High affinity Ca2+-binding site in the serine protease domain of human factor VIIa and its role in tissue factor binding and development of catalytic activity

Abstract

Factor VIIa, in the presence of Ca2+ and tissue factor (TF), initiates the extrinsic pathway of blood coagulation. The light chain (amino acids 1-152) of factor VIIa consists of an N-terminal γ-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor-like domains, whereas the heavy chain (amino acids 153-406) contains the serine protease domain. In this study, both recombinant factor VIIa (rVIIa) and factor VIIa lacking the Gla domain were found to contain two high-affinity (Kd ∼150 μM) Ca2+ binding sites. The rVIIa also contained ∼6-7 low-affinity (Kd ∼1 mM) Ca2+-binding sites. By analogy to other serine proteases, one of the two high affinity Ca2+-binding sites in factor VIIa may be formed involving Glu-210 and Glu-220 of the protease domain. In support of this, a synthetic peptide composed of residues 206-242 of factor VIIa bound one Ca2+ with Kd ∼230 μM; however, Ca2+ binding was observed only in Tris buffer (pH 7.5) containing 1 M NaCl and not in buffer containing 0.1 M NaCl. In both low or high salt ±Ca2+, the peptide existed as a monomer as determined by sedimentation equilibrium measurements and had no detectable secondary structure as determined by CD measurements. This indicates that subtle changes undetectable by CD may occur in the conformation of the peptide that favor calcium binding in high salt. In the presence of recombinant TF and 5 mM Ca2+, the peptide inhibited the amidolytic activity of rVIIa toward the synthetic substrate, S-2288. The concentration of the peptide required for half-maximal inhibition was ∼5-fold higher in the low salt buffer than that in the high salt buffer. From direct binding and competitive inhibition assays of active site-blocked 125I-rVIIa binding to TF, the Kd for peptide-TF interaction was calculated to be ∼15 μMin the high salt and ∼55 μM in the low salt buffer containing 5 mM Ca2+. Moreover, as inferred from S-2288 hydrolysis, the Kd for VIIa·TF interaction was ∼1.5 μM in the absence of Ca2+, and, as inferred from factor X activation studies, it was ∼10 pM in the presence of Ca2+. Thus, Ca2+ decreases the functional Kd of VIIa·TF interaction ∼150,000-fold. Furthermore, the amidolytic activity of VIIa·Ca2+, VIIa·TF, and VIIa·Ca2+·TF was increased ∼7-fold, ∼100-fold, and ∼350-fold, respectively, over that of factor VIIa alone; the half-maximal effect was observed at ∼200 μM Ca2+ when added. We conclude that TF can interact with the protease domain (and possibly other domains) of factor VIIa in the absence of Ca2+ and that the protease domain Ca2+-binding site, in part, enhances this interaction ∼105-fold.