The activation of prothrombin by the prothrombinase complex yields the protease thrombin (IIa) and the activation peptide fragment 1.2 (F1.2). IIa and F1.2 are established to interact noncovalently through the fragment 2 (F2) domain. Since membrane binding is mediated by the fragment 1 domain, the interaction between IIa and F1.2 could allow the newly produced protease to remain bound to the membrane surface and modulate its biological functions. In this case, the feedback action of thrombin to cleave the Arg155-Ser156 site between the F1 and F2 domains would be expected to play a key regulatory role in releasing newly formed thrombin into solution. We have investigated the ability of F1.2 to localize nascent IIa to the membrane surface and the role of IIa-mediated feedback cleavages in regulating this phenomenon. We used a recombinant prothrombin mutant in which the two thrombin cleavage sites were rendered uncleavable by mutation to Gln (IImut), a prothrombin derivative (IIS195A) that yields catalytically inactive product, and a prothrombin derivative IImut/Q271, which yields stable meizothrombin. The binding of IIa and prethrombin 2 (P2) produced from IIS195A to F1.2, examined by isothermal titration calorimetry, yielded: F1.2 plus IIaS195A (Kd 4.5±0.3 μM, n 0.93±0.02, ΔH −13.6±0.4 kcal.mol-1), and F1.2 plus P2S195A (Kd 156±11 nM, n 1.17±0.01, ΔH −28.6±0.2 kcal.mol-1). The relatively weak interaction between IIaS195A and F1.2 indicates that only a modest fraction of IIa will be bound to F1.2 even at the highest possible concentrations achievable in vivo. In contrast, binding of F1.2 to the zymogen P2 is of considerably higher affinity, implying that conversion of zymogen to protease results in a ∼25-fold change in affinity for F1.2 binding. Membrane retention of products during prothrombin activation catalyzed by prothrombinase was studied at the physiologic prothrombin concentration by assessing the fate of membrane-bound species using light scattering correlated with progress curves for cleavage established by SDS-PAGE and quantitative densitometry. In the absence of steps taken to inhibit thrombin produced in situ, prothrombin cleavage by prothrombinase and the associated cleavage of substrate and products by thrombin was accompanied by a rapid decrease in light scattering to a level expected for membrane-bound F1. Surprisingly, similar light scattering profiles were observed in the activation of IImut and IIS195A even in the absence of feedback proteolysis of F1.2. In these cases, the light scattering signal decreased to a level expected for F1.2 bound to membranes and correlated with the appearance of F1.2. In contrast, activation studies with IImut/Q271 revealed that formation of stable meizothrombin as a limiting product produced essentially no decrease in light scattering indicating that this species remains membrane-bound. Thus, covalent linkage with the F1 domain allows meizothrombin to remain membrane bound with potentially modulated function. In addition, the higher affinity interaction between P2 and F1.2 likely plays a very significant role in influencing prothrombinase function under conditions when P2 is produced as an intermediate. In contrast, as a result of the much weaker interaction between IIa and F1.2, membrane binding mediated by F1.2 likely plays a minor role in localizing IIa to the membrane surface during activation of physiological concentrations of prothrombin irrespective of feedback reactions catalyzed by newly formed thrombin.

Author notes

Disclosure: No relevant conflicts of interest to declare.