Mechanisms of regulated von Willebrand factor secretion from cultured endothelial cells

Abstract

The roles of phospholipase C and adenylate cyclase in thrombin-induced secretion of von Willebrand factor (vWf) were studied using cultured human umbilical vein endothelial cells (HUVEC). Additionally, the differential distribution of vWf in canine vessels was assessed to determine the best source of canine endothelial cells for use in future comparative studies. Immunohistochemistry and electron microscopy (EM) were used to evaluate canine vessels from several sites for the presence of vWf and Weibel-Palade bodies, the cytoplasmic storage organelles for vWf. Cultured endothelial cells were also evaluated for the presence of vWf and Weibel-Palade bodies. The canine jugular vein and thoracic aorta appear to be the best sources of endothelial cells for in vitro vWf secretion studies. In a second set of experiments, the effect of thrombin on intracellular calcium transients was evaluated using HUVEC. Endothelial cells were loaded with indo-1. a cell-permeable calcium sensitive dye, and ceils were monitored for changes in intracellular calcium concentration ([Ca]i) in response to thrombin after treatment with dibutyryl-cyclic adenosine monophosphate (B2cAMP), pertussis toxin (PT), nifedipine (Nif), verapamil (Verap), and thapsigargin. Thrombin induced the mobilization of intracellular calcium stores, as well as extracellular calcium influx, at relatively low concentrations (ED[50] = 0.052 U/ml). Treatment with Nif and Verap decreased thrombin-induced calcium influx at high thrombin concentrations, indicating indirectly that voltage-gated calcium channels are present in HUVEC. Treatment with PT prolonged the thrombin-induced rise in [Ca]i in endothelial cells in calcium-free medium during the export phase of the response, suggesting that pertussis toxin influences receptor-mediated calcium export through a PT-sensitive G-protein. Evaluation of thrombin-induced vWf secretion from HUVEC after pretreatment with B2cAMP and PT indicated that relatively high concentrations of thrombin ([greater than or equal to] 1.0 U/ml) are necessary to elicit vWf secretion from HUVEC. This concentration of thrombin is far greater than that necessary for mobilization of intracellular calcium, suggesting either that calcium is not the mediator that controls thrombin-induced vWf secretion, or that additional mediators are also involved.