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: Arg(51)-Thr(52)/Arg(54)-Asp(55) in the fragment 1 (F1) domain (k = 0.4 x 10(4) M-1 s(-1)), Arg(155)-Ser(156) in prothrombin (k = 2 x 10(4) M-1 s(-1)), and Arg(284)-Thr(285) in prethrombin 1 (k = 0.02 x 10(4) M-1 s(-1)). In the presence of 2.5 mM CaCl2, cleavage in fragment 1 (Arg(51)-Thr(52)/Arg(54)-Asp(55)) was not detectable, whereas cleavage at Arg(155)-Ser(156) (i.e., removal of F1) was inhibited 25-fold. Cleavage at Arg(284)-Thr(285) (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 (t(1/2) = similar to 30 s) of this reaction was independent of the meizothrombin concentration (0.1-1 mu M 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 Arg(284)-Thr(285) 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 x 10(-4) s(-1)) and intermolecular autocatalysis (k = 0.2 x 10(3) M-1 s(-1)). Intramolecular autocatalytic conversion of R155A-meizothrombin into thrombin was not affected by the presence of phospholipids (k = 0.8 x 10(-4) s(-1)), whereas intermolecular autocatalysis was accelerated 25-fold (k = 5.6 x 10(3) M-1 s(-1)) by phospholipid vesicles. Since factor Xa/Va-catalyzed conversion of meizothrombin into thrombin occurs with k = 5.5 x 10(8) 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.