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appears to be present in platelets and in endothelial cells. Site-directed mutagenesis has shown this to be a functional receptor whose mechanism of action involves a thrombin-mediated cleavage that then exposes a cellactivating thrombin-binding site. Thus the properties of this newly isolated and characterized receptor fit the previously published facts. These include (I) the report by Tam and Detwiler of a thrombin-induced platelet activation site separate from the thrombin-binding site on the same receptor 14and (2) reports from several investigators 13'15,19,23that the receptor is proteolytically cleaved by thrombin as part of the activation process, and that active site-inhibited thrombin binds to the receptor but does not activate the platelet. Conversely, since there is ample evidence that GPIb is not cleaved on platelet activation, and because the sequence of the receptor does not match that of GPIb, this recent publication reinforces the view that GPIb is not the high-affinity functional platelet thrombin receptor, as the concentrations used were < 10 nM. The size of the receptor, approximately 50,000 Da, is one-fourth that of the previously published values (approximately 200,000 Da), it remains to be seen whether this means that there is an assembly of these proteins in the membrane that is not dissociated by prolonged boiling in SDS (e.g., disulfide crosslinking).

[16] P l a t e l e t M e m b r a n e

Glycoprotein V Purification

By DAVID R. PHILLIPS and MICHAEL C. BERNDT Introduction

Glycoprotein (GP) V is a relatively minor glycoprotein of the platelet plasma membrane. Glycoprotein V ( M r 82,000) can be labeled on intact platelets by either the periodate- or the galactose oxidase-Iabeling procedures and has the distinguishing feature of being the only thrombin substrate yet detected on the platelet surface.l'2 Thrombin hydrolysis of GPV yields a soluble fragment termed GPVfl (Mr 69,500). The thrombin susceptibility of GPV indicates that it interacts with thrombin during platelet activation? GPV does not appear to be involved in thrombin-induced platelet activation, however, because the liberation of GPVn does not 1 D. R. Phillips and P. P. Agin, Biochem. Biophys. Res. Commun. 75, 940 (1977). 2 D. F. Mosher, A. Vaheri, J. J. Choate, and L. G. Gahmbery, Blood 53, 437 (1979). 3 M. C. Berndt and D. R. Phillips, in "Platelets in Biology and Pathology" (J. L. Gordon, ed.), Vol. 2, p. 43. Elsevier/North-Holland Biomedical Press, Amsterdam, 1981.

METHODS IN ENZYMOLOGY, VOL. 215

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

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correlate with platelet stimulation 4 and GPV antibodies fail to block platelet stimulation. 5 GPV may be either physically or metabolically associated with GPIb-IX, as these glycoproteins are all deficient in Bernard-Soulier platelets. 6,7 Amino acid sequencing of purified GPV has shown that this glycoprotein contains at least 11 leucine-rich repeats, each composed of the consensus sequence PXXLLXXXXXLXXLXLSXNXLXXL, which is also observed in GPIb~, GPIb~, and GPIX. 8 This chapter describes a procedure for isolation of homogeneous GPV from fresh platelets. 9 The purification procedure uses fresh washed platelets as the starting material because platelet membranes prepared by sonication or sucrose gradient centrifugation lack GPV, and because clinically expired platelets (>72 hr from venipuncture) contain little or no detectable GPV, as determined by periodate/sodium boro[3H]hydride labeling. Detection of Glycoprotein V The electrophoretic mobilities of GPV and G P V n on sodium dodecyl sulfate (SDS)-polyacrylamide gels are used to detect GPV during its purification. Platelets labeled by the periodate/sodium boro[3H]hydride procedure are used for these determinations. Glycoprotein V has an apparent molecular weight of 82,000 as determined by SDS-polyacrylamide gel electrophoresis of the labeled platelets and is identifiable by its disappearance when platelets are incubated with 2 units/ml ofthrombin for 15 min at 37°. The hydrolytic fragment GPVfl has an apparent molecular weight of 69,500 and is the new band that appears in solution after thrombin hydrolysis. Glycoprotein V-containing fractions are identified during purification by analysis of fractions using SDS-polyacrylamide gel electrophoresis before and after thrombin hydrolysis. Fractions (50/zl) are incubated in the absence and presence of a-thrombin (2.5 units/ml, final concentration) for 90 rain at 22°. The reaction is terminated by the addition of SDS [2% (w/v), final concentration], and the samples are subjected to SDS-polyacrylamide gel electrophoresis. Glycoprotein V is identified with thrombin treatment by the disappearance of one protein band and the appearance of another that corresponds in mobility to GPV and GPVtl, respectively, from periodate/sodium boro[3H]hydride-labeled platelets. Because GPV 4 E. B. McGowan, A. Ding, and T. C. Detwiler, J. Biol. Chem. 258, 11243 (1983). 5 D. Bienz, W. Schnippering, and K. J. Clemetson, Blood 68, 720 (1986). 6 K. J. Clemetson, J. L. McGregor, E. James, et al., J. Clin. Invest. 70, 304 (1982). 7 A. T. Nurden, D. Dupuis, T. J. Kunicki, and J. P. Caen, J. Clin. Invest. 67, 1431 (1981). 8 T. Shimomura, K. Fujimura, S. Maehama, et al., Blood 75, 2349 (1990). 9 M. C. Berndt and D. R. Phillips, J. Biol. Chem. 256, 59 (1981).

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TABLE I PURIFICATION OF HUMAN PLATELET GLYCOPROTEIN V

Step W a s h e d platelets Extract A m m o n i u m sulfate fraction (40 to 50%) Sephacryl S-200 Hydroxylapatite DEAE-cellulose CM-cellulose

Total protein ~ (A280) 12,000 960 91.5 10.5 4.45 1.86 0.45-1.00 b

A28o/A26o 1.46 1.69 1.45 1.38 1.28 1.29

" Values are per 100 units of platelet concentrate. b Values c o r r e s p o n d to 0.45 to 1.00 mg of purified G P V based on an EI~ mvalue to 10.0 (range in yield for four preparations).

stains poorly for protein, GPV-positive fractions are conveniently identified in the early steps of purification (i.e., before the Sephacryl S-200 column step, described below) by staining the gels for carbohydrate using the sensitive dansylhydrazine procedure.I° Purification Procedure Platelets from freshly drawn platelet concentrates (within 18 hr of venipuncture) are washed at ambient temperature as described, 11 except that the final washing solution contains 0.15 M sodium chloride, 1 mM EDTA, and 0.01 M HEPES, pH 7.6. Glycoprotein V is eluted from the platelet plasma membrane by equilibrating the platelets (4 x 109/ml) at 37 ° for 24 hr in 0.3 M NaC1, 1 mM EDTA, and 0.01 M HEPES, pH 7.6. All subsequent stages of the purification procedure are performed at 4 °. The supernatant obtained from centrifugation of the salt-extracted platelets at 800 g for 30 min is clarified further by centrifugation at 35,000 g for 60 min. The clear extract is dialyzed versus a buffer containing I mM EDTA and 0.05 M potassium phosphate, pH 6.8. The dialyzed extract is brought to 40% saturation with solid ammonium sulfate, stirred for 30 min, and centrifuged at 13,000 g for 60 min. The supernatant is brought to 50% saturation with ammonium sulfate and treated similarly. The pellet is taken up in a minimum of the 0.05 M potassium phosphate buffer and dialyzed versus the same buffer. 10 A. E. Eckhardt, C. E. H a y e s , and I. J. Goldstein, Anal. Biochem. 73, 192 (1976). u D. R. Phillips, L. A. Fitzgerald, L. V. Parise, and B. Steiner, this volume [22].

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GPVGp~'fl-

-43.5K

-29K

N

I

I

I

1

2

3

4

5

I

I

I

I

I

6

7

8

9

10

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified GPV and the thrombin hydrolytic product GPVfi. Lanes 1 to 4, periodate-labeled control and thrombintreated platelet pellets and supernatants: lane 1, control platelet pellet; lane 2, platelet pellet after thrombin treatment; lane 3, control platelet supernatant; lane 4, platelet supernatant after thrombin treatment. Lanes 5 to 10, GPV in 0,1 M sodium phosphate, pH 7.4, treated with buffer or an equal concentration of a-thrombin for 1 min at 22° (GPV = 40/zg/ml; a-thrombin = 50 U/ml): lanes 5 to 7, protein stained with Coomassie Brilliant blue; lanes 8 to 10, carbohydrate stained with dansylhydrazine; lanes 5 and 8, 2/zg of GPV; lanes 6 and 9, thrombin digest of 2/zg GPV; lanes 7 and 10, a-thrombin. Protein standards in decreasing order of molecular weight: phosphorylase a, bovine serum albumin, ovalbumin, and carbonate dehydratase. (Reproduced with permission from Berndt and Phillips. °)

The dialyzed sample is centrifuged at 35,000 g for 60 min to remove the flocculent precipitate that forms during dialysis. The clear supernatant is concentrated to about 10 ml by ultrafiltration using a Diaflo YM10 ultrafiltration membrane (Amicon, Danvers, MA). The concentrated sample is applied to a column (5 x 75 cm) of Sephacryl S-200 (Pharmacia, Piscataway, N J), equilibrated with the 0.05 M potassium phosphate buffer, and eluted at a flow rate of 20 to 30 ml/hr. All GPV-positive fractions are pooled and dialyzed versus a buffer containing 0.2 mM EDTA and 5 mM potassium phosphate, pH 6.8, and then loaded onto a column (1 x 20 cm) of hydroxylapatite (Bio-Rad, Richmond, CA) equilibrated with the same buffer. The protein is eluted with a 300-ml linear gradient of 0.005 to 0.2 M potassium phosphate, pH 6.8 (0.I mM in EDTA), at a flow rate of 12 ml/hr. Glycoprotein V-positive fractions are pooled, dialyzed versus a buffer containing 0.02 mM EDTA and 0.02 M sodium phosphate, pH 7.5, and loaded onto a column (0.7 z 14 cm) of DE-52 cellulose (Whatman,

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Clifton, N J) equilibrated with the same buffer. The flow-through is pooled, dialyzed versus a buffer containing 0.2 mM EDTA and 0.01 M sodium acetate, pH 4.5, and loaded onto a column (0.7 x 7 cm) of CM-52 cellulose (Whatman) equilibrated with the same buffer. The protein is eluted with a 100-ml linear gradient of 0 to 0.5 M NaCl at a flow rate of 12 ml/hr. The peak fractions containing GPV are pooled, dialyzed exhaustively against 0.05 M ammonium bicarbonate, lyophilized, and stored at - 2 0 °. The purification procedure for GPV is summarized in Table I. This procedure reproducibly yields 0.45 to 1.0 mg of purified glycoprotein per 100 units ofplatelet concentrate (about 6 x l012 platelets). Figure 1 shows purified GPV as detected by SDS-polyacrylamide gel electrophoresis and illustrates that the purified glycoprotein has electrophoretic properties identical to those of the glycoprotein in detergent-solubilized, periodatelabeled platelets and that the glycoprotein stains by Coomassie Brilliant blue and dansylhydrazine. Some physical properties of purified GPV have also been determined. 9 Glycoprotein V has an w~% ,-,1 cm value of 10.0 at 280 nm. It contains - 4 8 % carbohydrate by weight and is composed of neutral hexose, amino hexose, and sialic acid in a molar ratio o f - 8 : 2 : 1. Electro° phoresis of the purified glycoprotein according to O'Farrel112 showed that it contains at least eight distinct isoelectric forms with isoelectric points (pI) ranging from 5.86 to 6.55; the four major forms have pI values of 6.28, 6.20, 6.12, and 6.04. Acknowledgments The authors thank James X. Warger and Norma Jean Gargasz for graphics assistance, Michele Prator and Kate Sholly for preparation of the manuscript, and Barbara Allen and Sally Gullatt Seehafer for editorial assistance.

12 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975).