Identification Of A Cell-Surface Cofactor For Antithrombin III On Cultured Murine Endothelium

Cultures of mouse brain capillary endothelium grown on microcarrier beads were used as a model for the study of interactions of plasma proteins with the endothelium in the microcirculation, where the ratio of surface area to volume is large. Use of the beads increased the relative cell surface area about 100 fold over that in monolayer cultures. Cells obtained from capillary explants were grown to confluence on 140 μ (mean diameter) beads of cross-linked dextran. The cell-covered beads were packed into 0.3 ml columns in silicone-coated glass tubes, and were used within 2 hours. Cells remained viable during all experimental periods. Radioiodinated thrombin was bound to the cells, and unlabeled diisopropyl phosphoryl-thrombin eluted most of the bound radioactivity, which was identified by electrophoresis as α-thrombin. Likewise, bound thrombin was eluted with anti thrombin III, and most of the radioactivity was identified as thrombin-anti thrombin III complex. In contrast, when thrombin and anti thrombin III were incubated for comparable intervals in the absence of cells, little complex formation occurred. Similar enhancement of complex formation could not be readily detected over monolayer cultures because of the low ratio of surface area to volume coupled with a significant reaction rate without cells. It is concluded that a substance on the cell surface catalyzes the reaction of thrombin with anti thrombin III and that this catalyst becomes significant when cell surface concentration approaches that in the microcirculation.