?Membrane associated or soluble tissue factor (sTF) in complex with factor (F) VIIa activates FX to FXa and FIX to FIXa. In this process, the gamma-carboxyglutamic acid (Gla) domain and the epidermal growth factor-like domain1 (EGF1) of FX and that of FIX play important roles in interactions with TF/FVIIa complex. This activation process is regulated by tissue factor pathway inhibitor (TFPI), which consists of three Kunitz domains; the first domain (K1) binds to FVIIa and the second domain (K2) binds to FXa (but not to FIXa). Thus, formation of the quaternary TF:VIIa:Xa:TFPI inhibitory complex effectively shuts down the TF- induced FX activation. However, uninhibited TF/FVIIa continues to activate FIX for persistent coagulation. The molecular recognitions involved in the activation of FIX and FX by TF/FVIIa and in the formation of inhibitory complex (TF:VIIa:Xa:TFPI) are incompletely understood. Further, large discrepancies exist between the reported affinity values of sTF (but not for membrane TF) for zymogen FVII, FVIIa and active site inhibited- FVIIa. Our preliminary data indicate that the sTF affinity increases ~7-fold at each step during the conversion of zymogen FVII to FVIIa and to active site inhibited-FVIIa. Further, our NH2-Ile153-carbamylation data support that S1-subsite occupancy and sTF binding to FVIIa protease domain are thermodynamically linked.
In Specific Aim 1, we propose to obtain crystal structures of the zymogen FVIIR152E, and two naturally occurring activated mutants (FVIIaR304Q and FVIIaR304W) each in complex with sTF. We will further biochemically characterize the unactivated as well as activated FVIIR304Q and FVIIR304W mutants. Such data should provide the structure of zymogen FVIIR152E and role of Arg304 in relaying the allosteric signal to the active site of FVIIa for enhanced activity upon TF binding.
In Specific Aims 2 and 3, we propose crystallography and mutagenesis studies to delineate molecular interactions involved in binding of Gla and EGF1 domains of FXa and FIXa to TF/FVIIa, and in binding of FVIIa to the K1 domain and of FXa to the K2 domain of TFPI. We will use two- domain TFPI (TFPI161) that binds to FXa and to FVIIa. Wild-type FXa after binding to TFPI via its active site serine slowly cleaves TFPI. Thus we will use FXa in which its active site Ser195 is mutated to Ala (XaS195A). FXaS195A binds to TFPI very tightly. To understand interactions of Gla/EGF1 domains of FIXa with FVIIa/sTF, we will use a hybrid XaS195A in which its Gla and EGF1 domains are replaced from FIX, termed XaS195A-IXGE. For the proposed studies, we have expressed and purified zymogen FVIIR152E, FVIIR304Q and FVIIR304W. We have also successfully purified the quaternary complexes of (XaS195A)-VIIa-sTF-TFPI161 and (XaS195A-IXGE)-VIIa- sTF-TFPI161 and obtained crystals of (XaS195A)-VIIa-sTF-TFPI161 that diffract to 3.5 but decay after few frames. Crystallization trials/structure determinations of quaternary complexes and mutational studies in the Gla/EGF1 domains of FX and FIX will be carried out simultaneously. The knowledge gained from the proposed studies could aid in the development of new generation of antithrombotics and antihemophilic agents.
During clotting, blood protein factor VIIa complexes with tissue factor (TF) and activates factor X to Xa as well as factor IX to IXa. Activities of VIIa and Xa are shut down by a double headed inhibitor called tissue factor pathway inhibitor (TFPI) and imbalanced inhibition results in either bleeding or thrombosis. The principle aim of this proposal is to understand structural basis of the quaternary complex (VIIa/TF/Xa/TFPI) formation such that its formation could be controlled for prevention of bleeding and thrombosis.
