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

Abstract

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 primary cultures of human umbilical vein endothelial cells or in cultures pre-incubated with platelet-rich plasma. These studies suggest that primary cultures of human umbilical vein endothelial cells contain adherent platelets that contribute to thromboxane synthesis.