THE JOURNAL OF BIOLOC~CAL CHEMISTRY Vol. 251, No. 19, Issue of October 10, pp. 5950-5955, 1976 Printed in U.S.A. Platelet Glycocalicin II. PURIFICATION AND CHARACTERIZATION* (Received for publication, March 4, 1976) TADAYOSHI OKUMURA, CHRISTIAN LOMBART,~ AND G. A. JAMIESON From The American National Red Cross, Blood Research Laboratory, Bethesda, Maryland 20014 Glycocalicin, a major glycoprotein of the platelet glycocalyx, is obtained in soluble form following platelet homogenization and has been purified to homogeneity. Glycocalicin has a molecular weight of 148,000 (* 5,000) as determined by gel electrophoresis. It contains 60 grams % carbohydrate (46.1 mol %) comprising galactose, N-acetylgalactosamine, N-acetyl- glucosamine, and sialic acid as its principal sugars in a ratio of 2:1:1:2, but with a small amount of glucose (2.3 mol %), mannose (1.2 mol %), and fucose (1.9 mol %). The principal amino acids are serine and threo- nine (4.9 and 7.6 mol %), leucine (6.7 mol %), proline, (6.8 mol %), and aspartic and glutamic acids (4.7 and 5.8 mol %). Tryptic digestion of glycocalicin yielded a macroglycopeptide (M, = 118,000 + 5,000) identical with that previously obtained from intact platelets (Pepper, D. S., and Jamieson, G. A. (1970) Biochemistry 9, 3706-3713) and a peptide of molecular weight 45,000 (~2,000) which contained only 7 mol % carbohydrate. This peptide showed a significant enrichment of serine, glycine, and glutamic acids compared with glycocalicin and together these amino acids comprised over 50 mol % of the peptide. Purified glycocalicin gave a single precipitin line with antiserum prepared in chickens. It showed reactions of partial identity with both the macroglycopeptide and the (non-glyco)peptide obtained by tryptic digestion and these showed lines of partial identity with each other. These results suggest that at least three determinants are present in the intact molecule. Glycocalicin gave precipitin reactions with wheat germ agglutinin and with the lectin of Agaricus bisporus. Despite the tremendous interest in cell surface glycoproteins in the last decade, few have been isolated in anything approaching homogeneity and only in the case of red cell glycophorin has there been extensive chemical, structural, and immunological characterization (1, 2). This success has tended to lead to the assumption that the red cell membrane is a fixed prototype of membrane structures in general and to overlook the considerable variations which have developed reflecting functional specialization in individual cell types. Blood platelets play a central role in hemostasis and this is reflected in their ability to aggregate with a variety of agents such as thrombin, collagen, and ADP. In the preceding paper (3) we have shown that human blood platelets have on their outer surface a glycoprotein which appears to be loosely bound to the membrane and is released in soluble form on disruption of the platelet: we have termed this component glycocalicin because of its origin in the platelet glycocalyx. In this paper we describe the isolation and characterization of glycocalicin and demonstrate that it lacks the high content of lipophilic amino * This work was supported, in part, by United States Public Health Service Grants HL 14697 and AI 09017. Contribution No. 347 from the American National Red Cross, Blood Research Laboratory. $ Present address, Laboratoire de Biochimie, UER Biomedicale des Saint-Peres, 75270 Paris CEDEX 06, France. acids found in the terminal portion of glycophorin and thought to be required for the strong binding of that glycoprotein to the erythrocyte membrane. In work to be published elsewhere (4) we have obtained evidence that platelet glycocalicin is the receptor for platelet aggregation induced by thrombin or by a mixture of the antibiotic ristocetin and von Willebrand’s factor. Thus, platelet glycocalicin appears to play a major role in platelet function which may be reflected in its structural characteristics. EXPERIMENTAL PROCEDURES Analytical Procedures Unless otherwise stated analytical and preparative methods were as described in the preceding paper (3). In addition, sialic acid was determined by the thiobarbituric acid procedure (5) or by the resorcinol method (6), total hexose by the phenol/sulfuric acid method (7), and protein by the Folin procedure (8) except that column effluents were monitored by absorption at 280 nm. Total hexosamines were determined by the Elson-Morgan procedure (9). Analyses for specific components were carried out on automatic amino acid and carbohydrate analyzers (JEOL, Inc., Cranford, N.J.) following hydrol- ysis as indicated: amino acids (6 N HCl, 20 h/110”), hexosamines (3 N HCl, 16 h/102”); a time course of hydrolysis for neutral sugars in 1 N hydrochloric acid/l02” showed optimal release of fucose at 90 min and of hexoses at 3 h. 5950 by guest on February 29, 2020 http://www.jbc.org/ Downloaded from
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THE JOURNAL OF BIOLOC~CAL CHEMISTRY Vol. 251, No. 19, Issue of October 10, pp. 5950-5955, 1976
Printed in U.S.A.
Platelet Glycocalicin
II. PURIFICATION AND CHARACTERIZATION*
(Received for publication, March 4, 1976)
TADAYOSHI OKUMURA, CHRISTIAN LOMBART,~ AND G. A. JAMIESON
From The American National Red Cross, Blood Research Laboratory, Bethesda, Maryland 20014
Glycocalicin, a major glycoprotein of the platelet glycocalyx, is obtained in soluble form following platelet homogenization and has been purified to homogeneity.
Glycocalicin has a molecular weight of 148,000 (* 5,000) as determined by gel electrophoresis. It contains 60 grams % carbohydrate (46.1 mol %) comprising galactose, N-acetylgalactosamine, N-acetyl- glucosamine, and sialic acid as its principal sugars in a ratio of 2:1:1:2, but with a small amount of glucose (2.3 mol %), mannose (1.2 mol %), and fucose (1.9 mol %). The principal amino acids are serine and threo- nine (4.9 and 7.6 mol %), leucine (6.7 mol %), proline, (6.8 mol %), and aspartic and glutamic acids (4.7 and 5.8 mol %).
Tryptic digestion of glycocalicin yielded a macroglycopeptide (M, = 118,000 + 5,000) identical with that previously obtained from intact platelets (Pepper, D. S., and Jamieson, G. A. (1970) Biochemistry 9, 3706-3713) and a peptide of molecular weight 45,000 (~2,000) which contained only 7 mol % carbohydrate. This peptide showed a significant enrichment of serine, glycine, and glutamic acids compared with glycocalicin and together these amino acids comprised over 50 mol % of the peptide.
Purified glycocalicin gave a single precipitin line with antiserum prepared in chickens. It showed reactions of partial identity with both the macroglycopeptide and the (non-glyco)peptide obtained by tryptic digestion and these showed lines of partial identity with each other. These results suggest that at least three determinants are present in the intact molecule. Glycocalicin gave precipitin reactions with wheat germ agglutinin and with the lectin of Agaricus bisporus.
Despite the tremendous interest in cell surface glycoproteins in the last decade, few have been isolated in anything approaching homogeneity and only in the case of red cell glycophorin has there been extensive chemical, structural, and immunological characterization (1, 2). This success has tended to lead to the assumption that the red cell membrane is a fixed prototype of membrane structures in general and to overlook the considerable variations which have developed reflecting functional specialization in individual cell types.
Blood platelets play a central role in hemostasis and this is reflected in their ability to aggregate with a variety of agents such as thrombin, collagen, and ADP. In the preceding paper (3) we have shown that human blood platelets have on their outer surface a glycoprotein which appears to be loosely bound to the membrane and is released in soluble form on disruption of the platelet: we have termed this component glycocalicin because of its origin in the platelet glycocalyx. In this paper we describe the isolation and characterization of glycocalicin and demonstrate that it lacks the high content of lipophilic amino
* This work was supported, in part, by United States Public Health Service Grants HL 14697 and AI 09017. Contribution No. 347 from the American National Red Cross, Blood Research Laboratory.
$ Present address, Laboratoire de Biochimie, UER Biomedicale des Saint-Peres, 75270 Paris CEDEX 06, France.
acids found in the terminal portion of glycophorin and thought to be required for the strong binding of that glycoprotein to the erythrocyte membrane. In work to be published elsewhere (4) we have obtained evidence that platelet glycocalicin is the receptor for platelet aggregation induced by thrombin or by a mixture of the antibiotic ristocetin and von Willebrand’s factor. Thus, platelet glycocalicin appears to play a major role in platelet function which may be reflected in its structural characteristics.
EXPERIMENTAL PROCEDURES
Analytical Procedures
Unless otherwise stated analytical and preparative methods were as described in the preceding paper (3). In addition, sialic acid was determined by the thiobarbituric acid procedure (5) or by the resorcinol method (6), total hexose by the phenol/sulfuric acid method (7), and protein by the Folin procedure (8) except that column effluents were monitored by absorption at 280 nm. Total hexosamines were determined by the Elson-Morgan procedure (9). Analyses for specific components were carried out on automatic amino acid and carbohydrate analyzers (JEOL, Inc., Cranford, N.J.) following hydrol- ysis as indicated: amino acids (6 N HCl, 20 h/110”), hexosamines (3 N HCl, 16 h/102”); a time course of hydrolysis for neutral sugars in 1 N
hydrochloric acid/l02” showed optimal release of fucose at 90 min and of hexoses at 3 h.
FIG. 1. Summary of purification procedure for glycocalicin.
Preparation of Antisera
Freund’s complete adjuvant was emulsified with an equal volume of a solution of the purified glycoprotein (0.5 mg/0.5 ml of 0.85% NaCl solution). Female chickens were injected subcutaneously with 1 ml of the emulsion followed, after 1 week, by a second injection. Blood was collected by cardiac puncture on the 9th and 18th days following the second injection. The blood was allowed to clot and the antisera were stored at -20”.
Immunodiffusion
Appropriate samples were subjected to polyacrylamide gel electro- phoresis in the absence of SDS.’ The gel cylinder was then sectioned lengthwise and the half-gel placed on top of an agar-coated glass plate about 5 mm distant from and parallel to a slot cut in the agar and containing the chicken antiserum. After incubation overnight, the half-gel was removed prior to photography of the precipitin lines.
Purification of Glycocalicin
The starting material was platelet concentrates obtained from the Washington Regional Red Cross Blood Center and consisting of the platelets isolated from individual units of whole blood (450 ml) by differential centrifugation within a closed system followed by their resuspension in 15 to 25 ml of plasma. The platelets were further purified by low speed sedimentation at 900 x g for 1.5 min in tubes (2.5 x 10 cm) to remove residual red cells, and supernatant plasma was removed from the resulting platelet pellet. All steps were carried out below 4’. The procedure for the purification of glycocalicin is summa- rized in Fig. 1.
Step I-The sedimented platelets, freed from residual red cells and plasma as described above, were washed three times with 30-ml volumes of TrisiHCl buffer (0.01 M, pH 7.4, containing 0.8% NaCl and 1 mM EDTA). The platelets from 80 units were combined and suspended in 500 ml of the above buffer. The suspension was subjected to four 15-s bursts of ultrasound (Branson sonifier, Branson Sonic Power Co., Plainview, N.Y.; output control 7). The disrupted platelet suspension was then centrifuged at 127,000 x g (Sorvall A-641 rotor, 41,000 rpm) for 90 min and the clear supernatant solution separated by decantation.
Step II-Saturated ammonium sulfate solution, previously adjusted to pH 7.4, was added to the supernatant solution from Step I to give a final concentration of 30% saturation. The resulting precipitate was removed by centrifugation at 13,200 x g (Sorvall GSA rotor; 9,000 rpm) for 60 min and the supernatant solution was brought to 80% saturation with solid ammonium sulfate. The resulting precipitate was then removed by a further centrifugation at 13,200 x g.
FIG. 2. Gel filtration of partially purified glycoprotein on a column (1.9 x 180 cm) of Sephadex G-200 in 0.05 M potassium phosphate (pH 6.8) containing 1 mM EDTA. Protein (280 nm absorption) (---); NANA (resorcinol assay) (-). Unless otherwise indicated, all graphs follow this convention.
0.02M 0.03M 0.20M
I I I
‘. . . . 1’ ‘.’ <
0 20 40 60 80 100 120 fraction number
FIG. 3. Chromatography of partially purified glycoprotein on a column (2.5 x 25 cm) of hydroxylapatite using stepwise elution with increasing concentrations of dialysis buffer, as indicated under “Mate- rials and Methods.”
Step III-This precipitate was dissolved in Tris/HCl buffer (0:05 M, pH 7.4) containing 1 mM EDTA, and the solution was then carefully adjusted to pH 5.4 with 0.2 N HCl. The resulting precipitate was removed by centrifugation at 17,300 x g (Sorvall SS-34 rotor, 12,000 rpm) for 20 min. The clear supernatant solution was concentrated to 10 ml by pressure dialysis (Amicon membrane filter XM lOOA) and then applied to a column (1.9 x 180 cm) of Sephadex G-200 equilibrated with 0.05 M potassium phosphate buffer (pH 6.8) containing 1 mM
EDTA. Step IV-The fractions (40 to 50) comprising the sialic acid-posi-
tive peak which emerged at close to the void volume (Fig. 2) were pooled, concentrated to 10 ml by pressure dialysis and then dialyzed against 1 liter of potassium phosphate buffer (0.006 M, pH 6.8) con- taining 0.2 mM EDTA with 5 changes over 48 h. The resulting precipi- tate was removed by centrifugation.
Step V-The supernatant solution from Step IV was applied to a column (1.5 x 25 cm) of hydroxylapatite which had been equilibrated with the dialysis buffer. Stepwise elution was then carried out with the dialysis buffer at the following concentrations: 0.006 M, 0.02 M, 0.03 M, and 0.2 M. The glycoprotein fraction, which was eluted in the 0.02 M buffer concentration (Fig. 3), was then concentrated to 3 to 5 ml by pressure dialysis.
Step VI-Saturated ammonium sulfate solution was added to the above concentrate to a final concentration of 60% saturation. The pH was carefully adjusted to pH 3 with 0.2 N H,SO, and the precipitate
0) (ii) FIG. 4. SDS-gel electrophoresis of tryptic products. (i) and (iii)
glycocalicin, Coomassie blue and periodate-Schiff stains, respectively. (ii) tryptic (non-glyco) peptide, Coomassie blue stain. (io) macro- glycopeptide, periodate-Schiff.
removed by centrifugation. The supernatant solution was exhaustively dialyzed against distilled water and then freeze-dried.
RESULTS
Isolation of Glycocalicin-When isolated by the technique described under “Materials and Methods,” 5 to 6 mg of glycocalicin, corresponding to 3 to 3.6 pmol of sialic acid as determined by the thiobarbituric acid procedure, were obtained from 80 units of platelets.
The present method is a substantial modification of that previously published (10). It involves fewer steps and gives a more reproducible yield of glycoprotein and there is less frequent contamination of the glycoprotein by glycopeptide, probably because the general inclusion of EDTA reduces the activity of endogenous proteases in the platelet homoge- nate. In addition, the use of hydroxylapatite following gel fil- tration permits an excellent separation of glycocalicin from protein and glycoprotein contaminants (Fig. 3).
Glycocalicin prepared in this way migrated as a single band on SDS-polyacrylamide gels which gave a positive reaction with Coomassie blue (Fig. 4) and with the periodic acid-Schiff reagent (Fig. 4, iii).
Amino acid and carbohydrate analyses are shown in Table I in comparison with previous data. Glycocalicin contains a total of 46.1 mol % carbohydrate or 60% on a weight basis and is rich in sialic acid (12.5 mol %) and in hexosamines with approxi- mately equimolar amounts of glucosamine (7.2 mol %) and galactosamine (5.9 mol %), presumably in their N-acetylated forms (Table I, Column 2). Galactose (15.1 mol %) is virtu- ally the sole neutral sugar with very minor amounts of man- nose, fucose, and glucose. The ratio of the principal sugars, Gal:GlcNAc:GalNAc:NANA, is 2:1:1:2. The principal amino acids are aspartic and glutamic acids (4.7 and 5.8 mol %), serine and threonine (4.9 and 7.6 mol %), leucine (6.7 mol %), and proline (6.8 mol %).
TABLE I
Carbohydrate and amino acid analyses
The values in Columns 3, 6, and 8 are calculated as moles per cent of the amino acids only; all others include the carbohydrate components.
Glycocalicin Macroglycopeptide Tryptic peptide
Ref. 8 Present work Ref. 9 Present work Present work
Tryptic Digestion of Purified Glycoprotein-Following pro- longed treatment (6 h) with TPCK/trypsin (l/100) (Worthing- ton Biochemicals, 195 units/mg) glycocalicin was degraded to a high molecular weight component which was positive to the periodate-Schiff reagent and which had an electrophoretic mobility identical with that of the macroglycopeptide isolated from intact platelets (11). Gel filtration on Sephadex G-200 showed that, in addition to the glycopeptide fraction which eluted close to the V,, there was a peptide fraction of low molecular weight (Fig. 5, upper panel).
Carbohydrate and amino acid analysis (Table I, Columns 5 and 7) confirmed that virtually all the carbohydrate was found in the high molecular weight (macroglycopeptide) fraction and the relative molar proportions of monosaccharides in this fraction (Gal:GlcNAc:GalNAc:NANA = 2:1:1:2) were identi- cal with those in the intact glycoprotein. On the other hand, amino acid analysis of the glycopeptide showed a considerable decrease in the content of aspartic acid relative to the intact glycoprotein (8.8 mol % in the glycoprotein to 2.7 mol % in the glycopeptide), glycine (5.1, 2.0), valine (4.6, 1.8), and leucine (12.9, 6.7), with concomitant increases in the molar percent- ages of threonine (14.0, 23.1), serine (8.9, 14.1), and proline (12.6, 20.1).
At shorter times of tryptic digestion (2 to 4 min), another band was observed in addition to the macroglycopeptide; this band moved rapidly towards the cathode and stained with Coomassie blue (Fig. 4, i9 but gave a weak and variable staining with the PAS-reagent. The products of incomplete digestion were separated by gel filtration on Sephadex G-200
FIG. 5. Gel filtration of trypsin digest of glycocalicin on Sephadex G-200; glycocalicin was treated with TPCK trypsin (l/100) at pH 8.0 in 0.1 M NH,HCO, buffer at 37”. Column size, 1.4 x 86 cm. Eluting buffer, 0.1 M pyridine/acetate (pH 4.5). Upper panel, 6-h digestion, glycocalicin (2 mg); lowerpanel, 4-min digestion, glycocalicin (4 mg).
and showed the presence of a peptide fraction which eluted just behind the macroglycopeptide (Fig. 5, lower panel).
This tryptic peptide contained serine, glycine, and glutamic acid in approximately equimolar amounts (16 to 20 mol/lOO mol of amino acid) and these three amino acids comprised over
50% of the total peptide portion. Aspartic acid and glycine, in approximately half these amounts, were the next most preva- lent amino acids, while others were present in trace amounts only (Table I, Column 8). The peptide also contained 7 mol ‘9% carbohydrate; hexosamines were virtually absent and the principal monosaccharides were galactose, glucose, and sialic acid in the molar ratios 1:l:l.
Molecular Weight Determinations-When examined in SDS-polyacrylamide gels which had been calibrated with standard proteins (Fig. 6) the molecular weight of glycocalicin was 148,000 (~5,000), of the macroglycopeptide 118,000 (*5,000), and of the tryptic peptide, 45,000 (~2,000).
The presence of large amounts of carbohydrate in glyco- proteins has been thought to present problems in the determi- nation of molecular weights by SDS-polyacrylamide gel electro- phoresis (12). However, the excellent agreement between the molecular weight of the macroglycopeptide obtained by SDS- gel electrophoresis in the present work (118,000) and that ob- tained by ultracentrifugation in previous work (120,000; Ref. 11) provides an internal standard which lends confidence to the value of 148,000 determined for glycocalicin, itself, despite the high carbohydrate content of the glycoprotein.
Immunochemistry-Glycocalicin gave a single strong preci- pitin line following electrophoresis in polyacrylamide gels and
2
1 1 2 3 4 5
cm. from the origin FIG. 6. Molecular weight determinations. Standards: 2, fibrinogen,
bovine (A4, = 340,000); 2, albumin, human trimer (207,000); 3, y-globulin, human (160,000); 4, albumin, human dimer (138,000); 5, plasminogen, human (89,000); 6, albumin, human monomer (69,000); 7, y-globulin, human H chain (50,000); 8, ovalbumin (43,000); 9, y- globulin, human L chain (23,500); IO, trypsin, bovine (23,800). a, glyco- calicin; b, tryptic macroglycopeptide of glycocalicin; c, tryptic (non- glyco)peptide. Upper line, 10% gel (l/150 bis) without fl-mercapto- ethanol, Lower line, 7.5% gel (1.5/40 his) with @-mercaptoethanol. The electrophoretic mobility of the unknown samples was not affected by the presence or absence of @-mercaptoethanol.
subsequent diffusion in agar against the chicken antiserum (Fig. 7A,i). When examined by the same technique, brief tryptic digests gave a series of three intersecting arcs corre- sponding to undigested glycocalicin, the macroglycopeptide, and a broad diffuse arc corresponding to the tryptic peptide (Fig. 7A,ii).
The relationship between these determinants was brought out more clearly by the technique of double diffusion (Fig. 7B). Purified glycocalicin (Well I) gave a single line against the chicken antiserum which showed reactions of partial identity with the macroglycopeptide (Well 2) and with the tryptic peptide (Well 5). Both the tryptic peptide (Well 5) and the macroglycopeptide (Well 4) also showed reactions of partial identity with each other. The soluble supernatant solution from the platelet homogenate (Well 3) also showed a single pre- cipitin line with the antiserum.
Further information related to the immunochemistry of the purified glycoprotein was obtained by examining its reaction with lectins. A recent study (13) has shown that intact platelets and isolated platelet membranes are agglutinated by wheat germ agglutinin (WGA) and the lectins of Ricinus communis and Phaseolus coccineus at low concentrations (5 wg/ml) and by that of Agaricus bisporus at higher concentrations (100 wdml).
Neither glycocalicin nor its macroglycopeptide gave precipi- tin reactions in the immunodiffusion test with P. coccineus or R. communis. However, precipitin reactions were obtained with WGA and A. bisporus (Fig. 7C). The intensity of the precipitin line with WGA decreased markedly after treatment
FIG. 7. Precipitin reactions of glyco- calicin and its tryptic products. A, poly- a&amide gel electrophoresis with im- munodiffusion in agar against chicken antiglycocalicin antiserum. (i) purified glycocalicin; (ii) tryptic digest (2 min); (iii) macroglycopeptide. B, immunodif- fusion of purified fractions with chicken antiglycocalicin antiserum (center well). 1, glycocalicin; 2 and 4, tryptic macro- glycopeptide; 3, crude soluble superna- tant of platelet homogenate; 5, tryptic (non-glyco)peptide. C, with lectins. 1, intact glycocalicin; 2, Agaricus bisporus; 3 glycocalicin treated with neuramini- dase; 4, WGA.
of the glycoprotein with neuraminidase, indicating a role for sialic acid in the interaction with the lectin. In addition, spurring of the precipitin line suggested that WGA might be reacting against two determinants in the intact glycoprotein prior to treatment with neuraminidase. On the other hand, the intensity of the reaction with A. bisporus appeared to increase following treatment with neuraminidase suggesting that addi- tional terminal galactose receptors were being unveiled. No reaction was observed between the purified glycoprotein and concanavalin A.
DISCUSSION
The isolation and purification of glycocalicin is a necessary prerequisite to an understandink of the method of its binding to the platelet membrane and its possible role in platelet function. The isolation procedure described here gives a yield ranging from 70 to 100% corresponding to 10 to 15% of total platelet sialic acid. Tryptic digestion of the purified glycocali-
tin yields a glycopeptide of molecular weight 118,000 and a pep- tide of molecular weight 45,000. These data suggest, within the limits of experimental error, that these two components alone make up the intact glycoprotein. The glycopeptide obtained by proteolysis of the glycoprotein is virtually identical in molecu- lar weight and in carbohydrate and amino acid analysis with the purified macroglycopeptide isolated by trypsinization of intact platelets (11).
The presence of similar molar ratios of glucose in the glycoprotein, the tryptic peptide, and the macroglycopeptide is of particular interest. The presence of glucose in the macro- glycopeptide (13) and in glycopeptides isolated from other glycoproteins has previously been interpreted rather cautiously because of the possibility of their contamination with glucose from gel filtration and ion exchange media, sucrose gradients, etc. The similar molar ratios of glucose in glycocalicin and its macroglycopeptide, although isolated at different times and by very different techniques, strongly suggests that glucose is an integral part of this glycoprotein of the platelet surface.
Complementary differences are shown between the amino acid composition of the macroglycopeptide and tryptic peptide isolated from purified glycocalicin. In particular, the macro- glycopeptide is relatively enriched in threonine and proline while the peptide is relatively enriched in serine, glycine, and glutamic acid. Overall, this tryptic peptide obviously lacks the preponderance of hydrophobic amino acids present in the tryptic peptide isolated from red cell glycophorin (14, 15). However, Segrest and Feldmann (16) have emphasized that hydrophobic sequences as short as 10 residues may be suffi- cient for membrane penetration. Since the tryptic peptide of platelet glycocalicin (M, = 45,000) is about lo-fold larger than that from red cell glycophorin (M, = 4,000) it is possible that short hydrophobic domains may be detected in the former when complete amino acid sequence data become available. These authors (16) have also suggested that domains of intermediate hydrophobicity may be present in proteins that have dynamic or reversible binding with membranes. In the light of this, it is interesting to speculate whether there may be differences in the degree or extent of hydrophobic domains in glycocalicin as compared to glycoprotein I which remains bound to the membrane following platelet homogenization (3).
The immunochemistry of the isolated glycoprotein and its reaction with lectins is also of interest. Extensive studies on platelet immunochemistry have led to the production of monospecific antithrombocyte antisera (17, 18) using the soluble fraction of the platelet homogenate as antigen. The thrombocyte-specific antigen was thought to be a glycoprotein of molecular weight 117,000 and the platelet macroglycopep- tide has been found to inhibit the agglutinating activity of the thrombocyte-specific antisera (11). The identical source of glycocalicin and the previous two antigens and the similarity of the immunological data leaves little doubt but that our purified glycoprotein is the previously described thrombocyte- specific antigen of platelet homogenates. However, our study indicates that at least three individual determinants are associated with glycocalicin, the macroglycopeptide, and the tryptic peptide. Their combined role in the immunological reactivity of the platelet remains to be elucidated.
In the case of the reaction with lectins, WGA is known to cause an agglutination of platelets and platelet membranes which is inhibited by the platelet macroglycopeptide (13) and, in the present work, WGA has been found to give a precipitin reaction with purified glycocalicin. However, purified glycoca- licin does not give a precipitin reaction with the lectin of Phaseolus coccineus, which is also known to cause platelet agglutination but which is inhibited by the intermediate glycopeptide of platelets (glycopeptide II) (13). Thus, glycoca- licin is probably a receptor for WGA in effecting agglutination, but not for P. coccineus. This receptor appears to be affected
by the terminal sialic acid of the platelet heterosaccharide chains, since the precipitin reaction is lessened following treatment with neuraminidase. Although the lectin of A. bisporus gives a precipitin reaction with glycocalicin, its limited activity in the agglutination of platelets may reflect an insufficient number of galactose receptors in the intact platelet, since its precipitin activity is increased by treatment of purified glycocalicin with neuraminidase.
In summary, these experiments indicate (a) that glycocalicin is probably a receptor for the agglutination of platelets and platelet membranes induced by certain lectins such as WGA and A. bisporus; (b) that glycocalicin is a precursor of the macroglycopeptide isolated by trypsinization of intact plate- lets; and (c) that the amino acid composition of the (non- glyco)peptide portion of glycocalicin lacks the preponderance of lipophilic residues thought to be associated with membrane binding of red cell glycophorin. Experiments to be reported elsewhere (4) suggest that glycocalicin may also function as a receptor site on the platelet surface for various aggregation reactions affected by ristocetin and human factor VIII, by bovine factor VIII, and by thrombin.
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