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

被引:66
作者
SABHARWAL, AK
BIRKTOFT, JJ
GORKA, J
WILDGOOSE, P
PETERSEN, LC
BAJAJ, SP
机构
[1] ST LOUIS UNIV,SCH MED,DEPT MED,ST LOUIS,MO 63104
[2] ST LOUIS UNIV,SCH MED,DEPT BIOCHEM,ST LOUIS,MO 63104
[3] ST LOUIS UNIV,SCH MED,DEPT PATHOL,ST LOUIS,MO 63104
[4] WASHINGTON UNIV,SCH MED,DEPT BIOCHEM & MOLEC BIOPHYS,ST LOUIS,MO 63110
[5] WASHINGTON UNIV,SCH MED,HOWARD HUGHES MED INST,ST LOUIS,MO 63110
[6] NOVO NORDISK AS,DK-2880 BAGSVAERD,DENMARK
关键词
D O I
10.1074/jbc.270.26.15523
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (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+-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 K-d similar to 230 mu M; 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, 5-2288. The concentration of the peptide required for half-maximal inhibition was similar to 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 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 similar to 7-fold, similar to 100-fold, and similar to 350-fold, respectively, over that of factor VIIa alone; the half-maximal effect was observed at similar to 200 mu M 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 similar to 10(5)-fold.
引用
收藏
页码:15523 / 15530
页数:8
相关论文
共 51 条