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The preparation and properties of a glycoprotein from pig's blood platelets
282
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26o49 T H E P R E P A R A T I O N AND P R O P E R T I E S OF A GLYCOPROTEIN FROM PIGS' BLOOD P L A T E L E T S R. N. MULLINGEt¢~ AND (;. MANLEY N*~ffield Department of Clinical Biochemistry, Radcliffe Infirmary, Oxford ((;real Britai~z) (Received June 27th, 1968)
SUMMARY I. The two glycosaminoglycans in the platelets from pigs' blood have been isolated by papain digestion and ion-exchange chromatography. 2. The less highly charged component was shown to be resistant to testicular hyaluronidase but susceptible to neuraminidase. 3. The carbohydrate and amino acid content of this compound were examined. The analysis showed that the compound is a glycoprotein. No uronic acid could be detected. Galactose is the predonfinant neutral sugar; a small amount of fucose is also present. There is approximately twice as much glucosamine as galactosamine. Sialic acid composes 9.6 % of the dry weight. Aspartic and glutamic are the predominant anfino acids and relatively little serine and threonine are present. 4. A possible role in the cell surface is discussed.
INTRODUCTION The presence of two glycosaminoglycans in rat blood platelets was first demonstrated by A~XDEUSOX AND OI)ELLl. Electrophoretic and chromatographic studies enabled identification of the more highly charged component as chondroitin sulphate. This has been confirmed more recently by OLSSON AXD GARDELL2 who demonstrated that human blood platelets contain chondroitin 4-sulphate. The less highly charged component from platelets has been shown by MANLEYAND M U L L I N G E R :~ to migrate on electrophoresis at a slower rate than the glycosaminoglycans from the arterial wall. Thus identification of intact platelets deposited on the arterial wall would be possible from an examination of the glycosaminoglycan content. No fraction corresponding to that fronl platelets could be identified in atherosclerotic plaques; however the chemical composition of the arterial plaque suggested an increased proportion of a sialic acid containing component relative to the uronic acid characteristic of the glycosaminoglycans. The less highly charged component from platelets was tentatively identified as a sialoprotein because of its susceptibility to neuraminidase and so it was possible that the arterial plaque contained degraded platelets. This investigation has examined the carbohydrate and amino acid content of the less highly charged component isolated from pigs' blood platelets. Biochim. Biophys. Acta, 17o (1968) 282 288
PIG PLATELET
MATERIALS
283
GLYCOPROTEIN
AND METHODS
Preparation of platelets Io 1 of pigs' blood was mixed during collection with I 1 of 3.8 3/0 sodium citrate. The blood was spun at 600 x g for 15 rain. The supernatant was then spun at 2200 × g for 4 ° rain. The platelets sedimented as a tight button. A differential count of a stained smear from the button showed it consisted almost entirely of platelets ( > 99.95 %) with a small contamination of erythrocytes ( < 0.05 %), and leucocytes ( < 0.005 %). Tile preparation was dried in vac~to over silica gel.
Isolation of glycosaminoglycans The dried platelets were digested with twice recrystallized papain prepared as described b y KIMMEL AND SMITH4. The digestion was carried out at 600 for 24 h in pH 6.5 sodium phosphate buffer as described by MANLEY5. The digest was dialysed against distilled water for 48 h and centrifuged to remove protein which had precipitated during dialysis. The volume was reduced to approx. 30 ml by exposing the risking tubing to a forced draught. The sample was then layered on the top of a 4 c m x 45 cm colunm of Dowex 1-2 X4oo and eluted at 30 ml/h with an approximately linear gradient of NaC1 obtained by pumping 4 M NaCI into a constant volume mixing chamber containing initially 300 ml of o.I M NaC1. Fractions of IO ml were collected at 2o-min intervals and analysed for sialic acid by the orcinol method described by S V E N N E R H O L M 6, and for uronic acid as described by BITTER AND Mt~IR7. The platelet glycosaminoglycans were eluted at 0.3 M and I. 5 M NaC1 from the Dowex-I column (Fig. I). Appropriate fractions were pooled, dialysed against distilled water, and partially concentrated by exposing the risking tubing to a forced draught. Samples were finally freeze dried. The identity and homogeneity of the prepared samples was checked b y electrophoresis.
Analytical methods Amino acids were prepared by hydrolysis at IiO ° for 17 h under vacuum in
0.5
0.4
0.2
o.i
°
°
~ o.1~
il 300
1.o v
. 400 500 660 Effluent volume (ml)
. 700
~
0 ~) 800
F i g . I. T h e e l u t i o n of p l a t e l e t m a t e r i a l f r o m D o w e x I w i t h a c h l o r i d e g r a d i e n t . F r a c t i o n s w e r e analysed for sialic acid by the orcinol method 6 (O), and for uronic acid by a modified carbazole reaction ~ (O). The chloride concentration was determined with a conductivity meter.
Biochim. lgiophys. Acta, 17o (1968) 2 8 2 - 2 8 8
284
R. N. MULLINGER, G. MANLEY
twice distilled constant boiling HC1. After hydrolysis HC1 was removed under vacuum over NaOH pellets. Amino acids were determined on a Technicon Auto-analyser with a gradient of sodium citrate buffer over a period of 8 h. Free fatty acid was determined as described by DUNCOMBEs after preliminary hydrolysis in o.I M methanolic KOH at 4 °0 for 30 rain. Fucose was determined as described by TSIGANOSAND MUII~9. Galactose was determined by the non-specific anthrone reaction described by ROE1°. In both these methods appropriate blanks were used to account for interference by other sugars. Hexosamine was determined by the method of Boas 11. The relative proportions of glucosamine and galactosamine were determined on the Technicon Auto-analyser using the conditions described for amino acids. Hexuronic acid was determined on samples (3-4 o/,g uronic) by the method of BITTER AND MUIR7. Hexuronic acid in the glycoprotein was determined on 4o0 ~g samples with an appropriate blank to account for the brown colour which formed. Phosphate was determined as described by BARTLETT12. Sialic acid was released from the purified sample by treating with 0.025 M H2SO 4 for I h at 80 °. Sialic acid was determined as described by ~VARREN13. Sulphate was determined after hydrolysis in 30 % (v/v) formic acid at IOO° for 8 h by the method of SPENCER14 scaled up to analyse samples containing 1-5/~g sulphate.
Paper chromatography Neutral sugars were released by hydrolysis in o.I M HC1 at Ioo ° for 8 h. HC1 was removed under vacuum over NaOH and samples were examined by descending chromatography on Whatman No. I paper at room temperature for 24 h using the solvent systems ethyl a c e t a t e - p y r i d i n e - w a t e r (12o: 5o : 4o, by vol.) and isopropanol-water (I6o:4o, v/v). For amino sugar analysis samples were hydrolysed in 4 M HC1 at IOO° for 8 h. Descending chromatography was carried out using the solvent systems pyridineethyl a c e t a t e - w a t e r - a c e t i c acid (75:75:45:15, by vol.), described by FISCHER AXD NEBELaS. Ninhydrin degradation was carried out as described by STOFFYX AND JEANLOZ16, and the amino sugar composition confirmed by descending paper chromatography of the pentoses produced using the solvent system b u t a n o l - e t h a n o l water (4o: IO: Io, by vol.).
Electrophoresis Electrophoresis was carried out on cellulose-acetate strips at 3oo V in Michaelis's verona1 acetate buffer (pH 9.2) for 8o nfin. Glycosaminoglycans were located by staining with Alcian blue and measured by reflection densitometry as described by MANLEY5.
Enzymes Ovine testicular hyaluronidase (B.D.H.) was used as described by MATHEWS AN[) INOUYE 17.
Vibrio cholerae neuraminidase (B.D.H.) was used in aqueous solution in the presence of o.ooi M CaC12, at 37 ° for 16 h.
Gas chromatography Samples (I rag) were methanolysed at 75 ° in 0.5 M nlethanolic HC1 for 24 h as described by BOLTON, CLAMPAND HOUGH18. Using techniques described in the same t3iochim. Biophys. Acla, 17o (1968) 2b;2-2S8
PIG PLATELET GLYCOPROTEIN
285
article the hexosamine was N-acetylated and trimethyl-silyl derivatives were prepared at room temperature with pyridine containing hexamethyldisilazane and trimethylchlorosilane (5: I : I, by vol.). Samples were injected into a column of Apiezon M on 85/lOO Celite which was at 16o ° in a F and M gas chromatographic apparatus. The peak areas given by standard sugar samples were measured and the proportion of each sugar component determined in the sample. RESULTS
Electrophoresis of glycosaminoglycans prepared from human and pigs' blood platelets showed two fractions. The more rapidly migrating moved at the speed of chondroitin sulphate and was digested b y testicular hyaluronidase. The other moved more slowly than hyaluronic acid and was resistant to hyaluronidase, but its ability to stain with Alcian blue was totally destroyed by neuraminidase (Fig. 2). The relative proportions of the two Alcian blue positive fractions as determined b y reflection densitometry was approximately the same in human and pigs' blood platelets,
Fig. 2. Electrophoresis on cellulose acetate, b a r b i t u r a t e - a c e t a t e buffer (pH 9.2), 20 V / c m , 80 min, a t 2o °. Strips s t a i n e d w i t h Alcian blue a n d m e a s u r e d with a reflection d e n s i t o m e t e r . (a) Control platelet digest. (b) P l a t e l e t digest t r e a t e d w i t h n e u r a m i n i d a s e . (c) Platelet digest t r e a t e d w i t h hyaluronidase.
The more slowly migrating fraction was the major component isolated from pigs' blood platelets. From 5 g dry weight of pigs' platelets 35 mg of the less highly charged component and lO mg of chondroitin sulphate were prepared. This fraction was not significantly contaminated with nucleic acid since it contained less than o.2 ~g phosphate per mg dry weight. No uronic acid, sulphate or f a t t y acid were detected. It is consequently not an undersulphated chondroitin sulphate nor a hyaluronic acid. These conclusions are supported by the analysis described below which suggest the compound is a glycoprotein. Paper chromatography of neutral sugars showed that galactose was the major component; a small amount of fucose was also present. The presence of these two neutral sugars was confirmed by gas chromatography. Paper chromatography of the amino sugars showed the presence of both glucosamine and galactosamine. This was confirmed b y the ninhydrin degradation method of STOFFYN AND JEANLOZ16. Quantitative analysis of the sugars present showed that galactose and hexosamine were present in approximately equimolar amounts (Table I). Much less fucose was present; the molar ratio to galactose determined by cololimetric methods was i : 13.4. Gas chromatography gave a molar ratio of I : 13.o. There was nearly twice as Biochim. Biophys. Acta, 17o (1968) 282-288
286
R . N . MULLINGER, G. M:kNLEY
TABLE I THE
CARBOHYDRATE
CONTENT
OF
PIGS'
PLATELET
Sugar
itmole/mg
% dry weight
Galactose Fucose Hexosamine Sialic acid
o.9r 0.07 o.84 o.3E
r6.5 ~. 2 15.o 9.6
GLYCOPROTEIN
TABLE II THE
AMINO
ACID
CONTENT
Amino acid
l, mole~rag
Asp Thre Ser Glu Pro ,Gly Ala Val lie Leu Tyr Phe Lys His
0.52 o.26 o.2o 0.43 o.34 o. 16 o.I 7 o.2r o.o5 o.I 7 o.o 3 0.04 o.o9 o.o5
OF
PIGS'
PLATI~LET GLYCOPROTEIN
much glucosamine as galactosamine in the sample. The molar ratio of galactosanfine to glucosamine d e t e r m i n e d on the a m i n o acid a u t o a n a l y s e r was found to be 1:1.85. Sialic acid composed 9.6 % of the d r y weight of the m a t e r i a l a n d was present in a p p r o x i m a t e l y one third of the molar a m o u n t of galactose. The m o l a r ratio of galactosamine, glucosamine, a n d galactose to sialic acid using these figures is o . 9 5 : I . 7 6 : 2 . 9 3 : I . o o . If the sialic acid molecules are t e r m i n a l the c o m p o u n d will c o n t a i n short polysaccharide chains. Analysis of the a m i n o acids present shows aspartic acid to be the p r e d o m i n a n t a m i n o acid (Table II). If each polysaccharide chain is linked to an a m i n o acid in the p r o t e i n part of the molecule it would seem likely t h a t linkage is to aspartic or g l u t a n l i c acid since these are the only a m i n o acids present in sufficiently high concentration. DISCUSSION Previous reports have considered the less highly charged c o m p o n e n t of platelets to be a 'mucopolysaccharide' (glyeosanfinoglycan) or hyaluronic acidl,2; this investig a t i o n has shown t h a t it is a glycoprotein. RIDDELL AND BIER 10 e x a m i n e d the Alcian blue positive material released when J?iochim. t3iophys. Acla, I7o (I968) 282-288
PIG PLATELET GLYCOPROTEIN
28 7
platelets clumped in the presence of thrombin. Both chondroitin sulphate and the glycoprotein were released into the supernatant during clumping. The larger part, however, of the glycoprotein remained in the degraded platelets. During clumping with thrombin platelets lose most of their granules and other inner structures and the membranes are partly disintegrated 2°. Since a considerable amount of the glycoprotein is not released into the supernatant during clumping it is unlikely that the glycoprotein is a component of the granules. Some of the surface properties of platelets are dependant on sialic acid containing compounds. SPAET AND ZUCKER 21 and also MADOFF, E B B E AND BALDIN122 demonstrated that the electrophoretic mobility of platelets is reduced by treatment with neuraminidase. HOVlG"a demonstrated that neuraminidase-treated platelets quickly clumped in citrated platelet poor plasma. The glycoprotein described in this paper has a heterosaccharide structure similar to that found in the Le ;' blood group substance from the ovarian cyst fluids; as in these substances the protein backbone is partly resistant to proteolytic enzymes 24. However, the relatively high content of aspartic and glutamic acids found in the platelet glycoprotein is not found in blood-group substances 25, which have an alkali labile serine linkage to carbohydrate ~6. Although the location of this glycoprotein in the platelet can only be determined by fractionation of the cell components, it has properties which make a role in the cell surface possible. The high charge and susceptibility to neuraminidase suggest that this compound is responsible for the surface properties of platelets described above. ACKNOWLEDGEMENTS
The authors thank Mr. J. R. P. O'BRIEN for advice and facilities during t h e study, and Dr. V. PATTON for amino acid analyses. One of us (R.N.M.) is in receipt of an Medical Research Council training award. REFERENCES I 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20
B. ANDERSON AND T. T. ODELL, Proc. Soc. Exptl. Biol. Med., 99 (I958) 765 • I. OLSSON AND S. GARDELL, Biochim. Biophys. dcta, 141 (1967) 348. G. MANLEY AND R. N. MULLINGER, Brit. J. Exptl. Pathol., 48 (1967) 529 . J. R. K1MMEL AND E. L. SMITH, J. Biol. Chem., 207 (1954) 515 . G. MANLEY, Brit. J. Exptl. Pathol., 46 (19651 125. L. SVENNERHOLM, Arkiv Kemi, IO (19571 577. T. BITTER AND H. M. MUIR, Anal. Biochem., 4 (19621 33 o. W. G. DUNCOMBF, Biochem. J., 88 (19631 7. C. P. TSIGANOS AND H. MUIR, Anal. Biochem., 17 (19661 495. J. H. RoE, J. Biol. Chem., 212 (19551 335N. F. B o a s , J. Biol. Chem., 204 (1953) 553. C. H. BARTLETT, J. Biol. Chem., 234 (19591 466. L. ~ARREN, J. Biol. Chem., 234 (1959) 1971. B. SPENCER, Biochem. J., 75 (196o) 435. F. G. FISCHER AND H. J. NEBEL, Z. Physiol. Chem., 302 (19551 IO. P. J. STOFFYN AND R. W. JEANLOZ, Arch. Biochem. Biophys., 52 (1954) 373. M. B. MATHEWS AND M. INOUYE, Biochim. Biophys. Acta, 53 (1961) 509. C. H. BOLTON, J. R. CLAMP AND L. HOUGH, Biochem. J., 96 (1965) 5 C. P. E. RIDDELL AND A. M. BIER, Nature, 205 (19651 711. T. HovIG, Thromb. Diath. Haemorrhag., 8 (19621 455-
Biochim. Biophys. Acta, 17o (1968) 282-288.
288 21 22 23 24 25 26
R . N . MULLINGER, G. MANLEY
T. H. SPAET AND M. B. ZUCKER,Am. J. Physiol., 206 (1964) 1267. M. A. MADOFF, S. EBBE AND M. BALDINI, J. Clin. Invest., 43 (1964) 870. T. HOVlG, Thromb. Diath. Haemorrhag., 13 (1965) 84. W. T. J. MORGAN AND A. PUSZTAI, Biochem. J., 81 (1961) 648. A. PUSZTAI AND W. T. J. MORGAN, Biochem. J., 88 (1963) 546. B. ANDERSON, J. G. RILEY, I9. HOFFMAN AND K. MEYER, Proc. Intern. Congr. Biochem., 6th, New York, 2964, p. 138.
Biochim. I3iophys. Acta, 17o (1968) 282-288
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26o49 T H E P R E P A R A T I O N AND P R O P E R T I E S OF A GLYCOPROTEIN FROM PIGS' BLOOD P L A T E L E T S R. N. MULLINGEt¢~ AND (;. MANLEY N*~ffield Department of Clinical Biochemistry, Radcliffe Infirmary, Oxford ((;real Britai~z) (Received June 27th, 1968)
SUMMARY I. The two glycosaminoglycans in the platelets from pigs' blood have been isolated by papain digestion and ion-exchange chromatography. 2. The less highly charged component was shown to be resistant to testicular hyaluronidase but susceptible to neuraminidase. 3. The carbohydrate and amino acid content of this compound were examined. The analysis showed that the compound is a glycoprotein. No uronic acid could be detected. Galactose is the predonfinant neutral sugar; a small amount of fucose is also present. There is approximately twice as much glucosamine as galactosamine. Sialic acid composes 9.6 % of the dry weight. Aspartic and glutamic are the predominant anfino acids and relatively little serine and threonine are present. 4. A possible role in the cell surface is discussed.
INTRODUCTION The presence of two glycosaminoglycans in rat blood platelets was first demonstrated by A~XDEUSOX AND OI)ELLl. Electrophoretic and chromatographic studies enabled identification of the more highly charged component as chondroitin sulphate. This has been confirmed more recently by OLSSON AXD GARDELL2 who demonstrated that human blood platelets contain chondroitin 4-sulphate. The less highly charged component from platelets has been shown by MANLEYAND M U L L I N G E R :~ to migrate on electrophoresis at a slower rate than the glycosaminoglycans from the arterial wall. Thus identification of intact platelets deposited on the arterial wall would be possible from an examination of the glycosaminoglycan content. No fraction corresponding to that fronl platelets could be identified in atherosclerotic plaques; however the chemical composition of the arterial plaque suggested an increased proportion of a sialic acid containing component relative to the uronic acid characteristic of the glycosaminoglycans. The less highly charged component from platelets was tentatively identified as a sialoprotein because of its susceptibility to neuraminidase and so it was possible that the arterial plaque contained degraded platelets. This investigation has examined the carbohydrate and amino acid content of the less highly charged component isolated from pigs' blood platelets. Biochim. Biophys. Acta, 17o (1968) 282 288
PIG PLATELET
MATERIALS
283
GLYCOPROTEIN
AND METHODS
Preparation of platelets Io 1 of pigs' blood was mixed during collection with I 1 of 3.8 3/0 sodium citrate. The blood was spun at 600 x g for 15 rain. The supernatant was then spun at 2200 × g for 4 ° rain. The platelets sedimented as a tight button. A differential count of a stained smear from the button showed it consisted almost entirely of platelets ( > 99.95 %) with a small contamination of erythrocytes ( < 0.05 %), and leucocytes ( < 0.005 %). Tile preparation was dried in vac~to over silica gel.
Isolation of glycosaminoglycans The dried platelets were digested with twice recrystallized papain prepared as described b y KIMMEL AND SMITH4. The digestion was carried out at 600 for 24 h in pH 6.5 sodium phosphate buffer as described by MANLEY5. The digest was dialysed against distilled water for 48 h and centrifuged to remove protein which had precipitated during dialysis. The volume was reduced to approx. 30 ml by exposing the risking tubing to a forced draught. The sample was then layered on the top of a 4 c m x 45 cm colunm of Dowex 1-2 X4oo and eluted at 30 ml/h with an approximately linear gradient of NaC1 obtained by pumping 4 M NaCI into a constant volume mixing chamber containing initially 300 ml of o.I M NaC1. Fractions of IO ml were collected at 2o-min intervals and analysed for sialic acid by the orcinol method described by S V E N N E R H O L M 6, and for uronic acid as described by BITTER AND Mt~IR7. The platelet glycosaminoglycans were eluted at 0.3 M and I. 5 M NaC1 from the Dowex-I column (Fig. I). Appropriate fractions were pooled, dialysed against distilled water, and partially concentrated by exposing the risking tubing to a forced draught. Samples were finally freeze dried. The identity and homogeneity of the prepared samples was checked b y electrophoresis.
Analytical methods Amino acids were prepared by hydrolysis at IiO ° for 17 h under vacuum in
0.5
0.4
0.2
o.i
°
°
~ o.1~
il 300
1.o v
. 400 500 660 Effluent volume (ml)
. 700
~
0 ~) 800
F i g . I. T h e e l u t i o n of p l a t e l e t m a t e r i a l f r o m D o w e x I w i t h a c h l o r i d e g r a d i e n t . F r a c t i o n s w e r e analysed for sialic acid by the orcinol method 6 (O), and for uronic acid by a modified carbazole reaction ~ (O). The chloride concentration was determined with a conductivity meter.
Biochim. lgiophys. Acta, 17o (1968) 2 8 2 - 2 8 8
284
R. N. MULLINGER, G. MANLEY
twice distilled constant boiling HC1. After hydrolysis HC1 was removed under vacuum over NaOH pellets. Amino acids were determined on a Technicon Auto-analyser with a gradient of sodium citrate buffer over a period of 8 h. Free fatty acid was determined as described by DUNCOMBEs after preliminary hydrolysis in o.I M methanolic KOH at 4 °0 for 30 rain. Fucose was determined as described by TSIGANOSAND MUII~9. Galactose was determined by the non-specific anthrone reaction described by ROE1°. In both these methods appropriate blanks were used to account for interference by other sugars. Hexosamine was determined by the method of Boas 11. The relative proportions of glucosamine and galactosamine were determined on the Technicon Auto-analyser using the conditions described for amino acids. Hexuronic acid was determined on samples (3-4 o/,g uronic) by the method of BITTER AND MUIR7. Hexuronic acid in the glycoprotein was determined on 4o0 ~g samples with an appropriate blank to account for the brown colour which formed. Phosphate was determined as described by BARTLETT12. Sialic acid was released from the purified sample by treating with 0.025 M H2SO 4 for I h at 80 °. Sialic acid was determined as described by ~VARREN13. Sulphate was determined after hydrolysis in 30 % (v/v) formic acid at IOO° for 8 h by the method of SPENCER14 scaled up to analyse samples containing 1-5/~g sulphate.
Paper chromatography Neutral sugars were released by hydrolysis in o.I M HC1 at Ioo ° for 8 h. HC1 was removed under vacuum over NaOH and samples were examined by descending chromatography on Whatman No. I paper at room temperature for 24 h using the solvent systems ethyl a c e t a t e - p y r i d i n e - w a t e r (12o: 5o : 4o, by vol.) and isopropanol-water (I6o:4o, v/v). For amino sugar analysis samples were hydrolysed in 4 M HC1 at IOO° for 8 h. Descending chromatography was carried out using the solvent systems pyridineethyl a c e t a t e - w a t e r - a c e t i c acid (75:75:45:15, by vol.), described by FISCHER AXD NEBELaS. Ninhydrin degradation was carried out as described by STOFFYX AND JEANLOZ16, and the amino sugar composition confirmed by descending paper chromatography of the pentoses produced using the solvent system b u t a n o l - e t h a n o l water (4o: IO: Io, by vol.).
Electrophoresis Electrophoresis was carried out on cellulose-acetate strips at 3oo V in Michaelis's verona1 acetate buffer (pH 9.2) for 8o nfin. Glycosaminoglycans were located by staining with Alcian blue and measured by reflection densitometry as described by MANLEY5.
Enzymes Ovine testicular hyaluronidase (B.D.H.) was used as described by MATHEWS AN[) INOUYE 17.
Vibrio cholerae neuraminidase (B.D.H.) was used in aqueous solution in the presence of o.ooi M CaC12, at 37 ° for 16 h.
Gas chromatography Samples (I rag) were methanolysed at 75 ° in 0.5 M nlethanolic HC1 for 24 h as described by BOLTON, CLAMPAND HOUGH18. Using techniques described in the same t3iochim. Biophys. Acla, 17o (1968) 2b;2-2S8
PIG PLATELET GLYCOPROTEIN
285
article the hexosamine was N-acetylated and trimethyl-silyl derivatives were prepared at room temperature with pyridine containing hexamethyldisilazane and trimethylchlorosilane (5: I : I, by vol.). Samples were injected into a column of Apiezon M on 85/lOO Celite which was at 16o ° in a F and M gas chromatographic apparatus. The peak areas given by standard sugar samples were measured and the proportion of each sugar component determined in the sample. RESULTS
Electrophoresis of glycosaminoglycans prepared from human and pigs' blood platelets showed two fractions. The more rapidly migrating moved at the speed of chondroitin sulphate and was digested b y testicular hyaluronidase. The other moved more slowly than hyaluronic acid and was resistant to hyaluronidase, but its ability to stain with Alcian blue was totally destroyed by neuraminidase (Fig. 2). The relative proportions of the two Alcian blue positive fractions as determined b y reflection densitometry was approximately the same in human and pigs' blood platelets,
Fig. 2. Electrophoresis on cellulose acetate, b a r b i t u r a t e - a c e t a t e buffer (pH 9.2), 20 V / c m , 80 min, a t 2o °. Strips s t a i n e d w i t h Alcian blue a n d m e a s u r e d with a reflection d e n s i t o m e t e r . (a) Control platelet digest. (b) P l a t e l e t digest t r e a t e d w i t h n e u r a m i n i d a s e . (c) Platelet digest t r e a t e d w i t h hyaluronidase.
The more slowly migrating fraction was the major component isolated from pigs' blood platelets. From 5 g dry weight of pigs' platelets 35 mg of the less highly charged component and lO mg of chondroitin sulphate were prepared. This fraction was not significantly contaminated with nucleic acid since it contained less than o.2 ~g phosphate per mg dry weight. No uronic acid, sulphate or f a t t y acid were detected. It is consequently not an undersulphated chondroitin sulphate nor a hyaluronic acid. These conclusions are supported by the analysis described below which suggest the compound is a glycoprotein. Paper chromatography of neutral sugars showed that galactose was the major component; a small amount of fucose was also present. The presence of these two neutral sugars was confirmed by gas chromatography. Paper chromatography of the amino sugars showed the presence of both glucosamine and galactosamine. This was confirmed b y the ninhydrin degradation method of STOFFYN AND JEANLOZ16. Quantitative analysis of the sugars present showed that galactose and hexosamine were present in approximately equimolar amounts (Table I). Much less fucose was present; the molar ratio to galactose determined by cololimetric methods was i : 13.4. Gas chromatography gave a molar ratio of I : 13.o. There was nearly twice as Biochim. Biophys. Acta, 17o (1968) 282-288
286
R . N . MULLINGER, G. M:kNLEY
TABLE I THE
CARBOHYDRATE
CONTENT
OF
PIGS'
PLATELET
Sugar
itmole/mg
% dry weight
Galactose Fucose Hexosamine Sialic acid
o.9r 0.07 o.84 o.3E
r6.5 ~. 2 15.o 9.6
GLYCOPROTEIN
TABLE II THE
AMINO
ACID
CONTENT
Amino acid
l, mole~rag
Asp Thre Ser Glu Pro ,Gly Ala Val lie Leu Tyr Phe Lys His
0.52 o.26 o.2o 0.43 o.34 o. 16 o.I 7 o.2r o.o5 o.I 7 o.o 3 0.04 o.o9 o.o5
OF
PIGS'
PLATI~LET GLYCOPROTEIN
much glucosamine as galactosamine in the sample. The molar ratio of galactosanfine to glucosamine d e t e r m i n e d on the a m i n o acid a u t o a n a l y s e r was found to be 1:1.85. Sialic acid composed 9.6 % of the d r y weight of the m a t e r i a l a n d was present in a p p r o x i m a t e l y one third of the molar a m o u n t of galactose. The m o l a r ratio of galactosamine, glucosamine, a n d galactose to sialic acid using these figures is o . 9 5 : I . 7 6 : 2 . 9 3 : I . o o . If the sialic acid molecules are t e r m i n a l the c o m p o u n d will c o n t a i n short polysaccharide chains. Analysis of the a m i n o acids present shows aspartic acid to be the p r e d o m i n a n t a m i n o acid (Table II). If each polysaccharide chain is linked to an a m i n o acid in the p r o t e i n part of the molecule it would seem likely t h a t linkage is to aspartic or g l u t a n l i c acid since these are the only a m i n o acids present in sufficiently high concentration. DISCUSSION Previous reports have considered the less highly charged c o m p o n e n t of platelets to be a 'mucopolysaccharide' (glyeosanfinoglycan) or hyaluronic acidl,2; this investig a t i o n has shown t h a t it is a glycoprotein. RIDDELL AND BIER 10 e x a m i n e d the Alcian blue positive material released when J?iochim. t3iophys. Acla, I7o (I968) 282-288
PIG PLATELET GLYCOPROTEIN
28 7
platelets clumped in the presence of thrombin. Both chondroitin sulphate and the glycoprotein were released into the supernatant during clumping. The larger part, however, of the glycoprotein remained in the degraded platelets. During clumping with thrombin platelets lose most of their granules and other inner structures and the membranes are partly disintegrated 2°. Since a considerable amount of the glycoprotein is not released into the supernatant during clumping it is unlikely that the glycoprotein is a component of the granules. Some of the surface properties of platelets are dependant on sialic acid containing compounds. SPAET AND ZUCKER 21 and also MADOFF, E B B E AND BALDIN122 demonstrated that the electrophoretic mobility of platelets is reduced by treatment with neuraminidase. HOVlG"a demonstrated that neuraminidase-treated platelets quickly clumped in citrated platelet poor plasma. The glycoprotein described in this paper has a heterosaccharide structure similar to that found in the Le ;' blood group substance from the ovarian cyst fluids; as in these substances the protein backbone is partly resistant to proteolytic enzymes 24. However, the relatively high content of aspartic and glutamic acids found in the platelet glycoprotein is not found in blood-group substances 25, which have an alkali labile serine linkage to carbohydrate ~6. Although the location of this glycoprotein in the platelet can only be determined by fractionation of the cell components, it has properties which make a role in the cell surface possible. The high charge and susceptibility to neuraminidase suggest that this compound is responsible for the surface properties of platelets described above. ACKNOWLEDGEMENTS
The authors thank Mr. J. R. P. O'BRIEN for advice and facilities during t h e study, and Dr. V. PATTON for amino acid analyses. One of us (R.N.M.) is in receipt of an Medical Research Council training award. REFERENCES I 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20
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Biochim. I3iophys. Acta, 17o (1968) 282-288