Shear stress decreases endothelial cell tissue factor activity by augmenting secretion of tissue factor pathway inhibitor

Monolayers of human umbilical vein endothelial cells were activated with 50 U/mL interleukin-1 alpha (IL-1 alpha) for 3 hours and simultaneously condi tioned with shear stresses of 0, 0,68, or 13.2 dyne/cm(2) in a parallel-pla te 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 fu nctional tissue factor (TF), was determined as the product of outflow conce ntration 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 amplific ation products of TF mRNA by reverse transcription-polymerase chain reactio n. We found that sheer stress reduced factor Xa production (mean+/-SE; n=nu mber of experiments) from 13.33+/-1.14 (n=16) fmol/min x cm(2) at 0 shear s tress to 5.70+/-2.51 (n=5) and 0.54+/-0.54 (n=4) fmol/min x cm(2) at shear stresses of 0.68 and 13.2 dyne/cm(2), respectively. At the same time, immun ogold 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) x 10(-3) fmol/min x cm(2) at 0 shear stress to 23.7+/-7.3 (n=9) and 50.2+/-14.3 (n=4) x 10(-3) fmol/min x cm(2) at 0.68 and 13.2 dyne /cm(2), respectively. TF mRNA amplification products were not markedly chan ged by shear stress. We conclude that acute application of shear stress red uces functional, but not antigenic, expression of TF by intact, activated e ndothelial cell monolayers in a manner associated with shear stress-augment ed endothelial cell secretion of TFPI.