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.
INSERM U428, Faculté de Pharmacie, Université Paris V, 75270 Paris Cedex 06, France.
Elsa P
EP
Bianchini
Robert N
RN
Pike
Bernard F
BF
Le Bonniec
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