Background— The endothelium is a major source of tissue factor pathway inhibitor (TFPI), the endogenous regulator of TF-induced coagulation, and a significant proportion of the expressed TFPI remains associated with the endothelial surface. Methods and Results— Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment reduced TFPI at the surface of cultured endothelial cells by ≈80%, and at least a portion of the TFPI released by PI-PLC contained an intrinsic glycosylphosphatidylinositol (GPI) anchor that is recognized by anti-crossreactive determinant antibodies. Endothelial cells express both of the alternatively spliced forms of TFPI mRNA at a ratio of TFPIβ/TFPIα mRNA of ≈0.1 to 0.2. In Chinese hamster ovary (CHO) cells, TFPIα is predominantly secreted, whereas TFPIβ is a GPI-anchored membrane protein. Like TFPIβ, the small proportion of the TFPIα expressed by CHO cells that remains surface associated is also released by PI-PLC treatment, suggesting that it is bound to a separate GPI-anchored protein(s) at the surface of the cells. Conclusions— Both direct (TFPIβ) and indirect (TFPIα) GPI-mediated membrane anchorage is involved in localizing TFPI to the surface of cells.

Summary. Background: Tissue factor pathway inhibitor (TFPI) is mainly produced by endothelial cells and alternative mRNA splicing generates two forms, TFPIa and TFPIb. A portion of expressed TFPI remains associated with the cell surface through both direct (TFPIb) and indirect (TFPIa) glycosylphosphatidyl-inositol (GPT)-mediated anchorage. Objective: Compare the structure and properties of TFPIa and TFPIb. Methods: TFPIa and TFPIb, with protein molecular masses of 36 and 28 kDa, respectively, migrate similarly (46 kDa) on SDS-PAGE. Experiments using specific glycosidases were carried out to determine the different glycosylation pattern of the two forms. ECV304 cells, a cell line with some endothelial properties, were stimulated with IL-lb, LPS, and TNFa for up to 24 hrs and mRNA levels and protein synthesis were determined. Stable clones of ECV304 cells that express reduced levels of TFPIa, TFPIb or both were produced using a plasmidbased small-interfering RNA technique. Surface TFPI activity was determined by a two-stage chromogenic assay based on the ability of each form to inhibit FXa activation by FVIIa on cells with comparable amount of tissue factor (TF). Results and conclusions: The deglycosylation studies show that the difference in molecular masses is due to a greater degree of sialylation in O-linked carbohydrate in TFPIb. The mRNA and protein levels of neither form of TFPI were affected by stimulation of cells with inflammatory stimuli. Although TFPIa comprises 80% of the surface-TFPI, TFPIb was responsible for the bulk of the cellular FVIIa/TF inhibitory activity, suggesting a potential alternative role for cell surface TFPIa.

Background-Tissue factor pathway inhibitor (TFPI)-␣, a key regulator of tissue factor-induced coagulation, contains 3 tandem Kunitz-type inhibitory domains. Kunitz-1 binds and inhibits factor VIIa in the factor VIIa/tissue factor complex, and Kunitz-2 binds and inhibits factor Xa. The role of the Kunitz-3 domain of TFPI-␣, however, has remained an enigma. Methods and Results-To determine the structures within TFPI-␣ involved in its binding to cell surface, altered forms of TFPI-␣ were expressed in C127 (mouse mammary) cells: C-terminal truncated forms TFPI-␣ (252), TFPI-␣ (242), and TFPI-␣ (181), which also lacks the third Kunitz domain (K3); TFPI-␣ (desK3), which lacks only the K3 domain; and TFPI-␣ (R199L), in which the putative P1 site in K3 is changed from arginine to leucine. By flow cytometry (fluorescence-activated cell sorting), the altered forms 252, 242, and R199L showed significantly reduced binding, whereas the forms 181 and desK3 completely failed to bind to the cell surface. Transient expression of WT-, desK3-, and K3/K2-TFPI-␣ (in which K3 is replaced with K2) in another cell line (b-end3, mouse endothelial) produced comparable results. Exogenously added C-terminal truncated and R199L forms of TFPI-␣ bound poorly and desK3 did not bind at all to the surface of ECV304 cells in which TFPI-␣ expression had been "knocked down" by RNA interference. Conclusions-Optimal

Oxygen free radicals induce de novo synthesis of tissue factor (TF), the initiator of the extrinsic pathway of coagulation, within the coronary vasculature during postischemic reperfusion. In the present study we wanted to assess whether TF expression might cause myocardial injury during postischemic reperfusion. Anesthetized rabbits underwent 30 min of coronary occlusion followed by 5.5 h of reperfusion. At reperfusion the animals received 1) saline (n = 8), 2) human recombinant, active site-blocked activated factor VII (FVIIai, 1 mg/kg, n = 8), or 3) human recombinant activated FVII (FVIIa, 1 mg/kg, n = 8). FVIIai binds to TF as native FVII, but with the active site blocked it inhibits TF procoagulant activity. The area at risk of infarction (AR), the infarct size (IS), and the no-reflow area (NR) were determined at the end of the experiment. FVIIai resulted in a significant reduction in IS and NR with respect to control animals (28.1 +/- 11.3 and 11.1 +/- 6.1% of AR vs. 59.8 +/- 12.8 and 24.4 +/- 2.7% of AR, respectively, P < 0.01), whereas FVIIa resulted in a significant increase in IS and NR to 80.1 +/- 13. 1 and 61.9 +/- 13.8% of AR, respectively (P < 0.01). In conclusion, TF-mediated activation of the extrinsic coagulation pathway makes an important contribution to myocardial injury during postischemic reperfusion.