Uremic Serum and Solutes Increase Post-Vascular Interventional Thrombotic Risk Through Altered Stability of Smooth Muscle Cell Tissue Factor

Abstract

Background—Stent thrombosis (ST), a postinterventional complication with a mortality rate of 50%, has an incidence that rises precipitously in patients at risk. Chronic renal failure and end-stage renal disease have emerged as particularly strong ST risk factors, yet the mechanism remains elusive. Tissue factor (TF) is a crucial mediator of injury-related thrombosis and has been implicated for ST. We posit that uremia modulates TF in the local vessel wall to induce postinterventional thrombosis in patients with end-stage renal disease. Methods and Results—As a model of the de-endothelialized, postinterventional state, we exposed primary human vascular smooth muscle cells (vSMCs) pretreated with uremic serum (obtained from ESRD patients on hemodialysis) to coronary-like blood flow. vSMC TF expression, activity, stability, and posttranslational modification were examined after vSMCs were treated with uremic serum or solutes. We found significantly greater clot formation after uremic serum exposure, which was substantially reduced with the prior treatment with anti-TF neutralizing antibody. Uremic sera induced 2- to 3-fold higher TF expression and activity in vSMCs independent of diabetes mellitus. Relevant concentrations of isolated uremic solutes such as indole-3-acetic acid (3.5 μg/mL), indoxyl sulfate (25 μg/mL), and uric acid (80 μg/mL) recapitulated these effects in cell culture and the flow loop model. We show further that TF undergoes ubiquitination at baseline and that uremic serum, indole-3-acetic acid, and indoxyl sulfate significantly prolong TF half-life by inhibiting its ubiquitination. Conclusions—The uremic milieu is profoundly thrombogenic and upregulates vSMC TF levels by increasing TF stability and decreasing its ubiquitination. Together, these data demonstrate for the first time that the posttranslational regulation of TF in uremia may have a causative role in the increased ST risk observed in uremic patients. These data suggest that interventions that reduce vSMC TF may help to prevent ST and that uremic solutes should be considered as novel risk factors for ST in patients with chronic renal failure.