This application is based upon the conviction that activation of factor X by the tissue factor/factor VIIa (TF/VIIa) complex represents a key step in the initiation of coagulation by the extrinsic pathway. Further, factor VIIa has virtually no enzymatic activity prior to its complexation with TF. Tissue factor pathway inhibitor (TFPI) contains two functional Kunitz domains (K1 and K2) and it first inhibits factor Xa via its K2 domain. The resulting Xa TFPI complex then inhibits TF/VIIa via its K1 domain; the light chain of factor Xa also appears to be important for this interaction. Detailed molecular bases for these interactions are only partially understood. In the first two specific aims, we will define the structural basis for the K2 domain to inhibit factor Xa and for the K1 domain to inhibit factor VIIa. By molecular modeling, we have identified the putative residues to be mutated, and the expression systems for factor VII, factor X and TFPI are well established in the applicant's laboratory. Further, we hypothesize that EGF1 domain contained in the light chain of factor Xa plays an important role in binding of Xa TFPI to TF/VIIa. This will be evaluated in this proposal.
In Specific Aim 3, we will define the linkage between TF binding, Na+ binding, Ca2+ binding, and Zn2+ binding in the protease domain, as well as occupancy of the S1 site in the TF induced development of catalytic efficiency in factor VIIa.
In Specific Aim 4, we will crystallographically determine the structure of zymogen VII complexed with soluble TF (sTF), variously active-site inhibited factor VIIa/sTF (e.g., occupancy of S1 site, S1 and S3/S4 sites, S1, S2 and S3/S4 sites, and isolated K1 domain bound to VIIa), and of Gla domainless Xa-K2 complex. We have crystals of zymogen VII/sTF and several variously active-site inhibited VIIa/sTF complexes (including reversible inhibitors). We have collected x-ray intensity data on several of these crystals and are in the processes of refining some of the structures. Since our crystals are obtained in Ca2+/Mg2+/Zn2+-containing solution, we have located and are refining these sites in factor VIIa as well. The crystallographic studies will complement the biochemical studies proposed in the first three specific aims. Our integrated approach is expected to provide new valuable information regarding the assembly of the extrinsic coagulation complex and its regulation by TFPI. The knowledge gained from such studies is essential for developing new generations of antithrombotics.
Showing the most recent 10 out of 11 publications
