Proteolytic formation and properties of functional domains of thrombomodulin

Thrombomodulin is an endothelial cell surface protein which complexes with thrombin to accelerate protein C activation. To gain insight into the mechanisms of thrombomodulin-membrane association, limited proteolytic digestions of thrombomodulin with trypsin and elastase were performed. Trypsin digestion resulted in two major fragments (Mr = 54,000 and 27,000), both of which bound to phosphatidylcholine/phosphatidylserine vesicles. Elastase digestion also yielded two major fragments (Mr = 50,000 and 25,000), but only the smaller fragment bound to the phospholipid vesicles. The larger fragment obtained from both enzymatic digestions retained the ability to accelerate protein C activation. The Mr = 54,000 fragment from the trypsin digest retained a high affinity for thrombin (Kd less than or equal to 0.5 nM), a Km for protein C of approximately equal to 8 microM, and a half-maximal Ca2+ dependence of 0.3 mM. The Mr = 50,000 fragment from elastase digestion had a lower affinity for thrombin (Kd approximately equal to 6 nM) than intact thrombomodulin, and the Km for protein C was decreased to 0.3 microM in the presence of 0.3 mM Ca2+. The Ca2+ dependence of protein C activation with the Mr = 50,000 fragment was distinctly different from that of thrombomodulin or the active tryptic fragment. The active elastase fragment exhibited a Ca2+ optimum at 0.3 mM and activity rapidly decreased with further increases in Ca2+. At the Ca2+ optimum, the Km for protein C was similar to that observed on endothelial cell surfaces or with thrombomodulin reconstituted into liposomes. Our data demonstrate that thrombomodulin has one or more membrane-binding domains and that an active soluble form with catalytic activity can be generated by limited proteolytic digestion. Digestion with elastase appears to expose a site on thrombomodulin capable of recognizing the gamma-carboxyglutamic acid domain of protein C (residues 1-44 of the light chain). Whether this is the same site which is exposed on thrombomodulin upon incorporation into phospholipid vesicles (see accompanying manuscript) remains to be determined.