Factor VII (F.VII) is a vitamin-K-dependent serine protease required in the early stages of blood coagulation. We describe here a patient with severe F.VII deficiency, with a normal plasma F.VII antigen level (452 ng/mL) and F.VII activity less than 1%, who is homozygous for two defects: a G-->A transition at nucleotide 6055 in exon 4, which results in an Arg-->Gln change at amino acid 79 (R79Q); and a G-->A transition at nucleotide 8961 in exon 6, which results in an Arg-->Gln substitution at amino acid 152 (R152Q). The R79Q mutation occurs in the first epidermal growth factor (EGF)-like domain, which has previously been implicated in binding to tissue factor. The R152Q mutation occurs at a site (Arg 152-Ile 153) that is normally cleaved to generate activated F.VII (F.VIIa). Analysis of purified F.VII from patient plasma shows that the material cannot be activated by F.Xa and cofactors. In addition, in an in vitro binding assay using relipidated recombinant tissue factor, patient plasma showed markedly reduced binding to tissue factor at all concentrations tested. In an effort to separate the contributions of the two mutations, three recombinant variants, wild-type, R79Q, and R152Q, were prepared and analyzed. The R152Q variant had markedly reduced activity in a clotting assay, whereas R79Q showed a milder, concentration-dependent reduction. The R152Q variant exhibited nearly normal binding in the tissue factor binding assay, whereas the R79Q variant had markedly reduced binding. The time course of activation of the R79Q variant was slowed compared with wild-type. Our results suggest that the first EGF-like domain is required for binding to tissue factor and that the F.VII zymogen lacks activity and requires activation for expression of biologic activity.
The intrinsic pathway of coagulation is initiated when zymogcn factor VII binds to its cell surfxe receptor tissue factor to form a catalytic binary complex. Both the activation of factor VII to factor Vlla and the expression ofserine protuase activity of factor VlIa are dependent on factor VII binding to tissue factor lipoprotein. To better understand the molecular basis of these rate-limiting events, the intcractian of zymogcn factor VII and tissue factor was investigated using as probes both a murine monoclonal antibody and a monospecific rabbit antiserum to human factor VII. To measure fnctor Vllti functional activity, a two-stage chromogenic assay was used; an nsstly which measures the factor Xa generated by the activation of factor VII to factor Vlla. Purilicd immunoglobulin from murine moncclonal antibody 231-7. which was shown to bc reactive with amino acid residues 5l-SE of the first epidermal growth factor-like (EGF) domain of human factor VII, inhibited the activation of factor VII to factor VIla in a dose-dependent manner. The mechanism of this inhibition was demonstrated using a novel solid-phase ELISA which quantitatively measured the binding of purified factor VII zymogen to tissue factor adsorbed onto microtitcr wells. Thus, the binding of factor VII rymogcn to immobilized tissue factor was inhibited by antibody 231-7, again in a dose-dcpcndent manner, Similar results were obtained using a monospecific rabbit antiserum to human factor VII which also reacted with the fl-galactosidase fusion proteins containing amino acid residues 51-88 (exon 4) of human factor VII. WC conclude therefore that the CXOII 4.encoded amino acids of the first EGF domain of human factor VII constitute an essential domain participating in the binding of factor VII to tissue factor.
Several lines of evidence indicate that the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by microsomal membranes from carnation flowers is attributable to hydroperoxides generated by membrane-associated lipoxygenase (EC 22.214.171.124). As the flowers senesce, the capability of isolated microsomal membranes to convert ACC to ethylene changes. This pattern of change, which is distinguishable from that for senescing intact flowers, shows a close temporal correlation with levels of lipid hydroperoxides formed by lipoxygenase in the same membranes. Specific inhibitors of lipoxygenase curtail the formation of lipid hydroperoxides and the production of ethylene from ACC to much the same extent, whereas treatment of microsomes with phospholipase A2, which generates fatty-acid substrates for lipoxygenase, enhances the production of hydroperoxides as well as the conversion of ACC to ethylene. Lipoxygenase-generated lipid hydroperoxides mediate the conversion of ACC to ethylene in a strictly chemical system and also enhance ethylene production by microsomal membranes. The data collectively indicate that the in-vitro conversion ACC to ethylene by microsomal membranes of carnation flowers is not reflective of the reaction mediated by the native in-situ ethylene-forming enzyme.
ABSTRACrThe prospect that Ca+2 promotes senescence by activating al has been e d using cut pea (Pisan satiwm co Aska) foliasge a model system. Senescence was induced by severing 17-day-old plats from their roots and mainting them in aqueous test solu in the dark for an additiona 4 days. Treatment of the foliage with the Ca2+ ionophore (A23187) during the senescence-inductio period promoted a laterd phase separtion of the bulk lipids in mirosom membranes indicating that intention of Ca2+ failitates membrae deterioation. In addition, microsomal membres from ionophore-treated tissue displayed an in capacity to convert 1-aminocyclopropane-l-carboxylic acid to ethylene and an increased propensity to produce the superoxide anion (02-). Treatment (23). These compounds are able to translocate Ca2+ from an aqueous phase across the intact bilayer into another aqueous compartment, are permselective with respect to the two major divalent cations of physiological fluids (Ca > Mg), and do not lyse the membrane. This observation is of particular significance in the context of senescing tissues because there is now evidence that free radical-mediated peroxidation of membrane lipids is a characteristic feature of senescence (3). Moreover, products of membrane lipid peroxidation, which would include oxidized di-and trienoic fatty acids, apparently accumulate in membrane matrices with advancing senescence and contribute to lateral phase separations in the lipid bilayers of senescing membranes (1).Exogenous Ca2' has been shown to delay senescence of apple tissue slices, in particular ethylene production and the onset of lipid peroxidation (9). However, this effect has been attributed to the ability of Ca2" to rigidify the surfaces of lipid bilayers by acting as a divalent ligand (7)
The first epidermal growth factor-like domain (EGF-1) of factor VII (FVII) provides the region of greatest contact during the interaction of FVIIa with tissue factor. To understand this interaction better, the conformation-sensitive FVII EGF-1-specific monoclonal antibody (mAb) 231-7 was used to investigate the conformational effects occurring in this region upon both FVII activation and active site occupation. The binding affinity of mAb 231-7 was approximately 3-fold greater for the zymogen state than for the active state; a result affected by the presence of both calcium and the adjacent Gla domain. Once activated, active site inhibition of FVIIa with a variety of chloromethyl ketone inhibitors resulted in a 10-fold range of affinities of FVIIai molecules to mAb 231-7. Gla domain removal eliminated this variation in affinity, suggesting the involvement of a Gla/ EGF-1 interaction in this conformational effect. In addition, the binding of mAb 231-7 to FVIIa EGF-1 stimulated the amidolytic activity of free FVIIa. Taken together, these results imply an allosteric interaction between the FVIIa active site and the EGF-1 domain that is sensitive to variation in active site occupant structure. Thus, these present studies indicate that the conformational change associated with FVII activation and active site occupation involves the EGF-1 domain and suggest potential functional consequences of these changes.Blood coagulation is initiated when circulating FVII 1 in plasma binds to its essential cofactor, the trans-membrane lipoprotein receptor TF. Upon binding to cell surface TF, zymogen FVII can be activated to FVIIa by factor IXa (FIXa) (1), factor Xa (FXa) (2), or in an autocatalytic manner by endogenous FVIIa (3), thus allowing propagation of the coagulation cascade. Activation of FVII occurs upon proteolytic cleavage of the Arg 152 -Ile 153 bond, giving rise to a 152-amino acid light chain (ϳ20 kDa) linked by a disulfide bridge to a 254-amino acid heavy chain (ϳ30 kDa) (4). It is the light chain of FVII that contains the first epidermal growth factor-like domain (EGF-1), a stretch of 37 amino acids with characteristic structure that has been implicated as the principal site of FVII interaction with TF (5-8). The FVII molecule also requires calcium for the expression of optimum activity, one molecule of which is bound in each of the protease domains (9, 10) and the EGF-1 domain at a high affinity site (11), with seven more Ca 2ϩ molecules bound with variable affinity by the Gla domain (12).It has been shown recently that the activation of FVII to FVIIa, as well as occupation of the active site by pseudosubstrate inhibitors, results in conformational changes within the heavy chain with implications on cofactor and substrate interaction (13-15). To date, the conformational effects of these events on the TF-binding EGF-1 domain of the native FVII molecule have not been reported. Relevantly, experiments by Ambrosini et al. (16) have described conformational changes in the region of the EGF-1 domain of the highly homolo...
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