Mucosal Heparin Research Articles

Human platelet heparanase: purification, characterization and catalytic activity.

Heparan sulphate (HS) is an important component of the extracellular matrix (ECM) and the vasculature basal lamina (BL) which functions as a barrier to the extravasation of metastatic and inflammatory cells. Platelet-tumour cell aggregation at the capillary endothelium results in activation and degranulation of platelets. Cleavage of HS by endoglycosidase or heparanase activity produced in relatively large amounts by the platelets and the invading cells may assist in the disassembly of the ECM and BL, and thereby facilitate cell migration. Using a recently published rapid, quantitative assay for heparanase activity towards HS [Freeman, C. and Parish, C.R. (1997), Biochem. J., 325, 229-237], human platelet heparanase has now been purified 1700-fold to homogeneity in 19% yield by a five column procedure, which consists of concanavalin A-Sepharose, Zn2+-chelating-Sepharose, Blue A-agarose, octyl-agarose and gel filtration chromatography. The enzyme, which was shown to be an endoglucuronidase that degrades both heparin and HS, has a native molecular mass of 50 kDa when analysed by gel filtration chromatography and by SDS/PAGE. Platelet heparanase degraded porcine mucosal HS in a stepwise fashion from a number average molecular mass of 18.5 to 13, to 8 and finally to 4.5 kDa fragments as determined by gel filtration analysis. Bovine lung heparin was degraded from 8.9 to 4.8 kDa while porcine mucosal heparin was degraded from 8.1 kDa to 3.8 and finally to 2.9 kDa fragments. Studies of the enzyme's substrate specificity using modified heparin analogues showed that substrate cleavage required the presence of carboxyl groups, but O- and N-sulphation were not essential. Inhibition studies demonstrated an absolute requirement for the presence of O-sulphate groups. Platelet heparanase was inhibited by heparin analogues which also inhibited tumour heparanase, suggesting that sulphated polysaccharides which inhibit tumour metastasis may act to prevent both tumour cell and platelet heparanase degradation of endothelial cell surface HS and the basal laminar.

Differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of unfractionated and low molecular weight heparins as therapeutic agents.

The selectins are calcium-dependent C-type lectins that bind certain sialylated, fucosylated, sulfated glycoprotein ligands. L-selectin also recognizes endothelial proteoglycans in a calcium-dependent manner, via heparan sulfate (HS) glycosaminoglycan chains enriched in unsubstituted glucosamine units. We now show that these HS chains can also bind P-selectin, but not E-selectin. However, while L-selectin binding requires micromolar levels of free calcium, P-selectin recognition is largely divalent cation-independent. Despite this, HS chains bound to P-selectin are eluted by ethylenediamine tetraacetic acid (EDTA), but only at high concentrations. Porcine intestinal mucosal (mast cell-derived) heparin (PIM-heparin) shows similar properties, with no binding to E-selectin, calcium-dependent binding of a subfraction to L-selectin and to P-selectin, and calcium-independent binding of a larger fraction to P-selectin, the latter being disrupted by high EDTA concentrations. Analysis of defined heparin fragment pools shows a size dependence for interaction, with tetradecasaccharides showing easily detectable binding to L- and P-selectin affinity columns. L-selectin binding fragments include more heavily sulfated and epimerized regions and, as with the endothelial HS chains, they are enriched in free amino groups. The P-selectin binding component includes this fraction as well as some less highly modified regions. Thus, endothelium-derived HS chains and mast cell-derived heparins could play a role in modulating the biology of selectins in vivo. Notably, P- and L-selectin binding to sialyl-Lewisx and to HL-60 cells (which are known to carry the native ligand PSGL-1) is inhibited by unfractionated pharmaceutical heparin preparations at concentrations 12-50-fold lower than those recommended for effective anticoagulation in vivo. In contrast, two low molecular weight heparins currently considered as clinical replacements for unfractionated heparin are much poorer inhibitors. Thus, patients undergoing heparin therapy for other reasons may be experiencing clinically significant inhibition of L- and P-selectin function, and the current switchover to low-molecular weight heparins may come at some loss of this effect. Low-dose unfractionated heparin should be investigated as a treatment option for acute and chronic diseases in which P- and L-selectin play pathological roles.

Heparin-Induced Thrombocytopenia: Prevalence in a Large Cohort of Patients and Confirmed Role of PF4/Heparin Complex as the Main Antigen for Antibodies

We performed a retrospective study on the prevalence of heparin-induced thrombocytopenia (HIT) in 233 patients receiving hog mucosa heparin therapy. Of these, 82 patients received s.c. calcium heparin, 130 patient received unfractionated (UF) i.v. heparin, and 21 patients received low molecular weight heparins (LMWH). An additional four patients, referred to our consultation and diagnosed by us as having clinically active type II HIT (HIT-II) were also studied. The mean platelet count of the 233 patients receiving heparin showed a significant decrease after 2 days of heparin treatment and a following significant increase 6 days later (basal: 257 ± 147 x 109 platelets/L; day 2: 239 ± 122, p < 0.0002; day 6: 286 ± 119, p < 0.004). Of the 212 patients receiving UF heparin, 13 (6%) fulfilled the criteria for HIT-II: seven of these had received i.v. heparin (mean daily dose 26,600 ± 4,082 IU ± SD) and six had received s.c. heparin (mean daily dose 21,428:t 6,900 IU). Their mean basal platelet count was 226 ± 100 SD × 109 platelets/L and the nadir during heparin treatment was 78 ± 39 x 10 9 platelets/L. Thrombotic complications occurred in four (30.7%) of the 13 patients with HIT-II. Since the immunological mechanism has been demonstrated for HIT-II and since platelet factor 4 (PF4) was identified as the co-factor for the binding of heparin-related antibodies, we set up our own enzyme-linked immunosorbent assay (ELISA) for testing antibodies against PF4/heparin complex bound through electrostatic bridges to the solid phase. The highest binding capacity of HIT-related IgG to the multimolecular complex was obtained at 20 μg/ml for PF4 and 3 μg/ml for heparin, corresponding to 250 ng of PF4 and 42 ng of heparin in each microtiter well. Such binding was inhibited in a dose-dependent manner by increasing amounts of heparin, protamine hydrochloride, and a monoclonal antibody anti-human PF4 clone 1OB2. We observed that HIT-related antibodies bound also to PF4/LMWH complexes but the optimal PF4/glycosaminoglycan ratio appeared more critical for LMWH (enoxaparin, fraxiparin, and pamaparin) than for UF heparin. Sera from eight patients with HIT-II were tested by PF4/heparin ELISA; six of these had IgG against the complex PF4/heparin and three also had IgM. The persistence of HIT-related antibodies was investigated in three patients: in one such antibodies were still detectable 3 years after the acute episode, while in the other two, they disappeared after 6 months and 1 year, respectively. Key Words: Heparin-related anti body—Platelet factor 4 (PF4)—Heparin—Low molecular weight heparin—Thrombocytopenia—Thrombosis.

Comparison of ovine, bovine and porcine mucosal heparins and low molecular weight heparins by disaccharide analyses and 13C NMR

Ovine, porcine and bovine heparin were compared by NMR, disaccharide composition, optical rotation, elemental analyses, HPSEC and USP analyses. There were some differences found in the NMR and disaccharide composition of the heparins. Ovine heparin contained less monosulphated and disulphated disaccharides than porcine or bovine mucosal heparins. The heparins were approximately equivalent in USP activity. Low molecular weight ovine, porcine and bovine heparin were prepared by both nitrous acid and copper/peroxide depolymerisation. Products were compared to each other and to Fragmin® by disaccharide composition, optical rotation, HPSEC, NMR, elemental analyses, and the USP assay for anticoagulant activity. The low molecular weight products showed a greater similarity to each other than to the parent heparins and it was concluded that the species of origin of a low molecular weight heparin would be difficult to determine by these methods alone.

Biochemical and pharmacologic characteristics of Reviparin, a low-molecular-mass heparin.

The introduction of low-molecular-mass heparins (LMMHs) has added a new dimension to the prophylactic and therapeutic management of thromboembolic disorders. These agents are now globally accepted as drugs of choice for postsurgical prophylaxis of deep vein thrombosis (DVT). Currently, the LMMHs are being developed for various therapeutic and cardiovascular indications. Reviparin is an optimized LMMH prepared by controlled nitrous acid digestion of porcine mucosal heparin. This drug has been developed utilizing validated procedures and exhibits a relatively narrow molecular mass distribution in contrast to most other commercially available LMMHs. The specific activity in the anticoagulant assays is approximately 40 U/mg whereas the specific activity in amidolytic anti-Xa assays is approximately 100 anti-Xa U/mg. Reviparin is capable of producing dose- and time-dependent antithrombotic effects in animal models of thrombosis. Although ex vivo anticoagulant effects are initially observed at dosages that are antithrombotic, this agent has been found to produce sustained antithrombotic effects when ex vivo anticoagulant actions are not measurable. Repeated administration of this LMMH induces progressively stronger antithrombotic effects. This drug has also been found to release tissue factor pathway inhibitor (TFPI) following both intravenous (IV) and subcutaneous (SC) administration. The studies included in this article are designed to provide additional data on the molecular profile using new calibration methods and additional results on pharmacologic studies. In particular, the release of TFPI following IV and SC administration to nonhuman primates is described. The effect of repeated administration of Reviparin mimicking the postsurgical prophylaxis of DVT is also reported in terms of any augmentation of the antithrombotic or hemorrhagic effects of this agent.

Lactoferrin regulates the activity of heparin proteoglycan-bound mast cell chymase: characterization of the binding of heparin to lactoferrin.

Rat mast cell protease 1 (RMCP-1) is a secretory granule serine protease (chymase) that is recovered in vivo in a macromolecular complex with heparin proteoglycan (PG). We have previously shown that heparin activates RMCP-1 and that RMCP-1, when bound to heparin PG, is largely resistant to inhibition by a variety of macromolecular protease inhibitors. In the search for alternative mechanisms in the regulation of RMCP-1 activity, we hypothesized that heparin antagonists, by interfering with the RMCP-1/heparin PG interaction, might influence the activity of heparin-bound mast cell chymase. In the present study, lactoferrin (LF), a heparin-binding protein, was assessed for RMCP-1 inhibiting activity. LF proved to decrease the activity of heparin PG-associated RMCP-1, although a portion of the enzyme activity was resistant to regulation. The mechanism of regulation was shown to involve the displacement of RMCP-1 from heparin PG, and LF caused an approx. 6-fold increase in the apparent Km of the RMCP-1-heparin PG complex for the chromogenic substrate S-2586. The interaction of LF with heparin was characterized. Pig mucosal heparin and endogenous heparin PG were equally effective in binding LF, whereas heparan sulphate bound with lower affinity. None of dermatan sulphate, chondroitin sulphate or hyaluronan were effective in binding LF. Further, the 6-O-, 2-O- and N-sulphate groups in heparin were of approximately equal importance for binding. Octasaccharides were the smallest heparin oligosaccharides showing significant binding to LF.

Enzymatic Preparation of Heparin Oligosaccharides Containing Antithrombin III Binding Sites

Two new oligosaccharides were prepared from heparin by its partial depolymerization using heparin lyase I (EC 4.2.2.7) in an attempt to prepare oligosaccharides having intact antithrombin III binding sites. The oligosaccharides were purified by chromatography on the basis of both size and charge and demonstrated a high level of purity by capillary electrophoresis. One- and two-dimensional 1H NMR spectroscopy at 500 MHz revealed the structure of each oligosaccharide. The octasaccharide and decasaccharide are DeltaUAp2S(1-->4)-alpha-DGlcNpS6S(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D -GlcNpAc6S(1-->4)-betaD-GlcAp(1-->4)-alpha-D-GlcNpS 3S6S(1-->4)-alpha- L-IdoAp2S(1-->4)alpha-D-GlcNpS6S (where DeltaUAp is 4-deoxy-alpha-L-threo-hex-enopyranosyluronic acid, GlcNp is 2-amino-2-deoxy-glucopyranose, GlcAp is glucopyranosyluronic acid, S is sulfate and Ac is acetate) and DeltaUAp2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp++ +(1-->4)-alpha- D-GlcNpAc6S (1-->4)-beta-D-GlcAp(1-->4)-alpha-D-GlcNpS3S6S(1-->4)-alpha- L-IdoAp2S (1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha -D-GlcNpS 6S, respectively. A hexasaccharide containing a similar structural motif to that found in the antithrombin III binding site and having greatly reduced anticoagulant activity was also isolated. The structure of the hexasaccharide is DeltaUAp2S(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp++ +(1-->4)-alpha- D-GlcNpS3S6S(1-->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpS6S . The octasaccharide and decasaccharide correspond to the predominant structural motif found in porcine intestinal mucosal heparin. Sufficient quantities of the decasaccharide were obtained to examine its interaction with antithrombin III using microtitration calorimetry. This decasaccharide bound to antithrombin III with similar avidity as heparin and showed comparable anticoagulant activity, as determined using an antithrombin III dependent anti-factor Xa assay. Interestingly, while both decasaccharide and heparin bound to antithrombin with nanomolar affinity, very little heat of binding was observed.

Preclinical studies on a low molecular weight heparin

Some major developments in the area of antithrombotic therapy have occurred during the past decade. Of these, the concept of fractionation of heparin has resulted in the development of several products from this agent. The introduction of low molecular weight heparins (LMWHs) has added a new chapter to the prophylactic and therapeutic management of thromboembolic disorders. These agents are now globally accepted as drugs of choice for post-surgical prophylaxis of deep vein thrombosis (DVT). Currently, the LMWHs are being developed for various therapeutic and cardiovascular indications. Reviparin is an optimized LMWH prepared by controlled nitrous acid digestion of porcine mucosal heparin. This drug has been developed using validated procedures and exhibits a relatively narrow molecular weight distribution in contrast to most other commercially available LMWHs. The specific activity in anticoagulant assays is approximately 32 U/mg whereas the specific activity in terms of anti-Xa units is 120 anti-Xa U/mg. Reviparin is capable of producing a dose- and time-dependent antithrombotic effect in animal models of thrombosis. While the ex vivo effects initially occur at dosages that are antithrombotic, this agent has been found to produce sustained antithrombotic effects without any detectable ex vivo anticoagulant actions. This agent has also been found to release tissue factor pathway inhibitor (TFPI) after both intravenous and subcutaneous administration. Repeated administration of reviparin produces progressively stronger antithrombotic effects. The current studies are designed to provide additional data on its molecular profile using new calibration methods and additional results on the pharmacological studies in a dose-dependent manner. In particular, the release of TFPI following i.v. and s.c. administration in a primate model is described. The effect of repeated administration mimicking the post-surgical prophylaxis of DVT is also reported in terms of any increase in the antithrombotic or haemorrhagic effects of this agent. Comparative antithrombotic and pharmacological studies are also reported to compare the pharmacological profiles of reviparin, nadroparin and enoxaparin.

Structural Analysis of Heparin by Raman Spectroscopy

The Raman spectra of commercially available heparin disaccharide standards exhibit bands associated with the N-sulfate and the 6-O-sulfate groups of the glucosamine and the 2-O-sulfate of the iduronic acid. The N-sulfate has a strong band at 1039 cm–1. The 6-O-sulfate and the 2-O-sulfate exhibit bands at 1055 and 1065 cm–1, respectively. The pattern of these modes, which are assigned to the symmetric SO3 vibrations, was supported by semiempirical quantum mechanical calculations. The above bands were identified in the Raman spectrum of a commercial preparation of porcine mucosal heparin and were used to determine the relative proportion of the N-sulfate, 6-O-sulfate, and 2-O-sulfate groups in the heparin molecule. This information, which is complementary to that obtained by NMR spectroscopy, is of particular importance in relation to biological activity. This study also extends the usefulness of Raman spectroscopy to include structural details required for the quality assurance of pharmaceutical preparations of heparin.

Isolation and characterization of heparan sulfate from crude porcine intestinal mucosal peptidoglycan heparin

A method for the preparation of heparan sulfate from peptidoglycan heparin is described. The objective of this research was to provide a basis for the development and validation of an industrial process to support the preclinical development of heparan sulfate and/or heparan sulfate derivatives. In the preparation of heparan sulfate, heparin was recovered by alcohol fractionation and dermatan sulfate was isolated by selective precipitation. The remaining crude heparan sulfate was fractionated by anion-exchange chromatography into five subfractions. The biological activities of these subfractions were examined by anticoagulant and amidolytic assays. Molecular weight and molecular size were determined using capillary viscometry and polyacrylamide gel electrophoresis. Charge density and degree of sulfation were determined by cellulose acetate electrophoresis and elemental analysis. Oligosaccharide and disaccharide analysis relied on enzymatic depolymerization using heparin lyases followed by polyacrylamide gel and capillary electrophoresis. 1H NMR analysis provided detailed structural information on each subfraction. Crude heparan sulfate and its subfractions showed significant differences in physical, structural and biological properties.

Regulation of Rat Mast Cell Protease 1 Activity

Rat mast cell protease 1 (RMCP-1) is a chymotrypsin-like serine protease (chymase) that is specifically expressed by connective-tissue-type mast cells. It is stored in the secretory granules of the cells in a complex with heparin proteoglycan, and the chymase/heparin proteoglycan complexes are released following mast cell activation. The present study was undertaken to examine if the association with heparin proteoglycan influenced the regulation of RMCP-1 by various macromolecular protease inhibitors. Endogenous mast cell heparin proteoglycan was shown to significantly block the inhibition of RMCP-1 by the serpins alpha 1-protease inhibitor and alpha 1-antichymotrypsin, as well as the inhibition by alpha 2-macroglobulin, soybean trypsin inhibitor and plasma. The blocking of protease inhibition showed an optimum at a RMCP-1/proteoglycan ratio of 5:1 (by mass), corresponding to approximately 80 RMCP-1 molecules bound/proteoglycan molecule. Chymase activity present on intact peritoneal mast cells, i.e. present in its native complex with heparin proteoglycan, was also shown to be largely resistant to inhibition by alpha 1-antichymotrypsin and alpha 1-protease inhibitor. Heparin 10-saccharides and 20-saccharides were inefficient in preventing the interaction of RMCP-1 with alpha 1-antichymotrypsin, whereas pig mucosal heparin (approximately 50 monosaccharide units) blocked protease inhibition. We have previously shown that heparin potentiates the catalytic activity of RMCP-1 and, in the present study, we show that the mechanism for chymase activation involves a sixfold reduction of the Km,app value of RMCP-1 for the chromogenic substrate S-2586. Thus, the association of mast cell chymase with heparin proteoglycan may serve both to potentiate the catalytic activity of the enzyme and to increase the life-span of the chymases by preventing their inhibition after exocytosis.

Anticoagulant and antithrombotic actions of a semisynthetic β-1,3-glucan sulfate

Sulfation of the natural polysaccharide curdlan results in anticoagulantly active β-1,3-glucan sulfates whose activity depends on various structural parameters. In this study the anticoagulant and antithrombotic effects of one of these β-1,3-glucan sulfates (GS) were compared with those of a porcine mucosal heparin. GS produced a concentration dependent anticoagulant effect in all the global coagulation assays with the exception of the anti-Xa assay. The best activity was found in the APTT and the thrombin time assays indicating that protease generation and the direct inhibition of thrombin may be sites of actions of this agent. Whereas the anticoagulant activity of GS was approximately 5 fold lower compared to heparin, a 32 fold higher concentration (ED 50 = 550 μg/kg) was needed for an antithrombotic effect similar to heparin (ED 50 = 17.2 μg/kg) in a rabbit model of stasis thrombosis. In contrast to this, when a rat model of clamping induced jugular vein occlusion was used to produce vascular obstruction, GS produced similar antithrombotic actions to heparin. At a 250 μg/kg dosage, both agents doubled the number of clampings required for complete vascular obstruction. Since the mechanical injury to the blood vessel is the primary determinant of the thrombogenic response, GS may inhibit some of the pathophysiologic mechanisms responsible for the occlusion of the blood vessel. The current study also points to the fact that the global anticoagulant effects may not reflect the antithrombotic potential of newer sulfated carbohydrate derived drugs.

Pharmacologic validation of the clinical effects of an optimized low-molecular-weight heparin-reviparin.

Reviparin is a low-molecular-weight heparin (LMWH) prepared by controlled nitrous acid digestion of porcine mucosal heparin. The trade name designation for this agent is Clivarin. This agent has been released in Germany and France for the prophylaxis of deep venous thrombosis (DVT) in surgical patients. This agent is developed utilizing optimized procedures and exhibits a uniform, narrow-molecular-weight distribution in comparison to the other commercially available LMWHs. The specific activity in the anticoagulant assays is approximated to be 32 U/mg whereas the specific activity in terms of anti-Xa units is designated 120 aXa U/mg. Reviparin is capable of producing a dose- and time-dependent antithrombotic effect in animal models of thrombosis. While the ex vivo effects are initially presented at antithrombotically active dosages, this agent has been found to produce antithrombotic effects without any detectable ex vivo actions. This agent is also known to release tissue factor pathway inhibitor (TFPI) after both intravenous (IV) and subcutaneous (SC) administration. Repeated administration of Reviparin produces progressively stronger antithrombotic effects. Similarly, the bleeding as measured by rabbit ear blood loss is also progressively increased. However, the ratio between the dosage producing these effects is quite large. The current studies are designed to provide additional data on the molecular profile using new calibration methods and additional results on the pharmacologic studies in a dose-dependent manner. In particular, the release of TFPI following IV and SC administration in a primate model is described. The effect of repeated administration mimicking the post-surgical prophylaxis of DVT is also reported in terms of any augmentation of the antithrombotic or hemorrhagic effects of these agents.

Physical Characterization of Heparin by Light Scattering

Dynamic and electrophoretic light scattering were used to measure the size and charge heterogeneity of a commercial preparation of heparin. For this preparation of porcine mucosal heparin (Mr = 10–20 kDa), the diffusion coefficient was 1.2 ± 0.5×10−8cm2/s and the mobility was 4.4 ± 0.9×10−4cm2/V s for an unfiltered solution at 22°C in distilled water. This diffusion constant is 2 orders of magnitude smaller than expected for a molecule the size of heparin. A fast diffusion component of 5.8 ± 1.0×10−7cm2/s, corresponding to the individual molecule, was observed in the presence of 2M NaCl, where single molecule motion is better observed. This indicates that a portion of the heparin population is in an aggregated state, which produces a higher scattering intensity than individual heparin molecules. The weight percentage of the aggregates was 5–10 as measured by high performance exclusion chromatography. These aggregates were stable up to a temperature of 75°C as measured by light scattering. This suggests that the aggregates are made up of tightly bound heparin molecules. From the diffusion coefficients we estimate the average aggregate to be made up of about 50 heparin monomers, and from the mobility, we estimate the electrophoretic charge on the aggregate to be about 1000 or about 20 electrophoretic charges for each heparin monomer. The electrophoretic light‐scattering measurements also indicate that the aggregate scattering species have very similar surface charge densities resulting from the aggregates being formed from heparin molecules. These results can provide a framework to interpret future light‐scattering data for various preparations of heparin.

Strategy for the sequence analysis of heparin.

The versatile biological activities of proteoglycans are mainly mediated by their glycosaminoglycan (GAG) components. Unlike proteins and nucleic acids, no satisfactory method for sequencing GAGs has been developed. This paper describes a strategy to sequence the GAG chains of heparin. Heparin, prepared from animal tissue, and processed by proteinases and endoglucuronidases, is 90% GAG heparin and 10% peptidoglycan heparin (containing small remnants of core protein). Raw porcine mucosal heparin was labelled on the amino termini of these core protein remnants with a hydrophobic, fluorescent tag [N-4-(6-dimethylamino-2-benzofuranyl) phenyl (NDBP)-isothiocyanate]. Enrichment of the NDBP-heparin using phenyl-Sepharose chromatography, followed by treatment with a mixture of heparin lyase I and III, resulted in a single NDBP-linkage region tetrasaccharide, which was characterized as deltaUAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp -(1-->O-Ser-NDBP (deltaUAp is 4-deoxy-alpha-L-threo-hex-4-enopyranosyl uronic acid). Several NDBP-octasaccharides were isolated when NDBP-heparin was treated with only heparin lyase I. The structure of one of these NDBP-octasaccharides, deltaUAp2S(1-->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp(1-->3)-beta-D- Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp-(1-->O-Ser NDBP (S is sulphate, Ac is acetate), was determined by 1H-NMR and enzymatic methods. Enriched NDBP-heparin was treated with lithium hydroxide to release heparin, and the GAG chain was then labelled at xylose with 7-amino-1,3-naphthalene disulphonic acid (AGA). The resulting AGA-Xyl-heparin was sequenced on gradient PAGE using heparin lyase I and heparin lyase III. A predominant sequence in heparin at the protein core attachment site was deduced to be -D-GlcNp2S6S(or 6OH)(1-->4)-alpha-L-IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S (or60H) (1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNp2S6S( or 6OH)(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpAc (1- ->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-++ +GlcAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xyl-AGA.

Preparation and structural characterization of large heparin-derived oligosaccharides.

Porcine mucosal heparin was partially depolymerized with heparin lyase I and then fractionated into low-molecular-weight (< 5000) and high-molecular-weight (> 5000) oligosaccharides by pressure filtration. The high-molecular-weight oligosaccharide mixture (approximately 50 wt% of the starting heparin) also contained intact heparin. This intact polymer complicates oligosaccharide purification. Thus, the low-molecular-weight fraction was used to prepare homogeneous oligosaccharides for structural characterization. The low-molecular-weight oligosaccharide mixture was first fractionated by low-pressure gel permeation chromatography into size-uniform mixtures of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharides and higher oligosaccharides. Each size-fractionated mixture was then purified on the basis of charge by repetitive semi-preparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 14 homogeneous oligosaccharides from disaccharide to tetradecasaccharide. The purity of these heparin-derived oligosaccharides was determined by gradient polyacrylamide gel electrophoresis, analytical strong-anion-exchange high-performance liquid chromatography, capillary electrophoresis and one-dimensional nuclear resonance spectroscopy. The structure of these oligosaccharides was established using 600 MHz two-dimensional nuclear resonance spectroscopy. The spectral methods used included homonuclear correlation spectroscopy, nuclear Overhauser effect spectroscopy and heteronuclear multiple quantum coherence spectroscopy. The 1H/1H connectivities of the protons of each sugar residue in an oligosaccharide were established by two-dimensional homonuclear correlation spectroscopy, while 1H/13C assignments were made using 1H inverse detection. One- and two-dimensional nuclear resonance spectroscopic analysis of these heparin oligosaccharides showed two closely related groups of heparin-oligosaccharides are afforded by enzymatic depolymerization of heparin. One group is fully sulphated, having the structures delta UAp2S(1[-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S( 1]n-->4)-alpha- D-GlcNpS6S, where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid, GlcNp is 2-deoxy-2-aminoglucopyranose, IdoAp is idopyranosyluronic acid, S is sulphate and n = 0-6. The other group of oligosaccharides differ in that they contain beta-D-glucuronic acid in place of the alpha-L-iduronic acid residue nearest to the reducing end. The present study describes the isolation and structural elucidation of seven new oligosaccharides: an octasaccharide, two decasaccharides, two dodecasaccharides and two tetradecasaccharides. The utility of two-dimensional nuclear resonance spectroscopy to determine the structure of complex heparin oligosaccharides is also illustrated.

Differentiation of Beef and Pig Mucosal Heparins by NMR Spectroscopy

Differentiation of Beef and Pig Mucosal Heparins by NMR Spectroscopy

Different effects of mucosal, bovine lung and chemically modified heparin on selected biological properties of basic fibroblast growth factor.

Heparins from bovine mucosa and lung, and chemically modified heparins were assayed for their capacity to: (i) protect human recombinant basic fibroblast growth factor (bFGF) from tryptic cleavage; (ii) prevent 125I-bFGF binding to heparan sulphate proteoglycans present in the extracellular matrix and on the cell surface of fetal bovine aortic endothelial GM 7373 cell cultures; (iii) affect 125I-bFGF binding to high-affinity tyrosine kinase FGF receptors present on the cell membrane of GM 7373 cells; (iv) inhibit the mitogenic activity exerted by bFGF in the same cells. The results demonstrate that the potency shown by mucosal heparins in the different assays is a direct function of size, very-low-molecular-mass heparin (2.0 kDa) being significantly less effective on a molar basis than unfractionated heparin (13.6 kDa). Increased flexibility of the backbone structure, as observed in reduced/oxidized heparins of different size, does not affect the capacity of the polysaccharide to interact with bFGF. In contrast, selective 2-O-desulphation, but not 6-O-desulphation, drastically reduced the capacity of heparin to protect bFGF from proteolytic cleavage, to affect its interaction with low- and high-affinity sites, and to inhibit its mitogenic activity. Two preparations of bovine lung heparin, differing in molecular mass, were as effective as mucosal heparin in the bFGF-tryptic-digestion assay and the endothelial-cell proteoglycan-binding assay, but they were highly inefficient at inhibiting the capacity of bFGF to interact with its tyrosine kinase receptors. Bovine lung heparins were also less effective than mucosal heparin as bFGF antagonists in GM 7373-cell-proliferation assays. N-Desulphated/N-acetylated bovine lung heparin retained only a significant capacity to protect bFGF from tryptic cleavage. The results demonstrate that different chemical features of the heparin molecule, including decrease in molecular mass, selective desulphation, disaccharide composition and clustering, affect differently the capacity of the glycosaminoglycan to interact with bFGF and to influence its biological behaviour in different assays in vitro and in endothelial cell cultures. Our findings should aid the design of synthetic oligosaccharides aimed at improving the bioavailability of bFGF when administered in vivo as a therapeutic agent.

Fibrinolytic compromise by simultaneous administration of site-directed inhibitors of thrombin

Newly developed synthetic and recombinant thrombin inhibitors possess strong anticoagulant effects. Despite these effects, interactions of these agents with enzymes in the fibrinolytic network result in the modulation of such proteases as t-PA, u-PA and streptokinase. The inhibitory spectrum of several thrombin inhibitors [D-Phe-Pro-Arg-H (GYKI 14166), D-MePhe-Pro-Arg-H (GYKI 14766), Boc-D-Phe-Pro-Arg-H (GYKI 14451), Ac-D-Phe-Pro-boroArg-OH (DuP 714), recombinant hirudin (r-Hir) and unfractionated porcine mucosal heparin complexed with antithrombin III (Heparin/AT-III)] was studied towards various serine proteases such as tissue plasminogen activator (t-PA), plasmin, plasminogen/streptokinase complex, urokinase and kallikrein. Aprotinin was also studied in the same systems as the thrombin inhibitors. All four tripeptide derivatives were found to inhibit t-PA, plasmin and plasminogen/streptokinase complex at micromolar concentrations (IC 50: 0.57 mM - 3.3 μM). Boc-D-Phe-Pro-Arg-H and Ac-D-Phe-Pro-boroArg-OH also inhibited urokinase, while Ac-D-Phe-Pro-boroArg-OH inhibited kallikrein as well (IC 50: 0.15 mM - 16 μM). In contrast, r-Hir and Heparin/AT-III did not inhibit any of these enzymes at millimolar concentrations (IC 50≥ 1 mM). Aprotinin inhibited plasmin, plasminogen/streptokinase complex and kallikrein at micromolar concentrations (IC 50: 3.1-0.85 μM). In a rabbit thrombolysis model, where pre-formed clots are lysed by streptokinase, simultaneous administration of D-MePhe-Pro-Arg-H or Ac-D-Phe-ProboroArg-OH, at concentrations ∼ 1 μmol/kg, I.V. resulted in complete inhibition of the fibrinolytic process. Aprotinin at 0.1 μmol/kg, I.V. produced similar inhibition. These results demonstrate that thrombin inhibitors may exert significant antiprotease actions against various fibrinolytic enzymes.

Inactivation of thrombin by a complex between rat mast-cell protease 1 and heparin proteoglycan.

Rat peritoneal mast cells were shown to inactivate thrombin rapidly. The thrombin-inactivating activity was purified to homogeneity by a combination of anion-exchange chromatography and h.p.l.c. on a Superdex 75 column. The purified thrombin inactivator had an apparent molecular mass of 29 kDa and an N-terminal amino acid sequence identical to rat mast-cell protease 1 (RMCP-1). After labelling of the mast cells in vivo with 35SO4(2-), RMCP-1 was recovered in a macromolecular complex with [35S]heparin proteoglycans. Dissociation of RMCP-1 from the heparin proteoglycans by Superdex 75 chromatography in the presence of 2 M NaCl resulted in a marked loss of the thrombin-inactivating activity displayed by the enzyme. When RMCP-1 was reconstituted with either endogenous [35S]heparin proteoglycans or standard pig mucosal heparin, the enzyme regained its thrombin-inactivating properties. Affinity chromatography of endogenous [35S]heparin on matrix-linked RMCP-1 demonstrated that all of the heparin molecules contained high-affinity binding sites for the mast-cell protease. In contrast, the endogenous mast-cell heparin showed low affinity for antithrombin, a protease inhibitor involved in the regulation of coagulation enzymes.