Expression and Characterization of Recombinant Murine Factor VIII

Self Cite

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,

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Doering

Healey

Parker

et al. 2004

Self Cite

“…The faster human A2 subunit dissociation rate may be an evolutionary adaptation, perhaps associated with the development of an upright posture and venous stasis in the lower extremities. In this regard it is interesting to note that the half-life of murine fVIIIa at 23°C is greater than 30 min, compared with ϳ2 and 7 min for human and porcine fVIIIa, respectively (50).…”

Section: Discussionmentioning

confidence: 98%

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

Parker

Doering

Lollar

2006

Self Cite

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 ...

“…The transfected cells were cultured in serum free media. The medium was subjected to a two-step ion exchange chromatography (22). The factor VIII-containing medium was loaded on a HiPrep 16/10 SP FF column (Amersham Biosciences) and eluted using a linear gradient from 100 -650 mM NaCl in HEPES-CaCl 2 buffer containing 10 mM OPLS.…”

Section: Preparation Of Rfviii⅐opls Complexesmentioning

confidence: 99%

Lower Inhibitor Development in Hemophilia A Mice following Administration of Recombinant Factor VIII-O-Phospho-L-serine Complex

Purohit

Ramani

Sarkar

et al. 2005

Factor VIII is a multidomain protein composed of A1, A2, B, A3, C1, and C2 domains. Deficiency or dysfunction of factor VIII causes hemophilia A, a bleeding disorder. Administration of exogenous recombinant factor VIII as a replacement leads to development of inhibitory antibodies against factor VIII in 15-30% of hemophilia A patients. Hence, less immunogenic preparations of factor VIII are highly desirable. Inhibitory antibodies against factor VIII are mainly directed against immunodominant epitopes in C2, A3, and A2 domains.