Abstract
Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.
Highlights
From the $Howard Hughes Medical institute, §Departments of Medicine and Biochemistryand Molecular Biophysics, The Jewish Hospital of St
No binding inhibition was detected for meizothrombin S 2 0 5 A or human factor Xa thrombin binding to thrombomodulin [7]
In direct equilibrium binding experiments, Diisopropyl fluorophosphate (DFP)-thrombin, factor Xa, or recombinant mutant prothrombin S205A waslabeled with Na"'1 using chloramine T [18].Labeled mutant prothrombin S205A was activated to thrombin S205A with crude E. carinatus venom or t o meizothrombin S205A with ecarin, as described under "Activation of Prothrombin.'' Increasing concentrations of radiolabeled proteins were incubated with confluent monolayers of CV-l(l8A) cells or control CV-1 cells in 22-mm tissue culture wells for 2 h at 4 "C
Summary
EQUILIBRIUM BINDING OF HUMAN THROMBIN, MEIZOTHROMBIN, AND FACTOR XaTO RECOMBINANT THROMBOMODULIN*. '251-labeled product of prothrombin activation [8, 9], were reported to plasma thrombin and thrombiSn2 0 5 A bound to throm- catalyze thrombomodulin-dependent protein Cactivation (10, bomodulin with K d values of 4.0 f 1.9 and 6.9 f 1.2 11).It is notknown, whether human factor Xa and nM, respectively. Similar results were obtained with thrombin and Materials-Recombinant human thrombomodulin was expressed factor Xa when human umbilical vein endothelial cells were used as thesource of thrombomodulin.
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