Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
Add more filters










Publication year range
1.
Anal Biochem ; 406(2): 166-75, 2010 Nov 15.
Article in English | MEDLINE | ID: mdl-20670608

ABSTRACT

Irreversible inactivation of alpha-thrombin (T) by the serpin, heparin cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine-Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for heparin and DS were compared. Recombinant and deglycosylated HCII bound heparin with dissociation constants (K(D)) of 6+/-1 and 7+/-1 microM, respectively, approximately 6-fold tighter than plasma HCII, with K(D) 40+/-4 microM. Binding of recombinant and deglycosylated HCII to DS, both with K(D) 4+/-1 microM, was approximately 4-fold tighter than for plasma HCII, with K(D) 15+/-4 microM. Recombinant HCII, lacking N-glycosylation and tyrosine sulfation, inactivated alpha-thrombin with a 1:1 stoichiometry, similar to plasma HCII. Second-order rate constants for thrombin inactivation by recombinant and deglycosylated HCII were comparable, at optimal GAG concentrations that were lower than those for plasma HCII, consistent with its weaker GAG binding. This weaker binding may be attributed to interference of the Asn(169)N-glycan with the HCII heparin-binding site.


Subject(s)
Escherichia coli/metabolism , Glycosaminoglycans/metabolism , Heparin Cofactor II/metabolism , Amino Acid Sequence , Dermatan Sulfate/metabolism , Enzyme Activation , Fluorescence , Glycosylation , Heparin Cofactor II/chemistry , Heparin Cofactor II/isolation & purification , Humans , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptides/chemistry , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Thrombin/metabolism
2.
Glycobiology ; 16(8): 693-701, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16624894

ABSTRACT

Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three l-iduronic acid 2-O-sulfate (IdoA2SO3)-->N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO3) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS. Therefore, we sought to characterize oligosaccharides from porcine mucosal DS that interact with HCII. DS was partially depolymerized with chondroitinase ABC, and oligosaccharides containing 2-12 monosaccharide units were isolated. The oligosaccharides were then fractionated by anion-exchange and affinity chromatography on HCII-Sepharose, and the disaccharide compositions of selected fractions were determined. We found that the smallest oligosaccharides able to bind HCII were hexasaccharides. Oligosaccharides 6-12 units long that lacked uronic acid (UA)2SO3 but contained one or two GalNAc4,6SO3 residues bound, and binding was proportional to both oligosaccharide size and number of GalNAc4,6SO3 residues. Intact DS and bound dodecasaccharides contained predominantly IdoA but little D-glucuronic acid. Decasaccharides and dodecasaccharides containing one or two GalNAc4,6SO3 residues stimulated thrombin inhibition by HCII and prolonged the clotting time of normal but not HCII-depleted human plasma. These data support the hypothesis that modification of IdoA-->GalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate molecules with HCII-binding sites and anticoagulant activity.


Subject(s)
Acetylgalactosamine/chemistry , Dermatan Sulfate/metabolism , Heparin Cofactor II/metabolism , Mucous Membrane/chemistry , Sulfates/chemistry , Animals , Carbohydrate Sequence , Chondroitin Lyases/genetics , Chondroitin Lyases/metabolism , Dermatan Sulfate/chemistry , Heparin Cofactor II/isolation & purification , Humans , Monosaccharides/chemistry , Monosaccharides/isolation & purification , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Recombinant Proteins/metabolism , Swine , Thrombin/antagonists & inhibitors
3.
J Biol Chem ; 276(24): 20959-65, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11294849

ABSTRACT

Although fibrin-bound thrombin is resistant to inactivation by heparin.antithrombin and heparin.heparin cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate.heparin cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the heparin-catalyzed rate of thrombin inhibition by heparin cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by heparin.heparin cofactor II reflects heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary heparin.thrombin.fibrin complex. This complex, formed as a result of three binary interactions (thrombin.fibrin, thrombin.heparin, and heparin.fibrin), limits accessibility of heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate. thrombin.fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin.fibrin and thrombin. dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by heparin cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by heparin cofactor II in the presence of dermatan sulfate but not heparin.


Subject(s)
Dermatan Sulfate/pharmacology , Fibrin/metabolism , Heparin Cofactor II/metabolism , Heparin/pharmacology , Thrombin/chemistry , Thrombin/metabolism , Binding Sites , Dermatan Sulfate/isolation & purification , Fibrin/isolation & purification , Heparin/isolation & purification , Heparin Cofactor II/isolation & purification , Humans , Kinetics , Protein Binding , Spectrophotometry , Thrombin/isolation & purification
4.
Protein Expr Purif ; 6(6): 806-12, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8746633

ABSTRACT

Recombinant human heparin cofactor II (rHCII) was expressed as a fully active protein in the High-Five insect cell line. A maximal protein concentration of 6 micrograms/10(6) cells was achieved 2 days postinfection. Approximately 40 micrograms of partially purified rHCII was routinely recovered from 50 ml of media after sequential heparin and Q-Sepharose affinity adsorption. rHCII had a slightly lower apparent molecular weight than blood plasma HCII (pHCII) due to differences in N-glycosylation. Like pHCII, rHCII formed a stable bimolecular complex with thrombin when assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The thrombin and chymotrypsin inhibitory properties of rHCII and pHCII were quite similar. In the absence of glycosaminoglycan, the thrombin inhibition rate (k2 x 10(-4) M-1 min-1) was 2.29 +/- 0.36 for rHCII and 3.38 +/- 0.34 for pHCII. Chymotrypsin inhibition rates (k2 x 10(-5) M-1 min-1) were 6.2 +/- 2.0 for rHCII and 8.0 +/- 2.6 for pHCII. In the presence of glycosaminoglycans, the maximal thrombin inhibition rate (k2 x 10(-3) M-1 min-1) for rHCII was 10.4 +/- 2.5 at 100 micrograms/ml heparin and 16.0 +/- 4.3 at 1000 micrograms/ml dermatan sulfate compared to 9.0 +/- 0.7 at 200 micrograms/ml heparin and 18.5 +/- 5.3 at 1000 micrograms/ml dermatan sulfate for pHCII. HCII inhibition of thrombin was blocked by a synthetic sulfated hirudin peptide in both the presence and the absence of glycosaminoglycan. The present report describes for the first time the expression and characterization of HCII in a baculovirus system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rHCII for future structure-function studies.


Subject(s)
Heparin Cofactor II/genetics , Heparin Cofactor II/isolation & purification , Animals , Cell Line , Chromatography, Affinity , Chymotrypsin/antagonists & inhibitors , Gene Expression , Glycosaminoglycans/pharmacology , Glycosylation , Heparin Cofactor II/pharmacology , Hirudins/pharmacology , Humans , In Vitro Techniques , Kinetics , Molecular Weight , Nucleopolyhedroviruses/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Spodoptera , Thrombin/antagonists & inhibitors
5.
Biochemistry ; 33(12): 3632-42, 1994 Mar 29.
Article in English | MEDLINE | ID: mdl-7908224

ABSTRACT

Heparin cofactor II (HCII) is a glycoprotein in human plasma that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. Unexpectedly, we found that HCII activity in murine plasma is present in two proteins of 68 and 72 kDa. The two proteins have the same N-terminal amino acid sequence, and both react with an antibody raised against the C-terminal nine amino acid residues of murine HCII predicted from the cDNA sequence. Treatment of the two proteins with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase yields a single 54-kDa band. Thus, murine plasma contains two forms of HCII that appear to have identical amino acid sequences but differ in the composition of their N-linked oligosaccharides. HCII cDNA clones isolated from a murine liver library include a 1434 bp open reading frame following the first Met codon, a TAA stop codon, and 580 bp of 3'-untranslated sequence terminating in a poly(A) tail. The amino acid sequence deduced from the cDNA contains the N-terminal sequence of purified murine plasma HCII preceded by a 23-residue hydrophobic sequence presumed to be the signal peptide. The amino acid sequence of murine HCII is 87% identical to that of human HCII, the greatest variability occurring in the N-terminal portion of the protein. Northern blot analysis reveals a 2.3-kb HCII mRNA in murine and human liver, but no HCII mRNA is detectable in heart, brain, spleen, lung, skeletal muscle, kidney, testis, placenta, pancreas, or intestine. Southern blot analysis of restriction fragment length polymorphisms in progeny on interspecific and intersubspecific crosses indicates that mice have a single HCII gene (designated Hcf2), which maps to chromosome 16 between Prm-1 and Igl. The murine HCII gene is approximately 7.1 kb in size and consists of at least four exons and three introns. The intron/exon organization is identical to that of the human HCII gene except at the 5' end, where the murine gene may lack a large intron in the 5'-untranslated region. Our results indicate that HCII is more highly conserved than the human and murine homologues of other serpins such as alpha 1-antitrypsin and alpha 1-antichymotrypsin.


Subject(s)
DNA, Complementary/chemistry , Gene Expression , Heparin Cofactor II/genetics , Heparin Cofactor II/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Codon , DNA, Complementary/isolation & purification , Exons , Heparin Cofactor II/chemistry , Humans , Introns , Liver/chemistry , Mice , Molecular Sequence Data , Molecular Weight , Oligosaccharides/analysis , Organ Specificity , Polymorphism, Restriction Fragment Length , RNA, Messenger
6.
J Biol Chem ; 268(5): 3639-45, 1993 Feb 15.
Article in English | MEDLINE | ID: mdl-8429040

ABSTRACT

Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Thrombin is inhibited by the serpins antithrombin III and heparin cofactor II in a reaction that is dramatically accelerated by glycosaminoglycans. The structural basis of the interaction with these inhibitors was investigated by introducing single amino acid substitutions into the anion-binding exosite (R68E, R70E) and unique insertion loops (K52E, K154A) of thrombin. The rate of inhibition of these recombinant thrombins by antithrombin III and heparin cofactor II was determined in the absence and presence of glycosaminoglycan. The second order rate constant (k2) for inhibition by antithrombin III without heparin was 3.7 x 10(5) M-1 min-1 for wild-type thrombin; rates for the mutant thrombins varied less than 2-fold. For inhibition by antithrombin III with heparin, the rate constant was 4.5 x 10(8) M-1 min-1 for wild-type thrombin with no significant differences between any of the recombinant thrombins. In contrast, the rate constant for inhibition by heparin cofactor II without glycosaminoglycan was 4.3 x 10(4) M-1 min-1 for wild-type thrombin; rates were 10-fold slower for thrombin K52E and 2- to 3-fold slower for thrombins R68E and R70E. The rate constants for inhibition of wild-type thrombin by HCII in the presence of heparin or dermatan sulfate were 9.2 x 10(8) M-1 min-1 and 9.0 x 10(8) M-1 min-1, respectively. Compared to wild-type thrombin, the rate of inhibition by HCII with glycosaminoglycan was 5- to 15-fold slower for thrombins K52E and R70E and 50- to over 100-fold slower for thrombin R68E. Thrombin K154A was inhibited by heparin cofactor II with rates similar to wild-type thrombin in all assays. These results suggest that heparin cofactor II interacts with residue Lys-52 in the proposed S1' subsite and with residues Arg-68 and Arg-70 in the anion-binding exosite of thrombin, and that these interactions contribute to the molecular basis of heparin cofactor II specificity for thrombin.


Subject(s)
Antithrombin III/pharmacology , Heparin Cofactor II/pharmacology , Mutagenesis, Site-Directed , Thrombin/antagonists & inhibitors , Antithrombin III/isolation & purification , Cell Line , Chromatography, Affinity , Glycosaminoglycans/pharmacology , Heparin Cofactor II/isolation & purification , Humans , Kinetics , Plasmids , Prothrombin/biosynthesis , Prothrombin/genetics , Prothrombin/isolation & purification , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Thrombin/genetics , Transfection
7.
J Chromatogr ; 539(2): 493-500, 1991 Feb 22.
Article in English | MEDLINE | ID: mdl-2045458

ABSTRACT

Heparin cofactor II (HCII) is an inhibitor of thrombin in human plasma whose activity is enhanced by heparin and dermatan sulphate. HCII was purified to homogeneity from normal human plasma with an overall yield of 7.5%. After treatment with barium chloride, precipitation with 50% saturated ammonium sulphate and dialysis of the resuspended precipitate against 0.02 M Tris-HCl (pH 7.4), the sample was chromatographed on a heparin-Sepharose CL 6B affinity column, DEAE-Sepharose CL 6B ion-exchange gel and an AcA 34 gel permeation column. For the final steps, a high-performance liquid chromatographic system was used which included ion-exchange chromatography on a Mono-Q column and gel permeation using a Superose column. The purified protein was homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The specific activity of purified HCII was 12.2 U/mg. The HCII activity was evaluated as antithrombin dermatan sulphate cofactor activity. A specific antiserum against HCII was raised in the rabbit.


Subject(s)
Chromatography, Affinity/methods , Chromatography, High Pressure Liquid/methods , Chromatography, Ion Exchange/methods , Heparin Cofactor II/isolation & purification , Electrophoresis, Polyacrylamide Gel , Heparin Cofactor II/immunology , Humans , Immune Sera/immunology , Thrombin/antagonists & inhibitors
8.
J Chromatogr ; 539(2): 289-96, 1991 Feb 22.
Article in English | MEDLINE | ID: mdl-1646210

ABSTRACT

Dermatan sulphate (DS) is a glycosaminoglycan which catalyses specifically thrombin inhibition by a plasmatic inhibitor, Heparin cofactor II (HCII). DS was insolubilized on a silica matrix to study its interaction with HCII. The immobilization of DS was performed with a good yield on a silica previously coated with polysaccharides in order to neutralize the negatively charged silanol groups. The value of the affinity constant of insolubilized DS for HCII, measured by the adsorption isotherm, is consistent with the value obtained for soluble DS. The DS bound to the silica matrix was also tested as a chromatographic support for the purification of HCII from human plasma; the optimum conditions for HCII adsorption and desorption were determined. The eluted HCII was obtained with a good yield (21%) and with no contamination by antithrombin III, the other main plasmatic inhibitor of thrombin.


Subject(s)
Chromatography, Affinity/methods , Dermatan Sulfate , Heparin Cofactor II/isolation & purification , Silicon Dioxide , Chromatography, Affinity/instrumentation , Dermatan Sulfate/chemistry , Heparin Cofactor II/metabolism , Humans
9.
Thromb Res ; 59(2): 269-77, 1990 Jul 15.
Article in English | MEDLINE | ID: mdl-2122537

ABSTRACT

To investigate the involvement of heparin cofactor II (HC II) in fibrinolytic system, endothelial cells from human umbilical vein were cultured in the presence of HC II or antithrombin III (AT III) combined with or without thrombin. Although AT III significantly inhibited thrombin-induced increase in tissue plasminogen activator antigen (t-PA:Ag) release, HC II did not exhibit such a suppressive effect. In contrast, in the presence of dermatan sulfate, HC II inhibited thrombin stimulation of t-PA:Ag release more strongly than AT III did. The release of plasminogen activator inhibitor-1 antigen (PAI-1:Ag) was also stimulated by thrombin; this stimulation was inhibited only by the combination of HC II and dermatan sulfate. Comparatively high concentrations of HC II significantly suppressed thrombin stimulation of t-PA:Ag and PAI-1:Ag release but did not cause an obvious change of both release in the absence of thrombin. Based on these results, it was suggested that HC II may inhibit an increase in fibrinolytic activity mediated by thrombin-stimulated endothelial cells in the liquid phase through a suppression of thrombin stimulation of t-PA:Ag release, when plasma is exposed to vascular smooth muscle cells or fibroblasts which synthesize a significant amount of dermatan sulfate.


Subject(s)
Dermatan Sulfate/pharmacology , Endothelium, Vascular/drug effects , Heparin Cofactor II/pharmacology , Thrombin/antagonists & inhibitors , Tissue Plasminogen Activator/metabolism , Antithrombin III/pharmacology , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Heparin Cofactor II/isolation & purification , Humans , Plasminogen Inactivators/immunology , Plasminogen Inactivators/metabolism , Tissue Plasminogen Activator/immunology
10.
J Biol Chem ; 265(11): 6092-7, 1990 Apr 15.
Article in English | MEDLINE | ID: mdl-2318847

ABSTRACT

We investigated the interaction of the human plasma proteinase inhibitor heparin cofactor II (HC) with human neutrophil elastase and cathepsin G in order to examine 1) proteinase inhibition by HC, 2) inactivation of HC, and 3) the effect of glycosaminoglycans on inhibition and inactivation. We found that HC inhibited cathepsin G, but not elastase, with a rate constant of 6.0 x 10(6) M-1 min-1. Inhibition was stable, with a dissociation rate constant of 1.0 x 10(-3) min-1. Heparin and dermatan sulfate diminished inhibition slightly. Both neutrophil elastase and cathepsin G at catalytic concentrations destroyed the thrombin inhibition activity of HC. Inactivation was accompanied by a dramatic increase in heat stability, as occurs with other serine proteinase inhibitors. Proteolysis of HC (Mr 66,000) produced a species (Mr 58,000) that retained thrombin inhibition activity, and an inactive species of Mr 48,000. Amino acid sequence analysis led to the conclusion that both neutrophil elastase and cathepsin G cleave HC at Ile66, which does not affect HC activity, and at Val439, near the reactive site Leu444, which inactivates HC. Since cathepsin G is inhibited by HC and also inactivates HC, we conclude that cathepsin G participates in both reactions simultaneously so that small amounts of cathepsin G can inactivate a molar excess of HC. High concentrations of heparin and dermatan sulfate accelerated inactivation of HC by neutrophil proteinases, with heparin having a greater effect. Heparin and dermatan sulfate appeared to alter the pattern, and not just the rate, of proteolysis of HC. We conclude that while HC is an effective inhibitor of cathepsin G, it can be proteolyzed by neutrophil proteinases to generate first an active inhibitor and then an inactive molecule. This two-step mechanism might be important in the generation of chemotactic activity from the amino-terminal region of HC.


Subject(s)
Cathepsins/blood , Heparin Cofactor II/metabolism , Neutrophils/enzymology , Pancreatic Elastase/blood , Amino Acid Sequence , Cathepsin G , Cathepsins/antagonists & inhibitors , Drug Stability , Glycosaminoglycans/pharmacology , Heparin Cofactor II/antagonists & inhibitors , Heparin Cofactor II/isolation & purification , Hot Temperature , Humans , Kinetics , Molecular Sequence Data , Oligopeptides/pharmacology , Protease Inhibitors/pharmacology , Protein Binding , Serine Endopeptidases
11.
J Biol Chem ; 265(10): 5623-8, 1990 Apr 05.
Article in English | MEDLINE | ID: mdl-2138609

ABSTRACT

Heparin cofactor II (HCII), a member of the "serpin" family of serine protease inhibitors, is a 65,600-Da plasma glycoprotein that inhibits thrombin and chymotrypsin. The rate of thrombin inhibition is stimulated approximately 1000-fold by heparin or dermatan sulfate. Thrombin and chymotrypsin cleave the Leu444-Ser445 bond (designated P1-P'1) in the reactive site of HCII, forming a stable equimolar complex in which the protease is inactive. In this study, we have determined the effects of substituting an arginine for Leu444 in recombinant HCII (rHCII). The rHCII was expressed in Escherichia coli and partially purified by heparin-Sepharose chromatography. Apparent second-order rate constants (k2) for inhibition of thrombin, coagulation factor Xa, kallikrein, plasmin, and chymotrypsin by rHCII were determined using appropriate chromogenic substrates. In the absence of a glycosaminoglycan, rHCII(Leu444----Arg) inhibited thrombin at a 98-fold higher rate (k2 = 6.2 x 10(6) M-1 min-1) than native rHCII (k2 = 6.3 x 10(4) M-1 min-1). Dermatan sulfate accelerated thrombin inhibition by both forms of rHCII, but the maximum rate constant in the presence of dermatan sulfate was only 2-fold higher for rHCII(Leu444----Arg) (k2 = 5.3 x 10(8) M-1 min-1) than for native rHCII (k2 = 2.2 x 10(8) M-1 min-1). Heparin was less effective than dermatan sulfate in stimulating both forms of rHCII. Factor Xa, kallikrein, and plasmin were inhibited more rapidly and chymotrypsin more slowly by rHCII(Leu444----Arg) than by native rHCII. These effects are qualitatively similar to those observed with the natural mutant alpha 1-antitrypsin Pittsburgh (Met358----Arg at the P1 position) and strengthen the hypothesis that the P1 residue is a major determinant of protease specificity in the serpins. Furthermore, the rapid rate of inhibition of thrombin by rHCII(Leu444----Arg) in the absence of heparin or dermatan sulfate suggests that this variant may be useful as a therapeutic agent.


Subject(s)
Arginine , Heparin Cofactor II/pharmacology , Leucine , Thrombin/antagonists & inhibitors , Amino Acid Sequence , Base Sequence , Binding Sites , Chymotrypsin/antagonists & inhibitors , Dermatan Sulfate/pharmacology , Escherichia coli/genetics , Factor Xa Inhibitors , Fibrinolysin/antagonists & inhibitors , Gene Expression , Genetic Vectors , Heparin/pharmacology , Heparin Cofactor II/genetics , Heparin Cofactor II/isolation & purification , Kallikreins/antagonists & inhibitors , Kinetics , Molecular Sequence Data , Mutation , Recombinant Proteins/pharmacology , Structure-Activity Relationship , Transformation, Bacterial
SELECTION OF CITATIONS
SEARCH DETAIL
...