Mutant Factor VIII Research Articles

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.

A Thr359Met Mutation in Factor VII of a Patient With a Hereditary Deficiency Causes Defective Secretion of the Molecule

We elucidated the genetic basis responsible for factor VII deficiency in an Italian woman with a severe bleeding diathesis. In the allele inherited from the patient's father, we identified a G to A mutation at nucleotide 6070 at the 5' splice site of intron 4 and a G to A substitution at nucleotide 10976 resulting in the Arg353Gln polymorphism. The maternal allele demonstrated a C to T substitution at nucleotide 10994 resulting in Thr359Met. The mutation at nucleotide 6070 alters an invariant GT dinucleotide and disrupts normal mRNA processing. To investigate the mechanism by which Thr359Met reduces factor VIl levels, we expressed wild type factor VII cDNA (FVIIwt) and a mutant factor VII cDNA containing the base substitution resulting in Met359 (FVII359M) in Chinese hamster ovary cells (CHO). In cells transfected with the mutant factor VII cDNA, FVII359M accumulated intracellularly, and no factor VII was detected in the media after 3 hours of chase. The carbohydrate side chains associated with FVII359M were sensitive to Endo H digestion, which indicates that the protein is retained in the endoplasmic reticulum. Analysis of cell lysates also showed that FVII359M was associated with the 78 kD protein corresponding to GRP78/BiP. We conclude that a Thr359Met mutation in factor VII results in a severe secretion defect that probably results from abnormal folding of the molecule.

Impaired human tissue factor-mediated activity in blood clotting factor VIINagoya (Arg304–>Trp). Evidence that a region in the catalytic domain of factor VII is important for the association with tissue factor.

A 37-year-old man was found to have an inherited abnormal factor VII (FVII) (designated FVIINagoya) that has a lower FVII procoagulant activity when tested in a one-stage assay using rabbit, simian, or human tissue factor (TF), but surprisingly has a nearly normal activity using bovine TF. DNA sequence analysis of the propositus' FVII gene revealed a C to T mutation that results in a novel amino acid substitution of Arg304 to Trp. Restriction enzyme analysis of the amplified fragment obtained from each family member demonstrated that the patient is homozygous for the point mutation, and the mother and sister of the patient are heterozygous for this mutation. To elucidate the abnormality in FVIINagoya, we compared the recombinant mutant FVII with normal FVII. In the presence of human TF, kinetic analyses demonstrated that the apparent Km values of FVIINagoya for factor Xa-mediated FVII activation and for FVIIa-mediated factor X (FX) activation were 3.6- and 4.5-fold higher than those of normal FVII, respectively. The ligand binding assay to human TF also showed that FVIINagoya had a 5.2-fold larger apparent Kd than normal. In the presence of bovine TF, however, these values showed no difference between normal and mutant FVII. These findings indicate that the major abnormality in FVIINagoya is not a catalytic dysfunction but an impaired complex-formation of human TF.FVII.FX, suggesting the importance of Arg304 in the interaction with human TF.

Factor VIII S373L: Mutation at P1’ Site Confers Thrombin Cleavage Resistance, Causing Mild Haemophilia A

A novel CRM+ mutation, factor VIII position 373 serine to leucine substitution (FVIII 373-Leu) was identified during a survey of Factor VIII (FVIII) mutations. We have purified the variant protein from the patient's plasma in order to allow further characterisation of the molecule. The CRM+ plasma contained 120% Factor VIII antigen (FVIII:Ag) and 6% Factor VIII coagulant activity (FVIII:C). After purification the mutant FVIII was subjected to thrombin proteolysis, and was thereby activated 5.6-fold compared with 7-fold for wild type molecule. Subsequently, spontaneous inactivation of the mutant was much slower than noted for wild type FVIII. Western blot analysis using monoclonal antibodies demonstrated that thrombin cleavage of FVIII 373-Leu at positions 740 and 1689 were normal but that cleavage at position 372 was completely absent. Crystallographic coordinates of the active site of thrombin complexed to fibrinopeptide A were used to explore possible mechanistic reasons for the failure of thrombin to cleave the mutant FVIII at position 372. Steric hindrance between the mutant side chain and the side chain of the P1 residue was apparent. We conclude that the functional defect of FVIII 373-Leu results from the inability of thrombin to cleave the mutant at position 372-373, and propose that this is due to steric hindrance by the side chain of leucine 373, preventing correct formation of the enzyme substrate complex.

Factor VII Mie: Homozygous Asymptomatic Type I Deficiency Caused by an Amino Acid Substitution of His (CAC) for Arg(247) (CGC) in the Catalytic Domain

We found hereditary factor VII deficiency in a clinically asymptomatic family, and characterized their factor VII gene and the abnormal molecule using recombinant DNA techniques. The propositus was a 45-year-old woman who was noted to have a prolonged prothrombin time. The level of factor VII antigen of the patient was 25.9% of that of normal individuals and the level of factor VII activity was 28% and 24%, when tested using rabbit brain tissue factor and human placental tissue factor in a one-stage clotting assay, respectively. Two of her sisters had almost the same reduced levels of factor VII antigen and activity, and her parents who are first cousins, a son, a daughter and a niece had moderately reduced leves of both factor VII activity and antigen. To identify the mutation site, all the coding exons and exon-intron boundaries of the factor VII gene of the propositus were amplified using the polymerase chain reaction (PCR), then subcloned and sequenced. One missense mutation (G to A) was identified in exon VII of the gene resulting in an amino acid substitution of His(CAC) for Arg(247)(CGC) in the gene product. PCR using a mutagenic primer to introduce a new ApaL I site into the mutant allele of the patient's factor VII gene revealed that this allele was inherited in the affected individuals in the pedigree. Transient expression assays using BHK cells transfected with an expression vector containing the mutant factor VII cDNA suggested that this mutation leads to factor VII deficiency by impairing secretion of the mutated factor VII.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that an Arg79-->Gln substitution in human factor VII is not associated with a reduction in coagulant activity.

A recent report hypothesized that an Arg79-->Gln mutation in the first epidermal growth factor-like domain of human factor VII is the molecular basis for a severe (< 1%) factor VII functional deficiency. In the present study, a site-specific mutant human factor VII cDNA (Arg79-->Gln) was constructed, subcloned and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity and characterized with respect to gamma-carboxyglutamic acid content, ability to activate, tissue factor-dependent amidolytic activity and expression of factor VIIa proteolytic activity on tissue factor-bearing cells. Mutant factor VII was fully carboxylated and exhibited the same molecular weight and coagulant activity as plasma factor VII. Mutant factor VII was activated by factor Xa at the same rate, and to the same extent, as plasma factor VII. In the presence of tissue factor, mutant factor VII was converted to factor VIIa in an autocatalytic manner at a rate indistinguishable from that observed with plasma factor VII. In addition, the amidolytic activities of mutant factor VIIa and plasma factor VIIa towards S-2288 in the presence of relipidated tissue factor were identical. Finally, following complex formation with cell surface tissue factor, mutant factor VIIa activated factor X at essentially the same rate as plasma factor VIIa under comparable conditions. These results are not consistent with the notion that the arginine-79 residue in the first epidermal growth factor-like domain of human factor VII is essential for the expression of tissue factor-dependent factor VIIa proteolytic activity.

Epitope mapping of human factor VIII inhibitor antibodies by site-directed mutagenesis of a factor VIII polypeptide.

Previous epitope mapping studies of human factor VIII (FVIII) inhibitor antibodies with heavy chain specificity localized epitopes to the amino-terminal half of the FVIII A2 domain. In this report we have used unidirectional deletion analysis and site-directed mutagenesis to identify a minimum length polypeptide and amino acid residues that contribute to the FVIII conformation recognized by these antibodies. Bacterial expression plasmids were exploited to demonstrate that a FVIII polypeptide of approximately 150 residues is required to generate a common heavy chain epitope(s). Another series of plasmids were constructed that synthesize: a FVIII polypeptide containing an internal deletion; four polypeptides with single residue substitutions; two polypeptides with triple residue changes; and a quadruple amino acid replacement within one polypeptide. The relative reactivities of the wild-type and mutant FVIII polypeptides were tested by immunoblotting, inhibitor neutralization assays and ELISA with a variety of human FVIII inhibitor auto- and alloantibodies. These techniques illustrate that the internal deletion mutant and one of the relatively conservative amino acid substitution triple mutants, mutant 389, resulted in significantly decreased immunoreactivity. The data identify FVIII Glu389,390,391 as three critical components of an epitope for human FVIII inhibitor antibodies and identify a major inhibitory epitope involved in the immune response to FVIII.

Initiation of the extrinsic pathway of blood coagulation: evidence for the tissue factor dependent autoactivation of human coagulation factor VII

Previous studies demonstrated proteolytic activation of human blood coagulation factor VII by an unidentified protease following complex formation with tissue factor expressed on the surface of a human bladder carcinoma cell line (J82). In the present study, an active-site mutant human factor VII cDNA (Ser344----Ala) has been constructed, subcloned, and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity in a single step from serum-free culture supernatants by immunoaffinity column chromatography. Mutant factor VII was fully carboxylated, possessed no apparent clotting activity, and was indistinguishable from plasma factor VII by SDS-PAGE. Cell binding studies indicated that mutant factor VII bound to J82 tissue factor with essentially the same affinity as plasma factor VII and was cleaved by factor Xa at the same rate as plasma factor VII. In contrast to radiolabeled single-chain plasma factor VII that was progressively converted to two-chain factor VIIa on J82 monolayers, mutant factor VII was not cleaved following complex formation with J82 tissue factor. Incubation of radiolabeled mutant factor VII with J82 cells in the presence of recombinant factor VIIa resulted in the time-dependent and tissue factor dependent conversion of single-chain mutant factor VII to two-chain mutant factor VIIa. Plasma levels of antithrombin III had no discernible effect on the factor VIIa catalyzed activation of factor VII on J82 cell-surface tissue factor but completely blocked this reaction catalyzed by factor Xa. These results are consistent with an autocatalytic mechanism of factor VII activation following complex formation with cell-surface tissue factor, which may play an important role in the initiation of extrinsic coagulation in normal hemostasis.

Synthesis, purification, and characterization of an Arg152 .fwdarw. Glu site-directed mutant of recombinant human blood clotting factor VII

Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg152-Ile153. Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, we have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg152----Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. Purified mutant factor VII was indistinguishable from plasma-derived or recombinant wild-type factor VII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated as a single band with an apparent molecular weight of 50,000. The average specific activity of several mutant factor VII preparations was 0.00025 unit/micrograms, or 0.01% of that observed for recombinant wild-type factor VII preparations. The clotting activity of mutant factor VII was, however, completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease.(ABSTRACT TRUNCATED AT 250 WORDS)