Autocatalytic Peptide Bond Cleavages in Prothrombin and Meizothrombin

Abstract

During factor Xa-catalyzed prothrombin activation, several other reaction products accumulate as a result of proteolysis of prothrombin and its activation products by thrombin and meizothrombin. Gel electrophoretic analysis and N-terminal sequencing of reaction products showed that in the absence of Ca2+ ions thrombin cleaved the following peptide bonds: Arg51−Thr52/Arg54−Asp55 in the fragment 1 (F1) domain (k = 0.4 × 104 M-1 s-1), Arg155−Ser156 in prothrombin (k = 2 × 104 M-1 s-1), and Arg284−Thr285 in prethrombin 1 (k = 0.02 × 104 M-1 s-1). In the presence of 2.5 mM CaCl2, cleavage in fragment 1 (Arg51−Thr52/Arg54−Asp55) was not detectable, whereas cleavage at Arg155−Ser156 (i.e., removal of F1) was inhibited 25-fold. Cleavage at Arg284−Thr285 (formation of prethrombin 2 des-1−13) was not affected by the presence of Ca2+ ions. Meizothrombin rapidly converted itself into meizothrombin des-F1. The half-life (t1/2 = ∼30 s) of this reaction was independent of the meizothrombin concentration (0.1−1 μM meizothrombin), which is indicative for intramolecular autocatalysis (k = 0.02 s-1 in the presence of 2.5 mM Ca2+ ions). Since the rapid removal of fragment 1 precludes investigations of the cleavage at Arg284−Thr285 in intact meizothrombin, we analyzed the cleavage of this peptide bond in R155A-meizothrombin, a recombinant product that is resistant to autocatalytic removal of the fragment 1 domain. In the absence of phospholipids, R155A-meizothrombin converted itself into thrombin des-1−13 by a combination of intramolecular (k = 0.8 × 10-4 s-1) and intermolecular autocatalysis (k = 0.2 × 103 M-1 s-1). Intramolecular autocatalytic conversion of R155A-meizothrombin into thrombin was not affected by the presence of phospholipids (k = 0.8 × 10-4 s-1), whereas intermolecular autocatalysis was accelerated 25-fold (k = 5.6 × 103 M-1 s-1) by phospholipid vesicles. Since factor Xa/Va-catalyzed conversion of meizothrombin into thrombin occurs with k = 5.5 × 108 M-1 s-1, we conclude that in reaction systems containing purified proteins autocatalysis of meizothrombin hardly contributes to thrombin formation during factor Xa-catalyzed prothrombin activation.