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Platelets stimulated in vivo may cause endothelial injury. The question is whether a similar effect of platelet stimulation may be shown in vitro.  Endothelial cells were cultured from human umbilical veins in Medium 199 with 20 per cent heat-inactivated serum. Semiconfluent cultures, 100-400.000 cells per dish, were labelled with Na2Cr51O4. Twenty-four hours later human platelet suspension (final cone. 200.000 per mm3) and thrombin (final cone. 4 U/ml) were added to the medium and the culture dish shaken for 15 min. The percentage of cells detached from the culture dish and the percentage of Cr51 released from the endothelial cells into the ambient fluid during the shaking were determined and used as parameters of cell injury. Increased percentages of loosened cells and Cr51 into the ambient fluid were observed with platelet suspension and thrombin compared to controls without platelet suspension and/or thrombin. Platelet-free supernatant after reaction of thrombin with platelet suspension had a similar effect. Thrombin alone caused a moderat release of Cr51, but no increased loosening of cells. Both parameters increased with increasing dose of thrombin when the platelet count was kept constant. The percentage of loosened cells increased with increasing platelet number when the dose of thrombin was constant. A significant release of Cr51 occurred only at high concentrations of platelets. Scanning and transmission electron microscopy of endothelial cells exposed to thrombin-stimulated platelets confirmed the presence of loosening and injured cells in association with platelets. The endothelial cells were bulging, intercellular junctions opened up, the plasma membrane on the luminal side showed villi and breaks and the cytoplasm had increased electron density. Thus, platelets stimulated by thrombin may cause injury of endothelial cells also in vitro.

Injury Of Cultured Endothelial Cells By Thrombin-Stimulated Platelets