The active site of the thrombin-thrombomodulin complex. A fluorescence energy transfer measurement of its distance above the membrane surface.

The location of the active site of the membrane-bound anticoagulant complex of thrombin and thrombomodulin has been determined relative to the membrane surface using fluorescence energy transfer. Thrombin was reacted with 5-(dimethylamino)-1-naphthalenesulfonylglutamylglycylarginyl chloromethyl ketone (DEGR-CK) to yield DEGR-thrombin, an analogue of thrombin with a fluorescent dye covalently attached to its active site. When DEGR-thrombin was titrated with thrombomodulin that had been reconstituted into phospholipid vesicles containing octadecylrhodamine, singlet-singlet energy transfer was observed between the donor dyes, each in an active site of a DEGR-thrombin bound to thrombomodulin, and the acceptor dyes at the outer surface of the phospholipid bilayer. The extent of energy transfer reached a maximum when DEGR-thrombin and thrombomodulin were equimolar in the sample, as expected for the formation of a 1:1 complex between thrombin and thrombomodulin. This energy transfer was dependent upon the binding of DEGR-thrombin to thrombomodulin because no energy transfer was observed with vesicles that lacked thrombomodulin, and the extent of energy transfer was reduced greatly by the addition of excess unmodified nonfluorescent thrombin to compete with DEGR-thrombin for binding to the thrombomodulin. From the dependence of the energy transfer upon the acceptor density and assuming kappa 2 = 2/3, the distance of closest approach between a dye in the active site of the thrombin-thrombomodulin complex and a dye at the membrane surface was determined to average 66 A (65 +/- 3 A for phosphatidylcholine vesicles without and 67 +/- 5 A for those with 20% phosphatidylserine). This distance was also insensitive to the presence or absence of Ca2+. These direct measurements indicate that the active site of the membrane-bound thrombin-thrombomodulin complex is located far above the phospholipid surface, that the peptide bond cleaved during the activation of protein C is situated about 66 A above the membrane, that the thrombin binding site on thrombomodulin is positioned more than 45 A above the membrane, ant that thrombin, with a diameter near 40 A, is not positioned alongside thrombomodulin near the membrane to form the thrombin-thrombomodulin complex but is instead bound "on top" of thrombomodulin.