Human Antithrombin Research Articles

Separation between the α and β forms of human antithrombin by hydroxyapatite high-performance liquid chromatography

Human antithrombin (AT) inhibits several proteases in the coagulation system, including thrombin and factor Xa, and thus, plays an important role in the regulation of blood coagulation. The predominant form of AT in plasma is ATα, which contains four glycosylated asparagine residues, and the minor form is ATβ, which lacks the Asn-135 glycosylation. In this study, hydroxyapatite high-performance liquid chromatography, using a segmented sodium phosphate gradient, was utilized for the high-resolution separation of ATα and ATβ. The detection limit (signal-to-noise ratio of 3) for ATβ was 30 μg/mL, corresponding to 0.5% of the injected concentration of AT. Two analyzed commercial AT products both contained about 2% ATβ. This method is suitable for the determination of ATβ in pure samples of native AT.

Recombinant antithrombin attenuates pulmonary inflammation following smoke inhalation and pneumonia in sheep.

The interaction between coagulation and inflammation has become one of the major topics in critical care medicine. In the present study, we investigated the effect of posttreatment of sepsis with recombinant human antithrombin. Experimental laboratory in a university hospital. University laboratory. Female merino ewes (n = 16). After 1 wk of recovery from the surgical preparation, a tracheotomy was performed followed by insufflation of 48 breaths of cotton smoke (<40 degrees C). Afterward, a stock solution of live (5 x 10(11) colony-forming units) was instilled in the both lung lobes through a bronchoscope. All sheep were mechanically ventilated employing 100% oxygen. An infusion of recombinant human antithrombin (100 units x kg(-1) x 24 hrs(-1), intravenously; n = 6) or saline (n = 6) was started 1 hr after injury. Sham control animals (n = 4) were surgically prepared but not insufflated with smoke and bacteria. Lung histologic changes were evaluated by a scoring system. The infusion of recombinant human antithrombin maintained the baseline antithrombin activity throughout the study; in the saline-treated group, antithrombin activity decreased significantly. The lung wet/dry weight ratio and the histology score (combined scores for congestion, edema, inflammation, and hemorrhage) were significantly increased by the insult, but recombinant human antithrombin attenuated these responses. More than 30% of both bronchi and bronchioles were obstructed by cast formation after smoke inhalation and pneumonia. The cast was composed of epithelial cells, neutrophils, mucus, and fibrin. The obstruction was significantly improved by recombinant human antithrombin infusion. Arterial pressure and urine output were also attenuated in recombinant human antithrombin-treated animals. The increases in plasma nitrate/nitrite concentrations and pulmonary shunt fraction after the injury were not attenuated by recombinant human antithrombin. Posttreatment by recombinant human antithrombin was effective in treating acute lung injury after smoke inhalation and pneumonia in sheep. We hypothesize that the decrease in antithrombin activity during sepsis might induce severe airway obstruction and that supplementation with antithrombin inhibits this decrease.

Modulation of antithrombin-protease interactions by semisynthetic low-molecular-weight heparins with different sulfation patterns.

Heparin, a natural glycosaminoglycan (GAG), is widely used for the treatment of thrombotic diseases. Most of its side effects are related to its ability to bind to different proteins, thus interfering with its target biological activity. To gain insight into structure-activity relationships, we investigated the interaction of a homogeneous series of sulfated polysaccharides, derived from controlled desulfation of a supersulfated low-molecular-weight heparin (LMWH) with the target enzymes human antithrombin (AT) and thrombin (T). In addition, we analyzed the activation process of the serpin AT against T and factor Xa (FXa). A nonlinear correlation between the strength of the AT-heparin complex and the polysaccharide sulfation degree was observed, whereas only a modest modulation of T binding to heparin occurred. The efficiency of the LMWH derivatives in activating AT toward the proteases was generally high for derivatives exhibiting a low dissociation constant. Only the supersulfated heparin showed a serpin activation ability higher than expected from the affinity studies. Examination of the sulfation pattern in the light of the above results suggests a key role of the substitution of the iduronic acid residue in the heparin-mediated serpin binding and activation processes. Indeed, sulfation at position 2 of the uronic acid is beneficial, whereas 2,3-disubstitution generates unfavorable contacts between the GAG and AT. Glucosamine sulfation at position 6 appears to grant increased catalytic efficiency. These results indicate that chemical modification of the heparin sulfation pattern can be used to modulate binding specificity and activity toward its biological targets.

Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibration.

Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de-N-glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of +/-0.12 and +/-0.022%, respectively. The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT-III, namely AT-III(alpha) and AT-III(beta), were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135). After exhaustive de-N-glycosylation (by means of PNGase F) of the alpha- and beta-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (-0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N- and O-glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous gamma-carboxyglutamic acids). MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N-glycans.

Preparative conversion of native human antithrombin to the latent form

Human native antithrombin (AT) can be converted to a partially denaturated form of AT, known as latent AT (L-AT). This latent form of AT has been shown to exhibit strong antiangiogenic activity and also to suppress tumor growth in mice models. In the present work, a method is presented which induces the conversion of native AT to L-AT, using incubation at 60 °C, for 16 h, with 0.9 M ammonium sulfate, in 5 mM Hepes buffer, pH 7.4, giving a recovery of more than 70%. L-AT was determined by integration of the low heparin affinity peak when analyzed by the affinity chromatography method. Native polyacrylamide gel electrophoresis was used to show that the preparation contained no aggregates. Hydrophobic interaction chromatography was also used for the separation of AT and L-AT.

Characterization of N-linked oligosaccharides attached to recombinant human antithrombin expressed in the yeast Pichia pastoris.

We studied the structures of four N-linked oligosaccharide chains of the recombinant human antithrombin (rAT) expressed in the yeast Pichia pastoris. rAT was fully glycosylated at Asn 96 and Asn 155, whereas the glycosylation on Asn 135 and Asn 192 was partial. The glycosylation level on Asn 135 was only 12% and this reduction is assumed to be one of the reasons for a higher heparin-binding affinity of rAT than plasma-derived human antithrombin (pAT). In order to determine the sizes and electrostatic charges of the N-linked oligosaccharides, rAT was treated with PNGase F, and the reduced ends were labelled by pyridylamination followed by analysis using anion exchange and amide adsorption columns. The N-linked oligosaccharides were 78% neutral and 22% phosphomannosylated. The neutral oligosaccharides were thought to be Man(9-12)GlcNAc(2) as their major components. The phosphomannosylated oligosaccharides were then subjected to mild acid hydrolysis and/or digestion with alkaline phosphatase, and their charge shifts were analysed by the affinity to an anion exchange column. Among phosphomannosylated oligosaccharides, monophosphate diester type was predominant, whereas negatively charged diphosphate diester and monophosphate monoester types were minor components. The mannose residues at the non-reducing end(s) of Man(9-12)GlcNAc(2) were phosphomannosylated or phosphorylated and these are the major components. Because rAT is less negatively charged than pAT, which has disialyl biantennary N-glycans, it might be less repulsive to pentasaccharide-bearing anticoagulantly active heparan sulphate proteoglycan molecules exposed on the surface of the damaged vascular vessels.

Interaction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin III

Internally quenched fluorogenic (IQF) peptides bearing the fluorescence donor/acceptor pair o-aminobenzoic acid (Abz)/N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) at N- and C-terminal ends were synthesized containing heparin-binding sites from the human serpins kallistatin and antithrombin, as well as consensus heparin-binding sequences (Cardin clusters). The dissociation constant (K(d)), as well as the stoichiometry for the heparin-peptide complexes, was determined directly by measuring the decrease in fluorescence of the peptide solution. Experimental procedures were as sensitive as those used to follow the fluorescence change of tryptophan in heparin-binding proteins. The conformation of the peptides and the heparin-peptide complexes were obtained from measurements of time-resolved fluorescence decay and CD spectra. Kallistatin (Arg(300)-Pro(319))-derived peptide (HC2) and one derived from antithrombin III helix D [(AT3D), corresponding to Ser(112)-Lys(139)], which are the heparin-binding sites in these serpins, showed significant affinity for 4500 Da heparin, for which K(d) values were 17 nM and 100 nM respectively. The CD spectra of the heparin-HC2 peptide complex did not show any significant alpha-helix content, different from the situation with peptide AT3D, for which complex-formation with heparin resulted in 24% alpha-helix content. The end-to-end distance distribution and the time-resolved fluorescence-decay measurements agree with the CD spectra and K(d) values. The synthetic alpha-methyl glycoside pentasaccharide AGA*IA(M) (where A represents N,6-O-sulphated alpha-d-glucosamine; G, beta-d-glucuronic acid; A*, N,3,6-O-sulphated alpha-d-glucosamine; I, 2-O-sulphated alpha-l-iduronic acid; and A(M), alpha-methyl glycoside of A) also binds to AT3D and other consensus heparin-binding sequences, although with lower affinity. The interaction of IQF peptides with 4500 Da heparin was displaced by protamine. In conclusion, IQF peptides containing Abz/EDDnp as the donor/acceptor fluorescence pair are very promising tools for structure-activity relationship studies on heparin-peptide complexes, as well as for the development of new peptides as heparin reversal-effect compounds.

Poster Sessions AP02: Proteins, Amino Acids, Peptides and Metabolism. The effect of proline rich polypeptide on the interaction between activated factor X and anti‐thrombin III

The effect of the hypothalamic neurosecretory Proline Rich Polypeptide (PRP) on the interaction of human Factor Xa (Xa) with human antithrombin III (ATIII) was investigated by SDS–PAGE and Western Blot analysis. Xa runs as two bands with masses of ∼51 and ∼47 kDa. A fraction of the ATIII (∼55 kDa) undergoes a conformational change upon interaction with Xa to run slower at ∼58 kDa. Xa and ATIII form a 1° duplex complex of ∼109 and ∼104 kDa, a 2° duplex complex of ∼99 and ∼94 kDa, and a 3° duplex complex of ∼66 and ∼62 kDa. Additionally, a Xa degradation product (∼35 kDa) is noted. Pre‐incubation of 1.5 μg of either Xa or ATIII with 8 μg PRP for 15′ at R.T., or essentially simultaneous addition of all three, increases the 2° complex upper band, 3° complex lower band, ATIII conformational change, and the Xa degradation product. It also decreases the 3° complex upper band and the native ATIII. Xa/PRP premixtures cause a decrease in unreacted Xa. ATIII/PRP premixtures produce an increase in the 1° complex lower band and a decrease in the unreacted Xa upper band. Xa/ATIII/PRP mixtures increase both bands of the 1° complex. Thus, 8 μg PRP promotes 1° complex formation as well as formation of the 2° and 3° degradation complexes. It also promotes modification of ATIII and degradation of Xa, possibly by autolysis and/or promotion of proteolysis of the 1° complex by free Xa. Data obtained indicate the role of PRP in the molecular mechanisms of blood clotting. (Supported, in part, from a grant by the Office of Sponsored Programs and Research, BGSU.)

Protective Effects of Recombinant Human Antithrombin III in Pig-to-Primate Renal Xenotransplantation

Delayed rejection of pig kidney xenografts by primates is associated with vascular injury that may be accompanied by a form of consumptive coagulopathy in recipients. Using a life-supporting pig-to-baboon renal xenotransplantation model, we have tested the hypothesis that treatment with recombinant human antithrombin III would prevent or at least delay the onset of rejection and coagulopathy. Non-immunosuppressed baboons were transplanted with transgenic pig kidneys expressing the human complement regulators CD55 and CD59. Recipients were treated with an intravenous infusion of antithrombin III eight hourly (250 units per kg body weight), with or without low molecular weight heparin. Antithrombin-treated recipients had preservation of normal renal function for 4-5 days, which was twice as long as untreated animals, and developed neither thrombocytopenia nor significant coagulopathy during this period. Thus, recombinant antithrombin III may be a useful therapeutic agent to ameliorate both early graft damage and the development of systemic coagulation disorders in pig-to-human xenotransplantation.

Separation of active and inactive forms of human antithrombin by heparin affinity chromatography

During the manufacturing of an antithrombin preparation, it is necessary to define all steps that may damage or alter the target molecule, and thus decrease the biological activity of the inhibitor in blood coagulation. Pasteurization, commonly used procedure for viral inactivation of plasma derived antithrombin concentrates, was shown to partially alter the conformation of the active native antithrombin to an inactive latent form. To study intensively the different forms of inactive antithrombin that are formed upon heat treatment, human α-antithrombin, human β-antithrombin and an equimolar mixture of the two isoforms were incubated at 60 °C for 15 h in the presence of citrate as stabilizing agent. Using two subsequent heparin affinity chromatography steps, three different inactive fractions were separated. By comparison of the heparin binding capacities, isoelectric points and unfolding characteristics of these inactive forms, the α-latent and β-latent antithrombin isoforms could be identified. It was also shown that additional inactive forms such as proteinase cleaved and/or oxidized forms of antithrombin are formed during the heat treatment process. In four commercially available antithrombin preparations, all produced by pasteurization, the amount of inactive protein varied between 0.5% and 9.5%.

The interaction between captured human thrombin and antithrombin studied by surface plasmon resonance, and the effect of melagatran

The kinetics of the interactions between thrombin and antithrombin were studied by surface plasmon resonance. Although amine coupled thrombin binds low molecular weight active site inhibitors with a one‐to‐one stoichiometry hirudin only bound to 75% of the coupled active thrombin. However, antithrombin or thrombomodulin could not bind at all to amine‐coupled thrombin. To make it possible to follow the reaction with antithrombin in time thrombin was therefore captured on an antibody against thrombin. Alternatively, antithrombin was captured on an antibody against antithrombin to follow complex formation with thrombin. With these techniques the second‐order rate constant for the interaction of antithrombin with thrombin in the presence of heparin, was estimated to be ka≥0.4×106 M−1 s−1 with KD═ 50 nM for the formation of the initial complex. These values are similar to those found in solution. In the absence of heparin, the second‐order rate constant for the interaction of thrombin with captured antithrombin was only ka ═ 9×103 M−1 s−1. Thus, with this technique rate constants for the interaction of proteases with serpins can be determined in a convenient way, without the need for stopped‐flow. The rapid‒binding, active‐site‐directed thrombin inhibitor, melagatran, competed with the antithrombin‐thrombin interaction. Although melagatran, a reversible thrombin inhibitor, initially prevented formation of the complex, ultimately the irreversible thrombin‐antithrombin complex was always formed.

Cloned transgenic offspring resulting from somatic cell nuclear transfer in the goat: oocytes derived from both follicle-stimulating hormone-stimulated and nonstimulated abattoir-derived ovaries.

The use of nuclear transfer (NT) techniques to create transgenic offspring capable of producing valuable proteins may have a major impact on the pharmaceutical market. Our objective was to compare the in vivo developmental potential of NT embryos produced from the fusion of transgenic donor cells with cytoplasts prepared from either FSH-stimulated ovaries or nonstimulated abattoir-derived ovaries. Donor cells were prepared from a transgenic fetus carrying the gene for human antithrombin III as a marker and used within four to eight subpassages. Cells were serum deprived for 4 days prior to cytoplast transfer. Oocytes were enucleated by removing the metaphase plate using a DNA stain and epifluorescent illumination. Donor cells were fused to enucleated oocytes by electric pulse and then chemically activated. There was no difference in the number of transferable embryos produced from cytoplasts of FSH-stimulated ovaries or from the fusion of cytoplasts from abattoir ovaries, nor was there a difference in the number of pregnancies established per recipient with either treatment. All pregnancies from both groups culminated in the births of healthy female kids (five total). To our knowledge, this is the first report of cloned goats produced from NT using cytoplasts derived from abattoir ovaries.

Probing the molecular basis of factor Xa specificity by mutagenesis of the serpin, antithrombin

The molecular basis of the substrate and inhibitor specificity of factor Xa, the serine proteinase of the prothrombinase complex, was investigated by constructing two mutants of human antithrombin (HAT) in which the reactive site loop of the serpin from the P4–P4′ site was replaced with the corresponding residues of the two factor Xa cleavage sites in prothrombin (HAT/Proth-1 and HAT/Proth-2). These mutants together with prethrombin-2, the smallest zymogen form of thrombin containing only the second factor Xa cleavage site, were expressed in mammalian cells, purified to homogeneity and characterized in kinetic reactions with factor Xa in both the absence and presence of cofactors; factor Va, high affinity heparin and pentasaccharide fragment of heparin. HAT/Proth-1 inactivated factor Xa ∼3–4-fold better than HAT/Proth-2 in either the absence or presence of heparin cofactors. In the absence of a cofactor, factor Xa reacted with the HAT/Proth-2 and prethrombin-2 with similar second-order rate constants (∼2–3×10 2 M −1 s −1). Pentasaccharide catalyzed the inactivation rate of factor Xa by the HAT mutants 300–500-fold. A similar 10 4–10 5-fold enhancement in the reactivity of factor Xa with prethrombin-2 and the HAT mutants was observed in the presence of the cofactors Va and heparin, respectively. Factor Va did not influence the reactivity of factor Xa with either one of the HAT mutants. These results suggest that (1) in the absence of a cofactor, the P4–P4′ residues of HAT and prethrombin-2 primarily determine the specificity reactions with factor Xa, (2) factor Va binding to factor Xa is not associated with allosteric changes in the catalytic pocket of enzyme that would involve interactions with the P4–P4′ binding sites, and (3) similar to allosteric activation of HAT by heparin, a role for factor Va in the prothrombinase complex may involve rearrangement of the residues surrounding the scissile bond of the substrate to facilitate its optimal docking into the catalytic pocket of factor Xa.

Antithrombin reduces mesenteric venular leukocyte interactions and small intestine injury in endotoxemic rats.

We examined the hypothesis that recombinant human antithrombin would reduce mesenteric venule leukocyte adhesion and small intestine injury in endotoxemic rats. Endotoxemic (endotoxin 10 mg/kg, intravenously) rats were treated either with saline or recombinant human antithrombin (250 and 500 U/kg). In some rats, indomethacin (100 mg/kg, intraperitoneally) was injected 60 min prior to endotoxin and recominant human antithrombin (500 U/kg) treatment. Compared to controls, intravital videomicroscopy of the mesentric venule showed an increase of leukocyte rolling (55+/-17 versus 70+/-19 leukocytes/min; P < 0.05) and firm adhesion (1.1+/-0.3 versus 5.8+/-0.8 leukocytes/100 microm; P < 0.05) in endotoxemic rats. Recombinant human antithrombin attenuated endotoxin-induced venular endothelium leukocyte adhesive cascade. The beneficial effects of recombinant human antithrombin on leukocyte adhesion were inhibited by indomethacin (100 mg/kg, intraperitoneally) in endotoxemic rats. Endotoxin treatment increased fluorescein isothiocyanate (FITC)-labeled dextran 4,000 (FD4) gut lumen to plasma ratio and wet weight/dry weight ratio. Recombinant human antithrombin (500 U/kg) attenuated endotoxin-induced gut injury. These observations suggest that recombinant human antithrombin reduces endothelium-leukocyte interactions in endotoxemic rats by interacting with local prostacyclin production.

Separation of Native and Latent Forms of Human Antithrombin by Hydrophobic Interaction High-Performance Liquid Chromatography

Hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) was utilized for the separation of native human antithrombin (AT) and a partially denaturated form of AT, known as the latent form (L-AT). The AT used in this study is commercially available (Atenativ, Pharmacia & Upjohn, Sweden) and contains albumin as the main stabilizer. The AT was reconstituted and heat treated in order to generate L-AT. This latent form of AT has been shown to exhibit a strong antiangiogenic activity and also to suppress tumor growth. The HPLC system included a TSK Phenyl 5PW column and a segmented gradient, 4.5-0 mol/L sodium chloride. Antithrombin was eluted at about 13 min, and L-AT, at 30 min, corresponding to about 4.2 and 1.6 mol/L sodium chloride, respectively. A reference sample gave 42% L-AT when analyzed by the HIC method and 41% L-AT when analyzed by the heparin affinity chromatography method. The resolution between AT and L-AT was higher with the HIC method than with the heparin affinity method. Incubation of Atenativ at 45 degrees C for 15 h gave about 18% L-AT and was shown by native polyacrylamide gel electrophoresis to contain only monomeric AT. A good resolution between AT and L-AT, but not between albumin and L-AT, was also achieved by a linear gradient of 2-0 mol/L ammonium sulfate, in 25 mmol/L Tris/HCl, pH 8.0.

Effects of Sulfation on Antithrombin-Thrombin/Factor Xa Interactions in Semisynthetic Low Molecular Weight Heparins

Most of the biological effects of heparin and low molecular weight (LMW) heparins are related to their ability to bind to many different proteins. To gain insight into structure-activity relationships, we investigated quantitatively the interactions of a series of sulfated LMW heparins of similar molecular weights (derived from statistical desulfation of a supersulfated heparin) with the target enzymes human antithrombin (AT) and thrombin (T). In addition, we analyzed the activation of the protease inhibitor against T and factor Xa (FXa). A nonlinear correlation between the strength of the AT-heparin complex and the degree of sulfation of the LMW heparins was observed, whereas only a modest modulation of T binding to heparin occurred. The efficiency of the heparin derivatives in activating AT toward the proteases is generally high for derivatives exhibiting a low dissociation constant. Only the supersulfated LMW heparin showed serpin activation ability higher than expected from the affinity studies. These results indicate that chemical modification of the sulfation pattern of LMW heparin can be used to efficiently modulate binding affinity and activity toward biological targets.

Plasminogen and Tissue Plasminogen Activator Interact with Antithrombin III

Human antithrombin III was demonstrated to bind plasminogen specifically in a time and concentration-dependent manner. The above binding was also confirmed using ligand western blot assays. The interaction of plasminogen was significantly (>90%) inhibited by lysine, indicating the involvement of kringles in binding antithrombin III. Plasminogen also bound to heparin-antithrombin III complex. In converse experiments, antithrombin III also interacted with immobilized plasminogen. Using carboxypeptidase B digestion, the plasminogen-binding site of antithrombin III was localized to the carboxy-terminus lysine of the anticoagulant protein. Tissue plasminogen activator also interacted with antithrombin III in a time- and concentration-dependent manner and its binding was also significantly (>90%) inhibited by lysine. Moreover, the interaction of plasminogen and tissue plasminogen activator with antithrombin III was competitive. These results provide the first evidence for the interaction of antithrombin III with fibrinolytic factors and suggest that antithrombin III may serve to localize these factors at the site of clot formation.

Effect of aPMR1 disruption on the processing of heterologous glycoproteins secreted in the yeastSaccharomyces cerevisiae

TheSaccharomyces cerevisiae PMR1 gene encodes a Ca2+-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α1-antitrypsin (α1-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α1-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.

The Use of Human Antithrombin III Concentrate for Treatment of Heparin Resistance During Cardiopulmonary Bypass

We report the administration of biologic antithrombin III (AT III) concentrate for the treatment of heparin resistance in 44 patients undergoing cardiopulmonary bypass (CPB). During CPB, the amount of heparin required to maintain an activated clotting time greater than 480 sec was significantly reduced following the administration of AT III concentrate (p = .000). The average increase in the ACT was 176 sec following the administration of AT III concentrate (p = .000). Thirteen of 44 patients did not require any additional heparin for the duration of CPB after AT III therapy. AT III concentrate seemed to be efficacious in the treatment of heparin resistance attributable to presumed AT III deficiency. Moreover, the reduction in heparin requirements following administration of AT III concentrate may reduce postoperative bleeding associated with heparin rebound, protamine requirements, and its associated complications and threat of intravascular coagulation during CPB.

Recombinant human antithrombin III improves survival and attenuates inflammatory responses in baboons lethally challenged with Escherichia coli

Plasma-derived antithrombin III (ATIII) prevents the lethal effects of Escherichia coli infusion in baboons, but the mechanisms behind this effect are not clear. In the present study, we evaluated the effects of recombinant human ATIII (rhATIII) on the clinical course and the inflammatory cytokine and coagulation responses in baboons challenged with lethal dose of E coli. Animals in the treatment group (n = 5) received high doses of rhATIII starting 1 hour before an E coli challenge. Those in the control group were administered saline. Survival was significantly improved in the treatment group (P =.002). Both groups had similar hemodynamic responses to E coli challenge but different coagulation and inflammatory responses. The rhATIII group had an accelerated increase of thrombin-ATIII complexes and significantly less fibrinogen consumption compared to controls. In addition, the rhATIII group had much less severe thrombotic pathology on autopsy and virtually no fibrinolytic response to E coli challenge. Furthermore, the rhATIII group had a significantly attenuated inflammatory response as evidenced by marked reduction of the release of various cytokines. We conclude that the early administration of high doses of rhATIII improves the outcome in baboons lethally challenged with E coli, probably due to the combined anticoagulation and anti-inflammatory effects of this therapy. (Blood. 2000;95:1117-1123)