IMMUNOLOGICAL AND CHEMICAL DETECTION OF VAC IN CULTURED ENDOTHELIAL CELLS
Recently we reported the presence in human umbilical cord vessels of an anticoagulatory protein (VAC, Mr = 32,000) which inhibits phospholipid dependent procoagulant reactions through a high affinity binding, in the presence of calcium, to the phospholipid surface. The mechanism of anticoagulation differs fundamentally from those of the well-known physiological anticoagulants.Polyclonal antibodies, raised in rabbits against purified VAC, bind in cultured endothelial cell lysates to an antigen with Mr = 32,000, as was revealed with immunoblotting techniques. It is demonstrated with chemical techniques, that this antigen is identical to VAC.VAC appears to be an intracellular protein, attached to fine granular structures, which are located outside the nuclear area. Quiescent endothelial cells do neither secrete VAC in detectable amounts, nor contain detectable VAC on the extracellular side of their plasma membrane. In the presence of 1 mM calcium, endothelial VAC binds reversibly, as was indicated with EDTA, to the subcellular structures of the endothelial cell. Once VAC is bound to the subcellular structures, their apparent procoagulant activities are diminished, as was shown in a one-stage coagulation assay.Based on these findings, we propose that the presence of VAC in endothelial cells supplies the endothelium with an additional anticoagulatory mechanism, which can be activated after cell injury, when intracellular structures become exposed to plasma constituents. VAC then controls the formation of procoagulant complexes, localized to the subcellular structures.