Evidence for multiple conformational changes in the active center of thrombin induced by complex formation with thrombomodulin: an analysis employing nitroxide spin-labels

Abstract

Thrombomodulin (TM) is an endothelial cell surface protein that binds thrombin to form a reversible complex with altered enzyme specificity. The complex rapidly converts protein C to the anticoagulant enzyme activated protein C and has decreased fibrinogen clotting activity. To investigate whether formation of this complex elicits conformational changes in the active center of thrombin, we employed the following fluorosulfonyl spin-labelel inhibitors: N-(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-m-(fluorosulfonyl)benzamide (m-V); 0-(2,2,6,6-tetramethyl-1-oxy-4-piperidinyl) N-[m-(fluorosulfonyl)phenyl]carbamate(m-VI); N-[4-(fluorosulfonyl)phenyl]-2,2,5,5-tetramethyl-1-oxy-3-pyrroline-3-carboxamide (p-I); N-(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-p-(fluorosulfonyl)benzamide (p-V). To compare the spectra of the free thrombin with those of the complex, the viscosity of the solution was ajusted with sucrose to give similar tumbling rates (isokylindric spectra) or the macromolecular rotational contribution to the spectra was essentially eliminated with saturated sucrose. Both a buffer-soluble proteolytic derivative of TM and the intact molecule elicited changes in the electron spin resonance signals of many of the labeled thrombins employed. Two of the labels p-I and p-V had previously been shown to exhibit decreased mobility when indole derivatives were bound to thrombin. When TM complexes with thrombin, the mobility of the p-I label increases while the mobility of p-V label decreases. Two of the labels, m-V and m-VI, had previously been shown to be sensitive to conversion of .alpha.-thrombin to .gamma.-thrombin. The m-V label in thrombin exhibited decreased mobility upon complex formation with thrombomodulin, which is similar to the decrease in probe mobility in .gamma.-thrombin, while m-VI-labeled thrombin exhibited no detectable spectral change. Although Ca2+ is required for protein C activation, Ca2+ had no influence on the spectra, and Gd(III) produced no dipolar broadening of the p-I spectra, indicating that the binding site on TM is more than 12 .ANG. from the label. 6-Fluorotryptamine did not alter the mobility of the p-I-labeled thrombin-TM complex, indicating either that this site is blocked in the complex or that the probe was insensitive to the additional conformational changes. These studies demonstrate that complex formation between thrombin and TM alters the conformation in at least two distinct regions of the active center. Although some of the changes monitored by selected probes appeared similar to those induced by indole and those resulting from the conversion from .alpha.- to .gamma.-thrombin, the responses of other probes were clearly distinguishable.