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)
The Fab fragment of a monoclonal antibody (mAb) reactive to the N-terminal half (residues 180-310) of the protease domain of human factor IX has been previously shown to inhibit the binding of factor IXa to its cofactor, factor VIIIa. These data suggested that this segment of factor IXa may participate in binding to factor VIIa. We now report that the binding rate (ko.) of the mAb is 3-fold higher in the presence of Ca2' than in its absence for both factors IX and IXa; the half-maximal effect was observed at -300 ,uM Ca2+. Furthermore, the off rate (koff) of the mAb is 10-fold higher for factor IXa than for factor IX with or without Ca2 . Moreover, like the k0n for mAb binding, the incorporation of dansyl-Glu-Gly-Arg chloromethyl ketone (dEGR-CK) into factor IXa was -3 times faster in the presence of Ca2+ than in its absence. Since steric factors govern the ko. and the strength of noncovalent interactions governs the koff, the data indicate that the region of factor IX at residues 180-310 undergoes two separate conformational changes before expression of its biologic activity: one upon Ca2+ binding and the other upon zymogen activation.Furthermore, the dEGR-CK incorporation data suggest that both conformational changes also affect the active site residues. Analyses of the known three-dimensional structures of serine proteases indicate that in human factor IX a high-affinity Ca2+-binding site may be formed by the carboxyl groups of glutamates 235 and 245 and by the main chain carbonyl oxygens of residues 237 and 240. In support of this conclusion, a synthetic peptide including residues 231-265 was shown to bind Ca2+ with a Kd of -500 ,uM. This peptide also bound to the mAb, although with =500-fold reduced affinty. Moreover, like factor IX, the peptide bound to the mAb more strongly (-3-fold) in the presence of Ca2+ than in its absence. Thus, it appears that a part of the epitope for the mAb described above is contained in the proposed Ca2+-binding site in the protease domain of human factor IX. This proposed site is analogous to the Ca2+-binding site in trypsin and elastase, and it may be involved in binding factor IXa to factor VIIIa.Factor IX is a vitamin K-dependent protein that shares sequence homology with other serine protease zymogens of its class. The gene for human factor IX consists of eight exons that code for a leader sequence of 46 amino acids and a mature protein of 415 amino acids (1). A number of processing events occur during biosynthesis of the functional protein. These include cleavage and removal of the leader polypeptide, y-carboxylation of the first 12 glutamate residues, partial P-hydroxylation of Asp-64, and glycosylation of one serine and two asparagine residues (1, 2). The resultant factor IX protein circulates in blood as a zymogen of Mr 57,000 (3). Upon activation by factor XIa/Ca2+ or by factor VIIa/Ca2`/tissue factor, two peptide bonds (Arg-145-Ala-146 and Arg-180-Val-181) are cleaved to yield a two-chain disulfide-linked serine protease, factor IXa, and a 35-residue activat...
Previous studies have identified a putative calcium binding site involving two glutamic acid residues located in the protease domain of coagulation factor IX. Amino acid sequence homology considerations suggest that factor VII (FVII) possesses a similar site involving glutamic acid residues 210 and 220. In the present study, we have constructed site-specific mutants of human factor VII in which Glu-220 has been replaced with either a lysine (E220K FVII) or an alanine (E220A FVII). These mutants were indistinguishable from wild-type factor VII by SDS-PAGE but only possessed 0.1% the coagulant activity of factor VII. Incubation of E220K/E220A FVII with factor Xa resulted in a slower than normal activation rate which eventually yielded a two-chain factor VIIa molecule possessing a coagulant activity of approximately 10% that of wild-type rFVIIa. Amidolytic activity measurements indicated that E220K/E220A FVIIa, unlike wild-type factor VIIa, possessed no measurable amidolytic activity toward the chromogenic substrate S-2288, even at high CaCl2 concentrations. Addition of tissue factor apoprotein, however, induced the amidolytic activity of the mutant molecule to a level 30% of that observed for wild-type factor VIIa. This tissue factor dependent enhancement of E220K/E220A FVIIa amidolytic activity was calcium dependent and required a CaCl2 concentration in excess of 5 mM for maximal rate enhancement. This was in sharp contrast to wild-type factor VIIa which required CaCl2 levels of 0.5 mM for maximal enhancement of tissue factor dependent amidolytic activity. Competition binding experiments suggest that the decrease in amidolytic and coagulant activity observed in the factor VII mutants is a direct result of impaired tissue factor binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Factor EX consists of a y-carboxyglutamic acid-rich domain followed by two epidermal growth factor (EGF)-Iike domains and the C-terminal protease domain. To delineate the function of EGF1 domain in factor IX, we constructed three mutants: an EGF1 domain-deleted mutant (IXAEGF1), a point mutant (IXQsop) with a Gln-50 --Pro change, and a replacement mutant (IXPCEGF1) in which the EGF1 domain of factor IX was replaced by that of protein C. These mutants and wild-type (WT) factor IX (IXwT) were expressed in 293 kidney cells by using pRc/CMV vector. The purified proteins had the same y-carboxyglutamic acid content as the normal plasma factor IX (IXNP) and were activated normally by factor XlaCa2+. In contrast, IXAEGF1 could not be activated by factor VIa-tissue factor-Ca2+, and the activation of IXpcEGF1 in this system was markedly slow; however, IXQSP was activated at a normal rate. In additional studies, both IXwT and IX&EGF, were rapidly converted to their respective IXa forms by factor Xa-phospholipid-Ca2+. Since this reaction has an absolute requirement for phospholipid, it indicates that the mutants under study are not Impared in their interactions with phospholipid. Relative cogilant activities of factor XIa-activated proteins were IXNP, 100%; 1XwT, 75-85%; IXAEGF1, 51%; IXpCEGF1, <2%; and IXQ5pj 6-10%.We conclude that the EGF1 domain of factor IX is required for its activation by factor VIla-tissue factor and that the Gln-50 residue is not critical for this activation. Further, the EGF1 domain of factor IX is not essential for phospholipid binding and for its activation by factor XIa. In addition, the low coagulant activities of the activated mutants indicate that the EGF1 domain is also important in factor X activation by factor IXa-factor VIlaCa2-+-phospholipid complex.The blood coagulation cascade corresponds to an amplified sequence of zymogen to protease conversions that results in the formation of insoluble fibrin from the soluble fibrinogen (1). Factor IX is a vitamin K-dependent plasma protein that participates in the middle phase of blood coagulation. The complete DNA sequence of the human factor IX gene predicts that the protein is synthesized as a precursor molecule of 461 aa containing a 28-residue signal prepeptide and an 18-residue leader propeptide (2). Factor IX is synthesized by the hepatocytes, and both the signal and the propeptide sequences are removed prior to secretion of the protein into blood. During biosynthesis, the protein also undergoes several posttranslational modifications that include )-carboxylation of the first 12 glutamate residues, partial 3-hydroxylation of , and glycosylation of two serine (Ser-53 and Ser-61) and oftwo asparagine residues (Asn-157 and Asn-167)(1-5). The resultant 415-residue protein circulates in blood as a zymogen (Mr 57,000) of serine protease factor IXa and contains 17% carbohydrate by weight (6).During physiologic hemostasis, factor IX may be activated by factor VIIa-Ca2+-tissue factor (TF) complex and by factor XIa-Ca2+ (1). Activation by eit...
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