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 Zn²⁺. 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 Å and belonged to the space group P2₁2₁2₁. 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. Ca²⁺ 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 Zn²⁺-sites, one involving His71 and the other involving His117. The Zn²⁺-sites are unique to FVIIa since the His residues are not present in other proteases. To investigate the role of Na⁺, Ca²⁺, and Zn²⁺-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 Ca²⁺ increased it ~by 230-fold. In the presence of Ca²⁺, Na⁺ had virtually no effect on the hydrolysis of S-2288; however, in the presence of Na⁺, Ca²⁺ increased the kcat ~12-fold. Thus, the increase in kcat by Ca²⁺ in the presence or absence of Na⁺ was similar (~250-fold). Further, Na⁺ had no effect on Km whereas Ca²⁺ 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 (Ca²⁺-site mutant) showed no response to Ca²⁺ in hydrolyzing S-2288. These data indicate that the Na⁺ and Ca²⁺ effects observed are due to the occupancy of the protease domain Na⁺ and Ca²⁺ sites. Consistent with the Km data, Na⁺ had no effect on the binding of p-aminobenzamidine (pAB, S1 site probe) to FVIIa_WT. Interestingly, Ca²⁺ decreased the Ki for pAB binding by ~5-fold indicating that the increase in Km for S-2288 caused by Ca²⁺ is not related to the S1 site but rather to the S2 and/or S3/S4 sites in FVIIa. In further studies, Zn²⁺ 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 Ca²⁺. We conclude that the Na⁺-site in FVIIa is not linked to the synthetic substrate binding site(s), and that the Ca²⁺-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 Ca²⁺ are positive regulators for catalysis by FVIIa; whereas, Zn²⁺ exerts a negative effect. Conceivably, occupancy of the Na⁺-site and the protease domain Ca²⁺-site may render FVIIa in a conformation suitable for TF binding and substrate hydrolysis. The local Zn²⁺ concentration following release by activated platelets at the site of hemostasis could dampen coagulation as a regulatory mechanism.