Trans–Activated Expression of Fully Gamma–Carboxylated Recombinant Human Protein C, an Antithrombotic Factor

Abstract

Protein C, a vitamin K–dependent plasma protease involved in the regulation of hemostasis, has potent anticoagulant activity and may be useful as an effective antithrombotic agent. Because of the risk of infection by blood–borne viruses, such as HIV, a source of protein C other than from human plasma is desirable. We constructed a vector for the expression of recombinant human protein C (rHPC) using a hybrid promoter that could be activated in trans by the early region 1A (E1A) proteins of adenovirus. Stable cell lines secreting rHPC were isolated by introducing expression vectors into the adenovirus–transformed human kidney cell line 293 with a dominant selectable marker. The protein C was purified to homogeneity using a conformation (Ca²⁺) specific anti–HPC monoclonal antibody. Like plasma–derived HPC, the purified rHPC was 90% two chain and 10% one chain, and N–terminal sequence analysis indicated that the molecule was processed correctly. Upon activation of the recombinant zymogen with thrombomodulin–thrombin, the purified rHPC expressed anticoagulant activity at a level above that of plasma–derived human HPC. This high level of activity was reduced when the rHPC was produced in the presence of warfarin, indicating a vitamin K–dependence for the production of fully active rHPC from the 293 cell line. Biochemical analysis confirmed the presence of nine γ–carboxyglutamic acid residues per mole of rHPC.