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 gamma-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 approximately 150 microM) Ca2+ binding sites. The rVIIa also contained approximately 6-7 low-affinity (Kd approximately 1 mM) Ca(2+)-binding sites. By analogy to other serine proteases, one of the two high affinity Ca(2+)-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 approximately 230 microM; 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 approximately 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 approximately 15 microM in the high salt and approximately 55 microM in the low salt buffer containing 5 mM Ca2+. Moreover, as inferred from S-2288 hydrolysis, the Kd for VIIa.TF interaction was approximately 1.5 microM in the absence of Ca2+, and, as inferred from factor X activation studies, it was approximately 10 pM in the presence of Ca2+. Thus, Ca2+ decreases the functional Kd of VIIa.TF interaction approximately 150,000-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy

“…in the interaction with T F (Sakai et al, 1990;Wildgoose et al, 1990Wildgoose et al, , 1992Toomey et al, 1991;Clarke et al, 1992;Higashi et al, 1992Higashi et al, , 1994Kazama et al, 1993;Kumar & Fair, 1993;Petersen et al, 1994;Chang et al, 1995;Sabharwal et al, 1995). The recently published X-ray crystallographic structure of the fVI1a:TF complex corroborates the biochemical data and, in addition, identifies a small contribution from the second EGF-like domain (Banner et al, 1996).…”

Involvement of the hydrophobic stack residues 39–44 of factor VIIa in tissue factor interactions

“…Regions all over the factor VIIa molecule have been implicated as possible TF binding sites. The fact that the amidolytic activity of factor VIIa is stimulated by TF and Ca z+ [6] seems to indicate binding of these ligands to the catalytic domain, and substantiation of this interpretation is obtained by several independent observations [5,[7][8][9][10][11]. TF interaction with the catalytic domain does, however, not exclude additional involvement of other regions, and the EGF domains of factor VIIa have been implicated as possible TF binding sites [12][13][14].…”

“…It has been shown that binding of TF to factor VII~ in the presence of Ca 2+ induces a pronounced enhancement of the activity with peptidyl substrates [25], and this [5,16,22,[26][27][28] as well as other methods [29,30] have been used for the assessment of the dissociation constant, KTF. Our results (Table 1) on intact FVII, intreaction with detergent solubilized TF (KTF = 0.6 + 0.1 nM) and with TFl_21 s (Kaw = 6.0 -+ 0.4 nM) agrees reasonably well with previous values [22,27].…”

“…Ca2+-binding sites has been identified in the Gla-domain, the first EGF domain, and in the protease domain [4,5]. Binding of Ca2+-ions to one or more of these sites appears to be essential for factor VII interaction with TF.…”

Calcium‐Binding Properties of the Third and Fourth Epidermal‐Growth‐Factor‐Like Modules in Vitamin‐K‐Dependent Protein S

“…The isolated modules have been found to bind Ca2+ with low affinity (Kd 0.5-5 mM) [19,201. These proteins contain a second Gla-independent Caz+-binding site that is located in the serine-protease module and accounts for the Ca2+-induced fluorescence quenching observed in Gla-domainless protein C [21, 221, factors VII [23], IX [24], and X [25]. Determination of the structure of the EGF module from factor X, with and without Caz+, allowed identification of the side chains of Asp46, Gln49 and Hya63, and the backbone carbonyls of Gly47 and Gly64 as ligands to the Caz+ [26, 271. These residues correspond to positions 160, 163, 178 and 161 and 179, in the third EGF module of protein S (Fig.…”