not detect the single APC cleavage site mutant of FVIII. These results suggest that APC-mediated cleavage at either Arg336 or Arg562 partially inactivate FVIII.

Animals↗
PubMed1998

Cleavage requirements of factor V in tissue-factor induced thrombin generation.

Factor V (FV) activation is the result of cleavages at Arg709, Arg1018 and Arg1545 by thrombin or FXa. The relative importance of these cleavages in tissue factor (TF) induced thrombin generation in plasma and in a purified system was elucidated with recombinant FV in which the three sites had been eliminated one by one or in combinations. The mutants were analyzed with a clotting assay using FV-deficient plasma and in a TF induced thrombin generation system using plasma or purified components. Surprisingly, in the standard FV clotting assay, all mutants gave similar clotting activities and the thrombin generation curves obtained with wild-type and thrombin-resistant FV were similar. Differences in clotting activities and thrombin generation patterns between wild-type and thrombin-resistant FV were only observed when lower TF concentrations were used. The thrombin generation curve obtained in plasma containing wt FV was characterized by a short lag phase and a subsequent phase of rapid thrombin generation (propagation phase). The Arg709 to Gln mutation yielded a slightly prolonged lag phase and the rate of thrombin generation during the propagation phase was approximately 5-fold lower than that observed with wt FV. The Arg1018 to Ile mutation only slightly affected the thrombin generation curve, whereas the Arg1545 to Gln mutation yielded a prolonged lag phase and decreased maximum thrombin activity. Thrombin-resistant FV (mutated at all three sites) yielded a prolonged lag phase and poor thrombin generation during the propagation phase. The purified system further demonstrated the importance of the three cleavage sites for rapid and sustained thrombin generation. The results demonstrate that cleavages at positions 709, 1018 and 1545 are not required for assembly of a FXa-FV complex expressing low but significant prothrombinase activity but that all three sites in different ways are important for the creation of a FVa which maximally supports the FXa-mediated activation of prothrombin.

Arginine↗
PubMed1997

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa.

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690-2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

Enzyme Activation↗
PubMed1997

Identification and functional requirement of Cu(I) and its ligands within coagulation factor VIII.

Coagulation factor VIII (FVIII) is a heterodimer consisting of a light chain of 80 kDa (domains A3-C1-C2) in a metal ion-dependent association with a 220-kDa heavy chain (domains A1-A2-B). The nature of the metal ion-dependent association between the heavy and light chains was investigated using atomic absorption spectroscopy, electron paramagnetic resonance spectroscopy (EPR), and site-directed mutagenesis and expression of the FVIII cDNA. Whereas copper ion was not detected in intact recombinant FVIII, EDTA dissociation of the chains yielded an EPR signal consistent with 1 mol of Cu(I)/mol of active protein, supporting the hypothesis that a single molecule of reduced copper ion is buried within intact FVIII and is released and oxidized upon treatment with EDTA. Cu(I), and not Cu(II), was able to reconstitute FVIII activity from dissociated chains, demonstrating a requirement for Cu(I) in FVIII function. Three potential copper ion binding sites exist within FVIII: one type-2 site and two type-1 sites. The importance of these potential copper ion ligands was tested by studying the effect of site-directed mutants. Of the two histidines that compose the type-2 binding site, the His-1957 --> Ala mutant displayed secretion, light and heavy chain assembly, and activity similar to wild-type FVIII, while mutant His-99 --> Ala was partially defective for secretion and had low levels of heavy and light chain association and activity. In contrast, FVIII having the mutation Cys-310 --> Ser within the type-1 copper binding site in the A1 domain was inactive and partially defective for secretion from the cell, and the heavy and light chains of the secreted protein were not associated. Mutant Cys-2000 --> Ser within the A3 domain displayed secretion, assembly, and activity similar to that for wild-type FVIII. These results support the hypothesis that Cu(I) is buried within the type-1 copper binding site within the A1 domain and is required for FVIII chain association and activity.

Amino Acid Substitution↗
PubMed1997

Mutagenesis of a potential immunoglobulin-binding protein-binding site enhances secretion of coagulation factor VIII.

Coagulation factor VIII (FVIII) and factor V are homologous glycoproteins that have a domain structure of A1-A2-B-A3-C1-C2. FVIII is a heterodimer of the heavy chain (domains A1-A2-B) and the light chain (domains A3-C1-C2) in a metal ion-dependent association between the A1- and A3-domains. Previous studies identified a 110-amino acid region within the FVIII A1-domain that inhibits its secretion and contains multiple short peptide sequences that have potential to bind immunoglobulin-binding protein (BiP). FVIII secretion requires high levels of intracellular ATP, consistent with an ATP-dependent release from BiP. Site-directed mutagenesis was used to elucidate the importance of the potential BiP-binding sites in FVIII secretion. Mutation of Phe at position 309 to Ser or Ala enhanced the secretion of functional FVIII and reduced its ATP dependence. The F309S FVIII had a specific activity, thrombin activation profile, and heat inactivation properties similar to those of wild-type FVIII. However, F309S FVIII displayed increased sensitivity to EDTA-mediated inactivation that is known to occur through metal ion chelation-induced dissociation of the heavy and light chains of FVIII. The results support that Phe309 is important in high affinity heavy and light chain interaction, and this correlates with a high affinity BiP-binding site. Introduction of the F309S mutation into other secretion defective FVIII mutants rescued their secretion, demonstrating the ability of the this mutation to improve secretion of mutant FVIII proteins retained in the cell.

Adenosine Triphosphate↗
PubMed1997

Cleavage requirements for activation of factor V by factor Xa.

Coagulation factor V circulates in plasma as a single chain protein which expresses little procoagulant activity. After its activation by limited proteolysis by thrombin or factor Xa, factor Va functions as cofactor to factor Xa in the activation of prothrombin. Thrombin cleaves human factor V at Arg709, Arg1018 and Arg1545 and factor Va is formed by the heavy and light chains, which correspond to the N-terminal and C-terminal fragments, respectively. Factor Xa has been shown to cleave factor V at Arg1018 and at a second undefined position close to Arg709. The factor-Xa-mediated cleavage at Arg1018 has been proposed to be sufficient for expression of full factor Va activity. To study the activation of factor V by factor Xa, site-directed mutagenesis was used to convert Arg709 to Gln, Arg1018 to Ile, and Arg1545 to Gln. Constructs containing all possible combinations of native and mutated residues in these positions were expressed transiently in COS 1 cells. The various factor-V mutants were incubated with factor Xa or thrombin. The proteolytic cleavage pattern was analyzed by Western blotting, and the specific factor-Va activities determined in a prothrombinase assay. Control experiments using thrombin gave results which were in agreement with those on record, i.e. cleavages at both Arg709 and Arg1545 were required for expression of full factor-Va activity, whereas the cleavage at Arg1018 enhanced the rate of cleavage at Arg1545. Factor Xa was found to cleave factor V at all three thrombin cleavage sites, i.e. at Arg709, Arg1018 and Arg1545. An additional factor-Xa-cleavage site was found in the light chain region at Arg1765. Cleavage at Arg1018 by factor Xa was not sufficient for expression of full factor-Va activity. Full factor-Va activity was only obtained after cleavage at both Arg709 and Arg1545. The factor-Xa-mediated cleavage at Arg709 was kinetically favourable over that at Arg1545. Factor V which was mutated at all three sites (at positions 709, 1018 and 1545) was resistant to activation by thrombin. However, treatment with factor Xa yielded an increased factor-Va activity which was associated with the cleavage at Arg1765. Our study extends previously results on thrombin activation of factor V and elucidates the relative importance of the different cleavage sites for activation of factor V by factor Xa.

Factor V↗
PubMed1997

Gene induction in response to unfolded protein in the endoplasmic reticulum is mediated through Ire1p kinase interaction with a transcriptional coactivator complex containing Ada5p.

In eukaryotic cells, accumulation of unfolded protein in the endoplasmic reticulum induces transcription of a family of genes encoding endoplasmic reticulum protein chaperones through a conserved unfolded protein response element. In Saccharomyces cerevisiae, activation of a transmembrane receptor kinase, Ire1p (Ern1p), initiates signaling, although the mediators immediately downstream of Ire1 kinase are unknown. Here we demonstrate interaction of Ire1p with the transcriptional coactivator, Gcn5p (for general control nonrepressed; also known as Ada4p). Gcn5p associates with other Ada (for alteration/deficiency in activation) gene products in a heteromeric complex and has histone acetyltransferase activity. We show that the Gcn5/Ada complex is selectively required for the unfolded protein response but not for the heat shock response. A novel mechanism is proposed in which activation of a receptor kinase recruits a transcription coactivator complex to a specific chromosomal locus to mediate localized histone acetylation, thus making specific gene sequences accessible for transcription.

DNA-Binding Proteins↗
PubMed1997

Linkage of combined factors V and VIII deficiency to chromosome 18q by homozygosity mapping.

Combined Factors V and VIII deficiency is an autosomal recessive bleeding disorder identified in at least 58 families comprising a number of different ethnic groups. Affected patients present with a moderate bleeding tendency and have Factor V and Factor VIII levels in the range of 5-30% of normal. The highest frequency of the mutant gene is found in Jews of Sephardic and Middle Eastern origin living in Israel with an estimated disease frequency of 1:100,000. We sought to identify the gene responsible for combined Factors V and VIII deficiency using a positional cloning approach. Of 14 affected individuals from 8 unrelated Jewish families, 12 were the offspring of first-cousin marriages. After a genome-wide search using 241 highly polymorphic short tandem repeat (STR) markers, 13 of the 14 affected patients were homozygous for two closely linked 18q markers. Patients and all available family members were genotyped for 11 additional STRs spanning approximately 11 cM on the long arm of chromosome 18. Multipoint linkage analysis yielded a maximal log of the odds (LOD) score of 13.22. Haplotype analysis identified a number of recombinant individuals and established a minimum candidate interval of 2.5 cM for the gene responsible for combined Factors V and VIII deficiency. The product of this locus is likely to operate at a common step in the biosynthetic pathway for these two functionally and structurally homologous coagulation proteins. Identification of this gene should provide new insight into the biology of Factor V and Factor VIII production.

Chromosome Mapping↗
PubMed1997

Immunoglobulin binding protein (BiP) function is required to protect cells from endoplasmic reticulum stress but is not required for the secretion of selective proteins.

BiP/GRP78 is a lumenal stress protein of the endoplasmic reticulum (ER) that interacts with polypeptide folding intermediates transiting the secretory compartment. We have studied the secretion and the stress response in Chinese hamster ovary (CHO) cells that overexpress either wild-type immunoglobulin binding protein (BiP) or a BiP deletion molecule (residues 175-201) that can bind peptides and ATP but is defective in ATP hydrolysis and concomitant peptide release. Overexpressed wild-type BiP was localized to the ER and unique vesicles within the nucleus, whereas overexpressed ATPase-defective BiP was localized to the ER and cytoplasmic vesicles but was absent from the nucleus. Compared with wild-type CHO cells, overexpression of ATPase-defective BiP prevented secretion of factor VIII, a coagulation factor that extensively binds BiP in the lumen of the ER. Under these conditions factor VIII was stably associated with the ATPase-defective BiP. In contrast, the secretion of monocyte/macrophage colony stimulating factor, a protein that is not detected in association with BiP, was not affected by overexpression of ATPase-defective BiP. These results show that BiP function is not required for secretion of some proteins and suggest that some proteins do not interact with BiP upon transport through the ER. The presence of unfolded protein in the ER induces transcription of BiP and also elicits a general inhibition of protein synthesis. Overexpression of wild-type BiP prevented the stress-mediated transcriptional induction of BiP in response to either calcium ionophore A23187 treatment or tunicamycin treatment. In contrast, overexpression of ATPase-defective BiP did not prevent the stress induction of BiP, showing that the ATPase activity is required to inhibit transcriptional induction. Overexpression of wild-type BiP, but not ATPase-defective BiP, increased survival of cells treated with A23187. The increased survival mediated by overexpressed wild-type BiP correlated with reduced translation inhibition in response to the stress condition. These results indicate that overexpressed BiP alleviated the stress in the ER to prevent BiP transcriptional induction and permit continued translation of cellular mRNAs.

Adenosine Triphosphatases↗
PubMed1997

A model for the double-stranded RNA (dsRNA)-dependent dimerization and activation of the dsRNA-activated protein kinase PKR.

Binding of double-stranded RNA (dsRNA) to PKR induces autophosphorylation and activation. However, the requirement for dsRNA in promoting dimerization and the requirement for dimerization in PKR activation are controversial. We have studied the dsRNA binding and dimerization requirements for the activation of PKR in vivo. Co-expression and immunoprecipitation experiments detected an interaction between the K296P mutant and a bacteriophage T7-epitope-tagged K64E mutant of dsRNA binding domain. In contrast, the K64E/K296P double mutant did not form a detectable dimer with the wild-type dsRNA binding domain. These results support that dimerization of intact PKR with the isolated dsRNA binding domain requires dsRNA binding activity. Expression of the isolated PKR kinase domain (residues 228-551) reduced translation of the reporter mRNA even in the presence of PKR inhibitors. Furthermore, the isolated kinase domain (residues 228-551) undergoes autophosphorylation and sequentially transphosphorylates both mutant K296P PKR and wild-type eIF-2alpha in vitro. In contrast, the isolated kinase domain (residues 264-551) lacking the third basic region was not active. These observations lead us to propose that the dsRNA binding domains on intact PKR inhibit kinase activity and that dsRNA binding to intact PKR induces a conformational change to expose dimerization sites within the dsRNA binding domain thereby promoting dimerization and facilitating trans-phosphorylation and activation.

Animals↗
PubMed1997

DNA transfection to study translational control in mammalian cells.

Mammalian cells respond to changes in their environment by rapid and reversible covalent modification of the translational machinery. In most cases, these modifications involve the phosphorylation and dephosphorylation of translation initiation factors (for review see Ref. 1). The modification of translation initiation factors may affect translational activity of either specific mRNAs or general cellular mRNAs. To study the effect of a particular factor or its modification on the translational capacity of an mRNA, there are a number of potential approaches that include in vitro translation reactions as well as in vivo experiments. Generally, experiments initially report a covalent modification that correlates with altered translational capacity of either a specific or a general class of mRNAs. The modification and the particular amino acid residue involved are then identified. Then mutations are made at the modified residue to prevent modification (for example, a serine-to-alanine mutation to prevent phosphorylation) and the effect of the mutant factor on the translation of a target mRNA is tested. The most convenient method for monitoring the effect of a mutant translation factor on translation is the use of transient DNA transfection. However, in certain situations it is desirable to isolate stably transfected cell lines to study the effect of overexpression, underexpression, or expression of a particular mutant translation factor. This article reviews two methods that are routinely used to study translational control that involve either transient or stable DNA transfection.

Animals↗
PubMed1997

Biosynthesis, assembly and secretion of coagulation factor VIII.

Factor VIII is a large complex glycoprotein that is deficient in hemophilia A. It has a domain organization consisting of A1-A2-B-A3-C1-C2 where the B domain is a heavily glycosylated region that is dispensable for procoagulant activity. Factor VIII expression is 10-to 20-fold lower than the homologous coagulation factor V. Factor VIII expression is limited due to a low level of steady-state messenger RNA in the cytoplasm and inefficient transport of the primary translation product from the endoplasmic reticulum to the Golgi apparatus. Within the secretory pathway, factor VIII is processed to a heterodimer of the heavy chain (domains A1-A2-B) in a metal ion association with the light chain (domains A3-C1-C2). Upon secretion from the cell, von Willebrand factor binds the light chain of factor VIII and stabilizes the factor, preventing degradation. Protein folding within the mammalian secretory pathway is facilitated by molecular chaperones. Within the endoplasmic reticulum, factor VIII exhibits stable interaction with protein chaperones identified as the immunoglobulin-binding protein (BiP), calnexin and calreticulin. BiP is a peptide-dependent ATPase that interacts with exposed hydrophobic surfaces on unfolded proteins or unassembled protein subunits. A potential BiP binding site within factor VIII has been identified. Mutation of a single amino acid residue in the potential BiP binding site increased the secretion efficiency of factor VIII by threefold. Interestingly, the proposed BiP binding site is adjacent to a type-1 copper binding site within the A1 domain that is required for interaction between the factor VIII A1 domain and the A3 domain. We propose that Cu(I) binds the type-1 copper ion-binding site in the A1 domain and provides the essential requirement for a stable interaction between the heavy and light chains. Calnexin and calreticulin are transmembrane and lumenal proteins, respectively, localized to the endoplasmic reticulum, which associate transiently with many soluble and membrane glycoproteins during folding and subunit assembly. The calnexin and calreticulin interaction with factor VIII occurs primarily through amino-terminal linked oligosaccharides within the heavily glycosylated factor VIII B domain and this interaction appears to be required for factor VIII secretion. The findings suggest that factor VIII cycles through interactions with BiP, calnexin and calreticulin. Although the interaction with BiP does not appear to be required for factor VIII secretion, data suggest that the calnexin and/or calreticulin interaction is required for secretion. The observations suggest a unique requirement for carbohydrate processing and calnexin/calreticulin interaction that may limit the productive secretion of factor VIII and have implications for approaches towards somatic cell gene therapy for hemophilia A.

Animals↗
PubMed1996

Factor VIII C2 domain missense mutations exhibit defective trafficking of biologically functional proteins.

The half-life of coagulation factor VIII (FVIII) in plasma is prolonged by noncovalent interaction with von Willebrand factor (vWF). Antibody inhibition data indicate that epitopes within the carboxyl terminus of the FVIII light chain play a role in vWF binding. Analysis of hemophilia A patient DNA samples have identified missense mutations within this carboxyl terminus of the FVIII light chain at amino acid 2307 in which arginine is replaced with either glutamine or leucine. Patients with these mutations have reduced FVIII activity proportional to reduced cross-reacting material in their plasma. It was hypothesized that the reduced levels of FVIII in plasma due to these mutations may be related to a defect in vWF binding with resultant plasma instability. Wild-type and mutant FVIII cDNA expression vectors were prepared and expressed in COS-1 monkey cells by transient DNA transfection. FVIII mutants R2307Q and R2307L were synthesized at equal rates compared to FVIII wild-type but had greater than 10-fold reduced accumulation of antigen and activity levels in the conditioned medium. An additional mutation, Y2305F, also displayed a similar defect in protein accumulation, whereas Y2332F was secreted similarly to wild-type. The specific activity of immunoaffinity purified R2307Q was mildly reduced compared to FVIII wild-type, whereas vWF binding properties were retained. Inhibition of intracellular cysteine proteases resulted in intracellular accumulation of R2307Q protein, suggesting that the mechanism leading to hemophilia A is related to a block in secretion and subsequent degradation within the secretory pathway rather than extracellular instability.

Animals↗