THROMBOMODULIN INCREASES THE RATE OF THROMBIN INHIBITION BY BPTI

Thrombin undergoes allosteric modulation by thrombomodulin (TM) that r esults in a shift in macromolecular specificity, blocking fibrinogen c lotting while enhancing protein C activation. The TM enhancement of pr otein C activation involves both an 8-fold decrease in K-m and a 200-f old increase in k(cat). Although TM-mediated conformational changes in thrombin have been detected by many techniques, the nature of these c hanges remains obscure. Access to the active center of thrombin is rel atively restricted due to the presence of a large insertion loop at re sidue 60 (chymotrypsin numbering) that has been implicated in modeling studies as being responsible for poor inhibition by BPTI. Thrombin an d the E192Q mutant, which binds BPTI much more tightly than thrombin, are both inhibited very slowly by BPTI. TM increases the rate of throm bin or thrombin E192Q inhibition by BPTI similar to 10-fold. When anal yzed as slow tight binding inhibition, the TM effect on thrombin E192Q inhibition by BPTI is primarily on the first, reversible step in the reaction. Structural studies of the thrombin E192Q-BPTI complex have p reviously shown that the 60 loop lies over the BPTI, a position which requires 8 Angstrom movement at the apex of the 60 loop, and that BPTI is found in the same canonical orientation as in the trypsin complex. It follows that TM enhancement of the initial interaction of thrombin results in a conformation that favors interactions with BPTI, probabl y involving motion of the 60 loop.