THE SHAPE OF THROMBOMODULIN AND INTERACTIONS WITH THROMBIN AS DETERMINED BY ELECTRON-MICROSCOPY

Studies have been carried out to investigate aspects of the structure of thrombomodulin, an endothelial cell glycoprotein that binds thrombi n and accelerates both the thrombin-dependent activation of protein C and the inhibition of antithrombin III. We have determined the shape o f Solulin(TM), a soluble recombinant form of human thrombomodulin miss ing the transmembrane and cytoplasmic domains, by electron microscopy of preparations rotary-shadowed with tungsten. Solulin appears to be a n elongated molecule about 20 nm long that has a large nodule at one e nd and a smaller nodule near the other end from which extends a thin s trand. About half of the molecules form bipolar dimers apparently via interactions between these thin strands, Electron microscopy of comple xes formed between Solulin and human alpha-thrombin revealed that a si ngle thrombin molecule appears to bind to the smaller nodule of Soluli n, suggesting that this region contains the epidermal growth factor-li ke domains 5 and 6. Epidermal growth factor-like domains 1-4 comprise the connector between the small and large nodule, which is the lectin- like domain; the thin strand at the other end of the molecule is the c arbohydrate rich region. With chondroitin sulfate-containing soluble t hrombomodulin produced from either human melanoma cells Bowes or Chine se hamster ovary cells, a higher percentage of molecules bound thrombi n and, in some cases, two thrombin molecules were attached to one solu ble thrombomodulin in approximately the same region, These structural studies provide insight into the structure of thrombomodulin and its i nteractions with thrombin as well as aspects of the mechanisms of its actions.