Mechanism for the inhibitory effect of tetramethylpyrazine on the expression of tissue factor induced by tumor necrosis factor in human umbilical vein endothelial cells

Objective To investigate the role of PKC and transcription factor NF-κB in the regulation of TF expression induced by TNFα in human umbilical vein endothelium(HUVEC).Methods HUVEC were cultured in RPMI-1640.TF activity was determined with one-stage clotting assay measuring total cellular pro-coagulant activity.TF antigen was measured by ELISA.TF mRNA was examined by RT-PCR.Liquid scintillation count was used to check PKC activity by radioimmunoassay.Immunohistochemical analysis was performed to evaluate the activation of NF-κB.Results The addition of TMP or Staurosporin,significantly inhibited the stimulative effects of TNFα as well as PMA on TF activity,antigen and mRNA expression(P0.01).Addition of PMA or TNFα resulted in a sharp decrease in protein kinase C activity in the cytosolic fraction(P0.05),a great increase in the activity in the membrane fraction(P0.01) and NF-κB translocation from cytoplasm to the nucleus.Pretreatment with TMP,however,significantly decreased the PKC activity in the membrane fraction and inhibited TNFα-induced NF-κB translocation.Conclusion PKC activation and NF-κB translocation play an important role in TNFα-induced TF expression.The inhibitory effect of TMP on TF expression in HUVEC is involved in these signal transduction pathways.