Effect of Heterologous Factor V Heavy Chain Sequences on the Secretion of Recombinant Human Factor VIII

Thomas L Ortel; Karen D Moore; Mirella Ezban; William H Kane

doi:10.1055/s-0038-1650218

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1996; 75(01): 036-044
DOI: 10.1055/s-0038-1650218

Original Article

Schattauer GmbH Stuttgart

Effect of Heterologous Factor V Heavy Chain Sequences on the Secretion of Recombinant Human Factor VIII

Authors

Thomas L Ortel

¹The Division of Hematology-Oncology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina, USA
Karen D Moore

¹The Division of Hematology-Oncology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina, USA
Mirella Ezban

²The Division of Coagulation Research, Novo-Nordisk A/S, Copenhagen, Denmark
William H Kane

¹The Division of Hematology-Oncology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina, USA

Abstract

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Summary

Factor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIH/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIIIA1 domain dramatically reduced secretion of factor V ~ 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII ~3-fold, whereas substitution of the factor V A2 domain actually reduced secretion ~4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.

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References

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