Intracellular accumulation of factor VIII induced by missense mutations Arg<sup>593</sup>→Cys and Asn<sup>618</sup>→Ser explains cross‐reacting material‐reduced haemophilia A

Roelse, Joost; Laaf, R. T. M. De; Timmermans, Steven; Peters, Marjolein; Mourik, Jan A. van; Voorberg, Jan

doi:10.1046/j.1365-2141.2000.01834.x

Cited by 28 publications

(31 citation statements)

References 26 publications

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“…The ROS produced as a consequence of FVIII misfolding may exacerbate inflammatory responses, as well as stimulate production of anti-FVIII antibodies. We have shown that BHA feeding prevents apoptosis, suppresses ER stress, and increases the secretion of FVIII delivered by adenovirus, as well as a folding-defective functional FVIII mutant R593C that is known to cause hemophilia A (27). Therefore, antioxidants may provide a useful adjuvant to improve FVIII production in patients who receive gene therapy or who have mutations that disrupt FVIII folding.…”

Section: Discussionmentioning

confidence: 99%

“…The expression vectors for wtFVIII, BDD, and 226/N6 were previously described (20). Vectors for FVIII-BDD and R593C-BDD were kindly provided by J. Voorberg (Amsterdam, The Netherlands) (27). Plasmid DNA samples (100 g) were diluted in 2.5 ml lactated Ringer buffer and infused over 10 sec into the tail vein.…”

Section: Methodsmentioning

confidence: 99%

“…As antioxidant treatment improved secretion of wtFVIII and BDD, we asked whether BHA feeding could increase the secretion of a FVIII molecule having a missense mutation Arg593Cys (R593C) that causes protein misfolding and retention within the ER (27). This common mutation has been identified in patients with mild hemophilia A characterized by reductions in both FVIII antigen and activity.…”

Section: Antioxidant Treatment Improves Wtfviii and Bdd Secretion In mentioning

confidence: 99%

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Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Malhotra¹,

Miao

Zhang³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

606

564

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Protein misfolding in the endoplasmic reticulum (ER) contributes to the pathogenesis of many diseases. Although oxidative stress can disrupt protein folding, how protein misfolding and oxidative stress impact each other has not been explored. We have analyzed expression of coagulation factor VIII (FVIII), the protein deficient in hemophilia A, to elucidate the relationship between protein misfolding and oxidative stress. Newly synthesized FVIII misfolds in the ER lumen, activates the unfolded protein response (UPR), causes oxidative stress, and induces apoptosis in vitro and in vivo in mice. Strikingly, antioxidant treatment reduces UPR activation, oxidative stress, and apoptosis, and increases FVIII secretion in vitro and in vivo. The findings indicate that reactive oxygen species are a signal generated by misfolded protein in the ER that cause UPR activation and cell death. Genetic or chemical intervention to reduce reactive oxygen species improves protein folding and cell survival and may provide an avenue to treat and/or prevent diseases of protein misfolding.factor VIII ͉ oxidative stress ͉ unfolded protein response A lthough endoplasmic reticulum (ER) stress and oxidative stress are closely linked events, the molecular pathways that couple these processes are poorly understood (1). Reactive oxygen species (ROS) originate during oxygen-using cellular metabolic processes, such as oxidative phosphorylation within mitochondria. The ER provides a unique oxidizing environment for protein folding and disulfide bond formation before transit to the Golgi compartment. During disulfide bond formation ROS are formed as a product of electron transport from thiol groups in proteins through protein disulfide isomerase (PDI) and ER oxidoreductase 1 (ERO1) to reduce molecular oxygen to form the oxidant hydrogen peroxide. It has been suggested that oxidation of cysteine residues during disulfide bond formation in the ER may significantly contribute to oxidative stress (2, 3). The unfolded protein response (UPR) is an adaptive signaling pathway designed to prevent the accumulation of misfolded proteins in the ER lumen. Studies also suggest the UPR is designed to minimize the stress of oxidative protein folding (2). The UPR is signaled through the protein kinases inositolrequiring protein 1␣ and PKR-related ER kinase and the activating transcription factor 6␣ (4, 5). Chronic unresolved accumulation of unfolded proteins in the ER leads to apoptosis. To elucidate the relationship between unfolded protein accumulation in the ER lumen, oxidative stress, and apoptosis, we have analyzed the secretion of coagulation factor VIII (FVIII), a large glycoprotein that is deficient in the X chromosome-linked bleeding disorder hemophilia A. As FVIII is prone to misfolding in the ER lumen, FVIII expression provides a unique approach to manipulate the ER stress response that does not rely on pharmacological intervention, where any connection between ER stress and ROS would be difficult to dissect.FVIII is comprised of three domains in the ord...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Antioxidant Treatment Improves Wtfviii and Bdd Secretion In mentioning

confidence: 99%

See 1 more Smart Citation

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Malhotra¹,

Miao

Zhang³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

606

564

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“…In the haemophilia A mutation database, patients with Arg593Cys had similar FVIII antigen and activities levels pointing towards poor secretion of a functional protein. In addition, Roelse et al using a heterologous expression system found that an Arg593Cys substitution led to elevated accumulation of intracellular functional FVIII relative to wildtype FVIII [9]. The patient with the Arg593Cys had a polyclonal response that cleared both endogenous and exogenous FVIII.…”

Section: Discussionmentioning

confidence: 99%

Development of factor VIII inhibitors in two patients with moderate haemophilia A

2012

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IntroductionHaemophilia A is an X-linked disorder characterized by congenital deficiency of factor VIII (FVIII). The FVIII protein is secreted as a mixture of a single chain and heterodimer consisting of one heavy and one light chain. The heavy chain is comprised of the A1, A2 and B domains, whereas the light chain contains the activation peptide, A3, C1 and C2 domains [1]. Severe haemophilia A is defined by a FVIII activity level <1% of normal. Moderate and mild disease are defined by FVIII activity levels between 1% and 5%, and 5% and 40% of normal respectively [2]. Haemorrhage in severe haemophilia A may be spontaneous, whereas in mild and moderate disease, bleeding is usually a result of surgery or trauma. Patients are treated with either recombinant or plasma-derived FVIII concentrates which can induce the formation of inhibitory antibodies. The crude incidence of inhibitors in patients with severe haemophilia A is approximately 23%, whereas inhibitors in mild and moderate disease occur much less frequently [3]. When patients with mild and moderate haemophilia A (MMHA) develop inhibitors, antibodies are produced against the exogenous FVIII and occasionally against endogenous FVIII as well [4]. When both exogenous and endogenous FVIII are inhibited, the FVIII activity level falls to <1% which can increase the baseline bleeding frequency. Methods PatientsTwo adult patients with moderate haemophilia A seen at Vanderbilt University Haemostasis clinic who developed inhibitors were selected. Information on the clinical course during the presence of an inhibitor was obtained from the electronic medical record. Domain mapping ELISAPlasma specimens were available in the IRB approved inhibitor bank at The Emory University Haemophilia Treatment Center. Domainspecific anti-FVIII antibody mapping was carried out by direct ELISA in half-area 96-well plates using purified single human domain hybrid FVIII proteins as test antigens and B domain deleted (BDD) human FVIII and BDD porcine FVIII as positive control antigens. Patient plasma was diluted 1-20 in blocking buffer (0.15 M NaCl/ 20 mM HEPES/5 mM CaCl 2 /0.05% sodium azide/0.05% Tween-80/ 0.25% bovine serum albumin, pH 7.4) and then serially diluted down the ELISA plate. Domain specificity was evident by visual inspection of colour [5]. Cases Patient 1A 56-year-old man presented to establish care in comprehensive haemophilia clinic. He was diagnosed with FVIII deficiency in the 1980s and subsequently received factor replacement prior to invasive procedures. He had chronic hepatitis C infection presumed from blood product exposure. One month prior to presentation to clinic, he underwent a radical prostatectomy at another facility for a recently diagnosed prostate adenocarcinoma. Preoperative labs were significant for a prothrombin time (PT) of 16.4 s, INR 1.5 and partial thromboplastin time (PTT) of 63 s. He did not receive factor replacement prior to surgery. His postoperative course was complicated by hypovolemic shock felt secondary to mechanical bleeding. Following ...

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“…16 In addition, Roelse et al found that R593C and N618S A2-domain substitutions led to elevated accumulation of intracellular functional fVIII relative to wt-fVIII. 33 In these studies, the kinetic block in fVIII synthesis was not identified.In the present study, we used conformation-specific MAbs, which have been used as tools to study protein folding, including intracellular folding intermediates, 34 to detect folding intermediates in the biosynthesis of wt-fVIII and N1922S-fVIII. An intermediate containing incompletely folded A3 and C1 domains was identified after measurement of the apparent ratios of intracellular to secreted N1922S-fVIII ( Figure 6C).…”

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confidence: 95%

Factor VIII A3 domain substitution N1922S results in hemophilia A due to domain-specific misfolding and hyposecretion of functional protein

Summers

Meeks

Healey

et al. 2011

Blood

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A point mutation leading to amino acid substitution N1922S in the A3 domain of factor VIII (fVIII) results in moderate to severe hemophilia A. A heterologous expression system comparing N1922S-fVIII and wild-type fVIII (wt-fVIII) demonstrated similar specific coagulant activities but poor secretion of N1922S-fVIII. Immunocytochemical analysis revealed that intracellular levels of N1922S-fVIII were similar to those of wt-fVIII. The specific activity of intracellular N1922S-fVIII was 10% of that of wt-fVIII, indicating the presence of large amounts of a nonfunctional N1922S-fVIIIfolding intermediate. wt-fVIII colocalized with both endoplasmic reticulum (ER)-and Golgi-resident proteins. In contrast, N1922S-fVIII colocalized only with ERresident proteins, indicating a block in transit from the ER to the Golgi. A panel of conformation-dependent monoclonal antibodies was used to determine native or nonnative folding of N1922S-fVIII. Intracellular N1922S-fVIII but not secreted N1922S-fVIII displayed abnormal folding in the A3 and C1 domains, indicating that the A1, A2, and C2 domains fold independently into antigenically intact tertiary structures, but that folding is stalled in the mutant A3 and its contiguous C1 domain. In summary, the N1922S substitution results in poor secretion of a functional protein, and the domain-specific defect in folding and intracellular trafficking of N1922S-fVIII is a novel mechanism for secretion defects leading to hemophilia A. (Blood. 2011;117(11):3190-3198) IntroductionHemophilia A is an X-linked bleeding disorder caused by a deficiency of factor VIII (fVIII). It is classified as mild, moderate, or severe based on plasma fVIII levels of greater than 0.05-0.4 U/mL, 0.01-0.05 U/mL, or less than 0.01 U/mL, respectively. 1 With some exceptions, this classification correlates well with the bleeding diathesis such that patients with mild disease have abnormal bleeding only after trauma or surgery, whereas severe hemophilia A is characterized by spontaneous bleeding into joints or soft tissues. 2 FVIII is synthesized as an ϳ 300-kDa glycoprotein by hepatocytes, liver sinusoidal endothelial cells, and certain types of extrahepatic endothelial cells. [3][4][5][6] It contains a domain sequence designated A1-A2-B-ap-A3-C1-C2 and circulates as an A1-A2-B/ ap-A3-C1-C2 heterodimer bound noncovalently to von Willebrand factor (VWF), which protects it from rapid clearance. 7 The homologous ϳ 40-kDa A1, A2, and A3 domains are each made up of 2 cupredoxin-like subdomains that form an extensive interface. [8][9][10] During the activation of fVIII by thrombin to fVIIIa, the B domain and an ap activation peptide are released, and cleavage between the A1 and A2 domains produces an A1/A2/A3-C1-C2 fVIIIa heterotrimer. 11 FVIIIa is a cofactor for factor IXa during the proteolytic activation of factor X on relevant phospholipid membrane surfaces. At physiologic concentrations, the A2 subunit spontaneously dissociates, leading to loss of fVIIIa cofactor activity. 12 Mild and moderate hemophilia A are caused by ...

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Intracellular accumulation of factor VIII induced by missense mutations Arg⁵⁹³→Cys and Asn⁶¹⁸→Ser explains cross‐reacting material‐reduced haemophilia A

Cited by 28 publications

References 26 publications

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Development of factor VIII inhibitors in two patients with moderate haemophilia A

Factor VIII A3 domain substitution N1922S results in hemophilia A due to domain-specific misfolding and hyposecretion of functional protein

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Intracellular accumulation of factor VIII induced by missense mutations Arg593→Cys and Asn618→Ser explains cross‐reacting material‐reduced haemophilia A

Cited by 28 publications

References 26 publications

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Development of factor VIII inhibitors in two patients with moderate haemophilia A

Factor VIII A3 domain substitution N1922S results in hemophilia A due to domain-specific misfolding and hyposecretion of functional protein

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Intracellular accumulation of factor VIII induced by missense mutations Arg⁵⁹³→Cys and Asn⁶¹⁸→Ser explains cross‐reacting material‐reduced haemophilia A