Role of contaminating platelets in thromboxane synthesis in primary cultures of human umbilical vein endothelial cells

Previous studies suggested that cultured human endothelial cells metabolize arachidonic acid to thromboxane A 2. When primary cultures of human umbilical vein endothelial cells were incubated with 14 C -arachidonic acid and the 14 C -metabolites resolved by reverse phase high pressure liquid chromatography, radioactive products were observed that comigrated with 6-keto-prostaglandin F 1α and thromboxane B 2, the degradation products of prostacyclin and thromboxane A 2, respectively. Since platelets synthesize thromboxane A 2, the present study examined the hypothesis that adherent platelets may contaminate the primary cultures of human umbilical vein endothelial cells and be responsible for thromboxane B 2 production. Confluent primary cultures or passaged cells were stimulated with histamine (10 −5 M). Incubation buffer was analyzed by specific radioimmunoassays for 6-keto-prostaglandin F 1α and thromboxane B 2. The production of thromboxane B 2 decreased in the passaged cells (207±44 pg/ml versus 65±12 pg/ml; primary versus passaged cells). A moderate decrease in the yield of 6-keto-prostaglandin F 1α was measured in the passaged cells compared to the primary cultures (3159±356 pg/ml versus 1678±224 pg/ml, primary versus passaged cells). If the primary cultures were incubated with human platelet-rich plasma for 30 min prior to stimulation with histamine, the amount of thromboxane B 2 increased approximately 10-fold. In an additional experiment, sub-confluent primary cells were incubated with platelet-rich plasma for 30 min, washed to remove non-adherent platelets, and allowed to reach confluency. Confluent cells were then passaged and stimulated with histamine. The amount of thromboxane B 2 was not significantly different from that obtained with passaged cells that had not been incubated with platelet-rich plasma during the primary culture (83±15 pg/ml versus 65±12 pg/ml, respectively). If the cyclooxygenase inhibitor indomethacin was included in the incubations, the amounts of both thromboxane B 2 and 6-keto-prostaglandin F 1α decreased. In contrast, the thromboxane A 2 synthase inhibitor dazoxiben blocked thromboxane production and had no effect on the amount of 6-keto-prostaglandin F 1α. Light microscopy revealed the presence of adherent platelets in primary cultures with and without platelet-rich plasma but no platelets were observed in any group of passaged cells. Histofluorescence for platelet serotonin indicated the presence of platelets only in p