Differentiation of adherent human monocytes into macrophages markedly enhances tissue factor protein expression and procoagulant activity

Abstract

Circulating monocytes as well as resident monocyte-derived macrophages (MDMs) in the atherosclerotic plaque express tissue factor. We tested the hypothesis that differentiation of monocytes into macrophages increases their tissue factor expression. This was done by isolating monocytes from human blood and culturing them for 24 h or allowing their differentiation into macrophages for 8 days before assay. Tissue factor procoagulant activity was assessed by recalcification clotting assay while Western blotting quantitated tissue factor protein. Immunocytochemistry was applied to localize antigenic tissue factor in cells. The results showed that freshly-isolated monocytes expressed low baseline procoagulant activity (0.0004±0.0003 ng rhTF/10 6 cells) which increased 100-fold in 24-h cultured monocytes (0.04±0.01 ng/10 6 cells, P<0.0001) and was associated with detectable tissue factor protein by Western blotting. Furthermore, MDMs expressed 25-fold higher procoagulant activity (1.0±0.5 ng/10 6 cells, P<0.04) and increased tissue factor protein content compared with 24-h monocytes as corroborated by immunocytochemistry. LPS stimulation increased baseline binding activity of transcription factor κB three-fold ( P<0.03) and 2.6-fold ( P<0.09) in monocytes and in macrophages, respectively. The results demonstrate that differentiation of human monocytes into macrophages in culture enhances their tissue factor expression. The observed tissue factor increase that can be further stimulated by LPS may contribute to thrombogenicity of macrophage-rich atheroma. Activation of the TF-κB transduction pathway suggests its potential role in the transcriptional regulation of TF gene in response to acute stimulation in these cells. This cell-culture model may provide the opportunity to evaluate the effect of various interventions on macrophage procoagulant activity.