Haemopexin in sheep,mouflon and goat: genetic polymorphism,heterogeneity and partial characterization*

Benzidine staining of starch gels after electrophoresis of sera to which haematin was added revealed polymorphism of haemopexin in sheep, mouflon and goat. In sheep three phenotypes were observed, Hpx A, Hpx AB and Hpx B. Pedigree data support the hypothesis of codominant inheritance from a single locus by two alleles, Hpx^A and Hpx^B. Neuraminidase treatment of haemopexin preparations showed that Hpx B covered two variants, B1 and B2, thus indicating genetic control by three alleles (Hpx^A, Hpx^B1 and Hpx^B2). In sheep populations the frequency of Hpx^B is low. In mouflon, in addition to the two variants that are like those of sheep, absence of haemopexin was observed in some animals, by using starch gel electrophoresis as well as immunoelectrophoresis. In goat, three phenotypes were detected, Hpx A, Hpx AB and Hpx B, differing in migration from those of sheep. Haemopexins of the studied species are heterogeneous. Sialic acid is responsible for electrophoretic heterogeneity of sheep haemopexin. Chemical. composition (amino acid and carbohydrate), molecular weight (56 060) and N-terminal sequence (Leu-Pro-Pro-) of sheep haemopexin were also determined.     

Additional studies of sheep haemopexin: genetic control, frequencies and postnatal development

This study presents evidence that sheep haemopexin phenotypes are genetically controlled by three alleles, HpxA, HpxB1 and HpxB2, of a single autosomal locus. Frequencies of two alleles, HpxA and HpxB (HpxB encompasses two isoalleles, HpxB1 and HpxB2), were studied in eight sheep breeds in Czechoslovakia. The frequency of the HpxA allele was highest (ranging from 0.81 in Merino to 1.0 in East Friesian sheep). Qualitative and quantitative changes in haemopexin during postnatal development were studied by starch gel electrophoresis and rocket immunoelectrophoresis respectively. In electrophoresis, 1- or 2-day-old lambs had two very weak zones corresponding in mobility to two slower zones of adult animals. Later, the third more anodic zone appeared and gradually increased in intensity. In 1-month-old lambs the patterns were practically identical with those of adult animals. Using rocket immunoelectrophoresis, the level of haemopexin shortly after birth was practically zero. It rose sharply till the sixth day of life; then the level continued to rise slowly till about 1 month of age. The mean haemopexin level in adult sheep was 64.5 +/- 18.26 (SD) mg/100ml serum, ranging from 30.5 to 116.5 mg/100ml.     

Identification of potential serum biomarkers of inflammation and lipid modulation that are altered by fish oil supplementation in healthy volunteers

Long chain n-3 polyunsaturated fatty acids (n-3 LCPUFA) lower risk of coronary heart disease (CHD), but mechanisms are not well understood. We used proteomics to identify human serum proteins that are altered by n-3 LCPUFA. Such proteins could identify pathways whereby they affect CHD. Eighty-one healthy volunteers entered a double blind randomised trial to receive 3.5 g of fish oil or 3.5 g of high oleic sunflower oil daily. Serum was collected before and after 6 wk of intervention. Serum was analysed by proteomics using 2-DE. Proteins that were differentially regulated were identified by MS. We also analysed serum apolipoprotein A1 (apo A1), high-density lipoprotein (HDL) particle size and haptoglobin. Serum levels of apo A1, apo L1, zinc-alpha-2-glycoprotein, haptoglobin precursor, alpha-1-antitrypsin precursor, antithrombin III-like protein, serum amyloid P component and haemopexin were significantly downregulated (all p<0.05) by fish oil compared with high oleic sunflower oil supplementation. Fish oil supplementation caused a significant shift towards the larger, more cholesterol-rich HDL(2) particle. The alterations in serum proteins and HDL size imply that fish oil activates anti-inflammatory and lipid modulating mechanisms believed to impede the early onset of CHD. These proteins are potential diagnostic biomarkers to assess the mechanisms whereby fish oil protects against CHD in humans.     

Additional studies of sheep haemopexin: genetic control,frequencies and postnatal development*

Summary. This study presents evidence that sheep haemopexin phenotypes are genetically controlled by three alleles, Hpx^A, Hpx^B1 and Hpx^B2, of a single autosomal locus. Frequencies of two alleles, Hpx^A and Hpx^B (Hpx^B encompasses two isoalleles, Hpx^B1 and Hpx^B2), were studied in eight sheep breeds in Czechoslovakia. The frequency of the HpxA allele was highest (ranging from 0.81 in Merino to 1.0 in East Friesian sheep). Qualitative and quantitative changes in haemopexin during postnatal development were studied by starch gel electrophoresis and rocket immunoelectrophoresis respectively. In electrophoresis, 1- or 2-day-old lambs had two very weak zones corresponding in mobility to two slower zones of adult animals. Later, the third more anodic zone appeared and gradually increased in intensity. In 1-month-old lambs the patterns were practically identical with those of adult animals. Using rocket immunoelectrophoresis, the level of haemopexin shortly after birth was practically zero. It rose sharply till the sixth day of life; then the level continued to rise slowly till about 1 month of age. The mean haemopexin level in adult sheep was 64.5 ± 18.26 (SD) mg/100ml serum, ranging from 30.5 to 116.5 mg/100ml.     

Immunoglobulins and glycoproteins in lymphogranulomatosis]

In the serum of 27 patients with malignant lymphogranulomatosis the authors determined the serum level of glycoproteid-carbohydrate components (hexose, hexosamine, sialin acid, and seromucoid) and the concentration of 11 different glycoproteids. In the early stage of the disease the immunoglobulin level is moderately increased in the serum, whereas a diminution can be observed in stage IV. The concentrations of ceruloplasmin, alpha-2-macroglobulin and orosomucoid were already increased significantly in stage III. The increase did not continue in stage IV. In the final stage of the disease the concentrations of alpha-1-antitrypsin and haemopexin turned out to be increased considerably. A significant decrease in the transferrin level could be registered in stage III with this diminution also continuing in the further course. Changes of beta-C-globulin and haptoglobin concentrations could not be evaluated statistically. The content of carbohydrate components in the glycoproteids will already increase in the early stage of the disease with this increase continuing in the further course. Among histological types there was a more significant increase of immunoglobulins in those forms rich of lymphocytes.     

Characterization of human, bovine, and horse antithrombin III.

A comparison of the physical-chemical properties of human, bovine, and horse antithrombin III has been made. These three plasma proteins are strong inhibitors of bovine factor Xa and form a 1:1 molar complex with this coagulation enzyme. Human, bovine, and horse antithrombin III are glycoproteins containing hexose, hexosamine, and neuraminic acid. The total carbohydrate was 9, 12, and 16% for human, bovine, and horse antithrombin III, respectively. These proteins have a similar amino acid composition, although some monor variations were noted. Each antithrombin III is composed of a single polypeptide chain with an amino-terminal histidine residue. Of the first 17 amino-terminal residues, only three differences were noted between the three proteins. These occur in position 2 which is occupied by Gly, Arg, and Trp in human, bovine, and horse, respectively; position 6 which has a deletion in human antithrombin III; and position 8 where Ile in human and horse antithrombin III has been replaced by Val in the bovine preparation. The remainder of the first 17 residues is the same in all three proteins. The molecular weights for the bovine and horse preparation were 56 600 and 52 500, respectively, as determined by sedimentation equilibrium in the presence of guanidine hydrochloride. Some immunological cross-reactivity was also observed between the three different proteins.     

Isolation and characterization of antithrombin III from human, porcine and rabbit plasma, and rat serum.

1. Human, porcine, rabbit, and rat antithrombin III have been purified by affinity chromatography using heparin-agarose. The amino acid and carbohydrate compositions, amino-terminal sequences, immunological cross-reactivities, and inhibitions of human thrombin were studied. 2. Human, porcine, rabbit, and rat antithrombin III are single-chain glycoproteins containing hexose, glucosamine, and neuraminic acid. 3. The total carbohydrate contents were 17, 16, 14, and 15% for human, porcine, rabbit, and rat antithrombin III, respectively. 4. Molecular weights estimated from the migration in sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis were 59,000, 58,000, 63,000, and 63,000 for human, porcine rabbit, and rat antithrombin III, respectively. 5. These four proteins have similar amino acid compositions, although some minor differences were noted. 6. Human, porcine, and rabbit antithrombin III have a histidine residue at the amino-terminus, while rat antithrombin III contains an amino-terminal asparagine residue. 7. The amino-terminal sequences up to the first 17 residues showed high homology among the four proteins. 8. Some immunological cross-reactivity was observed only between human and porcine antithrombin III. 9. The apparent dissociation constants (KI) for the complexes between human thrombin and human, porcine, rabbit, and rat antithrombin III were about 1.2 x 10(-10) M, 9.5 X 10 (-9) M, 1.4 X 10(-7) M, and 2.8 X 10(-9) M, respectively.     

The heparin binding site of human antithrombin III. Selective chemical modification at Lys114, Lys125, and Lys287 impairs its heparin cofactor activity

Heparin binds to human antithrombin III and accelerates its inhibitory activity in the blood coagulation system. Previous reports (Rosenberg, R. D., and Damus, P. S. (1973) J. Biol. Chem. 248, 6490-6505; Pecon, J. M., and Blackburn, M. N. (1984) J. Biol. Chem. 259, 935-938) have shown that selective chemical modification of a limited number of lysine residues in antithrombin III causes drastic loss of its heparin cofactor activity. We have performed chemical modification of antithrombin III with trinitrobenzene sulfonic acid in order to determine the location of these lysine residues. When antithrombin III was treated with 100 M excess of trinitrobenzene sulfonic acid for 10 min, about 3.2 mol of amino group per mol of antithrombin III were modified. The heparin cofactor activity dropped to about 25%, whereas the progressive inhibitory activity (in the absence of heparin) remained essentially intact (about 95%). The modified amino groups were identified to be Lys114 (75%), Lys125 (94%), and Lys287 (96%). These results were obtained by comparing and analyzing the cyanogen bromide fragments derived from native antithrombin III and the 10-min modified antithrombin III. When antithrombin III was pretreated with heparin, followed by trinitrobenzene sulfonic acid modification, the extent of modification at Lys114 and Lys125 decreased from 75% and 94% to 20% and 40%, respectively, whereas the modification at Lys287 remained nearly quantitative (greater than 95%). Based on these results, we conclude that Lys114 and Lys125 are essential for the heparin cofactor activity of human antithrombin III.     

The serum protein content of human follicular fluid and its correlation with the maturity of oocytes

The authors determined, by an immunochemical method, the alpha-1-antitrypsin, Gc globulin, prealbumin, albumin, haemopexin, transferrin, haptoglobin, IgA, IgG, ceruloplasmin, alpha-2-macroglobulin contents of 98 follicular fluids, 10 peritoneal fluids and 24 blood sera. Out of these proteins analysed, change in the concentration of alpha-1-antitrypsin showed correlation with the maturity and fertilisation of the oocyte. The alpha-1-antitrypsin content was 1.6 +/- 0.26 g/l in the case of mature oocytes, and 3.1 +/- 1.12 g/l in the case of immature ones. Fertilisation was also concomitant of low alpha-1-antitrypsin levels.     

A general method for fractionation of plasma proteins. Dye-ligand affinity chromatography on immobilized Cibacron blue F3-GA.

The chromatographic behaviour of 27 different plasma proteins on fractionation of human plasma on immobilized Cibacron Blue F3-GA was studied. The column was eluted by using a three-step procedure. First, a low-molarity buffer (30 mM-H3PO4/Na3PO4, pH 7.0, I0.053) was used, then a linear salt gradient (0-1 M-NaCl in the buffer above) was applied, followed by a wash with two bed volumes of 1.0 M-NaCl. Finally, bound proteins were 'stripped' with 0.5 M-NaSCN. Up to 1 ml of whole plasma could be loaded per 5 ml bed volume. No denaturation of proteinase inhibitors or complement fractions was observed. The recovery of individual proteins ranged between 52 and greater than 95%. Enrichment of four individual plasma components (alpha 1-antitrypsin, caeruloplasmin, antithrombin III and haemopexin) was between 10-fold and 75-fold. These results indicate that chromatography on immobilized Cibacron Blue F3-GA can be a useful initial step in the purification of plasma proteins.     

Antithrombin III (AT3) polymorphism in the marsupialMonodelphis domestica: Identification and genetics

Antithrombin III polymorphism was observed in the gray short-tailed opossum, Monodelphis domestica, by either one-dimensional polyacrylamide gel electrophoresis (PAGE; pH 7.9), two-dimensional PAGE (agarose, pH 5.4; 12% T, pH 7.9), or isoelectric focusing (pH 4.2-4.9) followed by immunoblotting with rabbit antiserum to human antithrombin III. Family studies demonstrated an inheritance of three codominant autosomal alleles, AT3A, AT3B, and AT3C, and a population study revealed frequencies of 0.70, 0.10, and 0.20, respectively.     

Emerging strategies in microbial haem capture

Gram-negative pathogenic bacteria have evolved novel strategies to obtain iron from host haem-sequestering proteins. These include the production of specific outer membrane receptors that bind directly to host haem-sequestering proteins, secreted haem-binding proteins (haemophores) that bind haem/haemoglobin/haemopexin and deliver the complex to a bacterial cell surface receptor and bacterial proteases that degrade haem-sequestering proteins. Once removed from haem-sequestering proteins, haem may be transported via the bacterial outer membrane receptor into the cell. Recent studies have begun to define the steps by which haem is removed from bacterial haem proteins and transported into the cell. This review describes recent work on the discovery and characterization of these systems. Reference is also made to the transport of haem in serum (via haemoglobin, haemoglobin/haptoglobin, haemopexin, albumin and lipoproteins) and to mechanisms of iron removal from the haem itself (probably via a haem oxygenase pathway in which the protoporphyrin ring is degraded). Haem protein-receptor interactions are discussed in terms of the criteria that govern protein-protein interactions in general, and connections between haem transport and the emerging field of metal transport via metallochaperones are outlined.     

Structure and biological activity of finback-whale (Balaenoptera physalus L.) heparin octasaccharide. Chemical, carbon-13 nuclear-magnetic-resonance, enzymic and biological studies.

Finback-whale (Balaenoptera physalus L.) heparin was partially digested with a purified heparinase and an octasaccharide with high affinity for antithrombin III was isolated from the digest by gel filtration, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. This octasaccharide possessed high inhibitory activity for Factor Xa in the presence of antithrombin III, but was essentially inactive for thrombin-antithrombin III reaction. The anticoagulant activity determined by the activated-partial-thromboplastin-time method was very low (40-70 units/mg), although the initial whale heparin exhibited high activity (252 units/mg). On the basis of the results of chemical analyses, 13C n.m.r. spectrum and enzymic studies with purified heparinase, heparitinases 1 and 2, the predominant structure of the octasaccharide was proposed as follows: delta UA(2S) alpha 1 leads to 4GlcNS alpha 1 leads to 4IdUA alpha 1 leads to 4GlcNAc(6S) alpha 1 leads to 4GlcUA beta 1 leads to 4GlcNS(3S) alpha 1 leads to 4IdUA(2S) alpha 1 leads to 4GlcNS. Comparing this structure with those of the heparin octasaccharides so far reported, the presence of the critical structural elements for binding to antithrombin III was suggested in the pentasaccharide region situated at the reducing end of this octasaccharide. Binding to antithrombin III of the critical structural elements alone would appear to elicit the acceleration of the Factor Xa-antithrombin III reaction. Additional structural elements required for the acceleration of the thrombin-antithrombin III reaction and for the manifestation of high anticoagulant activity are discussed.     

Molecular weight analysis of antithrombin III-heparin and antithrombin III-thrombin-heparin complexes

The molecular interactions between components of the heparin-catalyzed antithrombin III/thrombin reaction were investigated by light scattering. When heparin was added to antithrombin III, the molecular weight increased to a maximum and then decreased to that of a 1:1 (antithrombin III X heparin) complex. The initial molecular weights at low heparin to antithrombin III ratios were consistent with the formation of a 2:1 (antithrombin III X heparin) complex in which only one antithrombin III molecule had undergone the conformational change measured by protein fluorescence enhancement. The peak molecular weight never reached that of a complete 2:1 complex. This behavior was observed for bovine and human antithrombin III in the presence of both unfractionated heparin and high molecular weight-high affinity heparin. Pentosane polysulfate also caused some multiple associations. Bovine antithrombin III and thrombin formed a 1:1 complex that underwent further aggregation within minutes, while the human proteins did not aggregate on this time scale after forming the 1:1 complex. In the presence of stoichiometric amounts of heparin, the bovine proteins formed an initial complex of Mr = 230,000 (corresponding to a dimer of heparin-antithrombin III-thrombin) which underwent further aggregation. The human proteins, however, formed a 1:1 (antithrombin III X thrombin) initial complex in the presence of heparin, followed by aggregation. These interactions of thrombin and antithrombin with heparin suggest complex interactions that could relate to heparin function.     

Monoclonal antibodies against antithrombin III. Identification of their epitopes and effects on antithrombin III activities

Four monoclonal antibodies with distinct epitopes were prepared against antithrombin III. None of them is directed against the heparin-binding region nor the active site, yet two mAb namely A36 and B108, interfere with antithrombin III inhibition of thrombin. The epitope of monoclonal antibody A36 is located within amino acid residues 1-393, at a site different from the active site since it recognizes antithrombin III and antithrombin-III-thrombin complexes with the same affinity. A36 partially prevents the intrinsic antithrombin III activity and has no effect on the heparin-enhanced antithrombin III activity when added to the antithrombin-III--heparin complex. If A36 is first reacted with antithrombin III and then heparin is added to the reaction mixture, A36 fixes the conformation of antithrombin III so that heparin binds to antithrombin III, but is not able to induce the conformational change in the antithrombin III molecule required for the enhanced activity. The epitope for monoclonal antibody B108 is located within residues 282-393, close to the active site. It does not recognize antithrombin-III-thrombin complexes by solid-phase radioimmunoassay. Its binding to antithrombin III induces a conformational change that enhances antithrombin III activity in a manner that resembles the heparin effect, but its effect is additive to the heparin effect, since when it was added to a reaction mixture which contained a saturating amount of heparin, inhibition of thrombin was enhanced. The epitope for monoclonal antibody A5 is located within residues 1-393, and its recognition of antithrombin III or antithrombin-III-thrombin is strongly dependent on the integrity of the disulfide bonds. A5 has no effect on antithrombin III activities. The epitope for monoclonal antibody A10 is well defined within a narrow range of 55 amino acid residues, 339-393, on the antithrombin III molecule, close to the active site, yet it has no effect on antithrombin III inhibitory activity. These monoclonal antibodies may be developed for various diagnostic or clinical purposes and offer a powerful tool for studying the conformational changes and structure/activity relationships in the antithrombin III molecule.     

Alterations of antithrombin III after acute myocardial infarction

The antithrombin III (AT III) activity and the AT III concentration were investigated in 62 consecutive patients with acute myocardial infarction (AMI). To identify the reactant pattern of AT III in postaggressive situations, we also determined labile and acute phase proteins. Firstly, 29 patients were nourished orally and then 33 patients were fed by i.v. hyperalimentation (additional caloric intake of approximately 1,000 cal). AT III activities and concentrations as well as prealbumin and retinol-binding protein decreased concomittantly and significantly whereas haptoglobin, C-reactive protein and fibrinogen increased significantly after AMI. The changes cannot be interpreted as being alterations of the haematocrit. The alterations of AT III correlated significantly with the changes of labile proteins but not with the acute phase reactant proteins. The AT III decrease in the postinfarctional phase may promote a prethrombotic state. In In addition it can be concluded from our results that AT III reacts as (nutritive-dependent) labile protein, which is lowered in postaggressive situation and does not increase as an acute phase reactant. This is in accordance with results from recent animal experiments.     

Properties of thrombin- and elastase-modified human antithrombin III

Proteolytically modified forms of human antithrombin III have been prepared by reaction of native antithrombin with thrombin, human neutrophil elastase, or porcine pancreatic elastase. These forms have two chains disulfide linked and are of the same molecular weight as native antithrombin III. 1H NMR spectroscopy has been used to characterize these proteins and to compare them to one another and to native antithrombin III. The three modified proteins have very similar NMR spectra and histidine residues with identical pH titration parameters, and they undergo the same spectral changes upon binding heparin. They differ from native antithrombin III in all of these respects. In addition, the proteins are much more stable than native antithrombin III. The three modified proteins behave identically as a function of temperature; at 372 K, 44 K above the unfolding temperature for native antithrombin III, the proteins are still folded and possess approximately 70 unexchanged amide protons even after several hours. The unfolding of the heparin binding domain at low concentrations of deuteriated guanidine hydrochloride seen in native thrombin III is absent in the modified forms. It is concluded that the thrombin- and elastase-modified forms of antithrombin have identical structures when allowance is made for the slightly different sites of cleavage by the two types of elastase and by thrombin. This structure is very different from that of native antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)     

A method to determine the affinity of heparin to thrombin and antithrombin III on equilibrium gel permeation chromatography

Equilibrium gel permeation chromatography was employed to determine the ability of heparin to form complexes with thrombin and antithrombin III. In the eluate from a Sephacryl S-200 column, heparin caused a peak and then a trough in the fluorescence of 48 nM antithrombin III or 63 nM thrombin. The peak-heights with known amounts of heparin were used for standard curves to determine the extent of complex formation by test heparin preparations. Only heparin species with high-affinity for antithrombin III specifically formed a complex with antithrombin III under the conditions used. The ability to form a complex of heparin preparations with different anticoagulant activities for thrombin and antithrombin III could be determined satisfactorily. The heparin species with different affinities for antithrombin III did not coincide those with different affinities for thrombin. Of 4 preparations with one low-affinity and three high-affinity subfractions of heparin for antithrombin III, the species with the lowest affinity for antithrombin III had the highest affinity for thrombin. All of these observations showed that the method could be used to determine the ability to form a complex of test heparin preparations.     

Net biosynthesis of antithrombin III by the isolated rat liver perfused for 12–24 hours compared with rat fibrinogen and α-2 (acute-phase) globulin,antithrombin III is not an acute phase protein

Antithrombin III-heparin cofactor has been isolated from normal rat plasma, purified to homogeneity on acrylamide gel electrophoresis and used to prepare a monospecific antiserum in rabbits. Measurements of rat antithrombin III were made by a single radial immunodiffusion assay.Net synthesis of antithrombin III was investigated during 12- or 24-h perfusions of the isolated rat liver. In perfusions performed under basal conditions cumulative synthesis of antithrombin-III was observed to occur at a rate sufficient to replace the total circulating plasma antithrombin III in about 6 h. In perfusions performed under full supplementation conditions which greatly enhanced synthesis of fibrinogen and α-2 (acute-phase) globulin (known acute-phase reactant proteins) net synthesis of antithrombin III was not significantly greater than that observed in control perfusions. Although these prolonged perfusion studies conclusively demonstrate net synthesis of antithrombin III by the isolated rat liver, they afford no evidence that this protein is an acute-phase reactant.     

Human haptoglobin structure and function--a molecular modelling study

Hemoglobin is the most prominent protein in blood, transporting O(2) and facilitating reactive oxygen and nitrogen species detoxification. Hemoglobin metabolism leads to the release of extra-erythrocytic hemoglobin, with potentially severe consequences for health. Extra-erythrocytic hemoglobin is complexed to haptoglobin for clearance by tissue macrophages. The human gene for haptoglobin consists of three structural alleles: Hp1F, Hp1S and Hp2. The products of the Hp1F and Hp1S alleles differ by only one amino acid, whereas the Hp2 allele is the result of a fusion of the Hp1F and Hp1S alleles, is present only in humans and gives rise to a longer alpha-chain. Haptoglobin consists of a dimer of alphabeta-chains covalently linked by a disulphide bond between the Cys15 residue of each alpha-chain. However, the presence of the Hp1 and Hp2 alleles in humans gives rise to HPT1-1 dimers (covalently linked by Cys15 residues), HPT1-2 hetero-oligomers and HPT2-2 oligomers. In fact, the HPT2 variant displays two free Cys residues (Cys15 and Cys74) whose participation in intermolecular disulphide bonds gives rise to higher-order covalent multimers. Here, the complete modelling of both haptoglobin variants, together with their basic quaternary structure arrangements (i.e. HPT1 dimer and HPT2 trimer), is reported. The structural details of the models, which represent the first complete view of the molecular details of human haptoglobin variants, are discussed in relation to the known haptoglobin function(s).