W0092

Crystal Structure of the Factor VIIa - Tissue Factor Complex Inhibited by a Kunitz-Type Inhibitor. R. St. Charles, E. Zhang, A. Tulinsky, Department of Chemistry, Michigan State University, East Lansing, MI

Binding of the freely circulating blood coagulation protein Factor VII (FVII) to the extracellular component of the integral membrane protein tissue factor (TF) exposed during vascular injury is the primary biochemical event initiating blood coagulation via the extrinsic pathway. Formation of the complex results in an activated form of FVII (FVIIa) which is then capable of efficiently catalyzing the conversion of factors IX and X to their biologically active forms. As a critical player during the early stages of blood coagulation, FVIIa represents a potentially important target for antithrombotic therapy, and the search for effective inhibitors of this multidomain serine protease is currently the focus of much research.

A ternary complex of FVIIa bound to both the soluble extracellular domain of tissue factor and the engineered Kunitz-type inhibitor, CVS 5L15, has been crystallized and its crystal structure solved to 2.1 Å resolution. CVS 5L15 is a derivative of bovine pancreatic trypsin inhibitor (BPTI) differing in sequence at eight positions. The inhibitor is bound to the active site of FVIIa in canonical fashion, similar to trypsin-bound BPTI, with its primary binding loop interacting in the active site cleft of the enzyme. Most of the interactions unique to the complex are polar in nature and occur peripheral to the active site. These interactions are capable of rationalizing the tight binding characteristics (Ki = 0.4 nM) and specificity of the inhibitor for FVIIa when compared with BPTI and other Kunitz-type inhibitors. Polar interactions with residues Asp 11, His 18, and Glu 46 of the inhibitor appear to be particularly important. These results offer insights in understanding the structural basis for the tissue factor pathway inhibitor-based inhibition of FVIIa.