Residues 484-508 Contain a Major Determinant of the Inhibitory Epitope in the A2 Domain of Human Factor VIII

Healey, John F.; Lubin, Ira M.; Nakai, Hiroaki; Saenko, Evgueni L.; Hoyer, Leon W.; Scandella, Dorothea; Lollar, Pete

doi:10.1074/jbc.270.24.14505

Cited by 153 publications

(185 citation statements)

References 28 publications

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“…These constructs contain a nucleotide sequence between the A2 and ap-A3 domains encoding a linker region of human, porcine, or murine origin (designated SQ, OL, or SQ, respectively), which includes the Arg-His-Gln-Arg recognition sequence for PACE/furin processing (24,25). HP and MP fVIII constructs were generated by splicing-by-overlap extension mutagenesis (26) using previously published procedures (27)(28)(29) and rh-fVIII, rpfVIII, and rm-fVIII as starting materials (Fig. 1).…”

Section: Methodsmentioning

confidence: 99%

Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion

Doering

Healey

Parker

et al. 2004

Journal of Biological Chemistry

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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. Factor VIII (fVIII)1 is a plasma protein that functions in proteolytically activated form as a cofactor within the intrinsic pathway of blood coagulation to increase the rate of proteolytic activation of factor X by activated factor IX. fVIII contains a domain structure designated A1-A2-B-ap-A3-C1-C2 that is defined based on internal sequence homology (1, 2). The fVIII A domains share homology with the copper-binding protein ceruloplasmin (3, 4), which has an A1-A2-A3 domain structure in which the three A domains are arranged along a pseudo-3-fold axis of symmetry (5). Before cell secretion, fVIII is cleaved at the B/ap-A3 domain junction into A1-A2-B (heavy chain) and ap-A3-C1-C2 (light chain) subunits. fVIII circulates in the plasma as an inactive heavy chain/light chain heterodimeric procofactor that is non-covalently bound to von Willebrand factor. Proteolytic activation of fVIII by thrombin results from cleavages at Arg-372 between the A1 and A2 domains, Arg-740 between the A2 and B domains, and Arg-1689 between the ap and A3 domains. During this process, the covalent linkage between the A1 and A2 domains is lost, and the B domain and 41-residue ap are released, producing a heterotrimeric,

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Section: Methodsmentioning

confidence: 99%

Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion

Doering

Healey

Parker

et al. 2004

Journal of Biological Chemistry

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“…Between 15% and 35% of severe hemophilia A patients will develop inhibitory responses to FVIII, presenting a major challenge in the clinical management of the disease (4). Based on systematic epitope mapping experiments, the anti-FVIII antibodies have been found to mainly target amino acid regions 484-508 of the A2 domain (5,6), 1811-1818 of the A3, and 2181-2312 of the C2 domains (7)(8)(9). Inhibition of FVIII activity results from antibodies against the A2 and A3 domains blocking interaction with factor IXa (7,10) while antibodies against the C2 domain interfere with binding to platelet membranes (9,11).…”

Section: Introductionmentioning

confidence: 99%

PEGylation of a Factor VIII–Phosphatidylinositol Complex: Pharmacokinetics and Immunogenicity in Hemophilia A Mice

Peng

Kosloski

Nakamura

et al. 2011

AAPS J

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Abstract. Hemophilia A is an X-linked bleeding disorder caused by the deficiency of factor VIII (FVIII). Exogenous FVIII is administered therapeutically, and due to a short half-life, frequent infusions are often required. Fifteen to thirty-five percent of severe hemophilia A patients develop inhibitory antibodies toward FVIII that complicate clinical management of the disease. Previously, we used phosphatidylinositol (PI) containing lipidic nanoparticles to improve the therapeutic efficacy of recombinant FVIII by reducing immunogenicity and prolonging the circulating half-life. The objective of this study is to investigate further improvements in the FVIII-PI formulation resulting from the addition of polyethylene glycol (PEG) to the particle. PEGylation was achieved by passive transfer of PEG conjugated lipid into the FVIII-PI complex. PEGylated FVIII-PI (FVIII-PI/PEG) was generated with high association efficiency. Reduced activity in vitro and improved retention of activity in the presence of antibodies suggested strong shielding of FVIII by the particle; thus, in vivo studies were conducted in hemophilia A mice. Following intravenous administration, the apparent terminal half-life was improved versus both free FVIII and FVIII-PI, but exposure determined by area under the curve was reduced. The formation of inhibitory antibodies after subcutaneous immunization with FVIII-PI/PEG was lower than free FVIII but resulted in a significant increase in inhibitors following intravenous administration. Passive transfer of PEG onto the FVIII-PI complex does not provide any therapeutic benefit.

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“…The hybrid human/porcine fVIII constructs HP66, HP67, HP68, HP69, HP70, and HP75 containing the human A2/ap-A3 linker region (Fig. 2) were produced by splicing-by-overlap extension mutagenesis (31) using previously published procedures (32)(33)(34). All PCR-generated sequences were confirmed by DNA sequencing.…”

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A1 Subunit-mediated Regulation of Thrombin-activated Factor VIII A2 Subunit Dissociation

Parker

Doering

Lollar

2006

Journal of Biological Chemistry

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Factor VIII (fVIII) is the plasma protein that is missing or deficient in hemophilia A. In contrast, elevated levels of fVIII are associated with an increased risk of arterial and venous thrombosis. fVIII is activated by thrombin to form a non-covalently linked A1/A2/A3-C1-C2 heterotrimer. At physiological concentrations, fVIIIa decays as a result of A2 subunit dissociation, which may help regulate the balance between hemostasis and thrombosis. A2 subunit dissociation is faster in human fVIIIa than in porcine fVIIIa, which may represent an evolutionary adaptation associated with the development of the upright posture and venous stasis in the lower extremities. To investigate the basis for the different decay kinetics of human and porcine fVIIIa, hybrid fVIII molecules representing all possible combinations of human and porcine A domains were isolated. The kinetics of fVIIIa decay were measured and fit to a model describing a reversible bimolecular reaction in which the dissociation rate constant, k, and dissociation constant, K d , were the fitted parameters. Substitution of the porcine A1 domain into human fVIIIa produced a dissociation rate constant indistinguishable from porcine fVIIIa. Subsequently, substitution of the second cupredoxin-like A1 subdomain resulted in a dissociation rate constant similar to porcine fVIIIa, whereas substitution of the first cupredoxin-like A1 subdomain resulted in a dissociation rate constant intermediate between human and porcine fVIIIa. We propose that cupredoxin-like A1 subdomains in fVIII contain inter-species differences that are a result of selective pressure on the dissociation rate constant.Factor VIII (fVIII) 2 contains internal homology that defines a sequence of domains designated A1-A2-B-ap-A3-C1-C2 (1). The B domain undergoes limited intracellular proteolysis at several sites prior to secretion of fVIII into the circulation. As a result, a series of 160 -300-kDa heterodimers are produced that contain a common ap-A3-C1-C2 light chain and A1-A2-B heavy chains in which the amount of B domain is variable. The B domain can be deleted with little or no change in the function of fVIII. fVIII circulates non-covalently bound to von Willebrand factor (vWf) and is proteolytically activated by thrombin, which catalyzes cleavages at Arg 372 between the A1 and A2 domains, at Arg 740 between the A2 and B domains, and at Arg 1689 between the ap and A3 domains (2). The product, fVIIIa, is a 160-kDa A1/A2/A3-C1-C2 heterotrimer whose subunits are held together non-covalently (3-5).fVIIIa functions in blood coagulation as a cofactor for factor IXa during the proteolytic activation of factor X on the phospholipid surface of platelets and monocytes. Cleavage at Arg 372 activates the factor IXa cofactor function of fVIIIa, as judged by naturally occurring mutations at this site that result in hemophilia A (6, 7) and by site-directed mutagenesis of this site, which produces loss of function (8). Cleavage at Arg 740 releases the B domain. However, removal of the B domain does not activate ...

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Residues 484-508 Contain a Major Determinant of the Inhibitory Epitope in the A2 Domain of Human Factor VIII

Cited by 153 publications

References 28 publications

Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion

Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion

PEGylation of a Factor VIII–Phosphatidylinositol Complex: Pharmacokinetics and Immunogenicity in Hemophilia A Mice

A1 Subunit-mediated Regulation of Thrombin-activated Factor VIII A2 Subunit Dissociation

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