THROMBIN INHIBITION BY CYCLIC-PEPTIDES FROM THROMBOMODULIN

Peptides corresponding to the loop regions of the fourth, fifth, and s ixth epidermal growth factor (EGF)-like domains of thrombomodulin (TM) have been synthesized and assayed for thrombin inhibition, as indicat ed by both inhibition of thrombin-mediated fibrinogen clotting and inh ibition of the association of thrombin with TM that results in protein C activation. Peptides from the fifth EGF-like domain showed signific ant inhibition of fibrinogen clotting and protein C activation, wherea s peptides from the fourth and sixth EGF-like domains were weak inhibi tors in both assays. Two structural features were important for inhibi tory potency of the peptides from the fifth EGF-like domain: cyclizati on by a disulfide bond and attachment of the ''tail'' amino acids C-te rminal to the disulfide loop. Linear control peptides did not signific antly inhibit clotting or protein C activation. The C-terminal loop al one, the ''tail'' peptide, or a mixture of the two were at least 10-fo ld less potent inhibitors of clotting or protein C activation. A more constrained peptide analog was designed by deletion of an isoleucine w ithin the C5-C6 disulfide loop, TM52-1+5(c). This analog was a better inhibitor in both assay systems, having a K-i for protein C activation of 26 mu M.