Fast detection of tissue factor and tissue factor pathway inhibitor messenger RNA in endothelial cells and monocytes by sensitive reverse transcription-polymerase chain reaction.

We developed fast and sensitive reverse transcription-polymerase chain reaction (RT-PCR) procedures to study the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI-1) mRNA in human endothelial cells and monocytes. The sensitivity of the technique was checked by performing RT-PCR with limited numbers of cells. Cells were stimulated either with tumor necrosis factor (TNF-alpha) or endotoxin to induce TF mRNA expression or with phorbol ester to increase TFPI-1 mRNA expression. Thus, RT-PCR specific for TF mRNA provided detection from as few as 10(3) TNF-alpha stimulated endothelial cells and 5 x 10(2) monocytes stimulated by endotoxin. TF mRNA expression was increased by TNF-alpha in endothelial cells and in monocytes stimulated by endotoxin. Elevated expression of TF mRNA in monocytes without stimulation by endotoxin was mainly related to cell adhesion. TFPI-1 mRNA was constitutively expressed in endothelial cells and was detected in only 5 x 10(2) unstimulated cells and 10(2) phorbol ester-stimulated cells. Expression was increased upon stimulation with phorbol ester. With this technique, TFPI-1 mRNA in monocytes was rather low even when cells were stimulated with phorbol ester or after adhesion.

Human allogeneic lymphocytes trigger endothelial cell tissue factor expression by a tumor necrosis factor-dependent pathway.

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.

Evolution of blood coagulation activators and inhibitors in the healthy human fetus.

Blood coagulation proteins were determined in 285 healthy fetuses from 19 to 38 weeks' gestation and compared with those of 60 normal full-term newborns and 40 adult controls. Prolongation of the coagulation screening tests, prothrombin time, activated partial prothrombin time, and thrombin clotting time, in fetuses throughout intrauterine life was explained by low levels of vitamin K-dependent factors (II, VII, IX, and X), contact factors (XI, XII, prekallikrein, and high-molecular-weight kininogen), factor V, factor VIII, and fibrinogen. Low levels of antithrombin III, heparin cofactor II, protein C and protein S, and tissue factor pathway inhibitor were also found, and these probably contributed to a satisfactory hemostatic balance. Some of these parameters were evaluated by both immunologic and functional assays to detect possible "fetal" proteins. An increase in factor levels was observed after the thirty-fourth week of intrauterine life for most of the coagulation activators and inhibitors, but only factors V and VIII reached adult values at birth. This study therefore showed that fetal hemostasis is a dynamic system that evolves gradually toward the neonatal state and then toward the adult state.

A simplified and low-cost one-stage chromogenic assay for tissue factor dependent procoagulant activity of endothelial cells.

We developed a sensitive tissue factor (TF) chromogenic assay on a limited number of endothelial cells (EC), performed in microtiter plates, and which uses a normal pooled human plasma instead of purified concentrates as a source of coagulation factors. Primary cultures of human umbilical vein EC (HUVEC), both unstimulated and stimulated by lipopolysaccharide (LPS) were incubated with 50 microliters of of diluted normal human plasma (NHP) and 50 microliters of Factor Xa-specific chromogenic substrate (CBS 31-39, Stago, France). Hirudin was added at 4 U/ml to the plasma/CBS 31-39 mixture to inhibit thrombin generation. Optical densities were read at 405 nm and corresponding amounts of generated factor Xa were expressed in mU Xa/well using a standard curve established with purified human Factor Xa. The following parameters were then defined: the number of EC to plate (10(4) EC/well of a 96-well plate), the plasma-test dilution (1:20), the concentration of CBS 31-39 (0.50 mM) and the incubation time of reagents with EC (2 hours). The procoagulant activity (PCA) measured was only dependent on TF since it was no longer detectable either when FVII-deficient plasma was tested instead of normal human plasma or when PCA assays were performed in the presence of a blocking anti-human TF monoclonal antibody. This method allowed detection of a TF-dependent PCA on as few as 1000 EC per well. In addition, TF expression equal to 50% of maximal values was measured with LPS concentrations as low as 1 ng/ml, supporting the high sensitivity of the assay.

Comparative study of the fibrinolytic system in human fetuses and in pregnant women.

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.