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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:157.)
© 2001 American Heart Association, Inc.

Thrombosis

Shear Stress Decreases Endothelial Cell Tissue Factor Activity by Augmenting Secretion of Tissue Factor Pathway Inhibitor

Eric F. Grabowski; Armin J. Reininger; Philip G. Petteruti; Olga Tsukurov; Roslyn W. Orkin

From the Cardiovascular Thrombosis Laboratory (E.F.G., A.J.R., P.G.T.) and the Vascular Surgery Research Laboratory (O.T., R.W.O.), Massachusetts General Hospital, Boston, Mass.

Abstract—Monolayers of human umbilical vein endothelial cells were activated with 50 U/mL interleukin-1 (IL-1) for 3 hours and simultaneously conditioned with shear stresses of 0, 0.68, or 13.2 dyne/cm² in a parallel-plate flow chamber. In the presence of an inflow buffer containing 100 nmol/L factor X and 10 nmol/L factor VII, production of factor Xa, a measure of functional tissue factor (TF), was determined as the product of outflow concentration of factor Xa (chromogenic assay performed under quasi-static flow conditions after the shear period) and flow rate. Similarly, production of TF pathway inhibitor (TFPI) was estimated as the product of antigenic TFPI (by enzyme-linked immunosorbent assay) in the supernatant and flow rate. In parallel experiments, total RNA was isolated for determination of amplification products of TF mRNA by reverse transcription–polymerase chain reaction. We found that shear stress reduced factor Xa production (mean±SE; n=number of experiments) from 13.33±1.14 (n=16) fmol/minxcm² at 0 shear stress to 5.70±2.51 (n=5) and 0.54±0.54 (n=4) fmol/minxcm² at shear stresses of 0.68 and 13.2 dyne/cm², respectively. At the same time, immunogold labeling showed that TF antigen on the endothelial surface increased >5-fold with shear stress, whereas TFPI antigen on the surface increased 2-fold. The secretion of TFPI (appearance of new supernatant TFPI) rose from 7.4±2.4 (n=12) x10^-3 fmol/minxcm² at 0 shear stress to 23.7±7.3 (n=9) and 50.2±14.3 (n=4) x10^-3 fmol/minxcm² at 0.68 and 13.2 dyne/cm², respectively. TF mRNA amplification products were not markedly changed by shear stress. We conclude that acute application of shear stress reduces functional, but not antigenic, expression of TF by intact, activated endothelial cell monolayers in a manner associated with shear stress–augmented endothelial cell secretion of TFPI.

Key Words: tissue factor • endothelial cells • shear stress • tissue factor pathway inhibitor

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