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D M Tollefsen

Publications and source records attributed to D M Tollefsen.

At least 37 records · Page 2Linked to original sources
PubMed1994

Murine heparin cofactor II: purification, cDNA sequence, expression, and gene structure.

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.

Amino Acid Sequence↗
PubMed1993

Specific glycosaminoglycans support the inhibition of thrombin by plasminogen activator inhibitor 1.

In the absence of accessory components, plasminogen activator inhibitor 1 (PAI-1) rapidly forms equimolar, inactive complexes both with tissue-type (t-PA) and with urokinase-type (u-PA) plamsinogen activator. In the presence of either the glycoprotein vitronectin or the glycosaminoglycan heparin, PAI-1 is endowed with additional, efficient thrombin-inhibitory properties (Ehrlich et al., 1990, 1991a). Here, we have investigated the interaction between PAI-1, thrombin, and glycosaminoglycans in more detail. Inhibition of thrombin by PAI-1 was quantitatively analyzed in the presence of a wide range of concentrations of heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, keratan sulfate, and hyaluronic acid by measuring residual amidolytic activity. In addition, a qualitative analysis was performed by determining the formation of SDS-stable, equimolar complexes between thrombin and PAI-1 in the presence of various glycosaminoglycans. Heparin, at concentrations between 0.1 and 1 microgram/mL, significantly promoted thrombin inhibition by PAI-1 as well as SDS-stable complex formation. Suboptimal inhibition was observed with dermatan sulfate, chondroitin 4-sulfate, and heparan sulfate at concentrations that are at least 1 order of magnitude higher than that required for optimal inhibition in the presence of heparin. Virtually no inhibition of thrombin and SDS-stable complex formation was detected with any of the other glycosaminoglycans at concentrations between 0.1 and 1 microgram/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

Mutagenesis of thrombin selectively modulates inhibition by serpins heparin cofactor II and antithrombin III. Interaction with the anion-binding exosite determines heparin cofactor II specificity.

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.

Antithrombin III↗
PubMed1993

Cross-competition for binding of alpha 1-antitrypsin (alpha 1 AT)-elastase complexes to the serpin-enzyme complex receptor by other serpin-enzyme complexes and by proteolytically modified alpha 1 AT.

The serpin-enzyme complex (SEC) receptor recognizes a pentapeptide neo-domain of alpha 1-antitrypsin (alpha 1 AT)-elastase complexes and, in so doing, mediates internalization and intracellular catabolism of the macromolecular complex, mediates an increase in synthesis of alpha 1 AT, and elicits neutrophil chemotactic activity. In previous studies we have shown that this pentapeptide domain is highly conserved among members of the serpin family and that binding of a synthetic peptide corresponding to this region (125I-peptide 105Y, SIP-PEVKFNKPFVYLI, based on alpha 1 AT sequence 359-374) to HepG2 cells is blocked by several serpin-enzyme complexes. To determine whether the SEC receptor is the primary HepG2 cell surface binding site for these serpin-enzyme complexes, we examined the capacity for serpin-enzyme complexes to compete with each other for binding to the SEC receptor. The results indicate that binding of 125I-elastase-alpha 1 AT complexes is blocked by thrombin-antithrombin III (ATIII), thrombin-heparin cofactor II, and cathepsin G-alpha 1-antichymotrypsin (alpha 1 ACT) complexes. Moreover, unlabeled elastase-alpha 1 AT complexes compete for binding of 125I-thrombin-ATIII, 125I-thrombin-heparin cofactor II, and 125I-cathepsin G-alpha 1 ACT complexes. Preformed soluble tissue plasminogen activator-plasminogen activator inhibitor 1 complexes also compete for binding of elastase-alpha 1 AT complexes to the SEC receptor but do so to a less effective extent, probably because of a less favorable pentapeptide sequence for binding to the SEC receptor. Under conditions in which these serpin-enzyme complexes would be expected to bind to the SEC receptor there is an increase in synthesis of alpha 1 AT but not in synthesis of ATIII or alpha 1 ACT. Proteolytically modified alpha 1 AT also competes for binding of 125I-elastase-alpha 1 AT complexes to the SEC receptor and vice versa. The purified 51-kDa amino-terminal fragment of alpha 1 AT does not compete for binding of 125I-elastase-alpha 1 AT complexes, indicating that the pentapeptide neodomain in the 4-kDa carboxyl-terminal fragment is sufficient for binding to the SEC receptor.

Amino Acid Sequence↗
PubMed1992

The interaction of glycosaminoglycans with heparin cofactor II: structure and activity of a high-affinity dermatan sulfate hexasaccharide.

The binding sites for dermatan sulfate and heparin in HCII overlap but are not identical. This may explain the observation that HCII binds non-specifically to heparin oligosaccharides but preferentially binds to a minor hexasaccharide isolated from dermatan sulfate having the structure shown in Fig. 4B. The tissue distribution of dermatan sulfate molecules containing the high-affinity HCII binding site may regulate HCII activity in vivo. Finally, in the presence of dermatan sulfate or heparin, the N-terminal acidic domain of HCII may interact with the hirudin-binding site of thrombin to produce maximal stimulation of the thrombin-HCII reaction.

Amino Acid Sequence↗
PubMed1991

The N-terminal acidic domain of heparin cofactor II mediates the inhibition of alpha-thrombin in the presence of glycosaminoglycans.

Heparin cofactor II (HCII) is a glycoprotein in human plasma that inhibits thrombin and chymotrypsin. Inhibition occurs when the protease attacks the reactive site peptide bond in HCII (Leu444-Ser445) and becomes trapped as a covalent 1:1 complex. Dermatan sulfate and heparin increase the rate of inhibition of thrombin, but not of chymotrypsin, greater than 1000-fold. The N-terminal portion of HCII contains two acidic repeats (Glu56-Asp-Asp-Asp-Tyr-Leu-Asp and Glu69-Asp-Asp-Asp-Tyr-Ile-Asp) that may bind to anion-binding exosite I of thrombin to facilitate covalent complex formation. To examine the importance of the acidic domain, we have constructed a series of 5' deletions in the HCII cDNA and expressed the recombinant HCII (rHCII) in Escherichia coli. Apparent second-order rate constants (k2) for inhibition of alpha-thrombin and chymotrypsin by each variant were determined. Deletion of amino acid residues 1-74 had no effect on the rate of inhibition of alpha-thrombin or chymotrypsin in the absence of a glycosaminoglycan. Similarly, the rate of inhibition of alpha-thrombin in the presence of a glycosaminoglycan was unaffected by deletion of residues 1-52. However, deletion of residues 1-67 (first acidic repeat) or 1-74 (first and second acidic repeats) greatly decreased the rate of inhibition of alpha-thrombin in the presence of heparin, dermatan sulfate, or a dermatan sulfate hexasaccharide that comprises the minimum high-affinity binding site for HCII. Deletion of one or both of the acidic repeats increased the apparent affinity of rHCII for heparin-Sepharose, suggesting that the acidic domain may interact with the glycosaminoglycan-binding site of native rHCII. The stimulatory effect of glycosaminoglycans on native rHCII was decreased by a C-terminal hirudin peptide which binds to anion-binding exosite I of alpha-thrombin. Furthermore, the ability of native rHCII to inhibit gamma-thrombin, which lacks the binding site for hirudin, was stimulated weakly by glycosaminoglycans. These results support a model in which the stimulatory effect of glycosaminoglycans on the inhibition of alpha-thrombin is mediated, in part, by the N-terminal acidic domain of HCII.

Amino Acid Sequence↗
PubMed1991

Cyclic AMP-dependent protein kinase phosphorylates serine378 in vitronectin.

We previously observed that Ser378 in the heparin-binding domain of vitronectin becomes phosphorylated by a protein kinase in plasma upon addition of ATP and divalent cations. We now report that purified plasma vitronectin contains approximately 2.5 mol of phosphate per mol of protein and that vitronectin becomes phosphorylated during biosynthesis in human hepatoma (HepG2) cells. In vitro, rabbit muscle cAMP-dependent protein kinase specifically phosphorylates Ser378 in single-chain (75 kDa) vitronectin but does not phosphorylate the two-chain (65/10 kDa) form cleaved at Arg379. Heparin affects neither the time course nor the extent of phosphorylation of Ser378 at neutral pH. The extent of phosphorylation of Ser378 achieved with cAMP-dependent protein kinase (greater than or equal to 0.3 mol phosphate per mol vitronectin) is greater than that obtainable in plasma and should enable comparisons to be made of the activities of the native and phosphorylated forms.

Adenosine Triphosphate↗
PubMed1991

Role of lysine 173 in heparin binding to heparin cofactor II.

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits alpha-thrombin in a reaction that is dramatically enhanced by heparin and other glycosaminoglycans/polyanions. We investigated the glycosaminoglycan binding site in HC by: (i) chemical modification with pyridoxal 5'-phosphate (PLP) in the absence and presence of heparin and dermatan sulfate; (ii) molecular modeling; and (iii) site-directed oligonucleotide mutagenesis. Four lysyl residues (173, 252, 343, and 348) were protected from modification by heparin and to a lesser extent by dermatan sulfate. Heparin-protected PLPHC retained both heparin cofactor and dermatan sulfate cofactor activity while dermatan sulfate-protected PLPHC retained some dermatan sulfate cofactor activity and little heparin cofactor activity. Molecular modeling studies revealed that Lys173 and Lys252 are within a region previously shown to contain residues involved in glycosaminoglycan binding. Lys343 and Lys348 are distant from this region, but protection by heparin and dermatan sulfate might result from a conformational change following glycosaminoglycan binding to the inhibitor. Site-directed mutagenesis of Lys173 and Lys343 was performed to further dissect the role of these two regions during HC-heparin and HC-dermatan sulfate interactions. The Lys343----Asn or Thr mutants had normal or only slightly reduced heparin or dermatan sulfate cofactor activity and eluted from heparin-Sepharose at the same ionic strength as native recombinant HC. However, the Lys173----Gln or Leu mutants had greatly reduced heparin cofactor activity and eluted from heparin-Sepharose at a significantly lower ionic strength than native recombinant HC but retained normal dermatan sulfate cofactor activity. Our results demonstrate that Lys173 is involved in the interaction of HC with heparin but not with dermatan sulfate, whereas Lys343 is not critical for HC binding to either glycosaminoglycan. These data provide further evidence for the determinants required for glycosaminoglycan binding to HC.

Amino Acid Sequence↗
PubMed1991

Complete nucleotide sequence of the gene for human heparin cofactor II and mapping to chromosomal band 22q11.

Heparin cofactor II (HCII) is a 66-kDa plasma glycoprotein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. Clones comprising the entire HCII gene were isolated from a human leukocyte genomic library in EMBL-3 lambda phage. The sequence of the gene was determined on both strands of DNA (15,849 bp) and included 1749 bp of 5'-flanking sequence, five exons, four introns, and 476 bp of DNA 3' to the polyadenylation site. Ten complete and one partial Alu repeats were identified in the introns and 5'-flanking region. The HCII gene was regionally mapped on chromosome 22 using rodent-human somatic cell hybrids, carrying only parts of human chromosome 22, and the chronic myelogenous leukemia cell line K562. With the cDNA probe HCII7.2, containing the entire coding region of the gene, the HCII gene was shown to be amplified 10-20-fold in K562 cells by Southern analysis and in situ hybridization. From these data, we concluded that the HCII gene is localized on the chromosomal band 22q11 proximal to the breakpoint cluster region (BCR). Analysis by pulsed-field gel electrophoresis indicated that the amplified HCII gene in K562 cells maps at least 2 Mbp proximal to BCR-1. Furthermore, the HCII7.2 cDNA probe detected two frequent restriction fragment length polymorphisms with the restriction enzymes BamHI and HindIII.

Amino Acid Sequence↗
PubMed1990

Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity.

Dermatan sulfate increases the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor and the protease bind. Dermatan sulfate is a linear polymer of D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) alternating with N-acetyl-D-galactosamine (GalNAc) residues. Heterogeneity in dermatan sulfate results from varying degrees of O-sulfation and from the presence of the two types of uronic acid residues. To characterize the HCII-binding site in dermatan sulfate, we isolated the smallest fragment of dermatan sulfate that bound to HCII with high affinity. Dermatan sulfate was partially N-deacetylated by hydrazinolysis, cleaved with nitrous acid at pH 4, and reduced with [3H]NaBH4. The resulting fragments, containing an even number of monosaccharide units with the reducing terminal GalNAc converted to [3H]2,5-anhydro-D-talitol (ATalR), were size-fractionated and then chromatographed on an HCII-Sepharose column. The smallest HCII-binding fragments were hexasaccharides, of which approximately 6% bound. Based on ion-exchange chromatography, the bound material appeared to comprise a heterogeneous mixture of molecules possessing four, five, or six sulfate groups per hexasaccharide. Subsequently, hexasaccharides with the highest affinity for HCII were isolated by overloading the HCII-Sepharose column. The high-affinity hexasaccharides were fractionated by strong anion-exchange chromatography, and one major peak representing approximately 2% of the starting hexasaccharides was isolated. The high-affinity hexasaccharide was cleaved to disaccharides that were analyzed by anion-exchange chromatography, paper electrophoresis, and paper chromatography. A single disulfated disaccharide, IdoA(2-SO4)----ATalR(4-SO4) was observed, indicating that the hexasaccharide has the following structure: IdoA(2-SO4)----GalNAc(4-SO4)----IdoA(2-SO4)---- GalNAc(4-SO4)----IdoA(2-SO4)----ATalR(4-SO4). Since IdoA(2-SO4)----GalNAc(4-SO4) comprises only approximately 5% of the disaccharides present in intact dermatan sulfate, clustering of these disaccharides must occur during biosynthesis to form the high-affinity binding site for HCII.

Animals↗
PubMed1990

The presence of methionine or threonine at position 381 in vitronectin is correlated with proteolytic cleavage at arginine 379.

Vitronectin (serum spreading factor, complement S protein, epibolin) is a glycoprotein that mediates cell adhesion and interacts with components of the complement, coagulation, and fibrinolytic systems. It circulates in plasma as a 75-kDa single chain polypeptide and as a two-chain form consisting of 65- and 10-kDa polypeptides linked by a disulfide bond. An individual may have a predominance of the single chain or the two-chain form inherited as a Mendelian trait or have approximately equal amounts of both forms. Inspection of published cDNA sequences suggests that either methionine or threonine can occur at position 381, which is adjacent to the presumed site of proteolytic cleavage (Arg379-Ala380) that gives rise to the two-chain form. We have determined the presence of the Met381 and/or Thr381 alleles of the vitronectin gene in 42 individuals by oligonucleotide hybridization to genomic DNA. To determine whether this polymorphism is correlated with the susceptibility to cleavage of the Arg379-Ala380 peptide bond in vivo, we have prepared immunoblots of plasma from the same group of individuals. Nineteen individuals homozygous for the Thr381 allele had 17 +/- 9% (mean +/- S.D.) single chain vitronectin in their plasma samples. Nine individuals homozygous for the Met381 allele had 66 +/- 4% single chain vitronectin. Fourteen heterozygous individuals had 38 +/- 13% single chain vitronectin. The differences in mean values were statistically significant (p less than 0.001). These results suggest that the presence of threonine rather than methionine at position 381 of vitronectin increases the susceptibility of the protein to cleavage of the Arg379-Ala380 peptide bond in vivo.

Alleles↗
PubMed1990

Substitution of arginine for Leu444 in the reactive site of heparin cofactor II enhances the rate of thrombin inhibition.

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.

Amino Acid Sequence↗
PubMed1990

Site-directed mutagenesis of arginine 103 and lysine 185 in the proposed glycosaminoglycan-binding site of heparin cofactor II.

Inhibition of thrombin by heparin cofactor (HCII) is accelerated approximately 1000-fold by heparin or dermatan sulfate. We found recently that the mutation Arg189----His decreases the affinity of HCII for dermatan sulfate but not for heparin (Blinder, M. A., Andersson, T. R., Abildgaard, U., and Tollefsen, D. M. (1989) J. Biol. Chem. 264, 5128-5133). Other investigators have implicated Arg47 and Lys125 of anti-thrombin (homologous to Arg103 and Lys185 of HCII) in heparin binding. To investigate the corresponding residues in HCII, we have constructed amino acid substitutions (Arg103----Leu, Gln, or Trp; Lys185----Met, Asn, or Thr) by oligonucleotide-directed mutagenesis of the cDNA and expressed the products in Escherichia coli. The recombinant HCII variants were assayed for binding to heparin-Sepharose and for inhibition of thrombin in the presence of various concentrations of heparin or dermatan sulfate. All of the Arg103 variants bound to heparin with normal affinity. Furthermore, inhibition of thrombin by the Arg103----Leu variant occurred at a normal rate in the absence of a glycosaminoglycan and was accelerated by normal concentrations of heparin and dermatan sulfate. These results indicate that HCII, unlike anti-thrombin, does not require a positive charge at this position for the interaction with heparin or dermatan sulfate. The Arg103----Gln and Arg103----Trp variants inhibited thrombin at about one-third of the normal rate in the absence of a glycosaminoglycan, suggesting that these mutations exert an effect on the reactive site (Leu444-Ser445) of HCII. All of the Lys185 variants bound to heparin with decreased affinity but inhibited thrombin at approximately the normal rate in the absence of a glycosaminoglycan. These variants required greater than 10-fold higher concentrations of heparin to accelerate inhibition of thrombin and were not stimulated significantly by dermatan sulfate, suggesting that heparin and dermatan sulfate interact with Lys185 of HCII. These results provide evidence that the glycosaminoglycan-binding site in HCII includes Lys185 but not Arg103, both of which were predicted to be involved by homology to anti-thrombin.

Amino Acid Sequence↗
PubMed1990

Laboratory diagnosis of antithrombin and heparin cofactor II deficiency.

Antithrombin activity should probably be determined in persons with early onset of recurrent thrombosis, especially if a family history is present. The initial screening test should be a heparin cofactor assay optimized to reduce the contribution of heparin cofactor II. If the heparin cofactor assay is low, an antigenic determination should be performed to rule out the possibility of an antithrombin variant. At the present time, there is insufficient evidence to recommend the routine determination of heparin cofactor II levels in patients with thrombosis.

Antithrombins↗
PubMed1990

Effect of low molecular weight heparin preparations on the inhibition of thrombin by heparin cofactor II.

1. Heparin molecules approximately 24 to 30 residues in length are required to catalyze the thrombin-HC II reaction. The requirement for heparin molecules of this length is consistent with a model for catalysis in which heparin binds HC II and thrombin simultaneously to form a ternary complex in a manner similar to that proposed for the thrombin-AT III reaction. Smaller molecules (18 or more monosaccharide units in length) are required to catalyze the thrombin-AT III reaction. 2. The specific AT III-binding pentasaccharide containing 3-O-sulfated glucosamine is not required for activity with HC II. 3. Some low molecular weight heparin preparations have significant activity with HC II (approximately 10 to 20% that of standard heparin). This is probably related to the presence of species with molecular weights greater than 6000 to 7500 (24 to 30 monosaccharide units) in these preparations.

Antithrombin III↗
PubMed1989

Antithrombin activity of a peptide corresponding to residues 54-75 of heparin cofactor II.

Heparin cofactor II (HCII) is a highly specific serine proteinase inhibitor, which complexes covalently with thrombin in a reaction catalyzed by heparin and other polyanions. The molecular basis for the thrombin specificity may be explained by the identification here of a segment of HCII including residues 54-75 that binds to thrombin. A synthetic peptide, HCII(54-75), based on this segment of HCII, Gly-Glu-Glu-Asp-Asp-Asp-Tyr-Leu-Asp-Leu-Glu- Lys-Ile-Phe-Ala-Glu-Asp-Asp-Asp-Tyr-Ile-Asp inhibited thrombin's cleavage of fibrinogen. Clotting activity of thrombin was inhibited 50% at a concentration of 28 microM. Polyacrylamide gel electrophoresis showed that HCII(54-75) inhibited thrombin's cleavage of both the A alpha and B beta polypeptides in fibrinogen. However, the peptide did not block thrombin's active site, as hydrolysis of chromogenic substrates was not inhibited. HCII(54-75) probably binds to the same site on thrombin as do carboxyl-terminal residues of hirudins, thrombin inhibitors of leeches. HCII(54-75) inhibited binding of thrombin to a synthetic peptide corresponding to residues 54-66 of hirudin PA, but the hirudin peptide was about 30-fold more potent in binding and clotting assays. Both synthetic peptides, as a result of their polyanionic character, might be expected to stimulate the reaction of HCII with thrombin. However, the hirudin-related peptide inhibited this reaction, suggesting that it blocked a site on thrombin required for interaction with HCII. HCII(54-75) had a net stimulatory effect on the thrombin-HCII reaction as a consequence of its lower affinity for thrombin and greater negative charge relative to the hirudin-related peptide. These studies suggest that residues 54-75 of HCII interact with a noncatalytic binding site on thrombin and that this interaction contributes to efficient inhibition of thrombin by HCII.

Amino Acid Sequence↗