THROMBIN-BINDING AFFINITIES OF DIFFERENT DISULFIDE-BONDED ISOMERS OF THE 5TH EGF-LIKE DOMAIN OF THROMBOMODULIN

The fifth EGF-like domain of thrombomodulin (TM), both with and withou t the amino acids that connect the fifth domain to the sixth domain, h as been synthesized and refolded to form several different disulfide-b onded isomers. The domain without the connecting region formed three d isulfide-bonded isomers upon refolding under redox conditions. Of thes e three isomers, the (1-2,3-4,5-6) bonded isomer was the best inhibito r of fibrinogen clotting and also of the thrombin-TM interaction that results in protein C activation, but all the isomers were inhibitors i n both assays. The isomer containing an EGF-like disulfide-bonding pat tern (1-3,2-4,5-6) was not found among the oxidation products. The dom ain with the connecting region amino acids (DIDE) at the C-terminus fo rmed two isolable products upon refolding in redox buffer. These produ cts had the same two disulfide-bonding patterns as the earliest and la test eluting isomers of the domain without the DIDE. In order to compa re the thrombin-binding affinities of these isomers to the isomer with the EGF-like disulfide bonds, acetamidomethyl protection of the secon d and fourth cysteines was used to force the disulfide bonds into the EGF-like pattern. Thrombin-binding affinity, measured as inhibition of fibrinogen clotting and as inhibition of protein C activation correla ted inversely with the number of crossed disulfide bonds. As was found for the domain without the connecting region, the isomer that was the best inhibitor of fibrinogen clotting and of protein C activation was the isomer with no crossing disulfide bonds (1-2,3-4,5-6). This isome r doubled the clotting time at a concentration of 200 nM and showed a K-i for protein C activation of 2 mu M, both an order of magnitude bet ter than the isomer with EGF-like disulfide bonds.