Preparation and properties of derivatives of bovine factor X and factor Xa from which the gamma-carboxyglutamic acid containing domain has been removed

Limited proteolysis of bovine blood coagulation Factor X by chymotrypsin produces a derivative in which the light chain is cleaved between Tyr 44 and Lys 45. Two peptide products, residues 1-44 of the Factor X light chain and a modified zymogen, Factor X(-GD) have been isolated and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, elution behavior on anion-exchange chromatography, amino acid composition, and by partial amino acid sequence determination. Factor X(-GD) no longer contains the 12 gamma-carboxyglutamic acid residues of the native zymogen and thus serves as a model for investigation of the properties conferred on Factor X by the presence of gamma-carboxyglutamic acid. Cleavage of Factor X at Tyr 44 by chymotrypsin is inhibited by Ca2+ and Mg2+ ions. Factor X(-GD) is activated by the coagulation factor activator of Vipera russellii venom, but at less than 1% of the rate of activation of native Factor X. The susceptibility of Tyr 44 to chymotryptic cleavage implies that this residue is on the surface of the light chain of Factor X. Factor Xa(-GD) is indistinguishable from native Factor Xa in its activity on Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, on prothrombin alone, and on prothrombin plus Factor Va. In the presence of phospholipid the rate of prothrombin activation catalyzed by Factor Xa(-GD) is the same as in the absence of phospholipid.     

Localization of the structural difference between bovine blood coagulation factors X1 and X2 to tyrosine 18 in the activation peptide

Bovine Factor X is isolated in two chromatographically separable forms, Factor X1 and Factor X2. Whereas only a single form of Factor Xa, the active protease, exists, the activation peptides also exist as two chromatographically distinct species. These peptides have been shown to differ at a tyrosyl residue by ultraviolet spectrophotometry, and in their composition after alkaline hydrolysis. On the basis of the spectral properties, and elution position of the modified tyrosine on Dowex 1 columns and on an amino acid analyzer, it has been concluded that Factor X2 contains a tyrosyl-O-SO4 residue at position 18 in the activation peptide whereas Factor X1 contains only tyrosine. Alternative explanations such as differences in carbohydrate composition, differences in phosphate content, or differences in the number of gamma-carboxyglutamic acid residues were demonstrated to be unrelated to the difference in chromatographic behavior between bovine Factors X1 and X2.     

Effect of propeptide mutations on post-translational processing of factor IX. Evidence that beta-hydroxylation and gamma-carboxylation are independent events

Post-translational processing of Factor IX includes glycosylation, cleavage of the signal peptide and propeptide, vitamin K-dependent carboxylation of specific glutamic acid residues to form gamma-carboxyglutamic acid, and beta-hydroxylation of aspartic acid at residue 64 to form beta-hydroxyaspartic acid. The human Factor IX cDNA coding sequence was modified in the propeptide region (residue -18 to -1) using oligonucleotide-directed site-specific mutagenesis, and the altered Factor IX cDNA was expressed in Chinese hamster ovary cells. The effects of the mutations on proteolytic processing, gamma-carboxylation, and beta-hydroxylation were assessed by direct structural analysis. After purification, the molecular weight of each of the recombinant Factor IX species and its NH2-terminal amino acid sequence were shown to be identical to those of plasma Factor IX. gamma-Carboxyglutamic acid and beta-hydroxyaspartic acid analyses revealed that recombinant wild-type Factor IX contained 9.2 gamma-carboxyglutamic acid and 0.3 beta-hydroxyaspartic acid residues/molecule compared with 11.4 gamma-carboxyglutamic acid and 0.39 beta-hydroxyaspartic acid residues in plasma Factor IX. When the 18-residue propeptide was deleted or when the cells were grown in the presence of sodium warfarin, secreted Factor IX contained no detectable gamma-carboxyglutamic acid but 0.36 and 0.40 residues of beta-hydroxyaspartic acid, respectively. Point mutations leading to substitution of alanine for phenylalanine at residue -16 or glutamic acid for alanine at residue -10 contained 0.2 and 1.7 gamma-carboxyglutamic acid residues, respectively, and 0.2 residues of beta-hydroxyaspartic acid. These data confirm that the propeptide mutations made do not interfere with proteolytic processing and that the Factor IX propeptide contains a recognition site that designates the adjacent glutamic acid-rich domain for gamma-carboxylation. In contrast, beta-hydroxylation of aspartic acid 64 is an independent process which does not require vitamin K and is mediated through a hydroxylation recognition site in the mature Factor IX, not in the propeptide.     

A conserved epitope on several human vitamin K-dependent proteins. Location of the antigenic site and influence of metal ions on antibody binding

A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 X 10(-8) to 1 X 10(-6) M. Other vitamin K-dependent proteins including Factor IX and protein S did not inhibit or inhibited only at the highest concentration binding of radiolabeled protein C to the immobilized antibody. Chemical treatment of prothrombin with a variety of agents including denaturation by sodium dodecyl sulfate, reduction with mercaptoethanol followed by carboxymethylation with iodoacetic acid, citraconylation of lysine residues, removal of metal ion with EDTA, or heat decarboxylation did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. Chymotrypsin digestion of prothrombin and isolation on QAE-Sephadex of the peptide representing amino-terminal residues 1-44 of prothrombin further localized the antigenic site recognized by the monoclonal antibody to the highly conserved gamma-carboxyglutamic acid-containing domain. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Antibody H-11 bound specifically to synthetic peptides corresponding to residues 1-12 of Factor VII and 1-22 of protein C. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. The glutamic acid residues in this peptide segment are the first 2 gamma-carboxyglutamic acid residues near the amino-terminal end in the native proteins. Increasing concentrations of Ca2+, Mg2+, or Mn2+ partially inhibited binding of 125I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of calcium ions in factor X activation by thrombin E192Q.

Despite considerable sequence similarities, blood coagulation serine proteases exhibit remarkable specificity with respect to which zymogen they activate. The basis for this specificity presumably involves recognition of a short sequence within the extended binding pocket of the enzyme, other interactions remote from the catalytic groove, and modulation by a definite protein cofactor. In addition, Ca2+ plays a major role in most activation processes, but, because both the enzyme and its substrate interact with Ca2+, whether Ca2+ influences the substrate, the enzyme, or both remains an open question. Thrombin is not a factor X-activating enzyme, but when Glu192, 3 residues remote from the active Ser195, is replaced with glutamine, the resultant serine protease (E192Q) becomes a bovine, but not human, factor X activator. Kinetic experiments with peptides corresponding to human and bovine factor X activating sites reveal that threonine at position P2 in human (versus a valine in bovine) accounts for the species specificity. Substitution of the threonine in P2 of the human sequence with valine allows E192Q to cleave the human peptide whereas substitution of the valine in P2 of the bovine sequence with threonine hinders E192Q catalysis. Thrombin has no high affinity Ca2+ binding sites, and E192Q proteolysis of these peptides is not altered by Ca2+. The influence of Ca2+ in E192Q-mediated factor X activation provides therefore new insights into the role of the different Ca2+ binding sites in factor X. With factor X as substrate, the addition of Ca2+ enhances Kcat 4-fold but increases Km 10-fold. When the vitamin K-dependent gamma-carboxyglutamic acid domain of factor X is removed, the Km remains high with or without Ca2+ whereas Kcat still increases upon addition of the metal ion. These results suggest that factor X undergoes two metal-dependent suggest that factor X undergoes two metal-dependent transitions that influence the presentation of the activation site to activators.     

Calcium binding to the isolated beta-hydroxyaspartic acid-containing epidermal growth factor-like domain of bovine factor X

Coagulation factor X is a vitamin K-dependent protein composed of discrete domains or modules. A proteolytically modified derivative of factor X that lacks the NH2-terminal gamma-carboxyglutamic acid (Gla)-containing region retains one Ca2+ binding site. To localize this Gla-independent Ca2+ binding site and to facilitate future studies aimed at elucidating structure-function relationship in the factor X molecule, we have devised a method to isolate the first beta-hydroxyaspartic acid (Hya)-containing epidermal growth factor (EGF)-like domain from proteolytic digests of bovine factor X performed under strictly controlled conditions. The EGF-like domain, corresponding to residues 45-86 in bovine factor X, was obtained in more than 50% recovery, and was at least 98% homogeneous as judged by NH2-terminal sequence analysis. Ca2+ binding to the isolated EGF-like domain was studied by 1H NMR spectroscopy. On binding of Ca2+ to the domain the resonances from Tyr-68 centered at 6.8 ppm were affected. The Ca2+ concentration dependence of the chemical shift was used to calculate the Ca2+ binding constant, resulting in a K alpha of 4 X 10(3) M-1 at pH 8.5 and 1 X 10(3) M-1 at pH 7.4, the higher value presumably reflecting an increase in negative surface charge due to deprotonation of a histidine residue with a pK alpha of 7.4. The NMR spectra gave no evidence of a conformational change in the EGF-like domain between pH 6 and 8.5.     

The effect of gamma-carboxyglutamate residues on the enzymatic properties of the activated blood clotting factor X. I. Activity towards synthetic substrates

The esterolytic and amidolytic properties of activated blood coagulation factor X (factor Xa) and the analogous decarboxy species were compared in order to find out if the gamma-carboxyglutamic acid residues influence the function of the active centre. It was found that the two proteins (1) showed similar kinetic parameters when titrated with p-nitrophenyl-p'-guanidinobenzoate hydrochloride (2) had a similar Km and kcat for various synthetic chromogenic tri- and tetrapeptides and (3) were inhibited in the same way by benzamidine. Further it was observed that (4) Ca2+ inactivates factor Xa, but has no influence on the amidase activity of decarbyxyfactor Xa (5) factor V prevents Ca2+-induced inactivation of factor Xa but does not influence the amidase activity of both factor Xa and decarboxyfactor Xa. We conclude that the interaction of the gamma-carboxyglutamic acid residues with Ca2+ in factor X has no measurable influence on the properties of the active site per se.     

Characterization of protein S, a gamma-carboxyglutamic acid containing protein from bovine and human plasma.

Protein S is a vitamin K dependent protein of unknown function, which is present in mammalian plasma. It was isolated from bovine plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, and column chromatography on DEAE-Sephadex, heparin-agarose, and polyhomoarginine-Sepharose. Bovine Protein S (Mr 64,200) is a single-chain glycoprotein with an amino-terminal sequence of Ala-Asn-Thr-Leu-Leu-. It contains 7.0% carbohydrate and 10 residues of gamma-carboxyglutamic acid per mol of protein. Human Protein S (Mr 69,000) is also a single-chain glycoprotein with an amino-terminal sequence of Ala-Asn-Ser-Leu-Leu-. It contains 7.8% carbohydrate and 10 residues of gamma-carboxyglutamic acid per mol of protein. These results indicate that Protein S from bovine or human plasma shows many similarities to the other vitamin K dependent proteins present in plasma.     

Recognition site directing vitamin K-dependent gamma-carboxylation resides on the propeptide of factor IX

Posttranslational processing of vitamin K-dependent proteins includes gamma-carboxylation of specific glutamic acid residues to form gamma-carboxyglutamic acids. To determine whether carboxylation is directed by the propeptide sequence, homologous among the precursors of these proteins, alterations were made in the Factor IX propeptide cDNA. The extent of gamma-carboxylation of recombinant Factor IX was assessed using conformation-specific antibodies directed against the gamma-carboxyglutamic acid-dependent, metal-stabilized structure. Deletion of the propeptide (residues -18 to -1) abolished carboxylation, but not secretion, of Factor IX. Substitution of alanine for phenylalanine -16 or glutamic acid for alanine -10 also impaired carboxylation. These results indicate that the Factor IX propeptide participates in defining a recognition site that designates an adjacent glutamic acid-rich domain for gamma-carboxylation. The association of the propeptide with the gamma-carboxylation recognition site provides the first demonstration of a specific function served by a propeptide in posttranslational protein processing.     

A new vitamin K-dependent protein. Purification from bovine plasma and preliminary characterization.

Four proteins active in blood coagulation have long been known to require vitamin K for their proper biosynthesis: factors II, VII, IX, and X. This paper describes the purification of a hitherto unrecognized vitamin K-dependent glycoprotein from bovine plasma. The biosynthesis of this protein is interfered with by the vitamin K antagonist Dicoumarol. The molecular weight of the protein is approximately 56,000 and, like factor X, it has two polypeptide chains. The light chain binds Ca2+. Its NH2-terminal amino acid sequence is homologous to the NH2-terminal sequences of the other vitamin K-dependent proteins and it contains vitamin K-dependent gamma-carboxyglutamic acid residues. The biological function of this protein is unknown.     

Activation of decarboxyfactor X by a protein from Russell's viper venom. Purification and partial characterization of activated decarboxyfactor X.

1. Incubation of decarboxyfactor X with the factor X-activating enzyme from Russell's Viper venom revealed the generation of amidase activity towards Bz-Ile-Glu-Gly-Arg-pNA, but not of activity in blood coagulation. 2. The rate of activation of both factor X and decarboxyfactor X depends on the ability of the zymogens to bind Ca2+. The relationship between Ca2+ concentration and velocity of the activation reaction is sigmoid in the case of factor X, but hyperbolic with decarboxyfactor X. 3. Activated decarboxyfactor X was purified by powder column electrophoresis. 4. Identical changes of primary structure accompanied the activation of factor X and decarboxyfactor X. Identical molecular weight and common antigenic determinants were found in factor Xa and decarboxyfactor Xa. The amino acid composition was identical except for 12 glutamic acid residues in decarboxyfactor Xa and gamma-carboxyglutamic acid residues in factor Xa. 5. Unlike factor X, activated factor X has a very low electrophoretic mobility in the presence of Ca2+ at pH 8.6. This is probably due to self association of factor Xa under the influence of Ca2+. The electrophoretic mobility of activated decarboxyfactor X is only slightly decreased compared to decarboxyfactor X in the presence of Ca2+.     

Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r.

The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r.     

Prothrombin fragment 1 X 2 X 3, a major product of prothrombin activation in human plasma

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.     

Mass-spectrometric identification and sequence location of the ten residues of the new amino acid (gamma-Carboxyglutamic acid) in the N-terminal region of prothrombin.

The detailed mass-spectrometric evidence for our original findings [Magnusson et al. (1974) FEBS Lett. 44, 189-193] of ten gamma-carboxyglutamic acid residues in the N-terminal calcium-binding polypeptide of prothrombin is presented. The identification and sequence location of gamma-carboxyglutamic acid was made by electron-impact and field-desorption studies on acetyl permethyl peptide derivatives, and on the free amino acid. Details of the derivatives formed, and how this new amino acid may be easily recognized and sequenced from the mass spectrum, are given as a basis for future work.     

Amino acid sequence of the peptide released from bovine factor XIII following activation by thrombin

The amino acid sequence of the peptide released during the conversion of bovine Factor XIII to the active enzyme by thrombin was determined. It contains N-terminal N-acetylserine and a total of 37 residues. The bovine peptide differs from the corresponding human peptide. There are 5 amino acid replacements and one deletion in the human peptide.     

Preparation of bovine blood coagulation factors X1 and X2

A method is described for the preparation of both Factor X1 and Factor X2 from citrated bovine blood. The proteins from the plasma were first adsorbed on barium citrate by adding barium chloride solution. The precipitate formed was stirred with citrate/NaOH pH 6.9 buffer; barium and other clotting factors were removed by adding ammonium sulphate (up to 30% saturation) to the suspension. The Factor X was then precipitated by 65% ammonium sulphate, after resolution in citrate buffer chromatographed on DEAE-Sephadex and purified by rechromatography on DEAE-Sephadex and DEAE-Sepharose, respectively. This yielded Factor X1 and Factor X2 with respective purifications of about 16 000 and 24 000-fold that of the plasma. The apparent molecular mass of both Factor X1 and Factor X2 was 55 kDa as estimated by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Factor X2 had a higher specific biological activity of about 340 000 units/mg compared to that of Factor X1 of about 230 000 units/mg.     

Mechanism of the calcium-dependent self-association of bovine prothrombin. Use of a covalent cross-linking reagent to study the reaction

The present study has made use of a covalent cross-linking agent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin and has demonstrated that the covalent dimerization reaction involves the gamma-carboxyglutamic acid region of prothrombin (1-42 of 582). An essential role for the gamma-carboxyglutamic acid residues of prothrombin in the association reaction was demonstrated by experiments that converted gamma-carboxyglutamic acid residues to gamma-methylene glutamic acid or glutamic acid and resulted in a prothrombin species that was inactive in our cross-linking assay. Other experiments showed that very high concentrations of calcium ion inhibit the cross-linkage of prothrombin. This result is most consistent with an essential gamma-carboxyglutamic acid-calcium ion-gamma-carboxyglutamic acid bridge(s) in the calcium-dependent self-associated form of prothrombin.     

Amino acid sequence of bovine protein Z: a vitamin K-dependent serine protease homolog

The amino acid sequence of protein Z has been determined from sequence analysis performed on fragments obtained by chemical and enzymatic degradations. The polypeptide consists of a single chain containing 396 amino acid residues (Mr 43 677). Comparison with the vitamin K-dependent plasma proteins reveals an extensive homology. The N-terminal part, containing 13 gamma-carboxyglutamic acid and one beta-hydroxyaspartic acid residue, is extensively homologous to and of similar length to the light chain of factor X. The remainder of protein Z is homologous to the serine proteases and of similar size to the heavy chain of factor Xa, but of the active site residues only aspartic acid-102 is present. Histidine-57 and serine-195 are replaced in protein Z by threonine and alanine, respectively. The physiological function of protein Z is still uncertain.     

Effects of gamma-carboxyglutamic acid and epidermal growth factor-like modules of factor IX on factor X activation. Studies using proteolytic fragments of bovine factor IX.

Factor IX is a vitamin K-dependent zymogen of a serine protease. The NH2-terminal half of the molecule consists of a Ca(2+)-binding gamma-carboxyglutamic acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. To elucidate the role of these non-catalytic modules of factor IXa beta in factor X activation, we have isolated and characterized fragments of bovine factor IX, containing one or both of the EGF-like modules as well as these modules linked to the Gla module. The fragments were used as inhibitors of factor IXa beta-mediated factor X activation in a plasma clotting system and in systems with purified components of the Xase complex. Fragments consisting of either the two EGF-like modules of factor IX linked together or the NH2-terminal EGF-like module alone were found to inhibit factor Xa generation both in the presence and absence of the cofactor, factor VIIIa. Moreover, a fragment consisting of the corresponding modules of factor X had a similar effect. We therefore propose that factor IXa beta and factor X interact directly through their EGF-like modules on or in the vicinity of a phospholipid surface. We have also found that the isolated Gla module of factor IX inhibits the formation of factor Xa both in the presence and absence of phospholipid but not in the absence of factor VIIIa. Our results are compatible with a model of the Xase complex, in which both the serine protease part and the Gla module of factor IXa beta interact with factor VIIIa.     

Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)