MODULATION OF ANTITHROMBIN III ACTIVITY AND ANTITHROMBIN III-THROMBIN COMPLEXES BINDING TO CULTURED CELLS BY MONOCLONAL ANTIBODIES AGAINST ANTITHROMBIN III
Two monoclonal antibodies (mAb's) against antithrombin III (ATIII) were characterized with respect to their ability to interfere with ATIII activity. AT III activity was measured by its ability to inhibit the amidolitic activity of thrombin on the substrate BCP-100. Incubation of 150 ng of ATIII with 28pg mAb A36R2 prior to addition of 50 ng thrombin totally abolishes the inhibitory effect of ATIII on thrombin. Incubation of 200ng of ATIII with 10 μg of mAb B26R4 prior to addition of 75 ng thrombin raises the inhibitory effects of ATIII from 37% to 100%. We examined the effect of these mAb's on binding of antithrombin III-thrombin (ATIII-Th) complexes to bovine corneal endothelial cells. 120 pg/ml mAb's are reacted with 2 μg/ml ATIII-Th complexes prior to their addition to the cells. mAb A36R2 completely blocks ATIII-Th complexes binding. In contrast, mAb B26R4 enhances binding up to 250% of the control binding.We conclude that mAb A36R2 prevents binding of thrombin to ATIII by recognizing an epitope on ATIII close to thrombin binding site or that its binding to ATIII induces a conformational change in the thrombin binding site thus it no longer recognizes thrombin. mAb B26R4 has a heparin-like effect on ATIII: Its binding to ATIII induces conformational changes which improve thrombin binding to ATIII. There is a correlation between inhibition and enhancement of thrombin binding to ATIII and of ATIII-Th complexes binding to cells by the two mAb's. These mAb's may provide a new tool to control the activity of ATIII and to identify the cellular binding site on the ATIII-Th complex.This research was supported by a grant from the National Council for Research and Development, Israel and G.S.F. München, Germany.