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At least 1,531 records · Page 85Linked to original sources
PubMed2000

Annexin 1 expression and phosphorylation are upregulated during liver regeneration and transformation in antithrombin III SV40 T large antigen transgenic mice.

We have used a transgenic animal model, which constitutively develops hepatocarcinoma (Antithrombin III SV40 T large Antigen: ASV), to study the involvement of Annexin 1 (ANX1) in liver regeneration and malignant transformation. Primary hepatocytes isolated from normal mice did not express ANX1. In contrast, ANX1 was strongly expressed in hepatocytes of transgenic mice during constitutive development of hepatocarcinoma. In ASV transgenic mice, an elevated ANX1 level preceded the appearance of the tumor, indicating that it could be a good marker in the diagnosis of cancer. One-third hepatectomy in normal mice resulted in stimulation of ANX1 synthesis and phosphorylation. This upregulation correlated with increased synthesis of EGF and consequently with increased phosphorylation of the EGF receptor (EGF-R). Stable transfection of a hepatocyte cell line derived from ASV transgenic mice (mhAT2) with antisense complementary DNA for ANX1 reduced the proliferation rate as well as cytosolic phospholipase A(2) (cPLA(2)) activity. Thus, ANX1 expression and phosphorylation could be a factor implicated in liver regeneration and tumorigenesis, either through modulation of cPLA(2) activity or EGF-R function.

Animals↗
PubMed1995

Interaction of antithrombin III with surface-immobilized albumin-heparin conjugates.

The interaction between antithrombin III (ATIII) and albumin-heparin conjugates covalently coupled onto carboxylated polystyrene beads either in buffer containing albumin or in plasma was studied using 14C-labeled ATIII. Binding isotherms of ATIII were modeled using a summation of two Langmuir equations. These equations describe the binding of ATIII to two different sets of binding sites, one with a high, the other with a low affinity of ATIII to these sites are 9 x 10(6) L/mol and 0.3 x 10(6) L/mol, respectively. The binding of ATIII to surface binding sites with a high affinity for ATIII was correlated with the presence of specific ATIII binding sites in the immobilized heparin. Binding of ATIII from albumin solutions to binding sites with a low affinity for ATIII was dominated by nonspecific binding of ATIII to the immobilized heparin. A third small fraction of the surface bound. ATIII is probably adsorbed to sites on the surface not covered with heparin. In the case of the binding of ATIII to the heparinized surface from plasma solutions, a fraction of initially adsorbed ATIII was desorbed by other plasma proteins. This desorption in combination with direct competition between ATIII and other plasma proteins resulted in lower ATIII surface concentrations using plasma as compared to the ATIII surface concentrations obtained using albumin solutions. The binding of ATIII to nonspecific binding sites was almost completely inhibited in the presence of plasma proteins. The amount of ATIII bound to immobilized heparin via specific ATIII binding sites was 30% lower in plasma solutions as compared to the specific binding of ATIII using albumin solutions. It is concluded that the accessibility of immobilized heparin for ATIII in plasma decreases by binding of heparin-binding proteins onto the immobilized heparin and/or adsorption of other plasma proteins on the heparinized surface.

Absorption↗
PubMed2003

Interactions of antithrombin and proteins in the plasma contact activation system with immobilized functional heparin.

The interactions of antithrombin (AT) and the contact phase clotting factors with two commercially available heparinized surfaces are reported. The Carmeda (CBAS) and Corline surfaces along with controls (a sulfonated polyethylene surface and a CBAS analog in which the heparin used was devoid of specific AT-binding sequences) were exposed to human plasma. Adsorbed proteins were eluted and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The CBAS and Corline surfaces adsorbed large amounts of AT, whereas adsorption on the controls was negligible. Immunoblots for the four contact phase clotting factors indicated less contact activation on the CBAS and Corline surfaces than on the controls. Determination of adsorbed functional AT using a FXa inhibition assay showed that the CBAS surface adsorbed about 4 times as much AT as the Corline surface. Adsorption of AT to the control surfaces was minimal. Assays for adsorbed FXII and FXIIa based on kallikrein generation showed that all four surfaces adsorbed similar amounts of FXII. However, on the controls, most of the FXII was in activated form, whereas on the CBAS and Corline surfaces very little activation occurred.

Antithrombins↗
PubMed1979

L-asparaginase effect on antithrombin-III levels.

The effect of L-asparaginase (L-asp) therapy on circulating antithrombin-III (AT-III) was monitored in three patients with acute lymphoblastic leukemia. Serial determination for AT-III by both a functional assay and an immunologic assay demonstrated a rapid decrease in serum AT-III levels, followed by a protracted recovery beyond the period of L-asp exposure. One of the three patients developed a reversible venous thrombosis of the arm. The mechanism of the L-asparaginase effect may be related to peripheral degradation of AT-III or to inhibition of production.

Adult↗
PubMed1988

Increased antithrombin III concentration in children with acute lymphatic leukaemia receiving L-asparaginase therapy.

Previous reports have observed a fall in antithrombin III (AT III) concentration in patients receiving L-asparaginase as part of acute lymphatic leukaemia (ALL) induction therapy. The aim of this study was to define any qualitative changes in AT III that might arise during the course of L-asparaginase therapy and predispose to coagulopathy. Serial AT III profiles were studied in 12 patients with ALL receiving a course of L-asparaginase for 21 consecutive days as part of induction therapy. AT III isoforms were examined by isoelectric focusing in polyacrylamide gels (IEF/PA) and immunoblotting, and no changes were observed throughout the study period. Contrary to expectation, AT III antigen was significantly increased on treatment, while AT III activity remained unchanged. Fibrinogen levels, in contrast, fell considerably by 1 week of therapy and had not reverted to pre-treatment values 1 week after completion of asparaginase. No coagulation or bleeding disorders were observed during or after the study period.

Antithrombin III↗
PubMed2005

Determination of the site-specific and isoform-specific glycosylation in human plasma-derived antithrombin by IEF and capillary HPLC-ESI-MS/MS.

The glycan structures of the major and more than ten minor populated isoforms of antithrombin (AT) were determined after separation of the isoforms by IEF using IPG strips. The bands excised from the gel were reduced, derivatized by iodoacetamide and submitted to tryptic digestion. The digest was analyzed by RP-HPLC-ESI-MS equipped with a quadrupole ion-trap mass analyzer. MS/MS experiments allowed establishing the monosaccharide compositions in the glycopeptides. For the major isoform of alpha-AT four identical biantennary glycans with two terminal sialic acids (SA) each, a total of eight SA, were found in full agreement with the literature. In the IEF-band containing this major isoform (pI 5.18) a further, much less abundant, isoform was detected showing a fucosylation on the glycan attached to Asn155 but being of otherwise identical structure as described above. The isoforms with pI 5.10 were found to include one triantennary glycan, all antennas carrying terminal SA. The occurrence of triantennary structure is site specific, involving the peptides with Asn(135) and Asn(155), alternately. At pI 5.24 we found those four isoforms that carry the glycans like the main-isoform of alpha-AT but missing one terminal SA. There was no site specificity found for the mono-sialo structure. The isoform at pI 5.31 is the major isoform of beta-AT containing three identical biantennary structures being fully sialylated. No isoforms (above 0.5% abundance) with two glycans only or three glycans other than beta-AT were detected. Fucosylation was found in the main isoform with an abundance of about 5%, and as expected with all the other isoforms with a comparable abundance.

Antithrombin III↗
PubMed1994

Heparin binding domain peptides of antithrombin III: analysis by isothermal titration calorimetry and circular dichroism spectroscopy.

The serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated oligosaccharides, such as heparin. A series of synthetic peptides have been prepared based on the proposed heparin binding regions of ATIII and their ability to bind heparin has been assessed by CD spectrometry, by isothermal titration calorimetry, and by the ability of the peptides to compete with ATIII for binding heparin in a factor Xa procoagulant enzyme assay. Peptide F123-G148, which encompasses both the purported high-affinity pentasaccharide binding region and an adjacent, C-terminally directed segment of ATIII, was found to bind heparin with good affinity, but amino-terminal truncations of this sequence, including L130-G148 and K136-G148 displayed attenuated heparin binding activities. In fact, K136-G148 appears to encompass only a low-affinity heparin binding site. In contrast, peptides based solely on the high-affinity binding site (K121-A134) displayed much higher affinities for heparin. By CD spectrometry, these high-affinity peptides are chiefly random coil in nature, but low microM concentrations of heparin induce significant alpha-helix conformation. K121-A134 also effectively competes with ATIII for binding heparin. Thus, through the use of synthetic peptides that encompass part, if not all, of the heparin binding site(s) within ATIII, we have further elucidated the structure-function relations of heparin-ATIII interactions.

Amino Acid Sequence↗
PubMed1998

Reactivities of the S2 and S3 subsite residues of thrombin with the native and heparin-induced conformers of antithrombin.

A pentasaccharide (PS) fragment of heparin capable of activating antithrombin (AT) markedly accelerates the inhibition of factor Xa by AT, but has insignificant effect on inhibition of thrombin. For inhibition of thrombin, the bridging function of a longer polysaccharide chain is required to accelerate the reaction. To study the basis for the similar reactivity of thrombin with the native or heparin-activated conformers of AT, several residues surrounding the active site pocket of thrombin were targeted for mutagenesis study. Leu99 and Glu192, the variant residues influencing the S2 and S3 subsite specificity of thrombin were replaced with Tyr and Gln. The Tyr60a, Pro60b, Pro60c, and Trp60d residues forming part of the S2 specificity pocket were deleted from the B-insertion loop of the wild-type and Leu99/Glu192 --> Tyr/Gln thrombins. Kinetic studies indicated that the reactivities of all mutants with AT were moderately or severely impaired. Although heparin largely corrected the defect in reactivities, it also markedly elevated the stoichiometries of inhibition with the mutants. Interestingly, PS also accelerated AT inhibition of the mutants 5-68-fold, suggesting that the mutants are able to discriminate between the native and activated conformers of AT. Based on these results and the recent crystal structure determination of AT in complex with PS, a model for thrombin-AT interaction is proposed in which the S2 and S3 subsite residues of thrombin are critical for recognition of the P2 and P3 residues of AT in the native conformation. In the activated conformation, other residues are made accessible for interaction with the protease, and the similar reactivity of thrombin with the native and heparin-activated conformers of AT may be coincidental. The results further suggest that the S2 and S3 subsite residues are crucial in controlling the partitioning of the thrombin-AT intermediate into the alternative inhibitory or substrate pathways of the reaction.

Antithrombins↗
PubMed1996

Structure-function relations of antithrombin III-heparin interactions as assessed by biophysical and biological assays and molecular modeling of peptide-pentasaccharide-docked complexes.

The serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated glycosaminoglycans, such as heparin. Peptide K121-A134, based on the sequence of ATIII in the D helix region, was previously shown by us (Tyler-Cross et al., Protein Sci. 3, 620-627, 1994) to encompass part (or all) of the purported high-affinity heparin binding region of ATIII. A series of peptide analogs has now been prepared whose sequences are identical to K121-A134 except that single cationic residues of K121-A134 have been successively replaced with Ala. In one case, the Arg residue of the reference peptide corresponding to R129 of ATIII has been replaced by Gln (R129deltaQ peptide), thus mimicking the naturally occurring mutant protein, ATIII Geneva. The heparin affinity of all peptides was quantitated by isothermal titration calorimetry and by peptide/ATIII competition binding assays. Replacement of any single cationic residue with Ala had a deleterious effect on heparin binding. The greatest reduction in heparin affinity (more than 30-fold) was observed with the R129deltaQ peptide (KD = 1.5 +/- 0.06 microM vs 51 +/- 2 nM for the reference peptide, K121-A134). Furthermore, each of the Ala-replacement peptides was a less-effective inhibitor of ATIII-heparin complex formation than the reference peptide. The poorest inhibitor was the R129deltaQ peptide which showed nearly 30% decrease in inhibition potency (60% inhibition at 100 microM peptide vs 90% inhibition with the reference peptide). The relative heparin affinities of the peptides measured by biological assay were the same as determined by titration calorimetry. Consequently, we modeled the complexes formed between the pentasaccharide unit structure and the R129deltaQ peptide or the reference peptide, K121-A134. In the "docked" complex, the assumed conformation of K121-A134 permitted juxtaposition of the cationic residues of the peptide with functional anionic groups of the pentasaccharide known to be involved in binding. A docked complex could also be formed between the R129deltaQ peptide and the pentasaccharide, but misalignment of critical peptide and saccharide functional groups was observed. The structure of the R129deltaQ-pentasaccharide complex was highly irregular because F123 and Y131 were completely surface exposed, likely yielding an unfavorable structure in aqueous solution. The observations from molecular modeling allow us to suggest that ATIII Geneva displays decreased heparin binding affinity due to its inability to form a productive binding complex in which essential electrostatic contacts are made between suitably juxtaposed saccharide anionic functional groups and cationic amino acid side chains.

Amino Acid Sequence↗
PubMed1999

Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of thrombospondin-1 with thrombin-antithrombin III.

The recent demonstration of a protein disulfide isomerase (PDI) on the surface of and secreted from blood platelets raises the possibility that proteins involved in hemostasis and wound healing are also substrates of this enzyme. In this study purified preparations of platelet PDI, thrombospondin-1 (TSP), alpha-thrombin, and antithrombin III (AT) were used to demonstrate that PDI catalyzes formation of a TSP-thrombin-AT complex consistent with previous results with supernatant platelet activation. Concentrations of 1.25 microg/ml of PDI were sufficient to convert almost 50% of thrombin to TSP-thrombin-AT complex. Complex formation requires low concentrations of a reduced thiol and the reaction can be prevented by N-ethymaleimide. The complex is dissociated by reducing agents such as mercaptoethanol. Absence of Ca2+ and the addition of EDTA increased the rate of complex formation, indicating that TSP in the Ca2+-free form is most effective. In the absence of AT a small amount of TSP-thrombin complex formed which was only 0-13% of maximal complex formation in the presence of AT. This result, in combination with kinetic studies showing rapid formation of thrombin-AT complex followed by conversion to ternary complex, suggests that the thrombin-AT complex is an obligatory intermediate in the reaction. Under optimal conditions over 70% of the thrombin is incorporated into the complex in 60 min. Heparin accelerated the reaction largely by enhancing formation of thrombin-AT complexes and had little effect on TSP. PDI coprecipitated with TSP from the supernatant solution of activated platelets, suggesting an association between PDI and its substrate. In summary, these data are consistent with a role for PDI-catalyzed formation of disulfide-linked complexes of TSP with other proteins.

Antithrombin III↗
PubMed2000

Boophilus microplus anticoagulant protein: an antithrombin inhibitor isolated from the cattle tick saliva.

An anticoagulant was isolated from saliva of the cattle tick Boophilus microplus. Crude saliva prolonged both recalcification time and prothrombin time in assays with bovine plasma. It also inhibited thrombin, but not fXa, amidolytic activity. We purified the antithrombin activity by a combination of gel filtration, anion exchange, and affinity chromatography. The purified inhibitor has a molecular weight of 60,000 Da, determined by SDS-PAGE. The anticoagulant IC50 varied from 100 nM to 1.1 microM, depending on the thrombin concentration and substrate used (fibrinogen or platelet receptor). The excess of inhibitor in relation to thrombin indicates that it is not a tight-binding inhibitor. Chromogenic assays using a panel of five serine-proteinases suggest that the inhibitor is specific against thrombin.

Acari↗
PubMed2000

Enzymatic modification of heparan sulfate on a biochip promotes its interaction with antithrombin III.

A heparan sulfate glycosaminoglycan chain, biotinylated at its reducing-end, was bound to a streptavidin-coated biochip. Surface plasmon resonance spectroscopy showed a low affinity interaction with antithrombin III (ATIII) when it was flowed over a surface containing heparan sulfate. ATIII bound tightly with high affinity when the same surface was enzymatically modified to using 3-O-sulfotransferase isoform 1 (3-OST-1) in the presence of 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The 3-OST-1 enzyme is involved in heparan sulfate biosynthesis and introduces a critical 3-O-sulfo group into this glycosaminoglycan affording the appropriate pentasaccharide sequence capable of high affinity binding to ATIII. This experiment demonstrates the specific structural modification of a glycosaminoglycan bound to a biochip using a biosynthetic enzyme, suggesting a new approach to rapid screening glycosaminoglycan-protein interactions.

Animals↗
PubMed1998

Receptor-mediated activation of murine peritoneal macrophages by antithrombin III acts as a costimulatory signal for nitric oxide synthesis.

We evaluated the effect of antithrombin III (ATIII), a serine protease inhibitor (SERPIN), on induction of nitric oxide (NO) synthesis in murine peritoneal macrophages. Incubation of macrophages with ATIII plus interferon-gamma (IFN-gamma) but not ATIII alone induced nitrite accumulation (a metabolite of NO) in a dose-dependent manner. Expression of the inducible nitric oxide synthase isoform was confirmed by Western blot. NO synthesis was inhibited by NG-monomethyl-l-arginine, by complexing ATIII with thrombin or by rabbit anti-human ATIII antiserum. Addition of polymyxin B to macrophage cultures failed to inhibit ATIII/IFN-gamma-induced NO synthesis, excluding lipopolysaccharide contamination. 125I-ATIII bound to macrophages in a dose-dependent, specific, and saturable manner, with a Km of approximately 7.1 nM. Our results demonstrate that ATIII, but not ATIII/thrombin complex, acts to costimulate macrophage activation and NO synthesis via a novel receptor mediated mechanism, which may indicate a role for SERPINs in macrophage activation.

Animals↗
PubMed2001

Antithrombin III enhances inducible nitric oxide synthase gene expression in vascular smooth muscle cells.

Evidence suggests that antithrombin III (ATIII) exerts anti-inflammatory properties in addition to its anti-coagulative mechanisms. In animal models of sepsis, ATIII affected cytokine plasma concentrations with a decrease of pro-inflammatory cytokines. In addition to cytokines, excessive production of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) might represent another important mediator of the cytotoxic events during sepsis. Regarding ATIII as a potential anti-inflammatory modulator, one may speculate that ATIII inhibits the synthesis of iNOS-derived NO. However, our data demonstrate that ATIII further stimulates iNOS gene expression when applied together with either interleukin-1 beta or the combination of lipopolysaccharide plus interferon-gamma. The most prominent synergistic effects on NO synthesis were found when ATIII was given at higher concentrations (1, 5, and 10 U/ml). Although the mechanisms of ATIII signal transduction remain to be established, intensification of interleukin-1 beta or interferon-gamma/lipopolysaccharide-induced NO synthesis by ATIII does not attribute to the anti-inflammatory properties of ATIII.

Animals↗
PubMed1996

Prothrombin fragment F1+2 and thrombin-antithrombin III complex (TAT) plasma levels in patients with gynecological cancer.

The pretreatment plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III complex (TAT) were measured in 56 consecutive patients with gynecological cancer and in 33 apparently healthy volunteer nonpregnant women as control. Prothrombin fragment F1+2 concentrations were significantly elevated in the 18 patients with ovarian cancer (median, 3.2 nmol/ liter; range, 0.9-17.0 nmol/liter, P < 0.0001), in the 24 patients with cervical cancer (median, 1.7 nmol/liter; range, 0.6-15.0 nmol/ liters, P < 0.0001), and in the 14 patients with endometrial cancer (median, 1.5 nmol/liter; range, 0.6-3.0 nmol/liter, P = 0.036) when compared to controls (median, 1.0 nmol/liter; range, 0. 1 -2.1 nmol/ liter). Similarly, TAT levels were significantly higher in patients with ovarian cancer (median, 5.3 microgram /ml; range, 1.3-65.0 microgram/ml , P < 0.0001), cervical cancer (median, 3.8 microgram/ml; range, 2.1 - 11.0 microgram / ml, P < 0.0001), and endometrial cancer (median, 2.7 microgram/ml; range, 1.9-19.0 microgram /ml, P = 0.008) when compared to controls (median, 2.2 microgram/ml; range, 1.0-5.0 microgram/ml). Prothrombin fragment F1+2 levels correlated with TAT levels (r = 0.659, P < 0.0001). Among ovarian cancer patients, prothrombin fragment F1+2 and TAT concentrations correlated with FIGO stage (III-IV versus 1, P = 0.01 and P = 0.003, respectively) and CA 125 levels (P 0.02 and P < 0.0001, respectively). The present data confirmed the occurrence of hemostasis activation in patients with gynecological cancer, and in particular in those with ovarian cancer.

Adenocarcinoma↗
PubMed1993

Crystallization and preliminary X-ray analysis of human antithrombin III.

Human antithrombin III has been crystallized from 18 to 21% (w/v) polyethylene glycol 4000 at pH 7.15. The spacegroup is P2(1) with cell parameters a = 89.8 A, b = 100.8 A, c = 70.0 A and beta = 106 degrees. The diffraction limit is 3.2 A. The asymmetric unit contains two protein molecules. Analysis of dissolved crystals for biological activity and by gel electrophoresis suggests that one protein molecule in the asymmetric unit is intact, while the other is cleaved.

Antithrombin III↗
PubMed1993

Crystal structure of cleaved bovine antithrombin III at 3.2 A resolution.

The crystal structure of cleaved antithrombin III (ATIII) has been determined to 3.2 A resolution by single isomorphous replacement, real space density modification and phase extension protocols. The heavy-atom sites and the first molecular envelope were determined owing to the molecular replacement solution previously reported and partially refined. Refinement of the two molecules of the asymmetric unit led to a crystallographic R-factor of 0.212 for all reflections between 8.0 and 3.2 A, without inclusion of water molecules. The root-mean-square deviation from ideal values is, respectively, 0.015 A and 3.6 degrees for bond lengths and bond angles. The topology of the molecule closely resembles that of cleaved serpins inhibitors with the two residues forming the reactive bond at opposite ends of the molecule. The most significant difference between ATIII and alpha 1-antitrypsin lies in the 45 residue N-terminal extension in ATIII which contribute to the definition of the heparin binding site. This loop region at the surface of the molecule is held by two disulphide bridges to the protein core and exhibits high temperature factor values. It forms a valley which restrains the possibilities for binding of heparin. Docking of the pentasaccharide unit which represents the minimum fragment of heparin able to bind to ATIII indicates a possible role for arginine 14 in the interaction of heparin and the protein.

Amino Acid Sequence↗
PubMed1994

Trinucleotide repeat polymorphism within the human antithrombin gene (AT3): allele frequency data for three population groups.

The fifth intron of the human antithrombin gene (AT3) of chromosome 1q23 contains a highly polymorphic short tandem repeat based on the trinucleotide repeat [ATT]. Alleles range in size from [ATT]5 to [ATT]18, and can be typed using the polymerase chain reaction. Here we report allele frequency population data for Caucasians, Blacks and Southeast Asians. Statistical analyses demonstrate consistency with Hardy-Weinberg equilibrium for all three databases, and that the allele frequencies of all three population groups are significantly different from each other. This locus reveals 76% to 87% heterozygosity and therefore may be useful for forensic identity testing and paternity determination.

Alleles↗