DESIGNED POLYANIONIC COILED-COIL PROTEINS - ACCELERATION OF HEPARIN-COFACTOR-II INHIBITION OF THROMBIN

Novel polyanionic proteins were designed to increase the rate of hepar in cofactor II (HC) inhibition of alpha-thrombin, an essential proteas e in the coagulation cascade. Two alpha-helical coiled-coil proteins, a 62-residue dimer containing 8 Glu residues (E8C) and a 104-residue d imer containing 14 Glu residues (E14C), plus two 31-residue control pe ptides containing 8 Glu residues each (E8A and E8B), were chemically s ynthesized, structurally characterized and enzymatically assayed, Circ ular dichroic spectrophotometry indicated that both E8C and E14C forme d stable two-chain alpha-helical coiled coils at pH 7 and 25 degrees C . The control peptides were only partially alpha-helical. E14C remaine d folded at 90 degrees C but E8C was half unfolded at 49 degrees C. Co iled-coil proteins E8C and E14C maximally accelerated by 35- and 33-fo ld, respectively, the rate of HC inhibition of alpha-thrombin. None of these compounds accelerated antithrombin inhibition of alpha-thrombin , and neither control peptide accelerated HC inhibition of alpha-throm bin. Acceleration of the HC inhibition of alpha-thrombin showed bimoda l dependence on the concentration of the polyanionic protein, which is consistent with formation of a HC-coiled-coil-thrombin ternary comple x. The results suggest that antithrombotic polyanionic alpha-helical c oiled-coil proteins can be designed and synthesized and that the occur rence of secondary structure can be correlated with biological activit y. (C) Munksgaard 1995.