Factor VII Central

Abstract

Factor VII is a vitamin K-dependent zymogen of a serine protease that participates in the initial phase of blood coagulation. A factor VII molecular variant (factor VII Central) was identified in a 24-year-old male with severe factor VII deficiency and whose plasma factor VII antigen was 38% of normal, but expressed <1% factor VII procoagulant activity. DNA sequence analysis of the patient's factor VII gene revealed a thymidine to cytidine transition at nucleotide 10907 in exon VIII that results in a novel amino acid substitution of Phe328 to Ser. The patient was homozygous for this mutation, whereas each parent of the patient was heterozygous for this mutation. To investigate the molecular properties of this variant, a recombinant F328S factor VII mutant was prepared and analyzed in relation to wild-type factor VII. F328S factor VII exhibited <1% factor VII procoagulant activity and a 2-fold decreased affinity for tissue factor and failed to activate factor X or IX in the presence of tissue factor following activation by factor Xa. In addition, F328S factor VIIa exhibited no detectable amidolytic activity in the presence of tissue factor. The rate of F328S factor VII activation by factor Xa was markedly decreased relative to the rate of wild-type factor VII activation as revealed by densitometry scanning of SDS gels. Temporal analysis of this reaction by SDS-polyacrylamide gel electrophoresis also revealed the formation of two novel F328S factor VII degradation products (40 and 9 kDa) resulting from factor Xa proteolysis of the Arg315-Lys316 peptide bond in intact F328S factor VII. Computer modeling and molecular dynamics simulations of the serine protease domain of factor VIIa suggested that the inability of F328S factor VIIa to cleave substrates may result from the apparent formation of a hydrogen bond between Tyr377 and Asp338, a residue at the bottom of the substrate-binding pocket important for the interaction of substrate arginine side chains with the enzyme. These findings suggest that Phe328, which is conserved in prothrombin, factor IX, factor X, factor VII, and trypsin, is important for factor VIIa catalysis. Factor VII is a vitamin K-dependent zymogen of a serine protease that participates in the initial phase of blood coagulation. A factor VII molecular variant (factor VII Central) was identified in a 24-year-old male with severe factor VII deficiency and whose plasma factor VII antigen was 38% of normal, but expressed <1% factor VII procoagulant activity. DNA sequence analysis of the patient's factor VII gene revealed a thymidine to cytidine transition at nucleotide 10907 in exon VIII that results in a novel amino acid substitution of Phe328 to Ser. The patient was homozygous for this mutation, whereas each parent of the patient was heterozygous for this mutation. To investigate the molecular properties of this variant, a recombinant F328S factor VII mutant was prepared and analyzed in relation to wild-type factor VII. F328S factor VII exhibited <1% factor VII procoagulant activity and a 2-fold decreased affinity for tissue factor and failed to activate factor X or IX in the presence of tissue factor following activation by factor Xa. In addition, F328S factor VIIa exhibited no detectable amidolytic activity in the presence of tissue factor. The rate of F328S factor VII activation by factor Xa was markedly decreased relative to the rate of wild-type factor VII activation as revealed by densitometry scanning of SDS gels. Temporal analysis of this reaction by SDS-polyacrylamide gel electrophoresis also revealed the formation of two novel F328S factor VII degradation products (40 and 9 kDa) resulting from factor Xa proteolysis of the Arg315-Lys316 peptide bond in intact F328S factor VII. Computer modeling and molecular dynamics simulations of the serine protease domain of factor VIIa suggested that the inability of F328S factor VIIa to cleave substrates may result from the apparent formation of a hydrogen bond between Tyr377 and Asp338, a residue at the bottom of the substrate-binding pocket important for the interaction of substrate arginine side chains with the enzyme. These findings suggest that Phe328, which is conserved in prothrombin, factor IX, factor X, factor VII, and trypsin, is important for factor VIIa catalysis. INTRODUCTIONFactor VII is a single-chain, vitamin K-dependent plasma glycoprotein (50 kDa) that plays a key role in the initiation of the extrinsic pathway of blood coagulation (2Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1616) Google Scholar). The gene for factor VII is located on chromosome 13 at q34-qter, spans 12.8 kilobase pairs, and contains nine exons (3De Grouchy J. Dautzenberg M.D. Turleau C. Beguin S. Chavin-Colin F. Human Genet. 1984; 66: 230-233Crossref PubMed Scopus (56) Google Scholar, 4O'Hara P.J. Grant F.J. Haldeman B.A. Gray C.L. Insley M.Y. Hagen F.S. Murray M.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5158-5162Crossref PubMed Scopus (262) Google Scholar). The gene organization and protein structure for factor VII are similar to other vitamin K-dependent coagulation proteins in that each is a multidomain glycoprotein containing a Gla domain, epidermal growth factor-like or kringle domains, and a C-terminal catalytic domain homologous to trypsin. Inherited factor VII deficiency is a rare autosomal recessive disorder and is phenotypically heterogeneous. The clinical features are variable with a rather poor correlation between reported procoagulant activity and bleeding tendency (5Ragni M.V. Lewis J.H. Spero J.A. Hasiba U.C. Am. J. Hematol. 1981; 10: 79-88Crossref PubMed Scopus (127) Google Scholar, 6Mariani G. Mazzucconi M.G. Hermans J. Ciavarella N. Failla A. Hassan H.J. Mannucci P.M. Nenci C.G. Orlando M. Romoli D. Mandelli F. Br. J. Haematol. 1981; 48: 7-14Crossref PubMed Scopus (32) Google Scholar, 7Triplett D.A. Brandt J.T. Batard M.A. Dixon J.S. Fair D.S. Blood. 1985; 66: 1284-1287Crossref PubMed Google Scholar). Factor VII deficiency is classified as either CRM− (type 1) or CRM+ (type 2) based upon the absence or presence, respectively, of a disparity between the activity and antigen levels (7Triplett D.A. Brandt J.T. Batard M.A. Dixon J.S. Fair D.S. Blood. 1985; 66: 1284-1287Crossref PubMed Google Scholar). In the majority of factor VII-deficient patients, plasma levels of factor VII activity diminish in parallel with immunoreactive factor VII antigen. A discrepancy between factor VII coagulant activity and factor VII antigen levels has been found in a few patients, suggesting the presence of a dysfunctional factor VII (reviewed in 8Tuddenham E.G.D. Pemberton S. Cooper D.N. Thromb. Haemostasis. 1995; 74: 313-321Crossref PubMed Scopus (55) Google Scholar). In this report, we describe the molecular basis of a severe factor VII deficiency in a 24-year-old male whose plasma factor VII activity was <1%, while his plasma factor VII antigen was 38% of normal. We have analyzed the factor VII gene sequence of this patient, designated as factor VII Central, and demonstrate that the patient was homozygous for a single point missense mutation in exon VIII in the catalytic domain, resulting in a Phe to Ser substitution at amino acid 328. We have expressed and purified this variant from baby hamster kidney cells transfected with the cDNA coding for this mutant and demonstrate that the isolated mutant exhibits <1% procoagulant activity, no demonstrable amidolytic or proteolytic activity following activation by factor Xa, and decreased affinity for tissue factor, thus recapitulating the patient's factor VII phenotype. In addition, the F328S factor VII mutant, as well as the isolated patient's factor VII, undergoes proteolytic degradation by factor Xa at Arg315-Lys316, suggesting that the Phe328 to Ser substitution creates an exposed surface loop in this region readily accessible for cleavage by factor Xa, further reducing the catalytic activity of this clotting factor.DISCUSSIONWe describe the molecular basis underlying a severe factor VII deficiency designated as factor VII Central. The factor VII Central propositus was homozygous for a single point missense mutation in exon VIII in the catalytic domain of the molecule, resulting in a phenylalanine to serine substitution at residue 328. As a result of this mutation, the patient's plasma factor VII clotting activity was <1% of normal, while his plasma factor VII antigen concentration was ∼38% of normal.In an effort to functionally characterize the patient's factor VII molecule, we constructed the F328S factor VII variant, expressed this mutant in baby hamster kidney cells, and purified it from serum-free conditioned media on a single-step, calcium-dependent immunoaffinity column. The specific clotting activity of the purified F328S factor VII preparation was <20 units/mg, in comparison with recombinant wild-type human factor VII, which exhibited a specific activity of ∼2000 units/mg. Incubation of F328S factor VII with a complex of factor Xa-calcium-phospholipids resulted in the cleavage of the Arg315-Lys316 peptide bond, in addition to cleavage of the Arg152-Ile153 bond, which leads to the activation of wild-type factor VII. An identical temporal cleavage pattern for F328S factor VII was observed when factor Xa was substituted with a purified preparation of a factor VII activator derived from Taipan snake venom (32Nakagaki T. Lin P. Kisiel W. Thromb. Res. 1992; 65: 105-116Abstract Full Text PDF PubMed Scopus (29) Google Scholar), strongly suggesting that cleavage of the Arg315-Lys316 peptide bond was not unique to factor Xa (data not shown). As revealed by densitometry scans of temporal aliquots of the incubation mixture, the rate of F328S factor VIIa formation was 10-20% of that observed for wild-type factor VII. At present, it is unclear as to whether this decreased rate of F328S factor VII activation by factor Xa relates to the apparent inability of factor Xa to cleave the 40-kDa fragment consisting of residues 1-315, inasmuch as no band corresponding to residues 152-315 (∼18 kDa) was observed in reduced samples of this incubation mixture. In spite of the formation of some two-chain F328S factor VIIa, no increase in coagulant activity was detectable in these incubation mixtures, whereas comparable incubation mixtures containing wild-type factor VII and factor Xa-calcium-phospholipids generated a 25-30-fold increase in coagulant activity that correlated with the conversion of zymogen factor VII to factor VIIa. Of additional importance, no amidolytic activity for S-2288 was observed in aliquots of F328S factor VIIa following incubation with soluble tissue factor apoprotein. Essentially identical results to those observed for F328S factor VII with respect to factor Xa-mediated cleavage and specific coagulant activity were observed using the factor VII (1-2 μg) preparation purified from small amounts of the patient's plasma by a combination of barium citrate adsorption, EDTA elution, and immunoaffinity chromatography, providing strong evidence that our F328S factor VII preparation recapitulated the structure-function characteristics of the patient's factor VII molecule.The structure-function basis for the inability of F328S factor VIIa to express proteolytic and particularly amidolytic activity remains enigmatic. While direct tissue factor binding assays revealed a 2-fold decrease in affinity of F328S factor VII for tissue factor apoprotein in relation to wild-type factor VII, these differences could not account for the complete absence of proteolytic and amidolytic activity in F328S factor VIIa. Our experimental evidence is consistent with a conformational change in the vicinity of the Cys310-Cys329 loop induced by the Phe to Ser mutation, resulting in the surface expression of the Arg315-Lys316 peptide bond and a mild disturbance of the factor VII-tissue factor interaction. In the latter case, crystallographic studies by Banner et al. (33Banner D.W. D'Arcy A. Chene C. Winkler F.K. Guha A. Konigsberg W.H. Nemerson Y. Kirchhofer D. Nature. 1996; 380: 41-46Crossref PubMed Scopus (685) Google Scholar) revealed that Asp309 in the heavy chain of factor VIIa directly interacts with Tyr94 in tissue factor apoprotein through three hydrogen bonds. Thus, it is entirely conceivable that the Phe to Ser substitution and the putative surface expression of the Arg315-Lys316 peptide bond impact on this particular molecular interaction and slightly decrease the affinity of F328S factor VII for tissue factor.To obtain insight into the complete lack of proteolytic and amidolytic activity of F328S factor VIIa, we used homology modeling, based on the published x-ray structure of human factor Xa (24Padmanabhan K. Padmanabhan K.P. Tulinsky A. Park C.H. Bode W. Huber R. Blankenship D.T. Cardin A.D. Kisiel W. J. Mol. Biol. 1993; 232: 947-966Crossref PubMed Scopus (401) Google Scholar), and performed dynamics simulations on both wild-type and F328S factor VII. The disposition of the critical residues (His193, Asp242, and Ser344) in the active site of factor Xa and VIIa structures appears to be virtually superimposable (data not shown). In wild-type factor VII, the catalytic triad is located immediately below a loop that separates it from Phe328. The aromatic ring of Phe328 stacks between Tyr377 and His348, while the aromatic hydroxyl of Tyr377 forms a strong hydrogen bond with Ser339. Upon inspection of the mutant, Ser328 is situated in a mostly hydrophobic environment without any hydrogen-bonding counterparts in the vicinity.In molecular simulation runs, the catalytic triad domain of wild-type factor VIIa appeared quite stable. The hydrogen bond between Ser344 and His193 weakened or broke occasionally, but was re-formed with favorable distances and acceptable dihedral angles. The hydrogen bond distances increased up to 5 Å, but the bond breakage did not last for more than 1 ps. However, this was not observed for the hydrogen bond between Ser344 and Asp242, as this interaction was retained throughout the simulations with very little deviation from the original distance and geometry. In addition, Tyr377 appeared to be rotating away from Ser339 in wild-type factor VII, thus resulting in a breakage of the hydrogen bond interaction with Ser339 (Fig. 5A). This motion of Tyr377, however, strengthens its stacking interaction with Phe328, for the phenol chromophore is oriented roughly perpendicular to the aromatic Phe328, which is typical for interactions of this type. The mutant enzyme displayed a similar behavior throughout the simulations, with transient weakening or breakage followed by re-formation. However, examination of the environment surrounding Ser328 provided some clues as to the local motions related to the single mutation. In this regard, Tyr377 no longer formed a hydrogen bond with Ser339, but rather interacted with Asp338, forming a strong linear hydrogen bond, while Ser328 rotated toward His348 (Fig. 5B). Thus, based upon these types of analyses, we speculate that the Phe to Ser substitution at residue 328 in factor VII Central results in the formation of a new conformation in the molecule such that Tyr377 interacts with Asp338, a critical residue at the bottom of the substrate-binding pocket of the enzyme (34Hagen F.S. Gray C.L. O'Hara P. Grant F.J. Saari G.C. Woodbury R.G. Hart C.E. Insley M. Kisiel W. Kurachi K. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2412-2416Crossref PubMed Scopus (318) Google Scholar), and thereby precludes substrate binding. Whether the interaction of F328S factor VII with tissue factor induces other changes in this conformation that reduce substrateenzyme interactions further is unknown and will require additional studies. INTRODUCTIONFactor VII is a single-chain, vitamin K-dependent plasma glycoprotein (50 kDa) that plays a key role in the initiation of the extrinsic pathway of blood coagulation (2Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1616) Google Scholar). The gene for factor VII is located on chromosome 13 at q34-qter, spans 12.8 kilobase pairs, and contains nine exons (3De Grouchy J. Dautzenberg M.D. Turleau C. Beguin S. Chavin-Colin F. Human Genet. 1984; 66: 230-233Crossref PubMed Scopus (56) Google Scholar, 4O'Hara P.J. Grant F.J. Haldeman B.A. Gray C.L. Insley M.Y. Hagen F.S. Murray M.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5158-5162Crossref PubMed Scopus (262) Google Scholar). The gene organization and protein structure for factor VII are similar to other vitamin K-dependent coagulation proteins in that each is a multidomain glycoprotein containing a Gla domain, epidermal growth factor-like or kringle domains, and a C-terminal catalytic domain homologous to trypsin. Inherited factor VII deficiency is a rare autosomal recessive disorder and is phenotypically heterogeneous. The clinical features are variable with a rather poor correlation between reported procoagulant activity and bleeding tendency (5Ragni M.V. Lewis J.H. Spero J.A. Hasiba U.C. Am. J. Hematol. 1981; 10: 79-88Crossref PubMed Scopus (127) Google Scholar, 6Mariani G. Mazzucconi M.G. Hermans J. Ciavarella N. Failla A. Hassan H.J. Mannucci P.M. Nenci C.G. Orlando M. Romoli D. Mandelli F. Br. J. Haematol. 1981; 48: 7-14Crossref PubMed Scopus (32) Google Scholar, 7Triplett D.A. Brandt J.T. Batard M.A. Dixon J.S. Fair D.S. Blood. 1985; 66: 1284-1287Crossref PubMed Google Scholar). Factor VII deficiency is classified as either CRM− (type 1) or CRM+ (type 2) based upon the absence or presence, respectively, of a disparity between the activity and antigen levels (7Triplett D.A. Brandt J.T. Batard M.A. Dixon J.S. Fair D.S. Blood. 1985; 66: 1284-1287Crossref PubMed Google Scholar). In the majority of factor VII-deficient patients, plasma levels of factor VII activity diminish in parallel with immunoreactive factor VII antigen. A discrepancy between factor VII coagulant activity and factor VII antigen levels has been found in a few patients, suggesting the presence of a dysfunctional factor VII (reviewed in 8Tuddenham E.G.D. Pemberton S. Cooper D.N. Thromb. Haemostasis. 1995; 74: 313-321Crossref PubMed Scopus (55) Google Scholar). In this report, we describe the molecular basis of a severe factor VII deficiency in a 24-year-old male whose plasma factor VII activity was <1%, while his plasma factor VII antigen was 38% of normal. We have analyzed the factor VII gene sequence of this patient, designated as factor VII Central, and demonstrate that the patient was homozygous for a single point missense mutation in exon VIII in the catalytic domain, resulting in a Phe to Ser substitution at amino acid 328. We have expressed and purified this variant from baby hamster kidney cells transfected with the cDNA coding for this mutant and demonstrate that the isolated mutant exhibits <1% procoagulant activity, no demonstrable amidolytic or proteolytic activity following activation by factor Xa, and decreased affinity for tissue factor, thus recapitulating the patient's factor VII phenotype. In addition, the F328S factor VII mutant, as well as the isolated patient's factor VII, undergoes proteolytic degradation by factor Xa at Arg315-Lys316, suggesting that the Phe328 to Ser substitution creates an exposed surface loop in this region readily accessible for cleavage by factor Xa, further reducing the catalytic activity of this clotting factor.