Prostacyclin analogues inhibit tissue factor expression in the human monocytic cell line THP-1 via a cyclic AMP-dependent mechanism.

Abstract

Increased expression of tissue factor procoagulant by peripheral blood monocytes has been implicated in a number of thrombotic disorders. The present studies were undertaken to determine whether stable analogues of prostacyclin, a potent endothelium-derived platelet inhibitor and vasodilator, could inhibit tissue factor expression by human monocytic cells. Exposure of monocytic tumor THP-1 cells to 100 ng/ml endotoxin, 2 units/ml interleukin-1 beta, or 5 ng/ml tumor necrosis factor-alpha for 4 hours led to increased tissue factor procoagulant activity. Preincubation for 30 minutes with iloprost, ciprostene, and carbacyclin led to a dose-dependent inhibition of tissue factor expression induced by all three challenging agents. Iloprost was the most potent: 50% inhibition occurred at 5 nM, a concentration close to the reported dissociation constant for iloprost binding to the platelet prostacyclin receptor. An orally active analogue, cicaprost, was equally effective against endotoxin-induced tissue factor expression. Carbacyclin and ciprostene were 100 times less potent. Iloprost prevented the endotoxin-induced expression of tissue factor antigen on the surface of THP-1 cells, as determined by flow cytometry. Iloprost (500 pM-50 nM) increased intracellular levels of cyclic AMP. This effect was potentiated by isobutylmethylxanthine, an inhibitor of phosphodiesterase. The inhibitory effects of iloprost on tissue factor expression were also potentiated by isobutylmethylxanthine and mimicked by forskolin and dibutyryl cyclic AMP but not dibutyryl cyclic GMP. These results suggest that prostacyclin may play a role in downregulating tissue factor expression in monocytes, at least in part via elevation of intracellular levels of cyclic AMP.

Molecular Mechanisms Regulating the Vascular Prostacyclin Pathways and Their Adaptation during Pregnancy and in the Newborn

“…In addition to inhibiting platelet aggregation, PGI 2 may inhibit thrombosis by inhibiting coagulation factors Va, VIIIa (Rabausch et al, 2005), tissue factor (Crutchley et al, 1992), and other proteins involved in coagulation, such as PAI-1 (Atsuta et al, 2009), von Willebrand factor, and D-dimer ). Iloprost and cicaprost increase mRNA expression and protein amount of thrombomodulin, which binds to thrombin with high affinity, resulting in activation of protein C, which in turn proteolytically degrades factors Va and VIIIa, thus inhibiting further thrombin generation and blood coagulation (Rabausch et al, 2005).…”

“…IP knockout mice demonstrate increased allergic responses and capillary permeability in the airways (Matsuoka and Narumiya, 2007), increased antigen-induced leukocyte accumulation in the lungs, increased Th2 inflammatory cytokines IL4 and IL5, and decreased Th2 anti-inflammatory IL10 (Hata and Breyer, 2004). Iloprost inhibits TNF␣ synthesis by leukocytes and macrophages, down-regulates MAPK in macrophages (Lo et al, 1998), and decreases tissue factor expression in monocytes (Crutchley et al, 1992;Mittag et al, 2001). Iloprost also decreases expression of ␣ M ␤ 2 integrin of phagocytes that has a key role in leukocyte-endothelium interactions in inflammation and thrombosis, inhibits chemotaxis, and modulates the expression of adhesion molecules on ECs, neutrophils, and monocytes (Mazzone et al, 2002).…”

Effects of prostacyclin analogues on human endothelial cell tissue factor expression.

“…19 The ability of iloprost to stimulate adenylate cyclase in endothelial cells and the relation of cAMP to endothelial tissue factor expression were therefore investigated. As shown in Table 3, 100 nmol/L iloprost had no effect on intracellular cAMP levels, either alone or in combination with the phosphodiesterase inhibitor IBMX.…”

“…In contrast, 5 nmol/L iloprost produced a 27-fold increase and 50 nmol/L iloprost produced a 95-fold increase in cAMP in THP-1 cells under similar conditions. 19 However, cAMP levels could be elevated in endothelial cells by other agents, such as forskolin and dibutyryl cAMP, and under these conditions tissue factor expression was inhibited.…”

“…18 - 19 Since vascular endothelial cells are a primary source of native prostacyclin, 20 - 22 it was of interest to determine whether prostacyclin analogues could inhibit tissue factor expression in these cells. Our results suggest that although the analogues do not directly affect endothelial tissue factor expression, they may do so indirectly by inhibiting monocyte cytokine secretion.…”