THE JOURNAL OF BIOLOGICAL CHEMISTRY (c) 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 16, Issue of June 5 , pp. 10518-10523,1991 Printed in U. S. A.

Characterization of a Heparan Sulfate and a Peculiar Chondroitin 4-Sulfate Proteoglycan from Platelets INHIBITION OF THE AGGREGATION PROCESS BY PLATELET CHONDROITIN SULFATE PROTEOGLYCAN*

(Received for publication, September 6, 1990)

Helena B. Nader From the Departmento de Bioquimica, Escola Paulista de Medicina, Caixa Postal 20372, CEP 04023, SGo Paulo, SP, Brazil

A high molecular weight chondroitin sulfate proteo- glycan (M, 240,000) is released from platelet surface during aggregation induced by several pharmacologi- cal agents. Some details on the structure of this com- pound are reported. &Elimination with alkali and bo- rohydride produces chondroitin sulfate chains with a molecular weight of 40,000. The combined results in- dicate a proteoglycan molecule containing 5-6 chon- droitin sulfate chains and a protein core rich in serine and glycine residues. Degradation with chondroitinase AC shows that a 4-sulfated disaccharide is the only disaccharide released from this chondroitin sulfate, characterizing it as a chondroitin 4-sulfate homopoly- mer. It is shown that this proteoglycan inhibits the aggregation of platelets induced by ADP. Analysis of the sulfated glycosaminoglycans not released during aggregation revealed the presence of a heparan sulfate in the platelets. Degradation by heparitinases I and I1 yielded the four disaccharide units of heparan sulfates: N,O-disulfated disaccharide, N-sulfated disaccharide, N-acetylated 6-sulfated disaccharide, and N-acety- lated disaccharide. The possible role of the sulfated glycosaminoglycans on cell-cell interaction is discussed in view of the present findings.

The presence of chondroitin sulfate in platelets was first described by Anderson and Ode11 in 1958 (1). Hagen (2) in 1972 showed that chondroitin sulfate is released during plate- let aggregation induced by thrombin and polystyrene latex particles. Also in 1972, Barber et al. (3) suggested that chon- droitin sulfate is released by thrombin as a type of proteogly- can. MacPhaerson (4), using radioactive electronmicroscope techniques, suggested that sulfated glycosaminoglycans seem to be present at the cell coat of these cells. Ward and Packham ( 5 ) have shown that rabbit platelets labeled in vivo with radioactive sulfate release chondroitin 4-sulfate during ADP- induced aggregation.

Among the animal cells, the blood platelets are typical examples of cells that exhibit cell-cell aggregation when spe- cifically stimulated. This aggregation can be induced by a variety of physiological agents including ADP, adrenalin, noradrenalin, serotonin, and thrombin. Since heparan sulfate and chondroitin sulfate have been implicated in cell-cell in-

* This study was aided by grants from Financiadora de Estudos e Projetos (FINEP), Conselho Nacional do Desenvolvimento Cientifico e Tecnol6gico (CNPq), and FundaGio de Amparo i Pesquisa do Estado de SBo Paulo (FAPESP). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

teractions (6-9), it became important to investigate these compounds in platelets and their possible role in the process of platelet aggregation induced by several pharmacological agents.

The present paper reports the presence of heparan sulfate in platelets and the structural details of a peculiar chondroitin 4-sulfate proteoglycan released from platelets when induced to aggregate by a variety of pharmacological agents. It also shows that platelet chondroitin sulfate inhibits platelet aggre- gation induced by ADP.

A preliminary communication of these findings has ap- peared (10).

EXPERIMENTAL PROCEDURES

Materials and Enzymes-Chondroitin 4-sulfate from whale carti- lage, chondroitin 6-sulfate from shark cartilage, dermatan sulfate from hog skin, chondroitinases AC and ABC, chondro-4-sulfatase and chondro-6-sulfatase were purchased from Miles Laboratories (Elkhart, IN). Hyaluronic acid from umbilical cord (M, of 230,000 daltons) was a kind gift from Drs. M. B. Mathews and J. A. Cifonelli (University of Chicago, IL). Beef pancreas heparan sulfate, low mo- lecular weight heparins, heparitinases I and 11, and chondroitinase C were prepared by methods previously described (11-14). Unsaturated disaccharides from chondroitin sulfates were prepared as described by Saito et al. (15) and unsaturated disaccharides prepared from heparan sulfates as previously described (14). 1,2-Diaminoethane and 1,3-diaminopropane were purchased from Aldrich. Bio-Gel A-1.5m (200-400 mesh), Bio-Gel A-50m (50-100 mesh), DEAE-cellulose (Cel- lex D, high capacity), and agarose (standard low electroendosmosis) were purchased from Bio-Rad and superase (a proteolytic enzyme from sporabacillus) from Pfizer. Thrombin (450 units/ml) was from the Bureau of Biologics, FDA (Bethesda, MD), collagen from Hormon Chimie (Munich, Federal Republic of Germany), and trypsin type I1 from Sigma. The following drugs were used (-)noradrenaline (arter- enol hydrochloride), adrenaline (L-epinephrine bitartrate), serotonin (5-hydroxytryptamine creatinine sulfate), and ADP from Sigma. Stock solutions were kept frozen and working solutions prepared daily before the experiments.

Preparation of Platelets-The internal carotid artery from rabbits, cats, and dogs was exposed through a small incision and cannulated with polyethylene tubing, and the blood was collected. For bovine and human platelet preparation, blood was collected by venipuncture. The human donors had not ingested any drugs, including aspirin, for 2 weeks prior to donation. Blood was collected directly on 0.1 volume of sodium citrate (3.8% w/v) and spun at 600 X g for 10-20 min. The supernatant (platelet-rich plasma, PRP) was then spun at 2,200 X g for 30 min. The platelets sedimented as a compact pellet. Human platelets were also provided by Dr. J. Rosenblitz from the Blood Transfusion Service of Hospital do Servidor Publico (SBo Paulo, SP, Brazil) and were isolated from the buffy coats of blood collected from normal volunteer donors utilizing acid citrate-dextrose as anticoagu- lant. The pellet of platelets was mixed with 10 volumes of acetone and, after standing for at least 4 h at 4 “C was centrifuged and dried.

Platelet Aggregation-The procedure consisted of centrifugation of blood (human, cat, and rabbit) for the preparation of PRP, followed by incubation in the presence of each of the following drugs: ADP

10518

Glycosaminoglycans from Platelets 10519

(lo-' to M ) , adrenalin to M ) , noradrenalin ( W 4 to M ) , serotonin (5 X to 5 X M), collagen (5 pg/ml), thrombin (0.05-0.2 units/ml), or trypsin (0.25%) a t 37 "C. After gentle stirring, the visible aggregate formed was separated by centrifugation and the pellet washed with acetone and dried. The supernatant was precipi- tated with 3 volumes of ethanol in the cold overnight, and the pellet was washed with acetone and dried. Both pellet and supernatant were subjected to proteolysis with superase prior to isolation of glycosa- minoglycans as described below. Aggregation induced by ADP, tryp- sin, adrenalin, and collagen were also performed using washed plate- lets (human and rabbit). The platelets were prepared from rabbit and human PRP according to Vargaftig et al. (16).

Extraction and Purification of Glycosaminoglycans and Proteogly- cans from Platelets-The acetone powder of platelets (1 g) was sus- pended in 10 ml of 0.05 M Tris-HCI buffer, pH 8.0, in the presence of 0.15 M NaCl containing 20 mg of superase. After incubation for 18 h a t 37 "C under a layer of toluene, trichloroacetic acid was added to the mixture to a final concentration of 5% and maintained at 4 "C for 15 min. The precipitate formed was removed by centrifugation, and the glycosaminoglycans were precipitated from the supernatant by the addition of 2 volumes of ethanol at 4 "C, overnight. The precipitate formed was collected by centrifugation, washed twice with 80% ethanol, and dried under vacuum. Intact proteoglycans were extracted essentially as described by Hardingham and Muir (17). The acetone powder of platelets (1 g) was suspended with 4 M guanidine hydrochloride (GdnHCl)' in the presence of 0.5 M sodium acetate, pH 5.8, 0.01 M EDTA, 5 X M benzamidine hydrochloride, 2.5 X

M pepstatin, and 5 X lX4 M phenylmethylsulfonyl fluoride for 24 h at 4 "C under agitation. After centrifugation, the clarified ex- tracts were dialyzed in the cold against distilled water with several changes for 24 h. The precipitate formed was solubilized in the presence of 3.0 M NaCl and the proteoglycans precipitated by the addition of 2 volumes of ethanol. The precipitate was collected by centrifugation, washed twice with 80% ethanol, and dried under vacuum. Alternatively, the proteoglycans were extracted from plate- lets by incubation with 4 M GdnHCl in the presence of protease inhibitors as described above, and after centrifugation, the clarified extract (1 ml) was applied directly to a Bio-Gel A-50m column (0.8 X 56 cm) equilibrated with 4 M GdnHCl. Fractions of 1 ml were collected at a flow rate of 5 ml/h a t room temperature using 4 M GdnHCl as eluent. The fractions were dialyzed exhaustively against distilled water, analyzed by agarose gel electrophoresis (13) and by the carbazole reaction (18), and those containing the proteoglycans were pooled and dried under vacuum. Both glycosaminoglycans and proteoglycans were further purified by Cetavlon precipitation (19), molecular sieving, and ion exchange chromatography as follows. About 10 mg of the compound were applied to a Bio-Gel A-1.5m column (0.9 X 10 cm) that was equilibrated and eluted with 0.05 M ammonium carbonate pH 7.5. Fractions of 2.0 ml were collected a t a flow rate of 8 ml/h a t 4 "C and analyzed as described above. The fractions were combined according to their elution volume, and the compounds were precipitated by the addition of 2 volumes of ethanol a t 4 "C overnight. The compounds were collected by centrifugation, washed twice with 80% ethanol, and dried under vacuum. About 10 mg of each compound obtained from the gel filtration were then submitted to an ion exchange chromatography in a DEAE-cellulose column (0.7 X 10 cm) previously equilibrated with water and eluted by increasing concentrations of NaCl (0.2, 0.4, 0.6, 0.8, 1.0, 1.3, 1.6, 2.0, and 4.0 M). Two fractions of 4.0 ml were collected for each molarity. The fractions were dialyzed against distilled water and the compounds precipitated by the addition of ethanol. The compounds were visualized by agarose gel electrophoresis and by the carbazole reaction as described above.

Identification and Quantitation of Sulfated Glycosaminoglycans- The sulfated glycosaminoglycans were identified and quantitated by

' The abbreviations used are: GdnHCI, guanidine hydrochloride; ADi-GS, 2-acetamido-2-deoxy-3-~-(D-g~uco-4-enepyranosyluronic acid)-6-O-sulfo-~-galactose; ADi-4S, 2-acetamido-2-deoxy-3-0-(~- gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose; ADi-OS, 2- acetamido-2-deoxy-3-O-(~-gluco-4-enepyranosyluronic acid)-D-ga- lactose; ADi-GlcNS,GS, 2-sulfamino-2-deoxy-4-O-(D-gluco-4-enepy- ranosyluronic acid)-6-0-sulfo-~-glucose; ADi-GlcNS, 2-sulfamino-2- deoxy-4-O-(D-gluco-4-enepyranosyluronic acid)-D-glucose; ADi- GlcNAc,GS, 2-acetamido-2-deoxy-4-O-(D-gluco-4-enepyranosylu- ronic acid)-6-O-sulfo-~-glucose; ADi-GlcNAc, 2-acetamido-2-deoxy- 4-O-(D-g~uco-4-enepyranosyluronic acid)-D-glucose; PRP, platelet- rich plasma.

agarose gel electrophoresis in 1,3-diaminopropane acetate buffer (pH 9.0), barbital buffer (pH 8.6), and the discontinuous barium/diami- nopropane buffer (pH 5.8/pH 9.0) as well as degradation with specific enzymes as previously described (13). Sulfated glycosaminoglycan quantitation was performed by densitometry of the agarose slides after electrophoresis in 1,3-diaminopropane buffer and toluidine blue staining. Standard error was approximately t-4.5%. The extinction coefficients of the sulfated glycosaminoglycans were calculated using standards of chondroitin 4-sulfate, dermatan sulfate, and heparan sulfate. The compounds were also quantitated by the carbazole method (18). Paper chromatography of the products formed after enzymatic degradation was performed in isobutyric acid, 1.0 M NHe (5:3, v/v). The products were visualized with the aid of an UV lamp and stained with silver nitrate reagent. Molecular weight determina- tions were performed by polyacrylamide gel electrophoresis (11) and by molecular sieving on a Bio-Gel A-1.5m column calibrated using standard glycosaminoglycans. Amino acid analysis was carried out by ion exchange chromatography using an amino acid analyzer after acid hydrolysis (4 M HCI for 24 h at 110 "C under N, in sealed ampules). Amino sugars were measured after acid hydrolysis (4 M HC1 for 6 h a t 100 "C in sealed tubes) by a modified Elson-Morgan reaction (20). Glucosamine, galactosamine, and galactose were analyzed after acid hydrolysis (4 M HCl for 6 h a t 100 "C in sealed tubes) by descending chromatography in Whatman No. 1 paper in ethyl acetate:n-butyl alcoho1:pyridine:butyric acidwater, 10:5:10:1:5, by volume for 18 h a t room temperature. The sugars were visualized by silver nitrate stain- ing. Glucosamine and galactosamine were also determined by means of an amino acid analyzer, and the results were comparable with those obtained by paper chromatography. Uronic acid was measured by the carbazole reaction (18). Total sulfate was measured after acid hydrolysis (8 M HC1 for 6 h a t 100 "C in sealed tubes) by a method previously described (21). P-Elimination in the presence of NaBH4 was performed essentially as described by Choi and Meyer (22).

RESULTS

Sulfated Glycosaminoglycans from Human Platelets-The crude mixture of sulfated glycosaminoglycans obtained from human platelets after proteolysis and Cetavlon complexing was separated by gel filtration (Fig. 1A). The fractions were combined into three pools as shown in the figure and subjected to agarose gel electrophoresis (Fig. 2). Pool I gave a single component in this system, whereas Pools I Z and IZI showed the presence of at least two components, one of them migrat- ing as chondroitin sulfate. In order to ascertain their compo- sition the fractions were incubated with chondroitinase ABC and heparitinases. The single component of Pool I and fast migrating components of Pools I1 and I11 were totally de- graded by chondroitinase ABC, characterizing the compound as a chondroitin sulfate. The fraction migrating as heparan sulfate of Pool I11 was partially degraded by the combined action of heparitinases I and 11, characterizing it as a heparan sulfate, whereas the slow moving component of Pool I1 was resistant to the action of the enzymes, even though the compound was precipitated by Cetavlon and stained with toluidine blue. These results show that the major sulfated glycosaminoglycan component of platelets is a chondroitin sulfate with a molecular weight of 240,000 daltons which was later characterized as a chondroitin sulfate proteoglycan (see below). It also shows that platelets contain a small molecular weight chondroitin sulfate (40,000 daltons) and a heparan sulfate with a molecular weight of 30,000 daltons (Fig. 1A).

Characterization of Sulfated Glycosaminoglycans from Hu- man Platelets-In order to further characterize the chondroi- tin sulfate proteoglycan, incubations with chondroitinases AC and ABC were carried out. The compound was totally de- graded by chondroitinase AC. Fig. 3A shows that unsaturated 4-sulfated disaccharide ( ADi-4s) is the only disaccharide ob- tained by the action of the enzyme. The formation of 6- sulfated or nonsulfated disaccharide was not observed. For comparison the degradation of the standards, chondroitin 4- sulfate (from whale cartilage) and chondroitin 6-sulfate (from

10520 Glycosaminoglycai OX HA CHIS HS

1000 230 I I 1001

60 40 nw x

A CHS

c 4 0 50 60 70 80

FRACTION NUHBER

FIG. 1. Gel filtration on Bio-Gel A-1.5m of the proteoglycan and glycosaminoglycans from human platelets. Panel A, the acetone powder of platelets after proteolysis and Cetavlon complexing was applied to a column of Bio-Gel A-1.5m (0.9 X 110 cm) equilibrated and eluted with 0.05 M ammonium carbonate, pH 7.5, as described under “Experimental Procedures.” The fractions were analyzed by agarose gel electrophoresis, quantitated by densitometry a t 525 nm, and pooled as indicated in the figure. Panel R, the proteoglycan extracted from platelets with 4 M guanidine hydrochloride in the presence of protease inhibitors, purified by Bio-Gel A-50m, was applied to Bio-Gel A-1.5m (0.9 X 110 cm) equilibrated with 0.05 M ammonium carbonate, pH 7.5. The elutions of dextran sulfate (DX), hyaluronic acid (HA), chondroitin 6-sulfate (CHGS), and heparan sulfate ( H S ) used as molecular weight markers are indicated. CHS (O), chondroitin sulfate from human platelets; HS (O), heparan sulfate from human platelets.

CHase AEC

CONTROL CHase AEC HEPARITINASES

. - ~~

I I I I I I I 1 I I I I I II Ill s I II Ill s I II Ill s POOLS

FIG. 2. Electrophoretic behavior and enzymatic character- ization of the sulfated glycosaminoglycans obtained from hu- man platelets. About 5-10 pg of the glycosaminoglycans present in the pools (1-111) obtained from Bio-Gel A-1.5m were incubated with 0.05 units of chondroitinase ABC (CHase ARC) with or without heparitinases as indicated. The incubation mixtures were subjected to gel electrophoresis in 0.05 M 1.3-diaminopropane acetate buffer, pH 9.0, for 30 min a t 120 V. The sulfated glycosaminoglycans were fixed in the gel with Cetavlon and stained with toluidine blue. S, standard mixture containing 5 pg of each glycosaminoglycan; C S , chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate; Or, origin.

shark cartilage), is shown in the same chromatogram; both glycosaminoglycans produce 4- and 6-sulfated disaccharides by the action of chondroitinase AC due to their copolymeric structure (23). The formation of only 4-sulfated disaccharide from human platelet chondroitin sulfate proteoglycan was further confirmed by degradation with chondro-4- and chon-

vs from Platelets

r 1

FIG. 3. Unsaturated disaccharides formed by action of chondroitinase AC and sulfatases upon chondroitin sulfates from platelets. Panel A, about 50 pg of chondroitin sulfate ( C H S ) from human and bovine platelets, as well as standards of chondroitin 4-sulfate (CH4S) and chondroitin 6-sulfate (CH6S) were digested with 0.1 units of chondroitinase AC. The incubation mixtures were spotted on Whatman No. 1 paper and chromatographed in isobutyric acid 1 M NH,, (5:3, v/v). The products formed were visualized with the aid of an UV lamp (circles drawn around blots) followed by silver nitrate staining. Panel R, about 50 pg of chondroitin sulfate from human platelets were incubated with 0.1 units of chondroitinase AC (CHase) in the presence or absence of 0.1 units of chondro-4-sulfatase (CHase + 4-sulfatase). The mixtures were subjected to paper chro- matography and the products visualized as described in panel A.

dro-6-sulfatases (Fig. 3 B ) . ADi-OS was formed only upon the action of chondro-4-sulfatase. Chondro-6-sulfatase was una- ble to degrade the sulfated disaccharide. Further evidence of this homogeneity of structure was revealed by its resistance to degradation by chondroitinase C (data not shown).

Analysis of the heparan sulfate from platelets was also performed. The combined action of purified heparitinases I and I1 on two different batches of heparan sulfate showed the presence of the characteristic disaccharide units found for other heparan sulfates, namely N-acetylated disaccharide ( ADi-GlcNAc), N-acetylated 6-sulfated disaccharide (ADi- GlcNAcGS), N-sulfated disaccharide ( ADi-GlcNS), and a di- sulfated disaccharide ( ADi-GlcNS,GS) (Table I).

Table I1 summarizes some chemical data on the major sulfated glycosaminoglycan from platelets. The chondroitin sulfate proteoglycan has a ratio of hexosamine, uronic acid, and sulfate of 1.

An acid hydrolysis for the study of the type of sugars present in the different pools (Table 111) revealed that galactosamine is the only amino sugar present in the chondroitin 4-sulfate proteoglycan (Pool I). It shows also significant amounts of galactose, xylose, and fucose (results not shown). Pool I11 showed the presence of glucosamine besides galactosamine (results not shown). The presence of glucosamine in this pool together with the data on enzymatic degradation (Table I) further confirms the presence of heparan sulfate in this frac- tion. Pool I1 contains galactosamine and glucosamine besides significant amounts of galactose (results not shown). Since this fraction contains a compound which is precipitable by Cetavlon, stains with toluidine blue, and is not degraded by

Glycosaminoglycans from Platelets

TABLE I Relative amounts of disaccharides in human platelet heparan sulfate

10521

Heparan sulfate

Disaccharides

ADi-ClcNS,fiS ADi-ClcNS ADi-GlcNAcfiS Ani-GlcNAc M ,

x IO"' Human platelet 30 16.7

0' /"

16.6 41.7 25.0

Bovine nancreas 24 16.3 25.6 11.6 46.5

TABLE I1 Rioch~mical analyses of human platelet chondroitin

sulfate oroteodvcan

Compound Molar ratio to galactosamine

Uronic acid Total sulfate M."

Chondroitin 4-sulfate pro- 240,000 1.06 1.01 teoglycan (human plate- lets)

(shark cartilage)

skin)

Chondroitin 4-sulfate 18,000 1.00 0.92

Dermatan sulfate (hog 12,000 0.40 1.00

" Mean value.

TABLE 111 Amino acid and amino sugar analyses of chondroitin sulfate

proteoglycan from human platelets Chondroitin sulfate

proteoglycan HeDarin

Amino acids Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Tyrosine Phenylalanine Lysine Histidine Arginine

Amino sugars Glucosamine Galactosamine

14 12 Trh Tr 29 24 6 7

35 36 7 11

Tr Tr Tr T r Tr Tr

10 10 8 8

T r Tr

89 1 892

2 1

24 1

Tr 27 1

T r 8 5

928 3

" From Horner (25). " Tr, trace.

chondroitinases and heparitinases, the presence of a keratan sulfate-like polymer in this fraction, aside from chondroitin sulfate, could be suggested.

Chondroitin 4-Sulfate Proteoglycan from Human Platelets- The chondroitin 4-sulfate proteoglycan prepared after prote- olysis of human platelets was subjected to alkaline degrada- tion. A chondroitin sulfate with a molecular weight of 40,000 daltons was obtained after this treatment. This result led to an inquiry as to the nature of the intact proteoglycan present in platelets. The proteoglycan was then extracted from plate- lets with protease inhibitors (see "Experimental Procedures,") purified by Bio-Gel A-50m, and subjected to Bio-Gel A-1.5m chromatography (Fig. 1B).

The molecular weight of this proteoglycan was essentially the same as that of the one extracted after proteolysis of the platelets. In addition, both compounds exhibited the same

, I-Or. - I I I I I I 1 2 3 4 5 s

FIG. 4. Electrophoretic behavior of glycosaminoglycans from platelets of different species. Ahout 3-5 pg of glycosami- noglycans extracted Irom cat (lane I), human (lane 2 ) , hovine (lane 3), dog (lane 4 ) , and rahhit (lane 5 ) platelets were applied to agarose gel electrophoresis in 0.05 M 1,3-diaminopropane acetate huffer, pH 9.0, for 30 min a t 120 V. The sulfated glycosaminoglycans were fixed in the gel with Cetavlon and stained with toluidine blue. S , standard mixture containing 5 pg of each glycosaminoglycan; CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate; Or, origin.

elution profile on DEAE-cellulose chromatography (1.0-1.2 M NaCl), and their Cetavlon complexes were solubilized using the same salt concentration (1.2 M NaCI). Amino acid analysis from the two types of chondroitin sulfates extracted from platelets (with and without proteolysis) is shown in Table 111. Both compounds have essentially the same amino acid com- position.

Chondroitin Sulfate Proteoglycans from Other Mammalian Platelets-The agarose gel electrophoresis of the sulfated glycosaminoglycans from cat, human, bovine, dog, and rabbit platelets is shown in Fig. 4. All of them show the same electrophoretic mobility between the standards dermatan sul- fate and chondroitin sulfate. This compound accounts for 1.5- 2.0% of the total acetone powder of the platelets obtained from the different species.

Enzymatic degradation and molecular weight determina- tions were also performed for chondroitin sulfate extracted from bovine, rabbit, cat, and dog platelets. Only ADi-4S was obtained from these chondroitin sulfates after the action of chondroitinase AC (Fig. 3A). All of them were resistant to the action of chondroitinase C. In addition, the molecular weight of these chondroitin 4-sulfates were over 150,000 dal- tons.

Release of Chondroitin 4-Sulfate Proteoglycan from Platelets by Action of ADP and Other Pharmacological Agents-When platelets are induced to aggregation, chondroitin 4-sulfate is the only sulfated glycosaminoglycan released to the medium. Some aspects of this release reaction were also investigated. Platelet aggregation induced by ADP, adrenalin, noradrena- lin, serotonin, collagen, thrombin, or trypsin on the PRP from human, cat, and rabbit has shown that about 1530% of the large M, chondroitin 4-sulfate is released during this process.

Similar results were also observed for aggregation induced by ADP, trypsin, adrenalin, and collagen on washed human and rabbit platelets. No differences in M, were found for the chondroitin 4-sulfate released by any of the agents.

Effect of Sulfated Glycosaminoglycans on Platelet ADP-in- duced Aggregation-Table IV shows that all sulfated gly- cosaminoglycans tested had no effect in the ADP-induced

10522 Glycosaminoglycans from Platelets

aggregation of human platelets, except for the chondroitin sulfate proteoglycan extracted from platelets. The chondroitin 4-sulfate proteoglycan from human platelets inhibited the aggregation of human platelets in a dose-dependent curve (Table IV, Fig. 5). The same results were observed for chon- droitin 4-sulfate proteoglycan from cat platelets upon aggre- gation of cat platelets (results not shown).

DISCUSSION

We have shown that platelets contain heparan sulfate aside from a chondroitin 4-sulfate proteoglycan. Procedures using gel filtration and ion exchange chromatography as well as enzyme and chemical treatments were used to purify and identify these compounds. The data we have reported indicate that platelets contain a peculiar chondroitin 4-sulfate proteo- glycan with a molecular weight of 240,000 daltons. The protein core is rich in aspartic acid, glutamic acid, glycine, and serine, which is typical of proteoglycans (24). This amino acid com- position is similar to the one reported by Horner for the heparin proteoglycan (25). The protein core of the chondroitin 4-sulfate proteoglycan from platelets seems to belong to the class of protease-resistant proteoglycans like the ones isolated from rat mast cells (26) and basophilic leukemia cells (27), differing from the chondroitin sulfate proteoglycan isolated from normal and neoplastic monocytes, which are almost completely degraded with proteases (28). The presence of fucose could suggest the occurrence of protein-linked oligo- saccharides. It remains to be established how this proteogly- can correlates with the one described by Huang et al. (29), which is the proteoglycan carrier of human platelet factor 4 and shows a molecular weight of 53,000. A question arises of whether the proteoglycan for PF4 is a proteolytic product of a larger proteoglycan, like the one described in this paper.

As judged by the molecular weight of the proteoglycan

TABLE IV Effect of sulfated glycosaminoglycans on platelet ADP-induced

aggregation

Glycosaminoglycans Platelet aggregation

d m 1 % None 100 Chondroitin 4-sulfate (whale cartilage)

100 95 500 93

Chondroitin 6-sulfate (shark cartilage) 100 93 500 85

Dermatan sulfate (pig skin) 100 97 500 96

Heparan sulfate (bovine pancreas) 100 98 500 95

Heparin (bovine lung) 100 93 500 90

Chondroitin 4-sulfate proteoglycan (hu- man platelet)

1 80 5 40

10 5 Chondroitin 4-sulfate proteoglycan (cat

platelet) 1 61 5 30

10 5

roo.*I 0 1 2 3 0 1 0 1 2 3

TIME ( minutes) FIG. 5. Effect of chondroitin 4-sulfate proteoglycan from

human platelets on human platelet ADP-induced aggregation. To 450 pl of platelet-rich plasma, 1 and 5 pg of chondroitin sulfate proteoglycan dissolved in 50 pl of saline were added, and after a 10- min incubation at 37 "C, 5 pl of M ADP were added and the resultant changes in transmittance recorded using a Payton Dual Channel aggregation module (Payton, Buffalo, NY). A , no additions; B , 5 pg of platelet chondroitin sulfate proteoglycan; C, 1 pg of platelet chondroitin sulfate proteoglycan. Arrows indicate the addition of 5 p1 of 10-4 M ADP.

(240,000) and the chondroitin sulfate chains (40,000) released after ,&elimination, one can conclude that this proteoglycan is composed of 5-6 chondroitin sulfate chains. These chains appear to be exclusively composed of chondroitin 4-sulfate, due to its total degradation to 4-sulfated disaccharides upon the action of chondroitinase AC. No 6- or nonsulfated disac- charides could be detected. Furthermore these chondroitin chains were resistant to chondroitinase C , confirming their homogenity of structure. This type of proteoglycan is also present in platelets obtained from five different species and accounts for 1.5-2.0% of their dry weight. The large molecular weight chondroitin 4-sulfate proteoglycan is released from platelets during aggregation induced by several pharmacolog- ical agents such as ADP, adrenalin, noradrenalin, serotonin, collagen, thrombin, and trypsin. Similar results were also observed by Ward and Packham (5) studying the aggregation induced by ADP and thrombin upon radioactive labeled rabbit platelets; they have shown that chondroitin 4-sulfate was the released glycosaminoglycan. Another interesting aspect of the aggregation phenomena is that among the sulfated glycosa- minoglycans tested, the chondroitin 4-sulfate proteoglycan from platelets was the only compound capable of inhibiting platelet aggregation.

By the use of the present methodology we were also able to show for the first time the unequivocal presence of heparan sulfate in platelets. This heparan sulfate shows an average molecular weight of 30,000 and is composed of the same four disaccharide units that we have described for heparan sulfates from other vertebrate as well as invertebrate sources (7, 13, 30).

It has been previously suggested that heparan sulfate from the surface of animal cells could play a role in cell-cell rec- ognition processes and that chondroitin sulfate would act as antirecognition molecules by intercalating between the hep- aran sulfate chains of viscinal cells (8).

The present findings that platelets, when submitted to aggregation, release chondroitin sulfate from their surface

Glycosaminoglycans from Platelets 10523

(probably exposing the heparan sulfate chains), combined 12. Michelacci, Y. M., and Dietrich, C. P. (1976) J. Biol. Chem. 251, with the observation that an excess of the platelet chondroitin 13, Nader, H, B., Dietrich, c, p., Buonassisi, v,, and Colburn, p, sulfate proteoglycan inhibits their aggregation, lead to the speculation that chondroitin sulfate in the platelets 14. Nader, H. B., Porcionatto, M. A., Tersariol, I. L. S., Pinhal, M. also function as antirecognition molecules. Its removal would A. S., Oliveira, F. W., Moraes, C. T., and Dietrich, C. P. (1990) then expose the heparan sulfate inducing platelet aggregation. J. Biol. Chem. 265, 16807-16813

15. Saito, H., Yamagata, T., and Suzuki, S. (1968) J. Biol. Chem.

1154-1158

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