Protease Domain Sodium, Calcium, and Zinc Sites in Factor VIIa: Crystal Structures and Kinetic Studies.

During physiologic coagulation, the factor VIIa (FVIIa)/tissue factor (TF) complex activates FIX and FX. FVIIa consists of a N-terminal γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like (EGF) domains, and a C-terminal serine protease domain. We obtained crystals of FVIIa/soluble TF in the presence of Na + , Rb + , or Choline + (Ch + ) under conditions containing micromolar concentrations of Zn 2+ . Rb + is a large monovalent ion and has been used to identify Na + -sites in several proteins; whereas, Ch + cannot substitute for Na + . The various crystals diffracted from 2.0 to 2.4 A and belonged to the space group P2 1 2 1 2 1 . In the crystal structures, Na + or Rb + in FVIIa coordinates to the carbonyl groups of residues 185 (chymotrypsin numbering), 185A, 221, and 224 as well as to two water molecules. Thus, the Na + -site in FVIIa is similar to that of FXa and activated protein C but not to that of thrombin. Ca 2+ in the protease domain of FVIIa is coordinated to the carboxylates of Glu70 and Glu80 as seen earlier by Banner and coworkers. Additionally, the crystal structures also showed two Zn 2+ -sites, one involving His71 and the other involving His117. The Zn 2+ -sites are unique to FVIIa since the His residues are not present in other proteases. To investigate the role of Na + , Ca 2+ , and Zn 2+ -sites in the protease domain of FVIIa, a series of biochemical and kinetic studies were performed. Na + increased the kcat for hydrolysis of S-2288 (H-D-Ile-Pro-Arg- p -nitroanilide) ~22-fold by FVIIa WT whereas Ca 2+ increased it ~by 230-fold. In the presence of Ca 2+ , Na + had virtually no effect on the hydrolysis of S-2288; however, in the presence of Na + , Ca 2+ increased the kcat ~12-fold. Thus, the increase in kcat by Ca 2+ in the presence or absence of Na + was similar (~250-fold). Further, Na + had no effect on Km whereas Ca 2+ increased it ~3.5-fold. However, the increase by in Km is biologically not pertinent since the Gla and EGF1 domains of FVIIa determine the Km for activation of FIX and FX. Moreover, FVIIa F225P (Na + -site mutant) showed little response to Na + and FVIIa E80V (Ca 2+ -site mutant) showed no response to Ca 2+ in hydrolyzing S-2288. These data indicate that the Na + and Ca 2+ effects observed are due to the occupancy of the protease domain Na + and Ca 2+ sites. Consistent with the Km data, Na + had no effect on the binding of p -aminobenzamidine ( p AB, S1 site probe) to FVIIa WT . Interestingly, Ca 2+ decreased the Ki for p AB binding by ~5-fold indicating that the increase in Km for S-2288 caused by Ca 2+ is not related to the S1 site but rather to the S2 and/or S3/S4 sites in FVIIa. In further studies, Zn 2+ inhibited the potentiation of S-2288 hydrolysis by FVIIa WT with Ki ~1 of μM in the absence and ~30 μM in the presence of Ca 2+ . We conclude that the Na + -site in FVIIa is not linked to the synthetic substrate binding site(s), and that the Ca 2+ -site is linked to the substrate binding site(s). These observations are in contrast to what has been previously observed for FXa and activated protein C. Thus, in the absence of TF, Na + and Ca 2+ are positive regulators for catalysis by FVIIa; whereas, Zn 2+ exerts a negative effect. Conceivably, occupancy of the Na + -site and the protease domain Ca 2+ -site may render FVIIa in a conformation suitable for TF binding and substrate hydrolysis. The local Zn 2+ concentration following release by activated platelets at the site of hemostasis could dampen coagulation as a regulatory mechanism.