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GLYCOCALICIN

289

[25] P l a t e l e t G l y c o c a l i c i n

By JOSEPH LOSCALZO and ROBERT I. HANDIN Glycocalicin is an Mr 110,000, hydrophilic, proteolytic fragment of the ieucine-rich two-chain integral membrane protein of the platelet glycoprotein lb. This heavily glycosylated surface protein serves as a receptor for ristocetin-dependent von Willebrand factor binding, 1 has been implicated as a thrombin receptor 2 and as a quinidine-dependent antibody receptor, 3 and comprises the IgG Fc receptor 4 on the platelet, although more recent data call into question the physiologic importance of these last three functions. These binding functions are believed to be localized to the glycocalicin domain. Platelets from individuals with Bernard-Soulier syndrome are deficient in glycoprotein Ib 5'6 and show defects in these binding functions. The topological relationship between glycocalicin and glycoprotein Ib is indicated in Fig. 1. Glycoprotein Ib consists of two glycoproteins, the a and/3 subunits, linked by one or more disulfide bonds. 7 In the platelet membrane, glycoprotein Ib forms a noncovalent complex with a second smaller glycoprotein, glycoprotein IX. Glycocalicin is derived from the larger a chain and itself consists of several domains as defined by proteolytic cleavage with trypsin. 8 A large, trypsin-resistant domain with an apparent molecular weight of 65,000 contains most of the glycosylation sites (the so-called macroglycopeptide), while an M r 45,000 trypsin-sensitive domain containing two internal disulfide bonds (Cys2°9-Cys 248 and Cys211-Cys264, 9 comprises the thrombin-binding site and the von Willebrand factor-binding region. Glycocalicin is 56.5% (w/w) carbohydrate, and these sugar residues are confined largely to the trypsin-resistant macroglycopeptide domain in which the major oligosaccharide is a hexasaccharide O-linked to one in

I T. O k u m u r a and G. A. Jamieson, Thromb. Res. 8, 701 (1976). 2 p. Ganguly and N. L. Gould, Br. J. Haematol. 42, 137 (1979). 3 T. J. Kunicki, M. M. J o h n s o n , and R. H. Aster, J. Clin. Invest. 62, 716 (1978). 4 A. Moore, G. D. Ross, and R. L. N a c h m a n , J. Clin. Invest. 62, 1053 (1978). 5 A. T. N u r d e n a n d J. P. Caen, Nature (London) 255, 720 (1975). 6 C. S. P. Jenkins, D. R. Phillips, K. J. C l e m e t s o n , D. Meyer, M.-J. Larrieu, and E. F. L u s c h e r , J. Clin. Invest. 57, 112 (1976). 7 D. R. Phillips and P. Poh Agin, J. Biol. Chem. 252, 2121 (1977). 8 T. O k u m u r a , M. Hasitz, and G. A. J a m i e s o n , J. Biol. Chem. 253, 3435 (1978). 9 D. H e s s , J. Schaller, E. E. Rickli, and K. J. Clemetson, Eur. J. Biochem. 199, 389 (1991).

METHODS IN ENZYMOLOGY,VOL. 215

Copyright © 1992by AcademicPress, Inc. All rights of reproduction in any form reserved.

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CHO

NH2

/ S I

OHC/

\CHO

S I

\ C H # H2

FIG. 1. Structure of platelet glycoprotein Ib and derived glycocalicin structure. Arrow indicates the site of calpain cleavage on the c~ subunit of glycoprotein Ib with the resulting release of glycocalicin from the membrane.

four amino acids. ~0The hexasaccharide is composed of two sialic acid, two galactose, one N-acetylglucosamine, and one N-acetylgalactosaminitol residues organized in the sequence shown in Fig. 2. ~ Capping of these side chains with sialic acid is responsible for much of the negative charge on the platelet surface. In addition to these O-linked oligosaccharides, four N-linked glycosylation sites have been identified.12 Glycocalicin was first purified by Lombart and colleagues 13'14and subsequently by Solum and co-workers, ~5 exploiting the intrinsic calciumdependent protease (calpain) of platelets 16 to which glycoprotein Ib is exquisitely sensitive.17'18 The former group of investigators used sonication, while the latter group used both freeze-thawing and high ionic strength to release stored platelet calcium and thereby activate the protease. Glycocalicin may also be hydrolyzed from glycoprotein Ib by extracelto p. A. Judson, D. J. Anstee, and J. R. Clamp, Biochem. J. 205, 81 (1982). 11 T. Tsuji, S. Tsunehisa, Y. Watanabe, K. Yamamoto, and T. Osawa, J. Biol. Chem. 258, 6335 (1983). i2 j. A. Lopez, D. W. Chung, K. Fujikawa, F. S. Hagen, T. Papayannopoulou, and G. J. Roth, Proc. Natl. Acad. Sci. U.S.A. 84, 5616 (1987). J3 C. Lombart, T. Okumura, and G. A. Jamieson, FEBS Lett. 41, 30 (1974). 14 T. Okumura, C. Lombart, and G. A. Jamieson, J. Biol. Chem. 251, 5950 (1976). t5 N. O. Solum, I. Hagen, C. Filion-Myklebust, and T. Stabaek, Biochim. Biophys. Acta 597, 235 (1980). 16 D. R. Phillips and M. Jakfibovfi, J. Biol. Chem. 252, 5602 (1977). 17 K. J. Clemetson, Blood Cells 9, 319 (1983). i8 E. B. McGowan, K.-T, Yeo, and T. C. Detwiler, Arch. Biochem. Biophys. 227, 287 (1983).

[25]

GLYCOCALICIN

291

NeuAc 7---5 Gal GalNAcol 613---iGIcNAc 413--5Gal ~

NeuAc

FIG. 2. Structure of the principal oligosaccharide of glycocalicin. NeuAc, sialic acid; Gal, galactose; GalNAcol, N-acetylgalactosaminitol; and GIcNAc, N-acetylglucosamine.

lular metalloproteases produced by gram-negative bacteria 19(e.g., S e r r a t i a m a r c e s c e n s metalloprotease) and by plasmin, z° Subsequent purification steps have employed chromatography on insolubilized lectins such as wheat germ lectin-Sepharose 6-MB, 14 thrombin-Sepharose 4B, 21 conventional gel-filtration columns, hydroxyapatite, or phenyl boronate followed by ion-exchange high-performance liquid chromatography (HPLC). 22 We have found that the following relatively simple procedure consistently yields sufficient quantities of purified glycocalicin for use in biochemical characterization and ligand-binding studies. Twenty units of outdated platelets obtained within 48-72 hr of collection is pooled and centrifuged at 3200 g for 30 min at 4 °. The sedimented platelets are suspended in a platelet washing buffer consisting of 10 mM Tris, pH 7.6, 0.15 M NaC1, 0.6 mM EDTA, and 5 mM at 4° glucose after which they are centrifuged at 250 g for 30 min to remove contaminating erythrocytes. The platelets are then washed twice by centrifugation at 3200 g for 30 rain, with the first resuspension performed in platelet washing buffer at 4 ° and the second in approximately 300 ml of warm (37 °) 3 M KC1. After the second resuspension, the platelets are incubated for 30 min at 37°, then centrifuged at 8000 g for 10 min. The supernatant from this centrifugation is dialyzed for 18 hr against several changes of 10 mM Tris, pH 7.6, 0.15 M NaC1, 0.6 mM EDTA, 0.01% (w/v) NaN 3 (buffer A) and applied to a 10-ml column of wheat germ lectin-Sepharose 6-MB. The column is washed with buffer A until the absorbance of the eluate is equivalent to that of buffer A. The column is then developed with 2.5% N-acetylglucosamine in buffer A and the fractions with maximal absorbance at 280 nm are pooled and dialyzed against 10 mM Tris, pH 7.6, 1 M NaC1, 0.6 mM EDTA, 0.01% (w/v) NaN3 (buffer B). After dialysis, the solution is concentrated to 15 ml by ultrafiltration (Diaflo PM30 membrane; Amicon, 19 H. A. Cooper, W. P. Bennett, A. Kreger, D. Lyerly, and R. H. Wagner, J. Lab. Clin. Med. 97, 379 (1981). 2o B. Adelman, A. D. Michelson, J. Loscalzo, J. Greenberg, and R. I. Handin, Blood 65, 32 (1985). 21 M. Moroi, A. Goetze, A. Dubay, C. Wu, M. Hasitz, and G. A. Jamieson, Thromb. Res. 28, 103 (1982). 22 R. DeCristofaro, R. Landolfi, B. Bizzi, and M. Castagnola, J. Chromatogr. 426, 376 (1988).

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Danvers, MA) and applied to a 2.5 x 90-cm Sepharose 6B column. The column is developed with buffer B and the purified glycoprotein elutes in 25-35 ml. The glycocalicin may then either be concentrated by ultrafiitration or dialyzed against water and lyophilized. The yield of purified glycoprotein from a typical preparation is 0.5 mg, appears as a single Mr 110,000 band on a 7.5% polyacrylamide gel in the presence of sodium dodecyl sulfate and 0.6 M 2-mercaptoethanol, and stains with both Coomassie blue and periodic acid-Schiff's reagent. Once isolated and purified, the characterization of glycocalicin by one of several possible biochemical or immunologic methods is essential to document unequivocally the identity of this protein. The compositional analysis of glycocalicin has been determined by two groups of investigatorsLZ3; the results of their analyses are listed in Table I as mole percentages, including carbohydrate constituents, and as residues per molecule. The most abundant amino acids are threonine, leucine, and proline. The presence of four tryptophan residues per molecule has been demonstrated by using the m-iodosobenzoic acid method and is supported by the substantial intrinsic fluorescence of the molecule. These direct compositional data are supported by the derived sequence of the molecule determined from an analysis of the cDNA of glycoprotein Ib~.~2 Inspection of the derived amino acid sequence also reveals the additional interesting features of 7 tandem, 24-amino acid leucine-rich glycopeptide (LRG) repeats located near the amino terminus of the polypeptide believed to contribute indirectly to function by participating in shear-dependent conformational changes within the molecule that expose the von Willebrand factor-binding site, 24as well as a charged "hinge" domain comprising the von Willebrand factor-binding site. The abundance of carbohydrate residues in the molecule and on the macroglycopeptide fragment produced by trypsin or chymotrypsin 25,26 is of interest in that it contains 64% of the total labile sialic acid of the platelet surface. ~5To date the only functional analysis of the carbohydrate portion of glycocalicin indicates that although removal of sialic acid and galactose residues does not affect ristocetin-dependent binding to von Willebrand factor, further removal of N-acetylglucosamine markedly reduces this binding. 27 By electron microscopy, glycocalicin appears to be a semiflexible rod 23 G. E. Carnahan and L. W. Cunningham, Biochemistry 22, 5384 (1983). 24 G. J. Roth, Blood 77, 6 (1991). 25 D. S. Pepper and G. A. Jamieson, Biochemistry 9, 3706 (1970). 26 A. J. Barber and G. A. Jamieson, Biochemistry 10, 4711 (1971). 27 A. D. Michelson, J. Loscalzo, B. Melnick, B. S. Coller, and R. I. Handin, Blood 67, 19 (1986).

[2 5]

GLYCOCALICIN

293

TABLE I COMPOSITIONAL ANALYSIS OF GLYCOCALICINa

Residues Lys His Arg Asp Thr Ser Glu

Pro Gly Ala l/2-Cys Val

Met Ile

Leu Tyr Phe

Mol%

Mol% (including CHO)

6.3 2.1 2.3 8.8 14.0 8.9 10.5 12.6 5.1 4.4 ND b 4.6 1.4 2.3 12.9 2.1 3.0

3.3 1.1 1.1 4.7 7.6 4.9 5.8 6.8 2.7 2.3 ND 2.5 0.8 1.3 6.7 1.2 1.6 12.5 1.2 1.9 15.1 2.3 7.2 5.9

NANA

Mannose Fucose Galactose Glucose GlcNAc GalNAc From

Ref.

23.

CHO,

carbohydrate;

NANA,

N-acetylneuraminic acid, GIcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine. h ND, None detected.

of 56.5 × 5.4 nm, with a thickening at one end of the molecule. 28It contains approximately 15% o~ helix, 17 and the native molecule contains sufficient tryptophan and tyrosine residues to generate a fluorescence spectrum; denaturation of the native molecule with 6 M guanidine hydrochloride markedly enhances intrinsic tryptophan fluorescence, suggesting that in the native structure these hydrophobic moieties are exposed to solvent or transfer fluorescence energy to other chromophores, thereby quenching fluorescence emission. The most sensitive, accurate, and convenient method for identifying glycocalicin in our hands is an enzyme-linked immunosorbent assay 28 j. W. Lawler, S. Margossian, and H. S. Slayter, Fed. Proc., Fed. Am. Soc. Exp. Biol. 39, 1895 (1980).

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(ELISA) method, 27 in which isolated glycocalicin competes with platelet surface glycoprotein Ib for a monoclonal antibody (see below) that recognizes glycocalicin epitopes. Microtiter wells (Immulon II) are incubated with l0/zg/ml poly(L-lysine) for 30 rain at room temperature, after which the uncomplexed polymer is removed by flicking and aspiration. One hundred microliters of washed platelets at 108/ml in 10 mM Tris, pH 7.4, 0.15 M NaCl, and 4 mM EDTA is added to each well and the plates are centrifuged for 5 min at 800 g. Fifty microliters of 0.5% (w/v) formaldehyde in 10 mM Tris, pH 7.4, 0.15 M NaC1, and 4 mM EDTA are added to each well and incubated for 15 min at room temperature, after which the wells are washed twice with l0 mM Tris, pH 7.4, 0.15 M NaCl. The wells are then washed three times with l0 mM Tris, pH 8.0, 0.05% (w/v) Tween 20 (washing buffer). Each well is filled with 5 mg/ml bovine y-globulin in l0 mM Tris, pH 7.4, 0.15 M NaCl and incubated for 30 rain at 37°. The assay is performed using a mouse monoclonal antibody (6DI) directed against an epitope of glycocalicin29 and sheep anti-mouse F(ab') 2 conjugated with horseradish peroxidase. After three washes of the wells with washing buffer, 25/zl of varying concentrations of glycocalicin in l0 mM Tris, pH 7.4, 0.15 M NaC1 and 25/zl of 175 ng/ml 6D1 are added to each well and the wells incubated for 60 min at 37 °. After three more washes with washing buffer, 50/zl o f a I : 250 dilution of sheep anti-mouse (F(ab') 2 is added to each well and incubated for 60 min at 37°. The wells are then washed six times with washing buffer, 50/zl o f a 1 : I : 18 (v/v) solution of 4% o-phenylenediamine, 0.3% (v/v) H202, and 17 mM citric acid in 65 mM phosphate, pH 6.3, is added to each well, and the wells incubated for 30 min at 37°. Color development is stopped by addition of 50/zl of 4.5 mM H E S O 4 . Optical density is read at 492 nm and expressed as a percentage of the optical density in assays with 6D1 without glycocalicin (i.e., 100% binding of monoclonal antibody to the fixed platelet surface). The optical density of wells without either 6D 1 or glycocalicin is defined as 0% binding.

29B. S. Coller, E. I. Peerschke, L. E. Scuder, and C. A. Sullivan,Blood 61, 99 (1983).