LOCALIZATION OF THE THROMBIN-BINDING DOMAIN ON PROTHROMBIN FRAGMENT-2

Co-crystallographic studies have shown that the interaction of human p rothrombin fragment 2 (F2) with thrombin involves the formation of sal t bridges between the kringle inner loop of F2 and anion-binding exosi te II of thrombin. When F2 binds to thrombin, it has been shown to evo ke conformational changes at the active site and at exosite I of the e nzyme. Using plasma, recombinant, and synthetic F2 peptides (F2, rF2, and sF2, respectively) we have further localized the thrombin-binding domain on F2. F2, rF2-(1-116), rF2-(55-116), and sF2-(63-116), all of which contain the kringle inner loop (residues 64-93) and the acidic C OOH-terminal connecting peptide (residues 94-116), bind to thrombin-ag arose. In contrast, analogues of the kringle inner loop, sF2-(63-90), or the COOH-terminal connecting peptide, sF2-(92-116), do not bind. Th us, contrary to predictions from the crystal structure, the COOH-termi nal connecting peptide as well as the kringle inner loop are involved in the interaction of F2 with thrombin. F2 and sF2-(63-116) bind satur ably to fluorescently labeled active site-blocked thrombin with K-d va lues of 4.1 and 51.3 mu M, respectively. The affinity of sF2-(63-116) for thrombin increases about 5-fold (K-d = 10 mu M) when Val at positi on 78 is substituted with Glu. F2 and sF2-(63-116) bind to exosite II on thrombin because both reduce the heparin-catalyzed rate of thrombin inhibition by antithrombin similar to 4-fold. In contrast, only F2 sl ows the uncatalyzed rate of thrombin inactivation by antithrombin. Lik e F2, sF2-(63-116) induces allosteric changes in the active site and e xosite I of thrombin because it alters the rates of thrombin-mediated hydrolysis of chromogenic substrates and displaces fluorescently label ed hirudin(54-65) from active site-blocked thrombin, respectively. Bot h peptides also prolong the thrombin clotting time of fibrinogen in a concentration-dependent fashion, reflecting their effects on the activ e site and/or exosite I. These studies provide further insight into th e regions of F2 that evoke functional changes in thrombin.