Release of Active Tissue Factor by Human Arterial Smooth Muscle Cells

Abstract

Tissue factor (TF), the initiator of coagulation, is thought to function predominantly at the cell surface. Recent data have suggested that active TF is present extracellularly in atherosclerotic plaques, the arterial wall, and the blood. This study was conducted to determine whether smooth muscle cells (SMCs), a major source of arterial TF, could generate extracellular TF. Active TF accumulated in the medium of cultured human SMCs, representing approximately 10% of that measured in the underlying cells at 24 hours. Platelet-derived growth factor, phorbol ester, and tumor necrosis factor-alpha caused approximately 3-fold increases in TF activity in the medium. Release of TF into the medium was dependent on the presence of the TF transmembrane domain but not the cytoplasmic domain. Antibodies to TF precipitated most of the activity from the culture medium, whereas antibodies to the beta(1)-integrin subunit precipitated approximately 33% of the activity. Treatment with detergent or phosphatidylserine:phosphatidylcholine did not increase activity, suggesting that all TF released by SMCs was in the appropriate lipid milieu and not encrypted. Western blotting showed that the medium contained full-length TF protein. Fluorescent cytometry showed that extracellular TF was present largely in particles < or =200 nm, which had a density of 1.10 g/mL. We hypothesize that active extracellular TF found in the injured arterial wall and atherosclerotic plaques derives, in part, from SMC microparticles.

Aggregated Low-Density Lipoprotein Uptake Induces Membrane Tissue Factor Procoagulant Activity and Microparticle Release in Human Vascular Smooth Muscle Cells

“…The secreted TF has a higher molecular weight than cellular TF, and it is mainly associated with MPs of 0.2 m, as reported previously. 22 Although lipid-loading induces TF release through apoptotic mechanisms in macrophages, 29,30 agLDL-lipid loading did not induce apoptosis of VSMCs, in agreement with the lack of apoptosis previously found in agLDL-loaded macrophages and VSMCs. 31 The present results are in agreement with those of Schecter et al, 22 who proposed that release of TF-containing MPs from VSMCs represents a constitutive property of VSMCs not related to apoptosis.…”

“…22,27 In the present study, we hypothesized that agLDLs were able to induce cellular TF expression and activity. We found that both nLDL and agLDL induced TF mRNA after 2 hours of LDL exposure to the same level, in agreement with previous studies showing that nLDL and oxidized LDL induce TF mRNA in different cell types, [12][13][14] which is likely unrelated to the receptor involved in LDL uptake.…”

“…22 The fraction overlying the 60% sucrose bed that contain MP was collected. One aliquot was dialyzed against 0.01 mol/L NH 4 HCO 3 , concentrated, and used for Western blot analysis and another fraction for flow cytometry analysis.…”

“…The culture medium was concentrated on a 60% sucrose cushion by centrifugation at 245 000g as described previously. 22 One aliquot of 200 L was used for lipid extraction. Lipid extraction was performed as described previously.…”

“…It has been demonstrated that most of TF is in an encrypted-inactivated state in the cell membrane. 28 Like nLDL, agLDL contributes to increase the pool of membrane TF, but it is also able to increase TF activity in a prolonged manner. Additionally, agLDL but not nLDL is able to increase TF release to the extracellular medium.…”