Protein Z (PZ) is a vitamin K-dependent protein isolated from human plasma, and acts as a cofactor for a serpin, called protein Z-dependent protease inhibitor (ZPI). A prothrombotic phenotype has been reported in PZ deficient mice, and PZ deficiencies have been observed in patients with arterial thrombotic events. PZ was immunologically detected in the endothelium of atherosclerotic arteries, suggesting that endothelial cells could be involved in the production of PZ. In this study we analyzed the synthesis and release of PZ and ZPI by human umbilical vein endothelial cells (HUVEC), representative of the macrovasculature, and by HMEC-1, a microvascular endothelial cell line. PZ was quantified by a specific ELISA in the supernatant and in the lysates of both cellular types. Western blotting of the supernatants showed the presence of a band of 62 kDa, identical to PZ synthesized by the hepatoma cell line HepG2. mRNA of PZ was also detected in each cellular type. PZ biosynthesis was unaffected by inflammatory cytokines in HUVEC, whereas a slight decrease of mRNA and PZ antigen (53.5 +/- 14.5% of protein synthesis as compared to the control, p < 0.01) and a modest increase (126 +/- 8.5% as compared to the control, p < 0.05) were induced respectively byTumor Necrosis Factor (TNF)-alpha (25 ng/ml) and oncostatin M (5 ng/ml) in HMEC-1. Immunological studies showed the presence of PZ near the nucleus and a possible expression of PZ at the membrane. In addition, PZ was present in the endothelial cells of both normal arterial and venous vessel sections. In contrast, neither ZPI nor its mRNA was detected in endothelial cells.