HIGH-AFFINITY CA2-BINDING SITE IN THE SERINE-PROTEASE DOMAIN OF HUMANFACTOR VIIA AND ITS ROLE IN TISSUE FACTOR-BINDING AND DEVELOPMENT OF CATALYTIC ACTIVITY()

Factor VIIa, in the presence of Ca2+ and tissue factor (TF), initiates the extrinsic pathway of blood coagulation. The light chain (amino ac ids 1-152) of factor VIIa consists of an N terminal gamma-carboxygluta mic acid (Gla) domain followed by two epidermal growth factor-like dom ains, whereas the heavy chain (amino acids 153-406) contains the serin e protease domain. In this study, both recombinant factor VIIa (rVIIa) and factor VIIa lacking the Gla domain were found to contain two high -affinity (K-d similar to 150 mu M) Ca2+ binding sites. The rVIIa also contained similar to 6-7 low-affinity (K-d similar to 1 mM) Ca2+-bind ing sites. By analogy to other serine proteases, one of the two high a ffinity Ca2+-binding sites in factor VIIa may be formed involving Glu- 210 and Glu-220 of the protease domain. In support of this, a syntheti c peptide composed of residues 206-242 of factor VIIa bound one Ca2+ w ith K-d similar to 230 mu M; however, Ca2+ binding was observed only i n Tris buffer (pH 7.5) containing 1 M NaCl and not in buffer containin g 0.1 M NaCl. In both low or high salt +/- Ca2+, the peptide existed a s a monomer as determined by sedimentation equilibrium measurements an d had no detectable secondary structure as determined by CD measuremen ts. This indicates that subtle changes undetectable by CD may occur in the conformation of the peptide that favor calcium binding in high sa lt. In the presence of recombinant TF and 5 mM Ca2+, the peptide inhib ited the amidolytic activity of rVIIa toward the synthetic substrate, 5-2288. The concentration of the peptide required for half-maximal inh ibition was similar to 5-fold higher in the low salt buffer than that in the high salt buffer. From direct binding and competitive inhibitio n assays of active site-blocked I-125-rVIIa binding to TF, the K-d for peptide-TF interaction was calculated to be similar to 15 mu M in the high salt and similar to 55 pM in the low salt buffer containing 5 mM Ca2+. Moreover, as inferred from S-2288 hydrolysis, the K-d for VIIa TF interaction was similar to 1.5 mu M in the absence of Ca2+, and, as inferred from factor X activation studies, it was similar to 10 pM in thepresence of Ca2+. Thus, Ca2+ decreases the functional K-d of VIIa- . TF interaction similar to 150,000-fold. Furthermore, the amidoIytic activity of VIIa . Ca2+, VIIa . TF, and VIIa . Ca2+-TF was increased s imilar to 7-fold, similar to 100-fold, and similar to 350-fold, respec tively, over that of factor VIIa alone; the half-maximal effect was ob served at similar to 200 mu M Ca2+ when added. We conclude that TF can interact with the protease domain (and possibly other domains) of fac tor VIIa in the absence of Ca2+ and that the protease domain Ca2+-bind ing site, in part, enhances this interaction similar to 10(5)-fold.