Kazuyoshi Sawada

A simple assay to detect endothelial cell injury; Measurement of released thrombomodulin from cells

A simple assay to determine the degree of endothelial cell injury has been developed using released thrombomodulin as the index. Thrombomodulin is a cell surface protein on endothelial cells which is released from the cell upon injury. In this assay, bovine arterial endothelial cells were cultured in serum free medium with the test substances and the amount of thrombomodulin released into the culture medium was measured by enzyme immunoassay. Substances which are known to injure cells such as H2O2, prostaglandin A2, lipopolysaccharide, and elastase released significant amounts of thrombomodulin. The sensitivity of this assay for mild injury was superior or at least equal to the traditional 51Cr release method. Since this method does not require the use of radioisotopes, it seems to be advantageous and suitable for the detection of endothelial cell injury during routine examination.

Changes in thrombomodulin level in plasma of endotoxin-infused rabbits

Changes in the plasma thrombomodulin (TM) level were examined in endotoxin-infused rabbits. The plasma TM level in normal rabbits was 143.8 +/- 8.4 ng/ml (n = 67) and the molecular weight of the major TM was about 55 kd. Endotoxin (lipopolysaccharide, LPS, E. Coli B8:0127) was intravenously infused. LPS infusion increased the plasma TM level dose-dependently between 0.2 mg/kg and 5 mg/kg. When 5 mg/kg LPS was infused, the plasma TM level started to increase immediately and was 2.3 times higher than the control value within 1 hr. The molecular weight of the major TM was about 75 kd. This rapid increase in TM occurred before the decrease in fibrinogen content and the prolongation of prothrombin time. To examine the effect of circulating leukocytes on the TM increase in endotoxin-infused rabbits, 5 mg/kg LPS was infused into rabbits with leukocytopenia induced by X-ray irradiation. The maximum plasma level of TM was significantly lower than in the untreated rabbits given LPS. These data suggest that the increase in plasma TM is caused by LPS-stimulated leukocytes prior to hemostaseological changes. It is well known that endothelial cells can be injured by stimulated leukocytes, so this increase in plasma TM probably reflects the deterioration of endothelial cells. This deterioration decreases the ability of endothelial cells to inhibit thrombosis, which would, in turn, contribute to the development of disseminated intravascular coagulation in endotoxin-infused rabbits.

Hypertension Associated with Reduced Plasma Thrombomodulin Levels and a Hypercoagulable State in Rats

The plasma thrombomodulin (TM) level, an indicator of systemic endothelial cell damage, was measured in spontaneously hypertensive rats (SHR), deoxycorticosteron acetate (DOCA)‐induced hypertensive rats and normotensive Wistar–Kyoto (WKY) rats to clarify its changes in hypertension. Plasma TM levels, measured by enzyme‐linked immuno‐sorbent assay, decreased with aging (5–20‐weeks‐old) in both SHR and WKY, and they were lower in SHR than age‐matched WKY in all ages examined. Deoxycorticosteron acetate‐induced hypertensive WKY also showed decreased TM levels compared with normotensive WKY. Accelerated coagulation and fibrinolysis shown by the increases in thrombin–antithrombin complex (TAT) and D‐dimer levels were observed in both groups of hypertensive rats. These results suggest that hypertension may decrease plasma TM levels and induce a hypercoagulable state in rats.