MECHANISM OF THROMBIN BINDING TO ENDOTHELIAL-CELLS

  • 1 January 1983
    • journal article
    • research article
    • Vol. 61  (2) , 368-372
Abstract
The interaction of human .alpha.-thrombin with mini pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated 2 populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of .apprx. 3 .times. 10-8 M. Modification of 2 lysine residues of thrombin with pyridoxal-5''-phosphte (PLP2-thrombin) destroyed the high-affinity binding and .apprx. 3/4 of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a 10-fold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin; it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. 3H-heparin apparently also binds to cells. This binding was characterized by a Kd of 3 .times. 10-7 M, .apprx. 106 sites/cell. Thrombin bound to endothelial cells was released by antithrombin IIII. A model is proposed for the mechanism of the binding of thrombin to endothelial cells.