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.

Factor VIII (FVIII) 1 functions as a cofactor in the blood coagulation cascade for proteolytic activation of factor X by factor IXa. It is synthesized as a single chain polypeptide of approximately 280 kilodaltons (kDa) with the domain structure A1-A2-B-A3-C1-C2 (1,2). The C domains are homologous to proteins that are capable of binding negatively charged phospholipids (3). Following synthesis, cleavage within the B do-main results in a 200-kDa heavy chain (A1-A2-B) and an 80-kDa light chain (A3-C1-C2) that are associated as a heterodimer through a divalent metal ion linkage between the A1 and A3 domains (4).
In human plasma, FVIII interacts through noncovalent interactions with von Willebrand factor (vWF) to form a complex with a molar ratio of 1:50. In addition to promoting FVIII secretion and stabilizing FVIII in plasma (4 -13), vWF also regulates FVIII activity by preventing activation and inactivation by factor Xa (14), inactivation by activated protein C (15,16), and by inhibiting FVIII binding to phospholipids (17,18). Thus the FVIII-vWF interaction is critical in the biology of FVIII.
Antibody inhibition data and site-directed deletion and mutagenesis studies indicate the acidic region at the amino terminus of the FVIII light chain is important for FVIII binding to vWF (19 -22). However, a synthetic peptide derived from the acidic region of the FVIII light chain did not compete for vWF binding to FVIII (22). Recent data suggest that additional amino acids within the C2 domain at the carboxyl terminus of the FVIII light chain are also important for vWF binding. Specifically, human inhibitor and monoclonal antibodies that recognize overlapping epitopes between amino acids 2303 and 2332 inhibit FVIII binding to vWF and/or phospholipid (23)(24)(25)(26). Thus, a model was proposed in which the acidic region and the C2 domain of the FVIII light chain together support high affinity binding to vWF (23). Once thrombin-cleaved FVIII has dissociated from vWF, the C2 domain of the light chain becomes available to interact with phospholipid membranes, and FVIII exerts its cofactor function with factor IXa (27)(28)(29)(30).
Recent clinical reports of hemophilia A have identified missense mutations in the FVIII gene that result in amino acid substitutions within the carboxyl terminus of the light chain (31)(32)(33)(34). They occur at amino acid 2307 in which arginine is replaced with either glutamine or leucine. The reported clinical data (Table I) show a range of phenotypes from mild to severe, and comparable antigen levels for the amount of plasma FVIII activity observed. The R2307L mutation tends to show a more severe clinical phenotype than the R2307Q mutation. Gitschier et al. (31) partially purified the R2307Q protein from plasma and found that it was able to clot FVIII-deficient plasma and respond to thrombin activation (31). It has been hypothesized that the mechanism leading to the reduced FVIII levels in these patients may be related to a defect in vWF binding affinity and destabilization of FVIII in plasma, similar to that observed in patients with severe type III von Willebrand's disease (31).
In order to address this hypothesis, FVIII cDNA expression constructs of R2307Q and R2307L were prepared and expressed in a mammalian transfection system. Two additional constructs were prepared in which tyrosine residues at amino acids 2305 and 2332 were mutated to phenylalanine. These epitopes are within the critical 2303-2332 region of the C2 domain and have been suggested to be involved in the phospholipid interaction (35,36). In addition, since factor V is highly homologous to FVIII (3) but is not known to interact with vWF, a construct was prepared in which the terminal amino acids of the factor V C2 domain were exchanged into the C2 domain terminal peptide region of FVIII. Functional characterization of the R2307Q mutant protein, expressed in transiently transfected COS-1 cells, demonstrates that the mutation only mildly impairs clotting factor activity and vWF binding properties are maintained. Evidence is presented that defective protein secretion and intracellular degradation of an apparently biologically functional molecule is responsible for the hemophilia A phenotype.
Plasmid Mutagenesis-Mutagenesis was performed within the mammalian expression vector pMT 2 (37) containing the FVIII cDNA. Mutant plasmids were generated through oligonucleotide site-directed mutagenesis utilizing the polymerase chain reaction as described previously (38). Codon 2305 was mutated from TAC to TTT predicting an amino acid change from tyrosine to phenylalanine, and the resultant mutant plasmid designated Y2305F. Codon 2307 was mutated from CGA to either CCC or CTC predicting an amino acid change from arginine to either glutamine or leucine, respectively, and the resultant mutant plasmids designated R2307Q and R2307L. Codon 2332 was mutated from TAC to TTT predicting an amino acid change from Tyr to Phe, and the resultant mutant plasmid designated Y2332F. The terminal 27 amino acids of the factor V cDNA were exchanged with the terminal 30 amino acids of the FVIII cDNA generating a FVIII/C2FV mutant plasmid. This was accomplished utilizing a 5Ј mutagenic oligonucleotide (which included FVIII WT sequence containing the XbaI restriction site at the terminus of the C2 domain up to but not including the sequence that codes for FVIII amino acid residue 2303), a 3Ј muta-genic oligonucleotide (which included the terminal sequence of factor V, a stop codon, and a restriction site for cloning into the pMT 2 vector polylinker), and factor V wild-type cDNA as a template. The plasmid designated 90/73 is wild-type FVIII cDNA sequence in which the B domain and the acidic region of the light chain have been deleted (del 741-1689) (17). The plasmid containing the wild-type FVIII cDNA sequence was designated FVIII WT. All plasmids were purified by centrifugation through cesium chloride and characterized by DNA sequencing over the mutagenized region.
DNA Transfection and Analysis-Plasmid DNA was transfected into COS-1 cells by the DEAE-dextran method as described previously (39). Conditioned medium was harvested at 64 h post-transfection in the presence or absence of 10% fetal bovine serum. FVIII activity was measured by one-stage APTT clotting assay on a MLA Electra 750. Protein synthesis and secretion were analyzed by metabolically labeling cells at 64 h post-transfection for 30 min with [ 35 S]methionine (300 Ci/ml in methionine-free medium), followed by a chase for 4 h in medium containing 100-fold excess unlabeled methionine and 0.02% aprotinin. Cell extracts were prepared by lysis in a Nonidet P-40 lysis buffer (39). For analysis of the effect of cysteine protease inhibition, increasing amounts of ALLN were included in the chase medium. Cell extracts and conditioned medium containing labeled protein were harvested as described previously (40). FVIII WT and C2 domain mutant proteins were immunoprecipitated from equal proportions of cell extract and conditioned medium with F-8 coupled to CL-4B Sepharose. Immunoprecipitates were washed as described previously (39), and resuspended in Laemmli sample buffer. Samples were analyzed by electrophoresis on a reducing SDS-low bis-8% polyacrylamide gel. The gels were treated with En 3 Hance and the proteins visualized by autoradiography. Bands were quantified using Intelligent Quantifier (Bio-Image, Ann Arbor, MI).
Protein Purification-Partially purified protein was obtained from 200 ml of conditioned medium from COS-1 cells transfected with FVIII WT, Y2332F, and R2307Q by immunoaffinity chromatography as described previously (40). The proteins eluted into the ethylene glycolcontaining buffer were dialyzed and concentrated against a polyethylene glycol (M r ϳ 15,000 -20,000) containing buffer (41) and stored at Ϫ70°C.
FVIII Activity Assay-FVIII activity was measured in a one-stage APTT clotting assay by reconstitution of human FVIII-deficient plasma or by the coamatic chromogenic assay according to the manufacturer.
FVIII Antigen Determination-FVIII antigen was quantified using a sandwich ELISA method utilizing anti-light chain antibodies ESH-4 and ESH-8 (42). Purified recombinant factor VIII protein was used as a standard.
FVIII-vWF Binding ELISA-Immulon 2 microtiter wells (Dynatech Laboratories, Inc., Chantilly, VA) were coated with F-8 antibody at a concentration of 2 g/ml overnight at 4°C in a buffer of 0.05 M sodium carbonate/bicarbonate, pH 9.6. Wells were washed with TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween 20) then blocked with 3% bovine serum albumin in TBST. Protein samples were diluted in TBST, 3% bovine serum albumin, 1% factor VIII-deficient human plasma, and incubated for 2 h at 37°C in 1.7-ml microcentrifuge tubes. Samples were then incubated for an additional 2 h in the blocked and washed microtiter wells. Wells were then washed in TBST containing 10 mM CaCl 2 . Anti-vWF-HRP antibody was diluted in TBST, 3% bovine serum albumin, 10 mM CaCl 2 and incubated in the wells for 2 h at 37°C. Following additional washing with TBST containing 10 mM CaCl 2 , O-phenylendiamine dihydrochloride substrate was added to the wells

RESULTS
Defective Secretion for FVIII C2 Domain Missense Mutants-FVIII wild-type (WT) and the C2 domain missense mutants were compared by transient DNA transfection of the cDNA expression vectors into COS-1 monkey cells. At 60 h following transfection, the rates of synthesis were analyzed by immunoprecipitation of cell extracts from [ 35 S]methionine pulse-labeled cells. Intracellular FVIII WT was detected in its single chain form and migrated at approximately 280 kDa (Fig. 1, lane 2). The mutant 90/73 migrated faster at approximately 160 kDa due to the 949-amino acid deletion from 741 to 1689 and had higher intensity indicating an increased rate of synthesis (Fig. 1, lane 4). Each of the C2 domain mutant proteins were detected as ϳ280-kDa single chain proteins and were of similar intensity to FVIII WT (Fig. 1, lanes 2, 6, 8, 10, 12, and  14). Thus there was no significant effect of these missense mutations on the rate of FVIII protein translation. Analysis of the cell extracts after a 4-h chase indicated that FVIII WT and all mutants disappeared from the cell extract at approximately similar rates. Additional bands in the cell extracts, that were not observed in cells that did not receive DNA (Fig. 1, lane 1), are FVIII-related polypeptides and/or associated proteins.
The secretion was analyzed by immunoprecipitation of conditioned medium from [ 35 S]methionine pulse-labeled transfected cells chased for 4 h in medium containing excess unlabeled methionine. FVIII WT was immunoprecipated from the conditioned medium as a 300-kDa single chain, 200-kDa heavy chain, and an 80-kDa light chain (Fig. 1, lane 17). 90/73 was recovered as a single chain of ϳ160 kDa (Fig. 1, lane 18). The amount of polypeptides from the mutants Y2305F, R2307Q, R2307L, and FVIII/C2FV were greatly reduced in the conditioned media (Fig. 1, lanes 19 -21 and 23). With prolonged exposures, single chain, heavy chain, and light chain forms of each mutant were detected and were of the same respective molecular weights as for the FVIII WT fragments (data not shown). The Y2332F mutant was secreted at a slightly in-creased level compared to FVIII WT (Fig. 1, lanes 17 and 22). The demonstration that the mutant polypeptides had similar molecular weights as FVIII WT suggested that these C2 domain missense mutations did not significantly interfere with post-translational modification of the secreted proteins.
90/73 was synthesized at a higher level than the B domain containing proteins but still had abundant protein present intracellularly following the 4-h chase period. Previous experience with this mutant has demonstrated reduced accumulation of the protein in the conditioned medium when collected over a longer period of time due to its inability to bind vWF (43). However, despite its defective vWF binding, it was demonstrated adequately in the conditioned medium in its single chain form in these 4-h chase experiments. This suggests that the 4-h chase reflects the actual rate of secretion rather than vWF-dependent stabilization in the conditioned medium. In contrast, FVIII WT and all C2 domain mutants demonstrated significantly reduced protein intracellularly at 4 h but only FVIII WT and Y2332F are detected in significant amounts in the conditioned medium. Therefore, for mutants Y2305F, R2307Q, R2307L, and FVIII/C2FV, the loss of labeled protein from the cell extract during the 4-h chase did not correlate with appearance in the conditioned medium. This is consistent with inefficient secretion and intracellular degradation of these mutants.
C2 Domain Missense Mutants Have Increased Accumulation in Conditioned Medium in the Presence of vWF-FVIII secreted by transiently transfected COS-1 cells binds to bovine vWF present in serum-containing conditioned medium. The vWF binding promotes association of the heavy and light chains, protects FVIII from proteolytic degradation, and thereby promotes stable accumulation of FVIII in the medium (4). When FVIII is expressed in COS-1 cells in conditioned medium supplemented with necessary growth factors but lacking vWF (i.e. absence of fetal bovine serum) there is a reduction in the amount of FVIII protein recovered from the medium. We hypothesized that accumulation in the medium of a mutant FVIII protein with defective vWF interaction would not be stimulated by the addition of serum, providing a source of vWF. Duplicate plates of COS-1 cells were transiently transfected with the FVIII cDNA expression vectors. At 40 h following transfection, fresh conditioned medium added contained either 10% heat-inactivated fetal bovine serum (FBS), as a vWF source, or serum-free medium (OptiMEM). Conditioned media from all plates were harvested at 64 h and analyzed for FVIII activity by the one-stage APTT clotting assay. FVIII WT demonstrated a severalfold increase in activity in the presence of 10% FBS (Fig. 2). Whereas, 90/73, with defective vWF binding, showed no significant increase in activity in the presence of 10% FBS. The mutants Y2305F, R2307Q, and R2307L, although having greater than 10-fold reduced activity as compared to FVIII WT, also showed a significant increase in activity in the presence of 10% FBS, suggesting that they may retain their vWF interaction (Fig. 2). The mutant Y2332F responded to the addition of serum with a fold-increase in activity similar to FVIII WT (Fig. 2). FVIII/C2FV had no detectable activity in the presence or absence of 10% FBS. Compared to FVIII WT, the amount of FVIII activity detected for all of the C2 domain mutants was proportional to the amount of secreted labeled protein observed (Figs. 1 and 2). FVIII antigen levels in the conditioned medium for Y2305F, R2307Q, R2307L and FVIII/ C2FV, as determined by a FVIII light chain ELISA method, were less than 5 ng/ml, compared to 26 ng/ml for FVIII WT and 27 ng/ml for Y2332F (data not shown).
Specific C2 Domain Mutations Alter FVIII Specific Activity as Compared to FVIII Wild-type-In order to characterize the mutant proteins further, FVIII WT, Y2332F, and the R2307Q mutant were partially purified and concentrated from conditioned medium by immunoaffinity chromatography. The very low levels of secreted protein from mutants Y2305F, R2307L, and FVIII/C2FV precluded their purification. FVIII activities were determined using both a chromogenic FVIII activity assay and a one-stage APTT clotting assay. Antigen determinations were made utilizing a FVIII light chain based ELISA. The specific activity of R2307Q was approximately 70 -75% of FVIII WT processed similarly (Table II). In contrast, the specific activity of Y2332F was not significantly different from FVIII WT.
C2 Domain Missense Mutants Retain Affinity for vWF-The immunoaffinity purified proteins were then assayed for their binding to vWF in solution. FVIII WT demonstrated saturable vWF binding as FVIII concentrations increased to 80 ng/ml (Fig. 3). The Y2332F mutant demonstrated vWF binding similar to FVIII WT. In addition, mutant R2307Q also displayed significant binding to vWF within the range of concentrations that could be tested. In a separate experiment, binding of vWF by the mutant 90/73 was not detectable within a similar range of protein concentration (data not shown).
R2307Q Accumulates Intracellularly in the Presence of Inhibitors of Intracellular Degradation-The role of intracellular proteases on the impaired secretion of the R2307Q mutant was analyzed in COS-1 monkey cells. At 64 h following transfection, duplicate plates of FVIII WT and R2307Q were pulse-labeled with [ 35 S]methionine for 20 min and chased for 4 h in conditioned medium containing excess unlabeled methionine and increasing concentrations of the cysteine protease inhibitor ALLN. FVIII was immunoprecipitated from equal amounts of cell extract and analyzed by SDS-PAGE. The treatment with ALLN showed an accumulation of R2307Q (Fig. 4, lanes 9 -12) that was not observed for FVIII WT (Fig. 4, lanes 3-6). Similar results were obtained in an independent experiment (data not shown). However, there was no increase in the accumulation of labeled protein in the conditioned medium with either FVIII WT or R2307Q (data not shown). After quantitating the amount of radioactivites in the cell extracts, it can be determined that greater than 60% of R2307Q was retained in the cell extract at an ALLN concentration of 250 M compared to ϳ20% of FVIII WT. The accumulation of the R2307Q mutant protein within the cell extract is consistent with a block in secretion. Since this accumulation was not observed in the absence of ALLN, we propose that rapid degradation by intracellular proteases is responsible for loss of the mutant FVIII from the cell extract.   3. FVIII-vWF binding of partially purified FVIII proteins as determined by ELISA. WT, R2307Q, and Y2332F proteins were purified through immunoaffinity chromotography and analyzed for binding to vWF after incubation with human FVIII-deficient plasma. Optical density represents the absorbance obtained through the detection of FVIII-vWF complexes by an anti-vWF-horseradish peroxidase conjugate antibody in the presence of O-phenylendiamine dihydrochloride. Ⅺ, FVIII WT; E and q, two independently purified preparations of R2307Q; f, Y2332F.

DISCUSSION
The study of missense mutations in the FVIII gene has led to further understanding of the role of specific amino acids in FVIII post-translational processing, protein-protein interactions, and functional activation. FVIII missense mutations leading to hemophilia A have been identified that alter cleavage by thrombin, N-glycosylation, vWF binding, and FVIII procoagulant activity (44). However, many other missense mutations have not yet been characterized as to their mechanism leading to hemophilia A.
In this report we have characterized the effect of two hemophilia A missense mutations within the C2 domain of FVIII, R2307Q, and R2307L. These mutations, leading to mild-moderate and severe hemophilia A, respectively, demonstrated quantitatively defective expression in transiently transfected COS-1 monkey cells that parallel their plasma levels observed in affected patients. FVIII activity and antigen levels of both mutants were reduced greater than 10-fold in COS-1 cell conditioned medium compared to FVIII WT. The reduced protein expression was not related to a defect in FVIII synthesis. However, despite the primary translation products disappearing from the cell extracts of labeled cells at a similar rate to FVIII WT, the amount of labeled protein in the conditioned media from cells transfected with R2307Q and R2307L was significantly reduced compared to FVIII WT. This suggested a defect in secretion associated with intracellular degradation. The role of intracellular proteases in this process was demonstrated by observing accumulation of the primary translation products of R2307Q in the cell extract in the presence of a membranepermeable cysteine protease inhibitor (ALLN).
Prior evidence suggested that Arg-2307 lies within a region important for vWF and phospholipid interaction (23)(24)(25)(26). Indeed, patients with mutation of Arg-2307 exhibit reduced FVIII activity and antigen, consistent with a defective vWF interaction and reduced plasma half-life. We have shown that the R2307Q mutant has a FVIII specific activity that was approximately 70 -75% of wild-type, consistent with the patient phenotype. The slightly reduced activity may either result from a mild impairment in FVIII interaction with either factor IXa and/or phospholipids, or it may also result from a partial loss of activity due to the low yield of the mutant FVIII protein from the conditioned medium, increasing the ratio of impurities to purified protein, and storage at lower concentrations as compared to FVIII WT and Y2332F. Significantly, the vWF binding of the R2307Q partially purified protein was not defective when analyzed by a FVIII-vWF ELISA. The ability to bind vWF for the R2307Q mutant is also consistent with the increased accumulation in conditioned medium containing serum. Previous studies identified vWF in serum as the primary factor for stabilizing FVIII upon secretion from the cell (4). In contrast, a deletion mutant (90/73) that did not bind vWF, did not display increased accumulation in conditioned medium containing serum. These findings support that the mutants retained the ability to bind and be stabilized by vWF in the conditioned medium similar to FVIII WT.
Previous studies with 90/73 demonstrated that vWF interaction is not necessary for secretion and that in the absence of vWF binding, FVIII appeared in the conditioned medium after 4 h and was not degraded extracellularly within the 4-h time course of the pulse-chase experiments (4). However, after longer periods of time (24 h) there was significant degradation of FVIII in the absence of vWF binding (4). Therefore, we think it is unlikely that extracellular instability of these FVIII C2 domain mutants accounts for the absence of labeled protein in the conditioned medium in the pulse-chase experiment. Rather, a mechanism can be proposed for these missense mutations in which an alteration in the primary translation product within this region leads to a block in secretion and the protein is targeted to intracellular degradation. This block in secretion was demonstrated when intracellular cysteine proteases were inhibited leading to accumulation of the mutant R2307Q primary translation product within the cell extract. Although Y2305F and FVIII/C2FV had similar phenotypes to the R2307 mutations, Y2332F was similar to FVIII WT, suggesting that not all mutations in this region lead to a defect in secretion, but rather some "quality control" mechanism exists within the cell.
One of the roles of the endoplasmic reticulum (ER) is to ensure quality control for the synthesis of proteins destined for the cell surface and the extracellular environment. Upon translocation of nascent polypeptide chains into the lumen of the ER a series of co-and post-translational modifications occur prior to exit from the ER to the Golgi complex and ultimately secretion to the cell surface (45). These modifications occur through interactions with resident ER proteins (46). Many aberrantly synthesized or improperly modified proteins are inefficiently secreted and retained in the ER (47). Others undergo lysosomal degradation. Recently, experimental observations have identified proteins which undergo degradation prior to exit from the ER. Whereas some of these proteins have no obvious abnormality in primary structure or assembly of subunits (e.g. acetylcholinesterase (48), apolipoprotein B (49), and cytochrome P 450 (50)), others undergo degradation related to specific mutations or defects in subunit assembly (␣ 1 -antitrypsin (51), class I major histocompatibility complex (52), immunoglobulin chains (53), and T-cell receptor (54)). These observations have led to the suggestion of a novel pathway for degradation of proteins retained within the ER. Although the exact nature of this quality control mechanism has not yet been elucidated, most evidence supports a non-lysosomal degradation within the ER or in an ER-related compartment (55)(56)(57), or alternatively, cytosolic translocation, and involvement with the ubiquitinproteasomal pathway (58).
Weak bases (e.g. ammonium chloride and chloroquine) and protease inhibitors (chymostatin and leupeptin) known to inhibit lysosomal degradation of secretory proteins have not been demonstrated to prevent degradation of proteins retained within the ER (59 -61). In contrast, compounds that alter the reducing environment within the ER (e.g. diamide and diethyl maleate), and specific cysteine protease inhibitors (ALLN and ALLM), can slow or block the degradation of certain mutant proteins (unassembled T-cell receptor chains (62) and protein C synthesized in the presence of warfarin (63)) resulting in accumulation of the proteins within the ER. Recently, inhibitors of the ubiquitin-dependent proteasome, including ALLN, were shown to inhibit the degradation of the cystic fibrosis transmembrane conductance regulator (64, 65) and major histocom-  ALLN (lanes 3-6 and 9 -12), cell extracts harvested, and equal volumes of each were immunoprecipated with an anti-FVIII-specific antibody for analysis by SDS-PAGE. Bands represent the single chain form of FVIII detected in the cell extracts. patibility complex class I heavy chains (58).
The observations from this report suggest that mutation of a single specific amino acid within the carboxyl terminus of the FVIII light chain, despite only mild alteration of functional activity, results in a protein that is recognized by ER resident proteins as a mutant and is targeted for degradation. Furthermore, this degradation can be inhibited by a membrane permeable cysteine protease inhibitor in a dose dependent manner, suggesting that the degradation process is associated with the ER. However, the reduced degradation was not associated with increased secretion, indicating that simply interfering with proteolysis is not sufficient to rescue the mutant protein toward productive secretion. The identification of these FVIII missense mutations associated with this quality control provides a novel mechanism by which hemophilia A may result. These observations do not refute any evidence that suggests that the carboxyl terminus of the FVIII light chain is important for interaction with vWF or phospholipid. Rather, it suggests that missense mutations at certain critical epitopes are not tolerated by the quality control mechanisms within the ER, even if they lead to only mild impairments in function. In addition, since sitedirected mutagenesis is often employed to identify the role of specific amino acids in protein processing and function, other missense mutations within this region may also be targeted for degradation precluding study of the secreted protein. These examples of FVIII mutations that yield secretion-defective molecules, that are otherwise biochemically functional, adds to a small but growing list of genetic deficiencies that result from defects in protein folding and/or transport (66).