The vascular endothelium, by virtue of its position at the interface between blood and the vessel wall, is known to play a critical role in the control of thrombosis and fibrinolysis. Thrombomodulin (TM) is a surface receptor that binds thrombin and is a potent activator of the protein C anticoagulant pathway. Although TM expression is known to be regulated by various cytokines, little is known about its response to ever-present biomechanical stimuli. We have explored the role of fluid shear stress, imparted on the luminal surface of the endothelial cell as a result of blood flow, on the expression of TM mRNA and protein in both bovine aortic endothelial (BAE) and bovine smooth muscle (BSM) cells in an in vitro system. We report in the present study that TM expression is regulated by flow. Subjecting BAE cells to fluid shear stress in the physiological range of magnitude of 15 (moderate shear stress) and 36 (elevated shear stress) dynes/cm(2) resulted in a mild transient increase followed by a significant decrease in TM mRNA to 37% and 16% of its resting level, respectively, by 9 hours after the onset of flow. In contrast, shear stress at the low magnitude of 4 dynes/cm(2) did not affect TM mRNA levels. The sensitivity of TM mRNA expression by flow was found to be specific to endothelium, since it was not observed in BSM cells exposed to steady laminar shear stress of 15 dynes/cm(2). Furthermore, unlike BAE cells, BSM cells did not exhibit altered cell shape nor align in the direction of flow after 24 hours of shear stress at 15 dynes/cm(2). In BAE cells, TM protein assessed by Western blot analysis also showed a decrease to 33% after 36 hours of laminar shear stress of 15 dynes/cm(2). The downregulation of TM mRNA in response to shear was found to recover completely to its static level within 6 hours after the cessation of the mechanical stimulus. Furthermore, the process was entirely reversible and without hysteresis, since further shear stimulus after recovery yielded the same behavior. The dynamic character of the shear was varied, and it was found that steady laminar, turbulent, and pulsatile shear stress with a mean magnitude of 15 dynes/cm(2) all resulted in a similar decrease in TM mRNA. Expression of tissue-type plasminogen activator (TPA) mRNA in the same BAE cells, previously reported to be affected by flow in human umbilical vein endothelial cells, was found to be increased 3-fold by 15 dynes/cm(2) and 22-fold by 36 dynes/cm(2) at 9 hours. The reciprocal behavior of TM and TPA to shear stress suggests a switch in endothelial phenotype from a predominantly antithrombotic state under static conditions, dominated by TM expression, to a fibrinolytic state governed by TPA under flow conditions, particularly under elevated shear stress (36 dynes/cm(2)). These findings suggest that TM may be playing a localized protective role against thrombosis in regions of stasis and low flow and identify flow as a novel regulator of endothelial TM expression.
