The elusive role of the potential factor X cation-binding exosite-1 in substrate and inhibitor interactions.
A number of studies suggest that blood-clotting factor X (FX) uses secondary site(s) to interact (as a substrate) with its activators. Numerous pieces of evidence also imply that, within prothrombinase (as an enzyme), activated FX (FXa) uses exosite(s) for cofactor Va and/or prothrombin recognition. Similarly, FXa exosite(s) seem to govern interaction with inhibitors. An obvious difference between FXa and thrombin resides within a region called exosite-1: positively charged in thrombin and clearly of opposite polarity in FXa. To investigate the role of this potential cation-binding exosite, we prepared a series of mutants within loops 34-40 and 70-80 of FX. Overall, the mutations induced relatively subtle, non-synergistic modulation. The potential exosite was dispensable for FX activation and is unlikely to constitute a critical region for factor Va binding, albeit it is clearly important for prothrombin activation. Our data also implicate loop 34-40 of FXa in the interaction with the tissue factor pathway inhibitor, in prevention of plasminogen activator inhibitor-1 binding, and in tempering inhibition by heparin-activated antithrombin. Compared with FX, mutants with reduced electrostatic potential potentiated thrombin production in FX-depleted plasma, whereas mutants with inverted electrostatic potential impeded clotting. Despite the definite consequences observed, disruption of the potential cation-binding exosite of FX had rather weak effects, far from what would be expected if this region was as crucial as in thrombin.
Bianchini
Elsa P
EP
INSERM U428, Faculté de Pharmacie, Université Paris V, 75270 Paris Cedex 06, France.
Pike
Robert N
RN
Le Bonniec
Bernard F
BF
D000595:Amino Acid Sequence; D000991:Antithrombins / Q000737:chemistry; D002412:Cations; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D015943:Factor Va / Q000737:chemistry; D005170:Factor X / Q000737:chemistry* / Q000502:physiology; D015951:Factor Xa / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000737:chemistry; D055672:Static Electricity; D013917:Thrombin / Q000737:chemistry*; D013997:Time Factors
D000991:Antithrombins; D002412:Cations; D018076:DNA, Complementary; D015943:Factor Va; D011516:Prothrombin; D005170:Factor X; D006493:Heparin; D013917:Thrombin; D015951:Factor Xa