Qualitative and quantitative studies of heparin and chondroitin sulfates in normal human plasma

BiochimicaL et Biophysics 4cta ELSEVIER

Biochimica

et Biophysics

Acta 1243 (1995) 49-58

Qualitative and quantitative studies of heparin and chondroitin sulfates in normal human plasma Nicola Volpi *, Mirella Cusmano, Tiziana Venturelli Department of ‘Biologia Animale’, Chair of Biological Chemistry, University of Modena, Via Berengario 14, 41100, Modena, Ital) Received 29 March 1994

Abstract Heparin was extracted and purified from normal human plasma, and full characterization of its structure and physico-chemical properties was achieved for the first time. Plasma was submitted to exhaustive proteolytic treatment with papain, trypsin, chymotrypsin, collagenase and pepsin, anion-exchange chromatography and precipitation with organic solvents. By this procedure, we recovered heparin (about 0.7 mg/lOO ml of plasma) and chondroitin sulfate (about 0.1 mg/lOO ml of plasma). Chondroitin sulfate has a peak molecular mass of about 15630, and it is composed of about 60% nonsulfated disaccharide, 3.5% disaccharide 6-monosulfate and about 40% disaccharide 4-monosulfate, with a sulfate-to-carboxyl ratio of 0.41. Heparin, identified by agarose-gel electrophoresis, is constituted by about 40% slow-moving component and about 60% fast-moving species. This glycosaminoglycan had a peak molecular mass of about 7000, and was identified as ‘typical’ heparin by its constituent disaccharide composition. About 70% of disaccharides were identified as trisulfated disaccharide, and about 18% as disulfated disaccharides, 3% as monosulfated disaccharides and 10% as nonsulfated disaccharide. Heparin extracted from normal human plasma has a high sulfate-to-carboxyl ratio (2.47) and in vitro anticoagulant activity of about 70 I.U. A more quantitative and statistical analysis performed on 10 ml of plasma obtained from 10 human healthy volunteers revealed a heparin level of 0.54 f 0.17 mg/lOO ml plasma (mean * standard deviation) with a coefficient of variation of about f 32%. These findings demonstrate for the first time the presence of heparin molecules in normal human plasma and confirm the importance of adequate extraction processes to purify a molecule that strongly interacts with plasma protein components. This is discussed in light of other authors that described a polysaccharide molecule named heparan sulfate in human plasma. Keywords: Glycosaminoglycan;

Heparin;

Chondroitin

sulfate; Human plasma

1. Introduction Glycosaminoglycans uranic acids and amino neous polysaccharides in (M,), charge density and

are alternating copolymers of sugars. They are very heterogeterms of relative molecular mass physico-chemical properties [l-

31. The backbone of sulfated glycosaminoglycans presents different uranic acids, different hexosamines and sulfate groups in varying amounts and O-linked in different positions [2-41. Heparin is an anticoagulant and antithrombotic drug also used as a thrombolytic and fat-clearing antiatherosclerotic agent [2]. Moreover, low-M, derivatives

Abbreviations: M,, relative molecular mass; APlT, activated partial thromboplastin time; HPSEC, high-performance size-exclusion chromatography; ADi-HA, 2-acetamido-2-deoxy-3-0~4-deoxy-a-L-threo-hex-4-enepyranosyluronic acid)-Dglucose; ADi-OS, 2-acetamido-2-deoxy-3-O-(4-deoxy-a-r_-threohex-4-enepyranosyluronic acid)-Dgalactose; ADi-4S, 2-acetamido-2-deoxy-3-0~4-deoxy-a-L-threo-hex-4-enepyranosyluronic acid)-Dgalactose 4-sulfate; JDi-6S, 2-acetamido-2-deoxy-3-0_(4-deoxy-a-L-threo-hex-4-enepyranosyluronic acid)-Dgalactose 6-sulfate; ADi-2,6diS, 2-acetamido-2-deoxy-3-O-(4-deoxy-a-L-three-hex-4-enepyranosyluronic acid 2-sulfate)-r>galactose 6-sulfate; ADi-2,4diS, 2-acetamido-2-deoxy-3-0-(4-deoxy-cY-L-threo-hex-4-enepyranosyluronic acid 2-sulfate)-D-galactose 4-sulfate; ADi-4,6diS, 2-acetamido-2-deoxy-3-0_(4-deoxy-cY-L-t~reo-hex-4-enepyranosyluronic acid)-Dgalactose 4,6-disulfate; ADi-2,4,6triS, 2-acetamido-2-deoxy-3-0~4-deoxy-a-L-threo-hex-4-enepyranosyluronic acid 2-sulfate)+galactose 4,6-disulfate; ADiHOS, 2-acetamido-2-deoxy-4-0_(4-deoxy-a-L-threo-hex-4-enepyr~osyluronic acid)-pglucose; ADM-NS, 2-deoxy-2-sulfamino-4-O-(4-deoxy-a-L-threo-hex4-enepyranosyluronic acid)-pglucose; ADiH-6S, 2-acetamido-2-deoxy-4-0~4-deoxy-a-L-threo-hex-4-enepyranosyluronic acid)-6-0-sulfo-Dglucose; ADiH-2,NdiS, 2-deoxy-2-sulfamino-4-0~4-deoxy-2-0-sulfo-a-L-r~reo-hex-4-enepyranosyluronic acid)-D-glucose; ADiH-N,6diS, 2-deoxy-2-sulfamino-40-(4-deoxy-a-L-three-hex-4-enepyranosyluronic acid)-6-0-sulfo+glucose; ADiH-triS, 2-deoxy-2-sulfamino-4-0~4-deoxy-2-0-sulfo-~-L-fhreo-hex-4-enepyranosyluronic acid)-6-0-sulfo-pglucose. * Corresponding author. Fax: + 39 (059) 581020. 0304-4165/95/%09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0304-4165(94)00123-5

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et Biophysics Acta 1243 (1995) 49-58

are used as drugs, and some derivatives of heparin devoid of anticoagulant action (with less N-sulfate and larger amount of N-acetyl groups) are used as anti-inflammatory, antithrombotic and antiatherosclerotic agents [2,3]. Glycosaminoglycans have been found in biological fluids such as plasma and urine [5,6]. In plasma, they interact with several proteins, such as protease inhibitors, coagulation factors, lipoproteins and complement proteins [7], cells such as lymphocytes [8], monocytes [9] and platelets [lo], and vascular endothelium [ll]. The presence of glycosaminoglycans in human plasma has been demonstrated in several studies [5,12,13]. However, the structures and physico-chemical properties of these glycosaminoglycans have not been fully identified and characterized due to their low blood concentration (about 2-6 pug of hexuronate/ml) and to a lack of adequate methodology. This is more evident for sulfated glycosaminoglycans such as chondroitin sulfates, heparan sulfate and heparin due to the complexity of their primary structures and to their strong interactions with plasma components. The sulfated glycosaminoglycans known in plasma are keratan sulfate, chondroitin sulfate and low charge chondroitin sulfate, and heparan sulfate [5,12]. However, heparan sulfate in human plasma was detected by indirect, nonspecific or insensitive measurements, such as lipoprotein lipase activation [5], degradation by lyases [12], sepa[ 141 and ration on cellulose polyacetate electrophoresis others [15,16]; no specific heparan sulfate (or heparin) extraction, purification and analysis processes were performed. Only few studies have detected heparin in normal human plasma [17,18]. Cavari et al. 1181 reported that anticoagulant activity, measured by thrombin time, appeared in human plasma after exhaustive proteolytic treatment, and this activity was identified as heparin due to its sensitivity to treatment with heparin lyase and nitrous acid. An accurate determination of primary structures of plasma glucosaminoglycans (heparan sulfate and heparin) is necessary due to the variability of chemical properties and biological functions of these glycosaminoglycans. Heparan sulfate and heparin have a very complex and heterogeneous primary structure depending on the source. In fact, heparin is localized in mast-cell granula whilst heparan sulfate, as a component of proteoglycans, is distributed both on plasma membranes and in the extracellular matrix [4]. On the other hand, specific kinds of heparan sulfate molecules are constituted by heparin-like sequences that impart these polysaccharides anticoagulant properties [19-211. These considerations suggest that a fine characterization of these molecules in normal human plasma could give us more information about their biological functions. Moreover fine structural characterization of the high levels of anticoagulant glycosaminoglycan which arise in plasma in certain pathological conditions would also be informative [22,23]. In this paper we report for the first time the extraction and the fine characterization of normal human plasma heparin after exhaustive proteolytic diges-

tion. Extraction and characterization of chondroitin in normal human plasma was also performed.

sulfates

2. Materials and methods

2.1. Materials Heparin from beef intestinal mucosa (peak M, = 11600) was prepared as reported elsewhere [24-261. Slow-moving and fast-moving species of heparin were purified by selective precipitation as barium salts at different temperatures [24]. Dermatan sulfate from beef intestinal mucosa (peak M, = 26000) and chondroitin sulfate from beef trachea (peak M, = 26000) were extracted and purified as reported elsewhere [24,26]. Papain (EC 3.4.22.2), trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.11, collagenase (EC 3.4.24.3) and pepsin (EC 3.4.23.1) were obtained from Sigma. Heparinase I (Hep I> from Flavobacterium heparinum (EC 4.2.2.7.) (2000 U/mg of protein; 1 U forms 1.0 nmol of unsaturated uranic acid per min at pH 7.0 at 37”(Z), Heparinase II (Hep. II) from F. heparinum (no assigned EC number) (47 U/mg of protein; one unit forms 0.1 pmole of unsaturated uranic acid per hour at pH 7.0 at 25”C), Heparinase III (Hep. III) from F. heparinum (EC 4.2.2.8) (215 U/mg of protein; 1 U forms 0.1 pmol of unsaturated uranic acid/h at pH 7.0 at 25Q chondroitinase ABC from Proteus uulgaris (EC 4.2.2.7.), chondroitinase AC I Flavo from F. heparinum (EC 4.2.2.5) and chondroitinase AC II Arthro from Arthrobacter aurescens (EC 4.2.2.5) were obtained from Sigma. Hyaluronidase from Streptomyces hyalurolyticus (EC 4.2.2.1) were obtained from Seikagaku Corporation, Tokyo, Japan. Unsaturated heparin/heparan sulfate disaccharides ( ADiH-OS, ADiH-NS, ADiH-6S, ADiH-2,NdiS, ADiHN,GdiS and ADiH-triS, see abbreviations), unsaturated chondroitin sulfate/dermatan sulfate disaccharides (ADiOS, ADi-4S, ADi-6S, ADi-2,6diS, ADi-2,4diS, ADi4,6diS and ADi-2,4,6triS, see abbreviations) and unsatured hyaluronate disaccharide ( ADi-HA, see abbreviations) were obtained from Seikagaku. Ecteola-cellulose was from Serva, Heidelberg, Germany. Column Protein Pak 125 (300 X 7.8 mm; particle size: 10 pm; molecular mass ranges: native globular from 2000 to 80 000 and random coil from 1000 to 30 000; cod. 84601) and Protein Pak 300 (300 X 7.5 mm; particle size: 10 pm; molecular mass ranges: native globular from 10000 to 400000 and random coil from 2000 to 150000; cod. T72711) were from Waters, Milford, USA. 10 pm Spherisorb SAX (trimethylammoniopropyl groups Si-CH,CH2-CH2-N+(CH& in Cl- form) was from Phase Separations, Deeside Industrial Park, Deeside Clwyd, UK. Multiphor II electrophoretic cell was from Pharmacia LKB Biotechnology, Uppsala, Sweden. High purity agarose and barium acetate were from BioRad. l,Zdiaminopropane and cetyltrimethylammonium bromide were from

51

N. Volpi et al./Biochimica et Biophysics Acta 1243 (1995) 49-58

Merck, Darmstadt, Germany. The densitometric unit was composed of Macintosh IIsi computer interfaced with Microtek Color Scanner from Microtek International Inc., Hsinchu, Taiwan. IMAGE processing and analysis program, Ver. 1.41 from Jet Propulsion Lab., NASA, Florida, U.S.A., was used for densitometric analysis of agarose-gel electrophoretic bands. Spectrapore dialysis tubing (molecular mass cut-off of 20001 were from Spectrum. All the other reagents were analytical grade. Proteases (papain, trypsin, chymotrypsin, collagenase and pepsin) were checked for the presence of heparin; 10 mg of each enzyme was solubilized in 1 ml of distilled water and 4 vol of ethanol saturated with NaCl were added. After standing at 4°C for 24 h, the solutions were centrifuged at 10000 X g for 15 min. The pellets were dried and dissolved in 10 ~1 of distilled water; 5 ~1 were analyzed by agarose-gel electrophoresis. This procedure revealed about 2 pg heparin/lO mg of protease. We can exclude the presence of heparin (< 2 pg/lO mg) in the proteases. 2.2. Plasma sampling for preparatiue

approaches

Blood samples were collected from human healthy volunteers with CPDA-1 (composed of citric acid, sodium citrate, sodium phosphate monobasic, dextrose and adenine) as anticoagulant. Plasma from different healthy volunteers was obtained by centrifuging whole blood, pooled and then stored at -80°C until used. 2.3. Extraction cosaminoglycans Three different

and purification of human plasma from pooled plasma preparative

approaches

gly-

were adopted.

Exhaustive proteolytic digestion Papain (50 mg) was added to 100 ml of plasma for 24 h at 60°C in a stirrer. After boiling for 10 min, trypsin, chymotrypsin, collagenase and pepsin (10 mg) were sequentially added at 24 h intervals at 37°C. The sample was boiled at the end of each incubation. The mixture was brought to pH 9.0 by adding 2 N NaOH. After 24 h at 40°C the product (brought to pH 6.0 with 2 N acetic acid) was centrifuged at 5000 X g for 15 min, and the pellet washed two times with distilled water. Two volumes of acetone were added to the pooled supernatants and stored at +4”C for 24 h. The precipitate was recovered by centrifugation at 5000 X g for 15 min and dried at 60°C for 6 h. The dried precipitate, 1.4 g, was dissolved in 15 ml of distilled water by prolonged mixing. After centrifugation at 5000 X g for 15 min, the supematant was applied to a column (1 cm X 8 cm) packed with about 6.5 ml of Ecteola-cellulosa previously washed with M NaOH and M HCl and equilibrated with 0.05 M NaCl. After washing the resin with 2 vol of 0.05 M NaCl, 20 ml of 3 M NaCl were added. Two volumes of acetone were added

to the eluate (20 ml) and stored at +4”C for 24 h. After centrifugation at 5000 X g for 15 min, the pellet was dried at 60°C for 6 h; 25 mg were recovered. Proteolytic digestion with nonspecific protease (papain) Papain (50 mg) was added to 100 ml of plasma for 24 h at 60°C in a stirrer. After boiling for 10 min, the mixture was brought to pH 9.0 by adding 2 N NaOH. After 24 h at 40°C the product (brought to pH 6.0 with 2 N acetic acid) was centrifuged at 5000 X g for 15 min and the pellet washed two times with distilled water. The pooled supernatants were applied to a column (1.5 cm X 10 cm> packed with about 18 ml of Ecteola-cellulosa previously washed with M NaOH and M HCl and equilibrated with 0.05 M NaCl. After washing the resin with 2 vol of 0.05 M NaCl, 40 ml of 3 M NaCl were flushed through. Two volumes of acetone were added to the eluate (40 ml) and stored at + 4°C for 24 h. After centrifugation at 5000 X g for 15 min, the pellet was dried at 60°C for 6 h; 257 mg were recovered. Fractionation on Ecteola-cellulose 100 ml of plasma were applied to a column (2.0 cm X 20 cm) packed with about 62 ml of Ecteola-cellulosa previously washed with M NaOH and M HCl and equilibrated with 0.05 M NaCl. The resin was washed with 2 volumes of 0.05 M NaCl, and 100 ml of 3 M NaCl were flushed through. The recovered eluate was dialysed, concentrated and freeze-dried; 350 mg were recovered from human plasma. The powder recovered from plasma was dissolved in 50 ml of distilled water and submitted to proteolytic digestion with papain (10 mg). After boiling for 10 min and centrifugation at 5000 X g for 15 min, the supernatant was applied to a column (1.0 cm X 8 cm) packed with about 6 ml of Ecteola-cellulosa previously washed with M NaOH and M HCl and equilibrated with 0.05 M NaCl. After washing the resin with 2 volumes of 0.05 M NaCl, 20 ml of 3 M NaCl were added. Two volumes of acetone were added to the eluate (20 ml) and stored at + 4°C for 24 h. After centrifugation at 5000 X g for 15 min, the pellet was dried at 60°C for 6 h; 50 mg were recovered. 2.4. Quantitatiue determination human healthy volunteers

of heparin

in plasma

of

Blood samples were collected in the morning from 10 human healthy volunteers with sodium citrate as anticoagulant. Plasma was immediately obtained by centrifugation at 3000 X g for 10 min. Papain (2 mg) was added to 10 ml of plasma for 24 h at 60°C in a stirrer. After boiling for 10 min, trypsin, chymotrypsin, collagenase and pepsin (0.1 mg) were sequentially added at 24 h intervals at 37°C. The samples were boiled at the end of each incubation. The mixtures were brought to pH 9.0 by adding 2 N NaOH. After 24 h at 40°C the

52

N. Volpi et al. /Biochimica et Biophysics Acta 1243 (1995) 49-58

products (brought to pH 6.0 with 2 N acetic acid) were centrifuged at 5000 X g for 15 min, and the pellets washed two times with 2 ml of distilled water. Two volumes of acetone were added to the pooled supernatants and stored at +4”C for 24 h. The precipitates were recovered by centrifugation at 5000 X g for 15 min and dried at 60°C for 6 h. The dried precipitates were dissolved in 10 ml of distilled water by prolonged mixing. After centrifugation at 5000 X g for 15 min, the supernatants were applied to columns (1 cm X 8 cm) packed with about 6.5 ml of Ecteola-cellulosa previously washed with M NaOH and M HCl and equilibrated with 0.05 M NaCl. After washing the resins with 2 volumes of 0.05 M NaCl, 20 ml of 3 M NaCl were added. Two volumes of acetone were added to the eluates and stored at +4”C for 24 h. After centrifugation at 5000 X g for 15 mitt, the pellets were dried at 60°C for 6 h. The dried precipitates were dissolved in 0.1 ml of distilled water by prolonged mixing and after centrifugation at 10000 X g for 10 mitt, the supernatants were analyzed by agarose-gel electrophoresis; 10 ~1 of plasma extracts from each human volunteer were deposited by micropipette on an agarose-gel plate and, after electrophoretic separation and toluidine blue staining, heparins were quantified by densitometric scanning against specific calibration curves (for slow-moving and fast-moving species) (Fig. 1). Software Regression Ver. Ml.23 for Macintosh computer was used for quantitative analysis.

HPLC equipment was manufactured by Jasco, Tokyo, Japan (pump mod. 880 PU; system controller mod. 801 SC; ternary gradient unit mod. 880-02; Rheodyne injector equipped with a 100 ~1 loop; U.V. detector, mod. 875 UV). The mobile phase was composed of 125 mM Na,SO, and 2 mM NaH,PO, adjusted to pH 6.0 with 0.1 M NaOH. Flow rate was 0.9 ml/min with a back pressure of 25 Kg/cm2. Column Protein Pak 125 and Protein Pak 300 were assembled in series. The peak it4, (mean value) was determined by a calibration curve plotted with glycosaminoglycan standards, as reported elsewhere [27].

2.5. Agarose-gel electrophoresis

2.7. Constituent disaccharide quantitation of heparin

Glycosaminoglycans extracted from plasma were analyzed by agarose-gel electrophoresis in barium acetate/ 1,Zdiaminopropane as reported elsewhere [24-261. A Pharmacia multiphor II electrophoretic cell was used. The

The degradation of human plasma heparin was performed with heparinase I alone, and with the three (I + II + III) heparinases simultaneously. 200 pg (20 mg/ml in H,O) of dried powder were incubated with 18.5 U of Heparinase I, 1.0 unit of Heparinase II and 1.0 unit of Heparinase III in 100 mM pH 7.0 acetate buffer in 2.5 mmoles of calcium acetate. The reactions were stopped, after 24 hours at 37°C by boiling 1 min. Constituent disaccharides were determined by HPLC at 232 nm. Isocratic separation was from run 0 to 5 min with 0.1 M NaCl, pH 4.00; linear gradient separation from 5 to 90 min with 100% 0.1 M NaCl, pH 4.00 to 100% 1.2 M NaCl, pH 4.00. Flow rate was 1.4 ml/min. Separation of non-sulfated and variously sulfated disaccharides was performed using the standards and retention times given by Seikagaku Kogyo Co. [28] (see Table 1 and structural formula). The amount of each identified disaccharide was determined by purified standards and reported as weight percent. Under experimental conditions, the treatment with heparinase I alone gives two sulfated disaccharides (ADiH2,NdiS and ADiH-triS) and oligosaccharides still resistent to heparin lyase I activity, according to Linhardt [29]. The simultaneous cleavage of plasma heparin with heparinase I, II and III produces about 90% disaccharides and 10% oligosaccharides. Thus, the percentage of each disaccha-

??SM

y = 11.0 + 29.5x R = 0.895 FM

SM CS

0

1

2

3

4

5

6

7

ug of gl ycosami nogl ycans Fig. 1. Calibration curves of slow-moving (SM) and fast-moving (FM) heparin species, and chondroitin sulfate (CS) performed by separation in agarose-gel electrophoresis, staining with toluidine blue and photodensitometric analysis. (At least six replications were carried out. The equations and the correlation coefficients are reported for each glycosaminoglycan).

first run was performed in 0.04 M barium acetate buffer pH 5.8 for 20 min at 60 mA, the second run was in 0.05 M 1,2-diaminopropane buffered at pH 9.0 with acetic acid for 45 min at 50 mA. After migration, the plate was soaked in cetyltrimethylammonium bromide (0.1% solution) for about 3 h, dried and then stained with toluidine blue (0.2% in ethanol-water-acetic acid, 50:49:1 v:v:v) for 30 min. After decoloration with ethanol-water-acetic acid 50:49:1 v:v:v, glycosaminoglycans were analyzed quantitatively in a densitometer, and the slow-moving and fast-moving heparin components and chondroitin sulfate amounts were evaluated by specific calibration curves as reported (Fig. 1). 2.6. Determination of peak M, s

N. Volpi et al. /Biochimica

Table 1 Disaccharide composition (expressed as weight percent), physico-chemical properties (peak M,, charge density and percentage of slow-moving and fast-moving species) and anticoagulant activity of purified human plasma heparin (HPHcp) compared with those of a heparin prepared from beef intestinal mucosa (Hep) [24-261. See formula for structures of the disaccharides

ADiH-OS ADiH-NS ADiH-6S ADiH-2,NdiS ADiH-N,6diS ADiH-2,N,6triS IV, ( x 1000) so,/coo-

R’

R=

R6

HPHep

Hep

COCH, so; COCH, so, SO, SO,

H H H SO; H so;

H H so; H so; so;

9.5 1.0 2.1 14.4 3.4 69.6 7.07 2.47 70 40 60

6.3 3.8 2.4 19.2 12.5 55.8 11.60 2.35 155 30 70

APlT (IU/mg) Slow-moving % Fast-moving %

considering ride masses as 100%. ride

was

calculated

2.8. Constituent sulfate

disaccharide

the sum of the disaccha-

quantitation

of chondroitin

200 pg (20 mg/ml in H,O) of dried powder were incubated with 25 mU of chondroitinase ABC in 50 mM pH 8.0 Tris-HCl buffer. The reaction was stopped, after 3 hours incubation at 37°C by boiling for 1 min. The con-

Table 2 Disaccharide composition (expressed as wt. %) and physico-chemical properties (peak IV, and charge density) of purified human plasma chondroitin sulfate (HPCS) compared with those of a chondroitin sulfate (CS) prepared from beef tissues [24-261. See formula for structures of the disaccharides

ADi-OS ADi-6S ADi-4S ADi-2,6diS ADi-4,6diS ADi-2,4diS ADi-2,4,6triS M, (x 1000) so;/coo-

R=

R4

R6

HPCS

CS

H H H SO; H so; so;

H H SO; H so; SO; so;

H so; H so; so; H so;

58.7 3.4 37.9 0.0 0.0 0.0 0.0 15.63 0.41

4.4 37.9 56.0 0.8 0.8 0.0 0.0 26.10 0.97

53

et Biophysics Acta 1243 (1995) 49-58

stituent disaccharides were determined by HPLC as reported above (see Table 2 and structural formula). 200 pg (20 mg/ml in H,O) of dried powder were also incubated with 25 mU of chondroitinase AC I Flavo and 25 mU of chondroitinase AC II Arthro in 50 mM pH 7.0 Tris-HCl buffer. The reactions were stopped, after 6 hours incubation at 37°C by boiling for 1 min. The constituent disaccharides were determined by HPLC as reported above. 200 pug (20 mg/ml in H,O) of dried powder were also incubated with 1 munit of hyaluronidase in 50 mM pH 6.0 Tris-HCl buffer. The reactions were stopped, after 6 h incubation at 37°C by boiling for 1 min. The presence of ADi-HA unsaturated disaccharide were determined by HPLC as reported above. The amount of each identified disaccharide was determined by purified standards and reported as weight percent. Under experimental conditions, cleavage of chondroitin sulfate by chondroitinase ABC or AC1 and II produces 100% disaccharides and no oligosaccharides resistant to treatment with lyases. 2.9. Determination

of sulfate-to-carboxyl

ratio

The sulfate-to-carboxyl ratio was determined by enzymatic degradation after HPLC separation of constituent disaccharides. The ratio was calculated considering the presence and the percentage of carboxyl and sulfate groups for each disaccharide [30,31]. 2.10. Determination tivity

of in vitro heparin anticoagulant

ac-

The anticoagulant activity of plasma heparin was determined as Activated Partial Thromboplastin Time activity (APTT activity) [32]. The AP’I’T activity was determined versus the WHO IV International Heparin Standard [33] and expressed as IU/mg.

3. Results The three different extraction procedures applied to purify plasma glycosaminoglycans produce various degrees of recovery. Exhaustive proteolytic digestion (procedure 1) yields a recovery of 25 mg/lOO ml plasma, whilst nonspecific proteolysis (procedure 21, and chargefractionation and further nonspecific proteolysis (procedure 3) provided 257 mg/lOO ml plasma and 50 mg/lOO ml plasma, respectively. Thus, proteolysis with papain permits a greater recovery then other two methods. Agarose-gel electrophoresis analysis of material purified by exhaustive proteolytic treatment (procedure 1) detected heparin (10 pg/lOO ml plasma, 30 pg/lOO ml plasma and 50 pg/lOO ml plasma of recovered material) (Fig. 2a). Agarose-gel electrophoresis with higher amount of extracted material (150 pg/lOO ml plasma, 250

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N. Volpi et al. /Biochimica

et Biophysics Acta 1243 (1995) 49-58

Fig. 2. A. Agarose-gel electrophoretic separation of human plasma glycosaminoglycans. 1. Glycosaminoglycan standards (A. Slow-moving species. B. Fast-moving species. C. Dermatan sulfate. D. Chondroitin sulfate). 2. Plasma extract, 10 pg. 3. Plasma extract, 30 pg. 4. Plasma extract, 50 pg. The arrow indicates the direction of electrophoretic migration.

pg/lOO ml plasma and 350 pg/lOO ml plasma) (Fig. 2b) leads to the presence of chondroitin sulfate. The identity of electrophoretic bands were confirmed by specific enzymatic degradation and agarose-gel electrophoretic separa-

tion. 200 pg of recovered material was treated with heparinases, or chondroitinase ABC, or chondroitinases AC. After electrophoretic separation, the sample treated with heparin lyases did not show the electrophoretic bands corresponding to slow-moving and fast-moving heparin species. Samples treated with chondroitin lyases AC or ABC produced an electrophoretic profile with no other bands than that corresponding to heparin. These results confirm no presence of dermatan sulfate or keratan sulfate in plasma extract. Nevertheless, we cannot exclude the presence of a lower amount of other sulfated glycosaminoglycans (in particular keratan sulfate as reported by other authors [5,12]) due to the quantitative limits of this analytical technique. Heparin or other glycosaminoglycans could not be detected in the material extracted with the other two methods, nonspecific proteolysis (procedure 2) and chargefractionation (procedure 31, even when 500 pg/lOO ml plasma were layed on the agarose plate. Quantitative analysis of glycosaminoglycans extracted by exhaustive proteolysis (procedure 1) performed by agarose-gel electrophoresis and densitometric scanning (Fig. 3) permits us to estimate a heparin content of about 0.7 mg/lOO ml of human plasma (a more accurate quantitative result is reported below) and about 0.1 mg of chondroitin sulfate/100 ml of plasma. Furthermore, plasma heparin is composed of about 40% slow-moving component and 60% fast-moving species (Fig. 3). Tables 1 and 2 report the physico-chemical properties of purified heparin and chondroitin sulfate from human plasma obtained by exhaustive proteolytic treatment (procedure 11, included the anticoagulant activity of heparin measured as APTI activity (70 I.U.1. Fig. 4 illustrates the HPSEC profile of plasma extract after exhaustive proteolysis (procedure 1). The identity of the two peaks was confirmed by specific degradation with chondroitin AEK lyase and heparinases. Small amounts of fractions were eluted with low retention time (great M,) and not further considered. The extimated peak M, was

4

SM

Human Plasma Hcparin

Fig. 2. B. Agarose-gel electrophoretic separation of human plasma glycosaminoglycans. 1. Glycosaminoglycan standards (A. Slow-moving species. B. Fast-moving species. C. Dermatan sulfate. D. Chondroitin sulfate). 2. Plasma extract, 150 pg. 3. Plasma extract, 250 wg. 4. Plasma extract, 350 pg. The arrow indicates the direction of electrophoretic migration.

I Migration

Dlrtonce

??

Fig. 3. Densitometric profile of purified human plasma heparin (50 pg of plasma extract) after agarose-gel electrophoretic separation. SM: slowmoving heparin, FM: fast-moving heparin.

N. Volpi et al. / Biochimica et Biophysics Acta 1243 (1995) 49-58

Human Plasma Heparrn degraded with heDarinases I+ll+lll

F

D A E 0

7

14

21

EC

28

Retention Time (min)

Fig. 4. High-performance size-exclusion chromatographic profile of purified human plasma heparin (Hep) and chondroitin sulfate (CS). Absorbance relative units.

about 15,630 for chondroitin sulfate and 7,070 for heparin. These values are appreciably lower of that of chondroitin sulfate and heparin purified from bovine tissues and of pharmaceutical grade [26] (Tables 1 and 2). Table 1 illustrates the percentage of constituent disaccharides of plasma heparin obtained by simultaneous cleavage with heparinase I, II and III (Fig. 5). Plasma heparin is constituted by large amounts of trisulfated disaccharide (about 70%), a disaccharide unit typical of the polysaccharide chains of heparin. Disulfated disaccharides represent about 18% and monosulfated disaccharides about 3%. Non-sulfated disaccharide occurs in amounts of about 10%. The sulfate-to-carboxyl ratio calculated by considering the percentage distribution of constituent disaccharides is about 2.47. The higher charge density of heparin purified from human plasma then that from beef mucosa and of pharmaceutical grade [26] is in accordance with the higher percentage of trisulfated disaccharide and amount of slow-moving species, the heparin component with higher M, and charge density [24]. Human plasma heparin degraded with heparinase I alone (Fig. 5) yields about 45% in weight of the trisulfated disaccharide and about 55% of oligosaccharides still resistent to the treatment with heparin lyase I [29]. Human plasma chondroitin sulfate is constituted by about 60% non-sulfated disaccharide, 3.5% disaccharide monosulfated in position 6 of N-acetyl-galactosamine and about 38% disaccharide monosulfated in position 4 of the hexosamine (Table 2 and Fig. 6) determined by chondroitinase ABC lyase. The sulfate-to-carboxyl ratio was calculated to be about 0.41. Simultaneous cleavage with chondroitinase AC I and II lyases, which specifically degrade chondroitin 4- and 6-sulfate 1341, produces about 95% of the disaccharides obtained by chondroitinase ABC. This indicates that this chondroitin sulfate is constituted by about 95-100% glucuronic acid, and that it cannot be considered dermatan sulfate. The possible presence of hyaluronic acid was detected by specific degradation with hyaluronidase from Streptomyces hyalurolyticus that specifically cleaved hyaluronic acid chains producing a

p 0

1

I

I

I

I

10

20

30

40

50

F

0

Human Platme Heparin degraded with hepari naee I

I

1

I

I

I

IO

20

30

40

50

Retention lime

(min)

Fig. 5. Chromatographic separations of constituent disaccharides of human plasma heparin obtained by cleaving with heparinase I +II+ III (upper) and heparinase I alone (lower). (A) ADiH-OS, (B) ADiH-NS, (C) ADiH-6S, (Dl ADiH-2,NdiS, (E) ADiH-N,6diS, (F) ADiH-triS. See Abbreviations and Table I for structures of the disaccharides. Absorbance relative units.

II

Human PlasmaChondroitin sulfate degraded with chondroitinase ABC

G

l_H

I

I

0

IO

I

I

20

30

Retention Time (min)

Fig. 6. Chromatographic separations of constituent disaccharides of human plasma chondroitin sulfate obtained by chondroitinase ABC lyase. (Gl ADi-OS, (H) ADi-6S, (I) ADi-4s. See Abbreviations and Table II for structures of the disaccharides. Absorbance relative units.

56

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unique kind of unsaturated disaccharide. This procedure reveals no hyaluronic acid. For a more accurate quantitative and statistical analysis, heparin was extracted and quantified from 10 ml of plasma obtained from 10 human healthy volunteers. Plasma was quickly obtained by drawing blood from each volunteer, and heparin was extracted by procedure 1 and quantified. By this procedure, heparin was detected in the plasma of all 10 volunteers, and the quantitative analysis (performed by calibration curves obtained by purified slow-moving and fast-moving heparin species) revealed levels of 0.54 _t 0.17 mg/lOO ml plasma (mean k standard deviation) with a coefficient of variation of about f32%. The slow-moving heparin percentage was 37 L- 4.4 and the fast-moving heparin percentage was 65 &- 10.3. Thus, this procedure allows an accurate quantitative determination of heparin in 10 ml of plasma even through it requires a long time (about a week) for analysis.

4. Discussion Plasma was digested with nonspecific protease, papain, and submitted to p-elimination in alkaline medium (procedure 2) a treatment known to produce complete /3elimination of all 0-glycosidic linkages to serine or threonine [35], and anion-exchange chromatography. These two steps were effectively very useful to purify glycosaminoglycans from tissues [26,35,36]. This method was not able to purify glycosaminoglycans from human plasma for agarose-gel electrophoresis detection and constituent disaccharides characterization, and large amount of contaminants, not further analyzed, were recovered. A second preparative approach was performed by filtering the untreated plasma on an anion-exchange resin and treating the recovered solution with papain (procedure 3) and further anion-exchange chromatography. This method was adopted in an effort to provide a preliminary purification of plasma components bearing glycosaminoglycans prior to the proteolytic treatment. Also in this case, no heteropolysaccharide was detected by the reported analytical techniques. These two approaches, together with other techniques, such as gel permeation and/or treatment with trichloroacetic acid [14,37,38], were used in several laboratories to extract and purify plasma glycosaminoglycans [5,17]. Moreover, the identification of plasma glycosaminoglycans was difficult due to the analytical techniques utilized, such as indirect measurements as biological activity [5,39-411 or insensitive determinations such as hexuronic acid content [5,38,42] and polyacetate cellulose electrophoresis [14,17]. Other analytical approaches were cleavage with specific polysaccharide lyases [12,43] and concanavalin A rocket electrophoresis [44]. These studies revealed the presence of keratan sulfate, low-charged chondroitin sulfate and heparan sulfate in normal human plasma; the

presence of dermatan sulfate or heparin remains an open question. Human plasma was submitted to exhaustive proteolytic digestion 1181, and further purified on anion-exchange resin and by precipitation with solvents (procedure 1). This procedure permitted us to recovery about 25 mg of powder/100 ml of plasma. Heparin was present at about 0.7 mg/25 mg of powder (0.7 mg/lOO ml of plasma) and chondroitin sulfate for about 0.1 mg/25 mg of powder (0.1 mg/lOO ml of plasma). Other components recovered from plasma, such as other glycosaminoglycans (keratan sulfate), proteins and contaminant low-M, molecules were not further analyzed and characterized. Quantitative analysis was performed by agarose-gel electrophoretic separation and densitometric scanning using calibration curves. However, the amount of chondroitin sulfate may be understimated due to the different primary structures of plasma chondroitin sulfate (rich in nonsulfated disaccharide) and chondroitin sulfate used to construct the calibration curve (Table 2). In fact, these two polysaccharides have very different charge density (0.41 for plasma chondroitin sulfate and 0.97 for trachea chondroitin sulfate), and this influences the capacity of toluidine blue (a cationic dye) to bind to polysaccharides [45]. This problem is irrelevant for quantitative analysis of heparin due to the similar primary structures and physico-chemical properties of plasma heparin and heparin used as standard (Table 1). An accurate quantitative analysis of heparin in plasma of 10 human healthy volunteers revealed levels of 0.54 + 0.17 mg/lOO ml plasma with a coefficient of variation of about f. 32%, confirming the previous value of 0.7 mg/lOO ml of pooled plasma. It is very difficult to compare the amounts of chondroitin sulfate and heparin measured in this study with those reported in the literature. In fact, the amounts of total or single glycosaminoglycan are generally evaluated as nmoles of hexuronic acid and not as weight. For heparin, a comparison can be suggested by its anticoagulant activity. Cavari et al. [18] reported a value of 0.1-0.2 I.U./ml of plasma after exhaustive proteolytic treatment, and Lewandowski et al. [39] calculated a value of 0.05 I.U./ml of untreated plasma. In our study, a value of about 0.5 I.U./ml was detected. These differences could be related to the variability of anticoagulant tests utilized or to the unavailability of heparin to inhibit thrombin activity in untreated plasma. The structural study of plasma chondroitin sulfate agrees with those reported elsewhere [14,37]. In fact, a large amount is constituted by non-sulfated chondroitin, and about 40% is formed by chondroitin-4-sulfate. Moreover, dermatan sulfate is not present or is detected in trace amounts. The peak M, of this glycosaminoglycans was about 15,630, a value close to that calculated by Juvani et al. [37], about 17000. Heparin purified from human plasma is constituted by a high percentage of trisulfated disaccharide, and this agrees

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with the study of Linhardt et al. [46], who find values of about 71% for heparin purified from human mast cells. Human plasma heparin also shows a lower amount of disulfated disaccharides and a greater sulfate-to-carboxyl ratio than beef mucosa heparin [24-261. The great amount of trisulfated disaccharide and charge density explains the high percentage of slow-moving species that forms human heparin. In fact, the slow-moving component is constituted by high-charge and high-iVr chains in comparison with fast-moving species [24], and it is responsible for the higher anticoagulant and lipoprotein lipase-releasing activities [47]. The peak M, of plasma heparin is appreciably lower than that of beef mucosa heparin [24-261 and of preparations of unfractionated heparin used as drugs [48], and it is closer to the values of low-M, heparin [49,50]. The in vitro anticoagulant capacity of human plasma heparin was calculated to be about 70 I.U., and it is markedly lower than the unfractionated heparin [24,48] used in therapy but similar to that of low-M, heparin [51]. This value is a compromise between the high charge density and slow-moving percentage and low peak h4,. In fact, these physico-chemical and structural properties are responsible for anticoagulant properties of heparin [25,31,47,52]. Heparin circulates in normal human plasma as low-M, molecules, and it is probabily released from a higher-Mr species from mast-cells [53]. This is supported by a similar primary structure, evaluated as disaccharide pattern of mast-cell heparin [46] and plasma heparin. On the other hand, this molecule shows peculiar characteristics such as low-M, and anticoagulant activity (similar to those of low-M, heparins) and, on the contrary, large amounts of slow-moving species and trisulfated disaccharide and high charge density typical of an unfractionated heparin [24-261. This study reports for the first time full evidence of the presence of heparin in normal human plasma and a fine characterization of its structure and physico-chemical properties. Previous papers have demonstrated the presence in human plasma of a molecule defined heparan sulfate but structural characterization was not performed. This is necessary as heparin and heparan sulfate are very heterogeneous macromolecules and several biological properties are common. Therefore, heparin is generally constituted by two components, fast-moving, less sulfated and with lower-iVr, and slow-moving, more sulfated and with higher-M, [24,46]. Besides, the fast-moving species is constituted by a lower amount of trisulfated disaccharide and higher percentage of disulfated and monosulfated disaccharides [24,54]. Without a fine characterization of primary structures, fast-moving heparin and heparan sulfate can be confused, as several analytical methods do not permit accurate differentiation. In particular, the heparan sulfate classified by other authors [5,14] as a glycosaminoglycan species present in human plasma could be the fast-moving component of heparin. On the other hand, we cannot exclude the aggregation of ‘typical’ heparan sulfate chains (high percentage of glucuronic acid, N-acetyl-glu-

57

cosamine and low-charged disaccharides) with ‘typical’ heparin chains (high percentage of iduronic acid, Nsulfate-glucosamine and high-charged disaccharides). The results obtained using different preparative approaches to purify plasma heparin reported in this study confirm the considerations of Cavari et al. [18] who indicate that this glycosaminoglycan is strongly bound to proteins. These characteristics are responsible for the difficulty in isolating heparin. In particular, the more sulfated heparin components, such as slow-moving species, responsible for additional biological properties [47], could covalently bind or interact with strong ionic linkages with proteins [7] having a regulative role in the biological availability of heparin. On the other hand, it is well known that heparin chains bind with endothelium [ll] and several blood cells [55]. Besides, low-charged heparin chains may interact with lower affinity with blood components and can be released by less drastic extraction procedure. In fact, Staprans et al. [5] reported that isolated heparan sulfate is not covalently linked to plasma proteins. This may explain the relatively easy extraction of low-charge heparin chains named heparan sulfate. The results described in this study permit us to make a few considerations. Heparin chains having anticoagulant activity are present in the normal circulation, and they are probabily released from mast-cells. Neverthless, plasma heparin is not readily disposable but, being linked to proteins or the vascular environment, it is subject to regulation of its biological capacity. Heparin circulating in blood can perform its anticoagulant and antithrombotic activity preventing thrombotic events and maintaining the antithrombogenic capacity of the vascular environment. Considering these points, we can speculate that pathological disorders could lead to conditions in which heparin is not more regulated or it is released in high amounts producing fatal bleeding [22,23]. On the other hand, conditions in which heparin is not produced or synthesized in defective form could involve thrombogenic or atherosclerotic conditions due to its incapacity to regulate the activity of protease inhibitors and to influence lipidic metabolism.

Acknowledgements The authors thank Prof. Bolognani L., Prof. Ronca G. and Prof. Conte A. for their collaboration. The human healthy volunteers are blood donors at the Laboratory of Chemistry and Clinical Analysis of the San Agostino Hospital, Modena. Research supported by a 60% grant from ‘Minister0 dell’Universit’a e della Ricerca Scientifica e Tecnologica (M.U.R.S.T.)‘.

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