Antithrombin III-binding Site Research Articles

Qualitative analysis of enzymatic and chemical depolymerized low molecular weight heparins by UHPLC coupled with electrospray ionization quadrupole time-of-flight-mass spectrometry.

Complete heparin digestion with heparin lyase I and II results in a mixture of hexasaccharides and tetrasaccharides with 3-O-sulfo group-containing glucosamine residues at their reducing ends. Because these tetrasaccharides are derived from antithrombin III-binding sites of heparin, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. Therefore, this paper presents a new low molecular weight heparin sample preparation method-chemical depolymerization. Qualitative analysis of the studied compounds and a comparison of their composition are an important contribution to the structural analysis of low molecular weight heparins, which has not been fully conducted so far. Qualitative on-line liquid chromatography-mass spectrometric analysis of these resistant oligosaccharides is also described in this paper.

Analysis of 3-O-sulfo group-containing heparin tetrasaccharides in heparin by liquid chromatography–mass spectrometry

Complete heparin digestion with heparin lyase 2 affords a mixture of disaccharides and resistant tetrasaccharides with 3-O-sulfo group-containing glucosamine residues at their reducing ends. Quantitative online liquid chromatography–mass spectrometric analysis of these resistant tetrasaccharides is described in this article. The disaccharide and tetrasaccharide compositions of seven porcine intestinal heparins and five low-molecular-weight heparins were analyzed by this method. These resistant tetrasaccharides account for from 5.3 to 7.3wt% of heparin and from 6.2 to 8.3wt% of low-molecular-weight heparin. Because these tetrasaccharides are derived from heparin’s antithrombin III-binding sites, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. The content of 3-O-sulfo group-containing tetrasaccharides in a heparin correlated positively (r=0.8294) to heparin’s anticoagulant activity.

Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site

Porcine mucosal heparin was fragmented into low-molecular-weight (LMW) heparin by treatment of periodate-oxidized heparin with sodium hydroxide, followed by reduction with sodium borohydride and acid hydrolysis. Gradient polyacrylamide gel electrophoresis analysis showed a mixture of heparin fragments with an average size of eight disaccharide units. 1D 1H NMR showed two-thirds of the N-acetyl groups were lost on periodate cleavage, suggesting cleavage had occurred at the glucopyranosyluronic acid (Glc pA) and idopyranosyluronic acid (Ido pA) residues located within and adjacent to the antithrombin III (ATIII) binding site. The N-acetyl glucopyranose (Glc pNAc) residue was lost on workup. The Glc pA residue, within the ATIII binding site, is on the non-reducing side of the N-sulfo, 3, 6- O-sulfo glycopyranosylamine (Glc pNS3S6S) residue. Thus, periodate cleaved heparin should be enriched in Glc pNS3S6S residues. Two-dimensional correlation spectroscopy (2D COSY) confirmed that LMW heparin prepared through periodate cleavage contained Glc pNS3S6S at its non-reducing end. As expected, this LMW heparin also showed reduced ATIII mediated anti-factor IIa and anti-factor Xa activities.

Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin.

Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III). Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobacterium heparinum, specifically heparinases I and II. Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.

Structures of Five Sulfated Hexasaccharides Prepared from Porcine Intestinal Heparin Using Bacterial Heparinase: STRUCTURAL VARIANTS WITH APPARENT BIOSYNTHETIC PRECURSOR-PRODUCT RELATIONSHIPS FOR THE ANTITHROMBIN III-BINDING SITE

Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography. Subfractionation of the hexasaccharide fraction by anion exchange high pressure liquid chromatography yielded 10 fractions. Six contained oligosaccharides derived from the repeating disaccharide region, whereas four contained glycoserines from the glycosaminoglycan-protein linkage region. The latter structures were reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de Beer, T., and Vliegenthart, J.F.G. (1995) J. Biol. Chem. 270, 22914-22923). In this study, the structures of one tetra- and five hexasaccharides from the repeat region were determined by chemical and enzymatic analyses as well as 500-MHz 1H NMR spectroscopy. The tetrasaccharide has the hexasulfated structure typical of heparin. The five hexa- or heptasulfated hexasaccharides share the common core pentasulfated structure delta HexA(2S) alpha 1-4GlcN-(NS, 6S) alpha 1-4IdoA alpha/GlcA beta 1-4GlcN(6S) alpha 1-4GlcA beta 1-4GlcN (NS) with one or two additional sulfate groups (delta HexA, GlcN, IdoA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S and NS stand for 2-O-, 6-O-, and 2-N-sulfate, respectively). Three components have the following hitherto unreported structures: delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS,6S), delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4IdoA alpha 1-4GlcNAc(6S)-alpha 1-4GlcA beta 1-4GlcN(NS, 3S), and delta HexA(2S) alpha 1-4GlcN-(NS,6S) alpha 1-4IdoA (2S) alpha 1-4GlcNAc(6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S). Two of the five hexasaccharides are structural variants derived from the antithrombin III-binding sites containing 3-O-sulfated GlcN at the reducing termini with or without a 6-O-sulfate group on the reducing N,3-disulfated GlcN residue. Another contains the structure identical to that of the above heptasulfated antithrombin III-binding site fragment but lacks the 3-O-sulfate group and therefore is a pro-form for the binding site. Another has an extra sulfate group on the internal IdoA residue of this pro-form and therefore can be considered to have diverged from the binding site in the biosynthetic pathway. Thus, the isolated hexasacharides in this study include the three overlapping pairs of structural variants with an apparent biosynthetic precursor-product relationship, which may reflect biosynthetic regulatory mechanisms of the binding site.

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.

Interaction of heparin with synthetic antithrombin III peptide analogues.

Heparin-binding proteins may contain specific patterns of basic amino acids, called consensus sequences, that interact with heparin. Small peptides were synthesized that contained consensus sequences (i.e. FAKLNCRLYRKANKSSK) or disrupted consensus sequences (i.e. K136-->A) based on the known sequence of antithrombin III (amino acid residues 123-139). These peptides were then examined in both competitive and non-competitive binding experiments using bioassays, fluorescence spectroscopy, affinity chromatography and n.m.r. spectroscopy. Both the consensus and disrupted-consensus peptide bound to heparin. Peptides with consensus sequences bound specifically to the pentasaccharide antithrombin III-binding site within heparin. In contrast, peptides with disrupted consensus sequences showed no specificity, binding to any sequence within heparin. Proton nuclear Overhauser enhancement spectroscopy demonstrated the proximity of leucine and tyrosine (within the consensus sequence) to the N-acetyl moiety found primarily within the pentasaccharide antithrombin III-binding site of heparin. This experiment confirmed the findings of the other techniques and helped to localize the binding sites in both peptides and heparin. A model is proposed for both specific and non-specific heparin interaction with consensus and disrupted-consensus peptides.

Structural studies on the tri- and tetrasaccharides isolated from porcine intestinal heparin and characterization of heparinase/heparitinases using them as substrates.

We prepared a series of oligosaccharides from porcine intestinal heparin after extensive digestion with a mixture of Flavobacterium heparinase as well as heparitinases I and II. Previously, we reported the structures of the two glycoserines derived from the carbohydrate-protein linkage region [Sugahara et al., J. Biol. Chem., 267, 1528-1533 (1992)] and three tetrasaccharides derived from the antithrombin III-binding site [Yamada et al., J. Biol. Chem., 268, 4780-4787 (1993)]. In this study, we determined the structures of 10 other tetrasaccharides and a trisaccharide by enzymatic digestion, fast atom bombardment mass spectrometry and 500-MHz 1H NMR spectroscopy. These tetrasaccharides share the common disulphated structure, delta HexA alpha 1-4GlcN(N-sulphate)alpha 1-4IdoA(2-sulphate)alpha 1-4GlcN (where HexA is hexuronic acid and IdoA is L-iduronic acid), and their structural variations are based upon the positions of additional sulphate groups. Eight among the 10 have never been isolated as discrete structures. The structure of the trisaccharide is GlcN(N-sulphate)alpha 1-4IdoA(2-sulphate) alpha 1-4GlcN(N,6-disulphate) and is derived from the non-reducing terminus of heparin chains. This structure may represent the terminus of a biosynthetically formed native heparin chain or a newly formed non-reducing terminus exposed by a tissue endo-beta-glucuronidase which may be involved in the intracellular post-synthetic fragmentation of macromolecular heparin. The 11 structures characterized in the present study and 6 additional tetrasaccharides were used to investigate the substrate specificities of heparinase, as well as heparitinases I and II. The results indicate that modification of the adjacent glucosamine on the reducing side of the disaccharide cleavage site influences the enzymatic action of the lyases, whereas the adjacent uronic acid on the non-reducing side is not recognized by these enzymes.

Binding of Heparin Fractions to von Willebrand Factor: Effect of Molecular Weight and Affinity for Antithrombin III

To investigate the influence of the structure of heparin on its binding to vWF, we compared heparin fractions of different molecular weight (MW) or affinity for antithrombin III (ATIII). We studied the interaction of purified 125I-vWF or plasma vWF, labeled with a pool of 125I-monoclonal antibodies to vWF, with unfractionated heparin immobilized on agarose beads. Fractions were compared as competitors of these interactions and their effect was quantitated by their half-maximal inhibition (IC50). When the MW of the fractions decreased, especially below 7500, their IC50 increased, indicating that the affinity of the fractions for vWF decreased with their MW. Using heparin-derived oligosaccharides, we also demonstrated that a minimal chain length of 18 monosaccharides was required for heparin binding to vWF. In addition, different fractions with low affinity for ATIII were compared as competitors of 125I-vWF binding to heparin-agarose. Despite a very low content of ATIII binding sites, some fractions retained a low IC50. Thus, heparin interaction with vWF is independent of the presence of the ATIII binding site and is mostly dependent on the length of the heparin chain. These data suggest that unfractionated heparin is a more potent inhibitor of vWF-dependent functions than low MW heparin fractions.

Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin.

Three discrete tetrasaccharide structures which are resistant to Flavobacterium heparinase and heparitinases I and II were isolated from porcine intestinal heparin after exhaustive digestion with a mixture of all the above enzymes, and the tri-, tetra-, and penta-sulfated structures were determined by negative ion mode fast atom bombardment mass spectrometry and 500-MHz 1H NMR analysis as delta 4,5GlcA beta 1-4GlcNAc (6-sulfate)alpha 1-4GlcA beta 1-4GlcN(N,3-disulfate), delta 4,5 GlcA beta 1-4GlcNAc(6-sulfate)alpha 1-4GlcA beta 1-4GlcN (N,3,6-trisulfate), and delta 4,5GlcA beta 1-4GlcN (N,6-disulfate)alpha 1-4GlcA beta 1-4GlcN(N,3,6-trisulfate). The three components share the 3-O-sulfated reducing GlcN and the 6-O-sulfated internal GlcN, indicating that they are structural variants derived from the nonreducing portion of the minimal pentasaccharide sequence required for binding to antithrombin III. Isolation of the pentasulfated component has never been reported. Their unexpected resistance to heparitinases I and II indicates that 3-O-sulfation of the reducing GlcN contributes to the resistant nature of these tetrasaccharides to the enzymes. The present study demonstrates that the nonreducing trisaccharide portion of the structural variants of the antithrombin III-binding pentasaccharide sequence can be isolated in tetrasaccharides resistant to heparinase/heparitinases I and II, while the rest of the repeating region is degraded into disaccharide units. The lyase treatment is applicable to evaluation of heparin/heparan sulfate preparations in terms of the presence or absence of the specific structure containing the 3-O-sulfated GlcN representing biosynthetic precursors, intermediates or final products of the binding site.

Search for the heparin antithrombin III-binding site precursor.

The last step of heparin biosynthesis is thought to involve the action of 3-O-sulfotransferase resulting in the formation of an antithrombin III (ATIII) binding site required for heparin's anticoagulant activity. The isolation of a significant fraction of heparin chains without antithrombin III-binding sites and having low affinity for ATIII suggests the presence of a precursor site, lacking the 3-O-sulfate group. Porcine mucosal heparin was depolymerized into a mixture of oligosaccharides using heparin lyase. One of these oligosaccharides was derived from heparin's ATIII-binding site. In an effort to find the ATIII-binding site precursor, the structures of several minor oligosaccharides were determined. A greater than 90% recovery of oligosaccharides (on a mole and weight basis) was obtained for both unfractionated and affinity-fractionated heparins. An oligosaccharide arising from the ATIII-binding site precursor was found that comprised only 0.8 mol % of the oligosaccharide product mixture. This oligosaccharide was only slightly enriched in heparin having a low affinity for ATIII and only slightly disenriched in high affinity heparin. The small number of these ATIII-binding site precursors, found in unfractionated and fractionated heparins, suggests the existence of a low ATIII affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step in heparin biosynthesis. Rather these data suggest that some earlier step, involved in the formation of placement of these precursor sites, may be primarily responsible for high and low ATIII affinity heparins.

Nonrandom structural features in the heparin polymer.

Computer simulation studies were used to prepare an ensemble of heparin number chains. The polydispersity of these chains was simulated by introducing a specific "fraction of terminators", and it closely resembled the experimentally observed polydispersity of a porcine mucosal, glycosaminoglycan heparin. The same percentage of simulated chains contained antithrombin III (ATIII) binding site sequences as are typically found to contain ATIII binding sites using affinity chromatography. Heparin lyase action was then simulated by using Michaelis-Menten kinetics. In one model, heparin chains were constructed from the random assembly of monosaccharide units using the observed mole percentage of each. After simulated depolymerization, the final oligosaccharides formed were compared to the observed oligosaccharide products. The simulation which assumed a random distribution of monosaccharide units in heparin did not agree with experimental observations. In particular, no ATIII binding site sequences were found in the simulated number chains. The results of this simulation indicate that heparin is not simply a random assembly of monosaccharide units. These results are consistent with the known, ordered biosynthesis of heparin. In a second model, heparin chains were constructed from randomly assembled oligosaccharides at the mole percentage in which each is found in the final product mixture. The action of heparin lyase was then simulated, and the distribution of the oligosaccharide products was measured throughout the simulated time course of the depolymerization reaction. The simulated rate of formation and final concentration of a particular oligosaccharide which contains a portion of heparin's ATIII binding site were similar to those observed experimentally. These results are consistent with the random distribution of ATIII binding sites within glycosaminoglycan heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping and quantification of the major oligosaccharide components of heparin.

A new method of determining the oligosaccharide composition of commercial glycosaminoglycan heparin is described in which heparin was first depolymerized using heparin lyase (EC 4.2.2.7), and then analysed by a single h.p.l.c. step. All 20 of the porcine and bovine heparins examined were found to contain a small number of major oligosaccharide components, which on average comprised 86% of their mass. The five most abundant oligosaccharides have defined chemical structures. Although the relative abundance of oligosaccharides varied, the heparins examined were surprisingly similar. Porcine, bovine, low-Mr, and high and low antithrombin III (ATIII)-affinity heparins, however, each had distinctly different proportions of these major oligosaccharide components. The concentrations of one of these five oligosaccharides, containing a portion of the ATIII binding site, correlated with the anticoagulant activity of the ATIII-affinity-fractionated porcine-mucosal heparins from which it was derived. An additional oligosaccharide of undetermined structure was found in significant quantities in both bovine heparin and high ATIII-affinity porcine-mucosal heparin. The correlation between oligosaccharide concentration and anticoagulant activity suggests that the oligosaccharide is derived from a structural variant of the ATIII-binding site. Finally, for the heparins examined chondroitin/dermatan sulphate formed 0.6-7.4% of their mass.

ChemInform Abstract: Conformational Analysis of Synthetic Heparin‐like Oligosaccharides Containing α‐L‐Idopyranosyluronic Acid.

High-field NMR spectroscopy of synthetic heparin-like di- and trisaccharides has afforded a complete set of inter-proton-coupling data on α-L-idopyranosyluronic acid, which have been used for conformational analysis. These data lead to the conclusion that α-L-iduronic acid may display considerable conformational freedom including 1C4, 4C1 and 2S0 (e.g. compound 1) conformers. The 2-O-sulphate substituent at iduronic acid tends to stabilize the 1C4 conformation (e.g. compounds 2 and 4). In addition carbohydrate substituents at position 4 hinder the 4C1 conformation (e.g. compound 3a,b versus 1). Furthermore, the conformation of α-L-idopyranosyluronic acid 2-sulphate is affected seriously by the substituent at position 2 of the non-reducing glucosamine residue. Thus the presence of an ammonium group (i.e. compound 7) leads to unexpected deformation of the 1C4 form of iduronic acid, whereas a sulphamino substituent at this position (e.g. compound 5a,b) brings about the 2S0 skew boat conformation. The iduronic acid moiety of a trisaccharide (compound 6), which represents a part of the anti-thrombin III-binding site of heparin, shows conformational preference for the 2S0 form in aqueous solution under low ionic conditions. However, increasing the ionic strength (e.g. 3 M NaCl) causes the conformational equilibrium to incline towards the 1C4 chair conformer. This feature may be indicative for the mechanism of conformational control as exerted by the AT-III protein upon heparin binding. Several molecular models of preferred conformers have been constructed; Nuclear Overhauser Enhancement data, exo-anomeric effect and Van der Waals interactions have been taken into account.

Structure and activity of a unique heparin-derived hexasaccharide.

A hexasaccharide representing a major sequence in porcine mucosal heparin has been enzymatically prepared from heparin. Its structure was determined by an integrated approach using chemical, enzymatic, and spectroscopic methods. Two-dimensional 1H homonuclear COSY, C-H correlation NMR, and selective irradiation were used to assign many of the NMR resonances. In addition, new techniques including sulfate determination by ion chromatography and Fourier transform IR and californium plasma desorption mass spectroscopy have been applied, resulting in an unambiguous structural assignment of delta IdoAp2S(1----4)-alpha-D-GlcNp2S6S(1----4)-alpha-L-IdoAp++ +(1----4)-alpha-D-GlcNA cp6S-(1----4)-beta-D-GlcAp(1----4)-alpha-D-GlcNp2S3S6S (where delta IdoA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, p represents pyranose, and GlcA and IdoA represent glucuronic and iduronic acid). This hexasaccharide contains a portion of the antithrombin III-binding site and has a Kd of 4 X 10(-5) M. Unlike other small heparin oligosaccharides, which are specific for coagulation factor Xa, it inhibits both factors IIa and Xa equally through antithrombin III. This hexasaccharide may have the unique capacity to act primarily through heparin cofactor II to inhibit thrombin (factor IIa) and shows over half of heparin's heparin cofactor II-mediated anti-factor IIa activity. These studies suggest the occurrence of contiguous binding sites on heparin for Xa, antithrombin III, and heparin cofactor II.

Heparin: Do We Understand Its Antithrombotic Actions?

Heparin: Do We Understand Its Antithrombotic Actions?

Cleavage and activation of human factor IX by serine proteases

Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated 125I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa- tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. Kallikrein did not cleave the zymogen. Nonactivation cleavage was noted by thrombin, but only in the absence of calcium. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In contrast, incubation of factor IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or chymotrypsin did not lead to generation of an antithrombin III-binding site, despite their digestion of 125I-factor IX into heavy and light chain-sized fragments. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

Cleavage and Activation of Human Factor IX by Serine Proteases

Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated 125I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa-tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. Kallikrein did not cleave the zymogen. Nonactivation cleavage was noted by thrombin, but only in the absence of calcium. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In contrast, incubation of factor IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or chymotrypsin did not lead to generation of an antithrombin III-binding site, despite their digestion of 125I-factor IX into heavy and light chain-sized fragments. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4.

Oligosaccharides of well-defined molecular size were prepared from heparin by nitrous acid depolymerization, affinity chromatography on immobilized antithrombin III (see footnote on Nomenclature) and gel chromatography on Sephadex G-50. High affinity (for antithrombin III) octa-, deca-, dodeca-, tetradeca-, hexadeca- and octadeca-saccharides were prepared, as well as oligosaccharides of larger size than octadecasaccharide. The inhibition of Factor Xa by antithrombin III was greatly accelerated by all of these oligosaccharides, the specific anti-Factor Xa activity being invariably greater than 1300 units/mumol. The anti-Factor Xa activity of the decasaccharide was not significantly decreased in the presence of platelet factor 4, even at high platelet factor 4/oligosaccharide ratios. Measurable but incomplete neutralization of the anti-Factor Xa activities of the tetradeca- and hexadeca-saccharides was observed, and complete neutralization of octadeca- and larger oligo-saccharides was achieved with excess platelet factor 4. The octa-, deca-, dodeca-, tetradeca- and hexadeca-saccharides had negligible effect on the inhibition of thrombin by antithrombin III, whereas specific anti-thrombin activity was expressed by the octadeca-saccharide and by the larger oligosaccharides. An octadecasaccharide is therefore the smallest heparin fragment (prepared by nitrous acid depolymerization) that can accelerate thrombin inhibition by antithrombin III. The anti-thrombin activities of the octadecasaccharide and larger oligosaccharides were more readily neutralized by platelet factor 4 than were their anti-Factor Xa activities. These findings are compatible with two alternative mechanisms for the action of platelet factor 4, both involving the binding of the protein molecule adjacent to the antithrombin III-binding site. Such binding results in either steric interference with the formation of antithrombin III-proteinase complexes or in displacement of the antithrombin III molecule from the heparin chain.