Abstract 642: Specific Protein Kinase C Isoforms Are Critical Mediators of Physiologic Downregulation of Perivascular Tissue Factor

Tissue factor (TF) is a procoagulant and transmembrane receptor for FVII(a) that is upregulated in pathological conditions. We recently demonstrated that perivascular TF is downregulated around angiogenic vessels near a cutaneous wound. The goal of this study was to identify mechanisms that mediate TF loss. Primary cultures of human pericytes express high levels of TF. TF expression was lost during culture with phorbol-12 myristate 13-acetate (PMA) for 8 hours, and was maintained for 24 hours. This model recapitulates the pattern of downregulation observed in vivo . Using qRT-PCR we assessed changes in TF gene expression in response to PMA. TF mRNA decreased 4- and 6-fold at 8 and 12 hours after treatment (p<0.01), and remained 2-fold lower in treated cells after 24 hours (p<0.05). Inhibiting de novo transcription with actinomycin D showed that degradation of TF mRNA was similar in PMA- and vehicle-treated groups (p=ns). Thus, downregulation of TF mRNA occurs primarily through inhibition of its synthesis. We next identified a physiologic mediator of TF downregulation using specific inhibitors against PMA-responsive signaling proteins. Two different inhibitors against Protein Kinase C (PKC) were used: Go6983, which inhibits isoforms α, β, δ, ε, μ, and ζ, and GFX, which inhibits α, β, ε, and γ. Both inhibitors significantly attenuated PMA-mediated transcriptional downregulation (p<0.001). Based on overlap of inhibited isoforms, this suggests a minimal role for ζ, μ, δ, and γ, while one or more of the α, β, and ε isoforms appear to be critical mediators of TF mRNA synthesis inhibition. Since the timing of TF protein loss is not fully explained by transcriptional inhibition, we investigated the role of protein degradation in TF loss. When protein synthesis was inhibited by cyclohexamide, addition of PMA shortened the half-life of pericyte TF from 11 hours to 5 hours (p<0.001). This indicates that increased protein degradation contributes to PMA-induced loss of TF expression. Both Go6983 and GFX significantly attenuated TF protein degradation also. Taken together, our data show that TF downregulation is mediated by transcriptional inhibition and protein degradation, and PKC α, β, and ε have emerged as potential mediators of both mechanisms.