Heparin inhibits thrombin binding to rabbit thoracic aorta endothelium.

Thrombin binding to freshly prepared sections of rabbit thoracic aorta was studied. After the sections had been exposed to a range of concentrations (0.1 to 3.8 IU/ml) of 125I-labeled thrombin for various periods of time at 37 degrees C, endothelial Häutchen preparations were obtained, and their radioactivity content was determined. Scatchard plot analysis of the data indicated that approximately 5.8 X 10(5) molecules of thrombin associated with each endothelial cell, with a KD of 2.6 X 10(08)M. By incubation with an excess of unlabeled thrombin, 50% of bound 125I-labeled thrombin was displaced from the endothelium in 7.3 min. Exposure of the endothelial surface to heparin (1 to 10 USP U/ml) did not significantly affect subsequent thrombin binding. However, incubation of the aorta in a thrombin solution containing 1 to 10 USP U/ml heparin did reduce enzyme binding to the endothelium by up to 60%. Similarly, the presence of heparin inhibited thrombin binding to the thoracic aorta of exsanguinated rabbits in situ. Endothelium, to which 125I-labeled thrombin was bound, lost 50% to 70% of the bound enzyme when suspended in a solution containing heparin (10 USP U/ml) and compared to the control incubated without heparin. These observations are consistent with the proposal that a major portion of endothelium-bound thrombin may be associated with pericellular heparan sulfate; heparin competes for thrombin with the heparan sulfate sites, and because of its higher affinity for thrombin, heparin displaces bound thrombin from, or inhibits binding of free thrombin by, the endothelium.