Effect of L-arginine on human coagulant factor VIII gene expression

To investigate the effect of L-arginine on expression of human FVIII gene. Plasmid pcDNA6/V5-HisA-BDDhF VIII containing B domain deleted human coagulant factor VIII cDNA (BDDhF VIII cDNA) was constructed and transfected into human umbilical vein endothelial cells (HUVEC). After 72 h incubation with L-arginine (final concentration was 10 mmol/L) , the supernatant was collected for determining the antigen and clotting activity of human FVIII (FVIII: Ag and FVIII: C ) with ELISA and one stage clotting assay respectively. HUVECs were harvested for detecting human FVIII mRNA by Northern blot analysis. The five functional domains of BDDhFVIII cDNA including A1, A2, A3, C1 and C2 were amplified with PCR and inserted into pcDNA6/V5-HisA to construct the expression plasmids pcDNA6/V5-Hi-sA-BDDhFVIII-A1, pcDNA6/V5-HisA-BDDhFVIII-A2, pcDNA6/V5-HisA-BDDhFVIII-A3, pcDNA6/V5-HisA-BDDhFVIII-C1 and pcDNA6/V5-HisA-BDDhFVIII-C2, respectively. HUVEC were transfected with the five plasmids respectively and incubated with L-argin