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Tissue factor pathway inhibitor-2 (TFPI-2) displays structural similarities with TFPI-1, the major inhibitor of tissue factor (TF)/, factor VIIa. It is synthesized mostly by syncytiotrophoblast in the placenta, but its physiological functions are not fully understood. We studied the synthesis of TFPI-2 mRNA and that of TFPI-1 and TF in three human trophoblast cell lines, JAR, BeWo, and JEG-3. We first developed specific competitive reverse transcription-polymerase chain reaction (RT-PCR) assays for each gene studied using human umbilical vein endothelial cells (HUVEC). The three trophoblast cell lines strongly synthesized TF mRNA whereas the synthesis of TFPI-1 mRNA was very low. TFPI-2 mRNA was not detected in unstimulated or stimulated JAR cells. In contrast, JEG-3 and, to a lesser extent, BeWo produced significant amounts of TFPI-2 mRNA, which were significantly increased after stimulation with phorbol 12-myristate 13-acetate (PMA). However, tumor necrosis factor-alpha (TNF-alpha) had no effect on this synthesis. JEG-3 and BeWo are thus two cell lines that could be used to study TFPI-2 gene regulation and to investigate the role of TF, TFPI-1, and TFPI-2 during trophoblast differentiation.

Lymphocyte adhesion to endothelial cells and the extravascular deposition of fibrin are 2 important processes during pathologic situations such as allograft rejection. Tissue factor (TF) expression was therefore measured on human umbilical vein endothelial cells (HUVECs) after coculture with allogeneic lymphocytes (PBLs) by a factor Xa generation assay. When cocultured with PBLs, HUVECs expressed strong procoagulant activity related to the TF/factor VII-dependent pathway, which was enhanced when endothelial cells were treated with interferon-gamma (IFN-gamma). The highest TF activity was measured when 10(5) lymphocytes were incubated with 10(4) HUVECs (ratio 10: 1) for 4 hours, a time-dependent course similar to that obtained with tumor necrosis factor-alpha (TNF-alpha), and direct contact between the 2 cell types was necessary. PBL-induced TF activity was inhibited by cycloheximide or actinomycin D, indicating active protein synthesis that was confirmed by the increase in TF mRNA detected by reverse transcription-polymerase chain reaction. It was then demonstrated that 1 of the primary signaling pathways leading to endothelial cell TF expression was a rapid initial interaction between membrane TNF expressed on PBLs and the 75-kd TNF receptor, with subsequent involvement of platelet-activating factor and P-selectin. Finally, we showed that the transduction of external signals involving the activation of protein kinase C and protein tyrosine kinases also contributed to the regulation of TF expression.

Levels of major parameters of fibrinolysis were measured in 50 normal human fetuses between 19 and 39 weeks of gestation and compared to those of 50 healthy normal pregnant women and 30 adult controls. In fetuses, euglobulin clot lysis time (ECLT) was significantly shortened, plasminogen level was low and histidine-rich glycoprotein undetectable. While t-PA and u-PA levels were slightly lower than in adult controls, a significant decrease in PAI activity was demonstrated and no PAI-2 could be detected in fetal plasma. In contrast with these findings, the fibrinolytic equilibrium of pregnant women exhibited a prolonged ECLT and a strong increase in both PAI activity and PAI-2 antigen levels, while only a moderate elevation in u-PA and t-PA levels was measured.