of biological. chemistry no. 5, 4882-4890,1988 by ... · plasma membrane has been termed the...

THE JOURNAL OF BIOLOGICAL. CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. Issue of April 5, pp, 4882-4890,1988 Printed in U.S.A.

Structure of the Glycoprotein Ib J X Complex from Platelet Membranes*

(Received for publication, September 8, 1987)

Joan E. B. FoxSP, Lawrence P. AggerbeckSll, and Michael C. BerndtII** From the $.Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute, Department of Pathology, University of California, San Francisco, California 94140-0608 and the IlDepartment of Medicine, University of Sydney Westmead Hospital, Sydney, New South Wales, Australia

The glycoprotein Ib*IX complex is a major component of the platelet membrane. It mediates the adhesion of platelets to exposed subendothelium and provides an attachment site for the membrane skeleton on the plasma membrane. The present study was designed to characterize the structure of the glycoprotein Ib*IX complex. Electron microscopy of purified glycoprotein Ib-IX complex in detergent showed that each complex existed as a flexible rod with a globular domain on either end. The overall length of the complex was -59.5 nm. The smaller globular domain had a diameter of -8.9 nm; the larger, a diameter of -15.9 nm. In the absence of detergent, the glycoprotein Ib*IX complexes tended to self-associate through the larger globular domain, suggesting that this domain contained the hydrophobic region that inserts into the membrane. Pro- teases known to cleave glycoprotein Ib, close to its membrane-insertion site released the larger globular domain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that this domain was composed of glycoprotein Ib,, glycoprotein IX, and a M, = 25,000 fragment of glycoprotein Ib,. Proteolysis at the external end of glycoprotein Ib, reduced the size of the smaller globular domain. This study shows that the glycoprotein Ib-IX complex has an elongated shape, with a globular domain on the end that inserts into the membrane and a smaller globular domain on the end of glycoprotein Ib, that is oriented external to the plasma membrane.

Glycoprotein Ib is one of the major platelet membrane glycoproteins; there are approximately 25,000 copies of glycoprotein Ib per platelet (1). The glycoprotein consists of two disulfide-linked subunits, glycoprotein Ib, (Mr = 145,000) and glycoprotein Ibo (Mr = 24,000) (2), that are complexed in a 1:1 ratio with another membrane glycoprotein, glycoprotein IX (M, = 22,000) (3, 4). The glycoprotein Ib. IX complex has

* This work was supported by Research Grants HL 30657-04 (to J. E. B. F.) and HL 18577-11 (to L. P. A.) from the National Institutes of Health, and 6K14443 from the National Health and Medical Research Council of Australia (to M. C. B.). 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 An Established Investigator of the American Heart Association. To whom correspondence should be addressed Gladstone Foundation Laboratories for Cardiovascular Disease, P. 0. Box 40608, San Fran- cisco, CA 94140-0608.

ll On leave from the Centre de Ginktique Moliculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.

** Recipient of a Wellcome Australian Senior Research Fellowship.

several known functions. It binds to von Willebrand factor on exposed vascular subendothelium and is therefore essential for the initial adhesion of platelets to the subendothelium at the site of injury (5, 6). In addition, glycoprotein Ib has been reported to be the receptor for quinine/quinidine drug-dependent antibodies (7) and to bind thrombin (8). The obser- vation that the binding of desialated von Willebrand factor initiates activation of platelets (9, 10) suggests that glycoprotein Ib may be involved in transmembrane signaling events. Finally, platelets contain a submembranous skeleton, composed in large part of actin and actin-binding protein, that determines the shape of the cell, stabilizes the membrane, and regulates the ability of glycoprotein Ib to bind von Willebrand factor (11-13). The glycoprotein Ib. IX complex functions as the attachment site for this membrane skeleton on the plasma membrane (11,12).

The amino acid sequence of glycoprotein Ib, has recently been obtained by cDNA and protein-sequencing techniques (14, 15). This glycoprotein contains 610 amino acids, 29 of which have been identified as a potential transmembrane domain. The putative cytoplasmic region contains 100-amino acid residues and is at the carboxyl end of the molecule.

Two regions of glycoprotein Ib, that are sensitive to proteolytic cleavage have been identified (16, 17). One region is close to the carboxyl end. It is susceptible to hydrolysis by Ca2+- dependent protease, Serratia marcescens, and trypsin (16-19). Cleavage at this site generates two fragments of glycoprotein Ibcr: a M , = 135,000 fragment that is heavily glycosylated and has been termed glycocalicin (20), and a M , = 25,000 fragment that is highly hydrophobic in nature (21) and contains the cysteine(s) that are disulfide-linked to glycoprotein I b p (16). On intact platelets, this site is susceptible to cleavage by externally added proteases (16, 18, 19, 21). Glycocalicin is released into the supernatant, while the M, = 25,000 fragment remains associated with glycoprotein Ib, in the plasma membrane (16).

The second protease-sensitive region of glycoprotein Ib, is also external to the membrane in platelets but is close to the other end of the molecule (16). This region is susceptible to hydrolysis by elastase and trypsin (16). Hydrolysis a t this region generates a poorly glycosylated M , = 45,000 amino- terminal fragment and a heavily glycosylated M , = 100,000 fragment that remains disulfide-linked to glycoprotein Ib, in the plasma membrane. Platelets lacking the terminal M , = 45,000 fragment of glycoprotein Ib, show reduced response to thrombin and von Willebrand factor (16), and antibodies against the M, = 45,000 fragment inhibit the response of platelets to these ligands (16,22). Thus, it has been suggested that the external M , = 45,000 fragment of glycoprotein Ib,, contains the binding sites for thrombin and von Willebrand factor (16, 22). The fragment of glycoprotein Ib, that remains

4882

Structure of the Platelet Membrane Glycoprotein I b . I X Complex 4883

after removal of the M , = 45,000 fragment a t the external end and the M, = 25,000 fragment at the end that inserts into the plasma membrane has been termed the macroglycopeptide (20).

Despite the relative abundance of the glycoprotein Ib.IX complex in the platelet membrane, the importance of the complex to platelet function, and the fact that the amino acid sequence of glycoprotein Ib,, has been determined, little is known about the tertiary structure of the glycoprotein Ib . IX complex. Until recently, the complex has been difficult to study because the methods for purification yielded either the hydrolytic fragment, glycocalicin (23), or the glycoprotein Ib. IX complex that remained in association with elements of the membrane skeleton (21). Recently, Berndt et al. (4) reported a method for purifying the glycoprotein Ib. IX complex intact and apparently free of contaminating proteins. In the present study, it has been shown that glycoprotein Ib-IX purified by the latter method is indeed free of other components of the membrane skeleton. This purified material has enabled us to make a detailed characterization of the glycoprotein Ib. IX complex. The complex isolated from washed platelet suspensions primarily contained intact glycoprotein Ib.IX, but a small amount of complex lacking the external M, = 45,000 region was also present, as were small amounts of glycocalicin. Visualized by electron microscopy, each intact glycoprotein Ib. IX complex has an elongated and flexible structure with a globular domain on either end. Examination of complexes subjected to limited proteolysis shows that the glycocalicin portion of glycoprotein Ib,, constitutes the elongated region of the molecule. The larger of the two globular domains contains glycoprotein Ib,,, glycoprotein IX, and the membrane-associated end of glycoprotein Ib'?. This domain contains the hydrophobic region of the complex that presumably constitutes the membrane-insertion region of the glycoprotein Ib-IX complex. The smaller globular domain contains the amino terminus of glycoprotein Ib,,, which is oriented external to the platelet plasma membrane.

MATERIALS AND METHODS

Purification of the Glycoprotein Zb. ZX Complex-The glycoprotein Ib.IX complex was purified from fresh platelet concentrates as described previously (4). In this method, the glycoprotein Ib . IX complex is dissociated from isolated membranes by incubation of the membranes in a buffer containing 0.1% Triton X-100. N-Ethylmaleimide is included in this buffer to inhibit the action of the Ca2'-dependent protease, an enzyme that can hydrolyze glycoprotein Ib,, generating glycocalicin. N-Ethylmaleimide serves the additional function of dis- rupting the interaction between the glycoprotein complex and the membrane skeleton. In some preparations, N-ethylmaleimide was omitted from the extraction buffer and the Triton X-100 concentration was raised to 1.0%. Although this solubilization buffer contained no inhibitors of the Caz+-dependent protease, glycoprotein Ib, was resistant to hydrolysis because of the protective effect of Triton X- 100 against the hydrolysis of glycoprotein Ib, (24). Since Triton X- 100 has no such protective effect against the hydrolysis of actin- binding protein by the Ca2'-dependent protease (12), actin-binding protein was hydrolyzed, resulting in dissociation of glycoprotein Ib. IX from the membrane skeleton (12).

The protein concentration of the purified glycoprotein Ib. IX complex was determined by quantitative amino acid analysis to be approximately 200 pg/ml. The purified protein was dialyzed at 4 "C against 150 mM sodium chloride, 0.1% Triton X-100, 0.02% sodium azide, and 10 mM Tris.HC1, pH 7.4, and stored at -70 "C. Purified glycoprotein was radiolabeled by the Iodo-bead method (25).

Purification of Glycocalicin-To purify glycocalicin, washed platelets were suspended in a Tyrode's buffer (12), incubated for 60 min at 37 "C with Caz'-dependent protease from chicken breast muscle (21 pglml; kindly provided by Dr. Darrel Go11 of the University of Arizona), and centrifuged at ambient temperatures for 1 min at 15,000 X g. Glycocalicin was purified from the resulting supernatant by

affinity chromatography on a column of wheat-germ lectin coupled to Sepharose 6MB (Sigma) (11).

Proteolytic Digestions of the Purified Glycoprotein Ib. ZX Complex- The purified glycoprotein Ib. IX complex was incubated with proteolytic enzymes at ambient temperatures (22 & 2 "C). Trypsin (Sigma) was used at a final concentration of 10 pg/ml. @-Lytic protease (purified from Lysobacter enzymogenes (26), kindly provided by Dr. Roger Bone of the University of California, San Francisco) was used at a final concentration of 0.15 pg/ml. Incubations were terminated by addition of a sodium dodecyl sulfate (SDS)'-containing buffer or by dilution into the buffers employed for rotary shadowing (see the following section). Because trypsin produced additional hydrolytic products a t times longer than those used for analysis of the products on SDS-polyacrylamide gels (16), incubations with trypsin were terminated by addition of soybean trypsin inhibitor (Sigma) (final concentration of 20 pg/ml) before samples were prepared for rotary shadowing.

Examination of Proteins by Rotary Shadowing-Low-angle rotary shadowing of molecules dried from glycerol-containing solutions was performed as adapted from Shotton et al. (27) and Tyler and Branton (28) using the mica sandwich technique described by Mould et al. (29). Samples were first diluted to a concentration of 10-20 pg of protein/ml either with a buffer containing 150 mM sodium chloride, 1 mM EDTA, 0.1% Triton X-100, and 10 mM Tris.HCI, pH 7.4, or with a buffer containing 0.5 mg of the detergent dodecyloctaoxyethylene glycol monoether (C,,E,)/rnl (Kouyoh Trading Co. Ltd., Tokyo) and 100 mM ammonium acetate, pH 7.0. Samples were then diluted 1:l with glycerol. Micrographs were taken on a JEOL 100 CX microscope. All dimensions were corrected by subtracting 2.5 nm, the width of the metal shell (30).

Analytical Procedures-For analysis of SDS-polyacrylamide gels, samples were solubilized in SDS-containing buffer (11) and electrophoresed on one-dimensional gels as described by Laemmli (31) or on two-dimensional nonreduced-reduced gels as described by Phillips and Agin (32). Polypeptides were detected with Coomassie Brilliant Blue or by silver staining (33, 34). Molecular weights of polypeptides were estimated by comparing their relative mobility on SDS-polyacrylamide gels with those of standard proteins (Bio-Rad M, = 14,500-92,500 and M , = 45,000-200,000) that were run in parallel.

Immunoblotting was performed as described by Towbin et al. (35). Glycoprotein Ib,, was detected by its reactivity with WM-23 (4). A monoclonal antibody against glycoprotein IIb-IIIa complex (7E3, kindly provided by Dr. Barry Coller of State University of New York at Stony Brook) was used as a control. Antibody-antigen complexes were detected using Vectastain" ABC Kit (Vector Laboratories, Bur- lingame, CA).

Sedimentation coefficients were determined as described previously ( l l ) , except that the buffer contained 1 mM EDTA, 150 mM sodium chloride, 0.05% Triton X-100, and 10 mM Tris.HC1, pH 7.4, and the gradients were fractionated into 33-35 fractions.

The apparent Stokes radii of glycoproteins were determined by gel filtration chromatography on a column of Sephacryl S-300 (Phar- macia Fine Chemicals, Uppsala, Sweden) as described previously (36, 37).

Amino-terminal sequences were determined by Edman degradation of protein that was reduced with P-mercaptoethanol, alkylated with iodoacetamide, and electroeluted from SDS-polyacrylamide gels (38, 39).

RESULTS

Characterization of the Glycoprotein Ib . I X Complex on So- dium Dodecyl Sulfate-Polyacrylamide Gels-The glycoprotein complex used in these studies was extracted from membranes by two methods. In the first method, the glycoprotein was dissociated from the membrane skeleton by inclusion of N - ethylmaleimide in the extraction buffer (4). In the second method, inhibitors of the Ca"-dependent protease were omitted from the extraction buffer, thus inducing hydrolysis of actin-binding protein (the linkage protein between the glycoprotein complex and submembranous actin (12)) and re- leasing glycoprotein Ib from the membrane skeleton. The

The abbreviations used are: SDS, sodium dodecyl sulfate; &E,, dodecyloctaoxyethylene glycol monoether.

4884 Structure of the Platelet Membram Glycoprotein Ib - IX Complex

polypeptide content of the purified glycoprotein complex prepared by the two methods was similar. Fig. 1 shows SDS- polyacrylamide gels of the glycoprotein Ib - IX complex purified in the presence of N-ethylmaleimide. Densitometry of Coomassie Brilliant Blue-stained gels (Fig. 1, left lane) showed that glycoprotein Ib,, glycoprotein Ibd, and glycoprotein IX constituted 100% of the detectable protein. Immunoblots showed that actin-binding protein and its hydrolytic fragments were absent from the glycoprotein preparation (data not shown), supporting the conclusion that glycoprotein Ib. IX had been purified free of components of the membrane skeleton.

Although the glycoprotein complex appeared homogeneous on Coomassie Brilliant Blue-stained gels, we wished to deter- mine whether any minor components were present. The gly-

GP Ib,-

GP Ibp- GP IX- z2r

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gels of purified glycoprotein (GP) Ib-IX. Purified glycoprotein was solubilized in an SDS-containing buffer in the presence of reducing agent either before (left and middle lanes) or after (right lane) radiolabeling of the protein by the Iodo-bead method (25). Polypeptides were detected by Coomassie Brilliant Blue staining (left lane), silver staining (middle lane), or autoradiography of the gels (right lane). 150K, IOOK, and 45K, polypeptides of apparent M, = 150,000,100,000, and 45,000, respectively.

coprotein preparation was therefore examined by the silver- staining method of Morrissey (33) (Fig. 1, middle lane), the silver-staining method of Wray et al. (34) (data not shown), and by autoradiography of SDS-polyacrylamide gels of radiolabeled glycoprotein preparations (Fig. 1, right lane). Four

t

1 2 FIG. 3. Western blots of purified glycoprotein (GP) lb*IX

obtained with a monoclonal antibody against glycoprotein Ibm. Purified glycoprotein Ib-IX was solubilized in an SDS-containing buffer in the presence of reducing agent. Samples were electrophoresed on SDS-polyacrylamide gels containing 5-20% acrylamide. Polypeptides reacting with WM-23, a monoclonal antibody against glycoprotein Ib, (lane 1 ), or with a control monoclonal antibody (lone Z ) , were detected by Western blotting. The bands indicated by 150K and IOOK are polypeptides that have migrated with apparent molecular weights of 150,000 and 100,000, respectively.

FIG. 2. Nonreduced-reduced two-dimensional gels of purified glycoprotein (GP) lb-IX. Purified glycoprotein was solubilized in an SDS-containing buffer in the absence of reducing agent either before (panel A ) or after incubation for 30 s with a-lytic protease (panel B ) or for 5 min with trypsin (panel C). Nonreduced samples were electrophoresed in the first dimension on SDS-polyacrylamide gels containing 5-20% acrylamide, reduced, and then electrophoresed in the second dimension, again on gels containing 5-20% acrylamide. Polypeptides were detected by silver staining. The dotted line represents the diagonal on which proteins fall if they migrate with the same apparent molecular weight in both the nonreduced and reduced direction; the line was determined by overlaying the two-dimensional gel with a gel containing total platelet protein that was electrophoresed simulta- neously. The broken-line ovals in panels B and C represent the position of the glycoproteins on the gel of unhydrolyzed glycoprotein Ib. IX complex. GC, glycocalicin (the hydrolytic product of glycoprotein %,lacking the cytoplasmic end of the molecule); MC, macroglycopeptide (the hydrolytic product of glycoprotein Ib, lacking both ends of the molecule); I50K, IOOK, and 45K represent polypeptides with apparent M, = 150,000, 100,000 and 45,000, respectively.

Structure of the Platelet Membrane Glycoprotein Ib. IX Complex 4885

minor components were detected. Analysis of these components on two-dimensional nonreduced-reduced gels identified three of them as hydrolytic fragments of glycoprotein Ib<,; these were glycocalicin and polypeptides of M , = 100,000 and 45,000. Glycocalicin was identified on the basis of its comi- gration with purified glycocalicin on two-dimensional nonreduced-reduced gels (Fig. 2 A ) and its reactivity with glycoprotein Ib,, antibodies on Western blots (data not shown). The M , = 45,000 polypeptide comigrated on two-dimensional nonreduced-reduced gels with the M , = 45,000 fragment cleaved from the external region of glycoprotein Ib,, during long incubations of the glycoprotein Ib. IX complex with trypsin (see Fig. 2C and Fig. 6) and was immunoprecipitated by five antibodies that react with epitopes on the external M, = 45,000 region of glycoprotein Ib., (AN51, Hipl, AK2, AP1, and SZ2) (40) (data not shown). The M , = 100,000 polypeptide migrated off the diagonal line and above glycoprotein Ibo (Fig. 2 A ) , suggesting that it is the M , = 100,000 fragment of glycoprotein Ib., that remains membrane-associated when the M , = 45,000 fragment is removed from the external end of the molecule. Like glycoprotein Ib,, the M , = 100,000 polypeptide stained with the periodic acid-Schiffs reagent (data not shown), reacted with WM-23 on Western blots (Fig. 3, lane 1 ), and was immunoprecipitated by AK3, AK1, SZ1, and FMC25 (four other monoclonal antibodies that react with epitopes on the membrane-insertion end of the glycoprotein Ib.IX complex) (40) (data not shown).

Although it is not known whether the M, = 100,000 fragment exists on platelets in the circulation, immunoblots with WM-23 revealed that it was present on suspensions of washed platelets (data not shown). The presence of this hydrolytic

fragment of glycoprotein Ib,, could explain the previous obser- vation that platelets isolated from patients with Bernard- Soulier syndrome lack a M, = 100,000 glycoprotein in addition to glycoprotein Ib, glycoprotein IX, and glycoprotein V (3).

The only other polypeptide present in the glycoprotein preparation was one of M , = 150,000. Its migration on two- dimensional nonreduced-reduced gels (Fig. 2 A ) , its retention on columns of wheat-germ lectin, and its isoelectric point (data not shown) were consistent with it being glycoprotein Ia (2, 23). However, as shown in Fig. 3, the M , = 150,000 polypeptide reacted with a monoclonal antibody against glycoprotein Ib,, ( l a n e 1) but not with a control antibody ( l a n e 2).

We conclude that the purified glycoprotein used in these studies was essentially homogeneous and free of components of the membrane skeleton. The contaminants present represented small amounts of hydrolytic fragments of glycoprotein Ib., and a very minor component that appeared to be immu- nologically related to glycoprotein Ib,.

Rotary-shadowed Images of the Glycoprotein Ib-IX Com- plex-Fig. 4A shows several examples of the glycoprotein Ib. IX complex as visualized by low-angle rotary shadowing. The complex appeared as a flexible rod with a globular domain on either end. The overall length of the complex was -59.5 nm. The globular domains had mean diameters of 15.9 f 1.8 nm (mean & S.D., n = 363) and 8.9 & 1.1 nm (mean f S.D., n = 363), while the rod-shaped domain connecting them had a length of -34.7 nm and a width of -6 nm. In some cases, the rod-shaped domain appeared to have substructure; it is con- ceivable that this is related to the presence of carbohydrate moieties, known to be enriched in the macroglycopeptide

FIG. 4. Electron micrographs of rotary-shadowed glycoprotein (GP) Ib-IX complexes. Purified glycoprotein was prepared in a buffer containing 0.1% Triton X-100 (panel A ) or in a buffer containing 0.5 mg of the detergent Cl2E8/ml instead of Triton X-100 (panels E and C). Panels A and E show purified glycoprotein Ib.IX before and panel C shows the glycoprotein after removal of the external amino terminus of glycoprotein Ib, by exposure of the complex to a-lytic protease for 30 s. In the presence of Triton X-100, the complex appears elongated and flexible, with a small globular domain at one end and a slightly larger globular domain at the other end (panel A, with selected examples of glycoprotein Ib-IX complexes and interpretive drawings of the micrographs beneath). In the absence of Triton X-100, glycoprotein Ib.IX complexes appear to self-associate through the larger globular domains (panel B, with selected examples of the complexes and interpretive drawings beneath). These domains form a central globule in the multimer from which the rod-like domains of the monomers extend outward in a radial fashion, terminating in the smaller globular domain. After hydrolysis with a-lytic protease, individual glycoprotein Ib.IX complexes still appear as flexible rods with a large globular domain on the end, through which self-association occurs. The smaller globular domain on the other end, however, appears to be decreased in size or absent (panel C, with selected examples of the complexes and interpretive drawings beneath). The arrows labeled I, 2,3, and 4 indicate monomers, dimers, trimers, and quatramers, respectively. The asterisk denotes a molecule that lacks one small globular domain.

4886 Structure of the Platelet Membrane Glycoprotein I b IX Complex

portion of glycoprotein Ib, (14,16,20). Some of the glycoprotein complexes lacked one or both of the globular domains, a finding consistent with there being a small amount of the hydrolytic products of glycoprotein Ib,, in the glycoprotein preparation. One such molecule is labeled with an asterisk in Fig. 4B.

To define the hydrophobic domain of the glycoprotein Ib- IX complex, purified complex was diluted until the Triton X- 100 was below its critical micellar concentration. Dilution was performed either with buffer lacking detergent or buffer containing the nonionic detergent CIPER. The latter detergent is milder than Triton X-100 in terms of its ability to dissociate interacting membrane proteins (41). Similar images were obtained under the two conditions. Fig. 4B shows that in the absence of Triton X-100, the glycoprotein Ib IX complex self- associated through the larger of the two globular domains. Dimers and higher-order aggregates appeared to have a roughly globular region from which the rod-like domains extended radially (like the spokes of a wheel), terminating in the smaller domain. These results suggest that the larger domain contains the hydrophobic membrane-insertion site and that the smaller domain represents the external region of the molecule.

Structure of the Glycoprotein Zb-ZX Complex Lacking the External Domain of the Glycoprotein Zb, Molecule-The rotary-shadowed images in Fig. 4, A and B, suggest that the glycoprotein Ib. IX complex has the structure illustrated sche- matically in Fig. 5. To test the hypothesis that the larger globular domain contains the hydrophobic membrane-insertion end of the molecule, while the smaller globular domain represents the external region of the molecule, the glycoprotein Ib. IX complex was subjected to limited proteolysis with a-lytic protease or trypsin. As shown in Fig. 2B, and sche- matically in Fig. 5, a-lytic protease hydrolyzed both intact glycoprotein Ib, and glycocalicin (which lacks the membrane- insertion region of glycoprotein Ib,). The molecular weight of

NH,- Terminus External

n t .-Lytic \

Protease-

GPlbm-

Ca'-'Dependent Protease Serratia marcescens -

TrypSin (early)

Attas M emt

c hm

FIG. 5. Schematic representation of the glycoprotein (GP) Ib*IX complex and its hydrolytic fragments. The vertical arrows indicate dimensions of glycoprotein Ib, and its hydrolytic fragments. The arrows on the left indicate the regions of the molecule a t which the proteases act. GC, glycocalicin; MG, macroglycopeptide; 145K, 120K, and lOOK indicate the apparent molecular weights of the polypeptides (145,000, 120,000, and 100,000, respectively) as determined on SDS-polyacrylamide gels under reduced conditions. S-S, disulfide bond.

both polypeptides was reduced by approximately 20,000. In contrast, the M, = 100,000 fragment of glycoprotein Ib,, (which already lacks the external portion of the molecule) was not hydrolyzed. These results show that a-lytic protease removes the external region of glycoprotein Ib,,. This conclusion was supported by the finding that glycoprotein Ib, and glycocalicin (which both contain the external portion of the molecule) had the same amino-terminal sequence (His-Pro- Ile-Cys-Glu-Val-Ser-Lys-Val-Ala) (initial yield >40% and re- petitive yield -90%). This sequence includes the amino-terminal sequence reported recently by Handa et al. (22) and is identical to the sequence predicted from cDNA clones of glycoprotein Ib, (14). In contrast, the M, = 120,000 fragment did not have this sequence. Several amino-terminal sequences were obtained for this fragment, suggesting that a-lytic protease cleaves glycoprotein Ib, at several sites close to the external amino-terminal end of the molecule.

Rotary shadowing showed that glycoprotein Ib. IX complex that had been exposed to a-lytic protease retained the larger globular domain and maintained the ability to self-associate (Fig. 4C). However, 58% of the molecules now completely lacked the smaller globular domain. Although the remainder of the molecules retained a smaller globular domain, the average size of these domains had decreased from 8.9 f 1.1 nm (mean f S.D., n = 579) before proteolysis to 8.3 & 1.4 nm (mean * S.D., n = 386) after proteolysis. This decrease in the mean size of the smaller domain was significantly different a t a level of p c 0.001.

Structure of Glycoprotein Zb . ZX Complex Lacking the Mem- brane-insertion End-To confirm that the larger globular domain contained the membrane-insertion region of glycoprotein Ib- IX, the structure of glycoprotein Ib. IX complex from which the membrane-insertion site had been removed was studied. As shown in Fig. 5, trypsin hydrolyzes glycoprotein Ib, close to the membrane-insertion end of the molecule. Hydrolysis at this site generated glycocalicin (Fig. 2C). The

GP Iba

lOOK

GP I b GP IX

A. B.

-MG-

-45K- - ..e -25K- -GP IbB-

fragment i* .'* w.-

1 2 3 4 1 2 3 4

FIG. 6. Sodium dodecyl sulfate-polyacrylamide gel showing hydrolysis of the glycoprotein (GP) Ib-IX complex by trypsin. Purified glycoprotein Ib.IX was solubilized in an SDS-containing buffer either immediately before (lane 1 ) or after incubation with 10 pg of trypsin/ml for 30 s (lane 2), 60 8 (lane 3), or 5 min (lane 4) . Samples were electrophoresed in the presence (panel A ) or absence (panel B ) of reducing agent on an SDS-polyacrylamide gel containing 5-20% acrylamide. Polypeptides were detected by silver staining. GC, glycocalicin (the hydrolytic product lacking the membrane-insertion end of glycoprotein IbJ; MG, macroglycopeptide (the hydrolytic product lacking both ends of glycoprotein IbJ; the bands indicated by IOOK, 45K, and 25K are polypeptides that have migrated with apparent molecular weights of 100,000, 45,000, and 25,000 respectively.

Structure of the Platelet Membrane Glycoprotein Ib - IX Complex 4887

small amount of M , = 100,000 fragment of glycoprotein Ib, (which already lacks the external region of the molecule) that was present in the glycoprotein preparation was hydrolyzed, generating macroglycopeptide (see Figs. 2C, 5, and 6). Both products migrated on the diagonal line of two-dimensional nonreduced-reduced gels, indicating that they were no longer disulfide-linked to glycoprotein Ib,,(Fig. 2C). Although trypsin can convert glycocalicin to macroglycopeptide and the M , = 45,000 fragment if hydrolysis continues long enough (see Figs. 5 and 6), the major hydrolytic product at the times used for rotary shadowing was glycocalicin (Fig. 2C). As reported previously by others (16, 17), glycoprotein Ib,, was also hydrolyzed (Fig. 6A). The M , = 22,000 product of hydrolysis of this glycoprotein remained disulfide-linked to the M , = 25,000 membrane-insertion end of the hydrolyzed glycoprotein Ib,, as indicated by the migration of glycoprotein Ib,, with a M , = 22,000 on reduced gels (Figs. 2C and 6A) but one of approximately M, = 45,000 on nonreduced gels (Figs. 2C and 6B).

The products of hydrolysis of glycoprotein Ib. IX by trypsin were separated on a column of wheat-germ lectin. As shown in the insert to Fig. 7A, the column flow-through was devoid of the high molecular weight fragment of glycoprotein Ib,. The two low molecular weight bands that were detected comprised the M , = 25,000 membrane-insertion end of glycoprotein Ib,, the M , = 22,000 fragment of glycoprotein Ib,,, and glycoprotein IX (MI = 22,000). Rotary-shadowed images of this material showed that it was globular (Fig. 7A) and comparable in size to the larger globular domains (monomers and higher-order aggregates) of the intact glycoprotein Ib - IX molecule (compare Fig. 7A with Fig. 4). The material that was retained by the column contained primarily glycocalicin (see inset to Fig. 7B). Images of this material showed that it was

an elongated molecule with a small globular domain on one end (Fig. 7B). These elongated molecules represented 88% of the structures seen ( n = 850). Approximately 12% of the structures had a globular morphology; we assume that these globular structures represent some membrane-insertion ends of the glycoprotein Ib . IX complex that bound to the column but were below the limit of detection on the SDS-polyacrylamide gels. The results shown in Fig. 7 suggest that glycoprotein Ib.IX complexes that lack the membrane-insertion region do not have the larger globular domain but retain the smaller one. Similar results were obtained when the membrane-insertion end of the complex was removed by S. m r - cescens (data not shown) or when glycocalicin was generated from intact platelets (Fig. 8).

Physical Properties of the Glycoprotein Ib. IX Compkx- The apparent sedimentation coefficient (s~~,,.,) of the glycoprotein Ib. IX complex was 6.1 S, while that of purified glycocalicin was 5.5 S (Fig. 9A).

The apparent Stokes radius of glycocalicin was 78 A. The Stokes radius of the purified glycoprotein Ib. IX complex was estimated to be 86 8, since it eluted slightly ahead of, but very close to, the largest standard protein. A frictional ratio of 2.3 was calculated from the sedimentation coefficient and Stokes radius of glycocalicin (using a partial specific volume of 0.66 based on the known amino acid composition and carbohydrate content of glycocalicin (42)). This is in agreement with previously published data obtained by analytical ultracentrifugation (42, 43). The hydrodynamic parameters of the glycoprotein Ib. IX complex are also suggestive of a highly asymmetrical molecule, although precise values of the frictional ratio could not be calculated because the amount of bound detergent was not known.

FIG. 7. Structure and composition of the products of hydrolysis of glycoprotein Ib-IX by trypsin. Purified glycoprotein Ib. IX was incubated for 5 min with trypsin. The incubation was terminated by addition of phenylmethylsulfonyl fluoride to a final concentration of 1 mM and the sample applied to a column of wheat-germ lectin. The flow-through (panel A ) or the material that bound to the column and was subsequently eluted with N - acetyl-glucosamine ( p a n e l E ) was solubilized in SDS-containing buffer in the presence of reducing agent or was prepared for rotary shadowing in a buffer containing C12ER. The gel shown as an inset in panel A demonstrates that the flow-through from the wheat-germ column contained the membrane attachment end of the glycoprotein Ib. IX molecule. In panel A, the rotary-shadowed images (shown also in selected examples and interpretive drawings beneath) demonstrate that this material had a globular morphology. The material that bound to the column contained the glycocalicin portion of glycoprotein Ib, (inset in panel E ) . This material appeared as flexible rods with a small globular domain on one end. These rods (also shown as selected examples with interpretive drawings beneath) demonstrated no tendency to self-associate.

4888 Structure of the Platelet Membrane Glycoprotein Ib . IX Complex

FIG. 8. Electron micrograph of rotary-shadowed glycocalicin. Glycocalicin, prepared by exposure of intact platelets to Ca'+- dependent protease, was dialyzed into 100 mM ammonium acetate and examined by rotary shadowing. The molecules appear as rod-like structures that lack the larger globular domain but contain the smaller one. These molecules (also shown as selected examples with interpretive drawings beneath) demonstrated no tendency to self-associate.

DISCUSSION

There is considerable evidence that glycoprotein Ib is complexed 1:l with glycoprotein IX in the platelet membrane (3, 444). Until recently, intact glycoprotein Ib. IX complex could not be purified in a form that was free of membrane skeletal proteins and thus amenable to structural examination. In the present study, we have purified glycoprotein Ib in a form that remained associated with glycoprotein IX and appeared homogeneous on Coomassie Brilliant Blue-stained SDS-gels. Glycoprotein Ib-remained predominantly intact. The absence of actin-binding protein (the linkage protein between the glycoprotein complex and the membrane skeleton) indicated that the glycoprotein complex had been isolated free of membrane skeleton components. In support of this conclusion, the minor contaminants that were detected on silver-stained SDS-gels could be accounted for as being structurally related to glycoprotein Ib,.

Rotary shadowing of the purified glycoprotein complex revealed that glycoprotein Ib - IX existed as a flexible rod with a small globular domain on one end and a larger globular domain on the other end. The molecule was -59.5 nm long; the smaller globular domain had a diameter of -8.9 nm and the larger a diameter of -15.9 nm. Glycocalicin also appeared as a highly asymmetrical rod-shaped molecule. In a prelimi- nary report, Lawler et al. (43) have described a similar structure for glycocalicin. The high (2.3) frictional ratio of glycocalicin obtained in the present study is the same as that which can be calculated from previously reported (42,43) analytical ultracentrifugation results (sedimentation equilibrium and sedimentation velocity) and is consistent with glycocalicin

A. 15 -

s tn g 10-

5 -

I I

5 10 15 20 25 30 Fraction Number

2o t I I I I I

.1 .2 .3 .4 KO

FIG. 9. Determination of hydrodynamic parameters of glycoprotein (GP) Ib-IX. Purified glycoprotein Ib. IX or glycocalicin (GC) was sedimented through linear sucrose gradients (panel A ) or applied to a column of Sephacryl S-300 (panel B ) . The sucrose gradients were calibrated with the following proteins, represented by the filled circles: 1 , catalase, sm.,,.= 11.3 S; 2, y-globulin, sm.,,.= 7.12 S; 3, lactoperoxidase, sz0.* = 5.37 S; 4, bovine serum albumin, s20.v = 4.6 S; 5, ovalbumin, sm.,,,= 3.66 S. The gel filtration column was calibrated with the following proteins, represented by the fil+d circles: 1, thy- roglobulin, R, = 86 A; 2, @-galactosidase, R, = 69.A; 3, ferritin, R, = 63 A; 4, catalase R, = 52 A; 5, aldolase, R. = 46 A; 6, transferrin, R, = 36 A. R,, Stokes radius; &, partition coefficient; GC, glycocalicin.

being an asymmetrical molecule. The hydrodynamic parameters of the purified glycoprotein Ib. IX complex were indic- ative of the fact that the glycoprotein Ib. IX complex was also an asymmetrical molecule. We conclude from these results that the glycoprotein Ib IX complex is a highly asymmetrical molecule whose elongated portion is made up of the glycocalicin portion of glycoprotein Ib,.

The use of proteolytic enzymes enabled us to characterize the globular domains of the glycoprotein Ib IX complex. The larger globular domain was identified as the one containing the hydrophobic region of the complex that inserts into the membrane. This interpretation was suggested by the tendency of this domain to self-associate in aqueous solution and was confirmed in experiments in which removal of the membrane- insertion end of the glycoprotein Ib. IX complex resulted in loss of the larger domain and in the inability of the remaining portion of the molecule to self-associate. The size and hydrophobic characteristics of the larger globular domain are consistent with the biochemical demonstration that this domain contains three polypeptides of approximate M, = 20,000. Two of these three polypeptides (glycoprotein Ibo (45) and the membrane-insertion end of glycoprotein Ib., (14, 21)) are as- sumed to possess hydrophobic transmembrane domains and cytoplasmic extensions.

The smaller globular domain was identified as the end of the glycoprotein Ib, molecule that is external to the plasma membrane. This identification was based on the modification of the smaller domain by a-lytic protease, an enzyme that

Structure of the Platelet Membrane Glycoprotein Ib. I X Complex 4889

hydrolyzed glycoprotein Ib, at its external end. I t is well known that glycoprotein Ib,, has a protease-sensitive region at the external end of the molecule (Fig. 5) . Hydrolysis of the molecule at this site by enzymes, such as trypsin and elastase, removes amino-terminal fragments of approximate M, = 45,000 that have been implicated as containing the binding sites for both thrombin and von Willebrand factor (16, 22). The hydrolysis of glycoprotein Ib,, by a-lytic protease has not previously been reported. Since a-lytic protease cleaves glycoprotein Ib,, closer to the amino terminus of the molecule than the previously described proteases, this enzyme may prove useful in further characterizing the functional domains of glycoprotein Ib,.

We have previously reported that glycoprotein Ia coisolates in the membrane skeleton with glycoprotein Ib,, and is copre- cipitated with glycoprotein Ib,, from platelet lysates incubated with actin-binding protein or glycoprotein Ib, antibodies (1 1, 12). We have interpreted these results as showing that glycoprotein Ia is complexed with glycoprotein Ib.IX or, like glycoprotein Ib. IX, is associated with actin-binding protein. The M , = 150,000 polypeptide that copurified with glycoprotein Ib. IX complex on WM-23 in the present study had characteristics similar to those of glycoprotein Ia (molecular weight, retention on wheat-germ lectin columns, and isoelectric point); however, it reacted with WM-23 on Western blots. While this finding suggests that there is a relationship between glycoprotein Ia and glycoprotein Ib,, the possibility that WM-23 is directed against common carbohydrate moieties cannot be excluded. Nieuwenhuis et al. have provided evidence that glycoprotein Ia may be the receptor for collagen (46). It is of interest in this regard that a monoclonal antibody against glycoprotein Ib, has recently been shown to inhibit the response of platelets to collagen (47), supporting the idea that glycoprotein Ia and the glycoprotein Ib . IX complex may be physically close or structurally related.

The present study describes the structure of the glycoprotein Ib. IX complex from platelet membranes. The other major glycoprotein complex in platelet membranes, the glycoprotein IIb. IIIa complex, is 20-30 nm long (48). The greater length of the glycoprotein Ib a IX complex (-60 nm) suggests that a considerable amount of the glycocalyx seen on the outside of platelet plasma membranes in electron-microscope images may represent the glycoprotein Ib. IX complex. The length of the glycoprotein Ib,. molecule would orient it so that the amino-terminal end, which contains the binding site for von Willebrand factor (16, 22), would be ideally situated to make the initial contact with von Willebrand factor on exposed subendothelium. Similarly, the attachment of the other end of the glycoprotein Ib . IX complex to the submembranous membrane skeleton would provide an anchor for the molecule, which presumably must withstand enormous shear forces as blood flows past adherent platelets in the circulation.

The glycoprotein Ib. IX complexes used in the present study were extracted from platelet membranes by two different methods. In one method N-ethylmaleimide was included in the extraction buffer as an inhibitor of the Ca2'-dependent protease. The finding that N-ethylmaleimide dissociated the glycoprotein Ib.IX complex from the membrane skeleton indicates that an essential sulfhydryl group may be involved in the interaction between the glycoprotein complex and actin-binding protein. While the nature of the sulfhydryl group is not known, a potential candidate is the single sulfhydryl known to be on the cytoplasmic domain of glycoprotein Ib, (45). The second method used to dissociate glycoprotein Ib.IX from the membrane skeleton was to induce hydrolysis of actin-binding protein by the Ca"-dependent protease. As reported previously (12), the degradation products of actin-

binding protein did not remain associated with the glycoprotein Ib. IX complex, as indicated by their failure to coisolate with the glycoprotein Ib. IX complex. Although considerable evidence implicates the cytoplasmic domain of the gIycopro- tein Ib. IX complex as the binding site for the glycoprotein complex to actin-binding protein on the membrane skeleton, the details of this interaction have not yet been established. The availability of glycoprotein IbeIX complex isolated free of membrane-skeletal components should prove useful in establishing the molecular details of the interaction between glycoprotein Ib. IX and the membrane skeleton.

Acknowledgments-We thank Dr. Stanley Rall, Jr., for determining amino acid sequences, Clifford Reynolds, Pamela Steffen, and Cheryl Gregory for technical assistance, James X. Warger, Norma Jean Gargasz, and Sandra Barlow for graphics, A1 Averbach and Sally Gullatt Seehafer for editorial assistance, and Kate Sholly and Michele Prator for manuscript preparation.

REFERENCES

1. Coller, B. S., Peerschke, E. I., Scudder, L. E., and Sullivan, C. A.

2. Clemetson, K. J. (1985) in Platelet Membrane Glycoproteins (George, J. N., Nurden, A. T., and Phillips, D. R., eds) pp. 51- 85, Plenum Press, New York

3. Berndt, M. C., Gregory, C., Chong, B. H., Zola, H., and Castaldi,

4. Berndt, M. C., Gregory, C., Kabral, A., Zola, H., Fournier, D., and Castaldi, P. A. (1985) Eur. J . Biochem. 151 , 637-649

5. George, J . N., Nurden, A. T., and Phillips, D. R. (1984) N . Engl. J. Med. 311 , 1084-1098

6. Berndt, M. C., and Caen, J. P. (1984) in Progress in Hemostasis and Thrombosis (Spaet, T. H., ed) Vol. 7, pp. 111-150, Grune & Stratton, Orlando

7. Berndt, M. C., Chong, B. H., Bull, H. A., Zola, H., and Castaldi, P. A. (1985) Blood 6 6 , 1292-1301

8. Harmon, J. T., and Jamieson. G. A. (1986) J. Biol. Chem. 2 6 1 ,

(1983) Blood 6 1 , 99-110

P. A. (1983) Blood 6 2 , 800-807

15928115933 . ,

9. De Marco, L., and Shapiro, S. S. (1981) J. Clin. Invest. 6 8 , 321- 328

J. Clin. Invest. 75 , 1198-1203 10. De Marco, L., Girolami, A., Russell, S., and Ruggeri, 2. M. (1985)

11. Fox, J . E. B. (1985) J. Clin. Invest. 7 6 , 1673-1683 12. Fox, J. E. B. (1885) J. Biol. Chem. 260 , 11970-11977 13. Fox, J. E. B., and Boyles, J. K. (1985) Blood 6 6 , 304a 14. Lopez, J. A., Chung, D. W., Kujikawa, K., Hagen, F. S., Papay-

annopoulou, T., and Roth, G. J. (1987) Proc. Natl. Acad. Sci.

15. Titani, K., Takio, K., Handa, M., and Ruggeri, Z. M. (1987) Proc.

16. Wicki, A. N., and Clemetson, K. J. (1985) Eur. J. Biochem. 153,

17. Berndt, M. D., Gregory, C., Dowden, G., and Castaldi, P. A.

18. Yoshida, N., Weksler, B., and Nachman, R. (1983) J. Biol. Chem.

19. McGowan, E. B., and Detwiler, T. C. (1985) Blood 65,1033-1035 20. Okumura, T., Lombart, C., and Jamieson, G. A. (1976) J. Biol.

Chem. 261,5950-5955 21. Wicki, A. N., and Clemetson, K. J. (1987) Eur. J. Biochem. 163 ,

43-50 22. Handa, M., Titani, K., Holland, L. Z., Roberts, J. R., and Ruggeri,

2. M. (1986) J. Biol. Chem. 2 6 1 , 12579-12585 23. Clemetson, K. J., Pfueller, S. L., Luscher, E. F., and Jenkins, C.

S. P. (1977) Biochim. Biophys. Acta 464,493-508 24. Solum, N. O., Olsen, T. M., Gogstad, G. O., Hagen, I., and

Brosstad, F. (1983) Biochim. Biophys. Acta 729,53-61 25. Markwell, M. A. K. (1982) Anal. Biochem. 125,427-432 26. Hunkapiller, M. W., Smallcombe, S. H., Whitaker, D. R., and

27. Shotton, D. M., Burke, B. E., and Branton, D. (1979) J. Mol.

28. Tyler, J . M., and Branton, D. (1980) J. Ultrastr. Res. 71 , 95-102 29. Mould, A. P., Holmes, D. F., Kadler, K. E., and Chapman, J. A.

30. Slayter, H. S. (1976) Ultramicroscopy 1, 341-357

U. S. A. 84.5615-5619

Natl. Acad. Sci. U. S. A . 8 4 , 5610-5614

1-11

(1986) Ann. N . Y. Acad. Sci. 4 8 5 , 374-386

2 5 8 , 7168-7174

Richards, J . H. (1973) Biochemistry 12,4732-4743

Biol. 1 3 1,303-329

(1985) J. Ultrastr. Res. 9 1 , 66-76

4890 Structure of the Platelet Membrane Glycoprotein I b . I X Complex

31. Laemmli, U. K. (1970) Nature 227,680-685 32. Phillips, D. R., and Agin, P. P. (1977) J. Biol. Chem. 252, 2121-

33. Morrissey, J. H. (1981) Anal. Biochem. 117,307-310 34. Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. (1981)

35. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad.

36. Ackers, G. K. (1967) J. Biol. Chern. 242,3237-3238 37. Jennings, L. K., and Phillips, D. R. (1982) J. Biol. Chem. 257,

38. Stephens, R. E. (1975) Anal. Biochem. 65, 369-379 39. Weisgraber, K. H., Rall, S. C., Jr., and Mahley, R. W. (1981) J.

40. Du, X., Beutler, L., Booth, W. J., Castaldi, P. A., and Berndt, M.

2126

Anal. Biochem. 118,197-203

Sci. U. S. A. 76,4350-4354

10458-10466

Biol. Chem. 256,9077-9083

C. (1987) A u t . N. Z. J. Med. 1 7 , 146 (abstr.)

41. Houssin, C., Le Maire, M., Aggerbeck, L. P., and Shechter, E.

42. Carnahan, G. E., and Cunningham, L. W. (1983) Biochemistry

43. Lawler, J. W., Margossian, S., and Slayter, H. S. (1980) Fed.

44. Du, X., Beutler, L., Ruan, C., Castaldi, P. A., and Berndt, M. C.

45. Kalomiris, E. L., and Coller, B. S. (1985) Biochemistry 24, 5430-

46. Nieuwenhuis, H. K., Akkerman, J. W. N., Houdijk, W. P. M.,

47. Ruan, C., Du, X., Xi, X., Castaldi, P. A., and Berndt, M. C. (1987)

48. Carrell, N. A., Fitzgerald, L. A., Steiner, B., Erikson, H. P., and

(1985) Arch Biochem. Biophys. 240, 593-606

22,5384-5389

Proc. 39, 1895 (abstr.)

(1987) Blood 69, 1524-1527

5436

and Sixma, J. J. (1985) Nature 318,470-472

Blood 69,570-577

Phillips, D. R. (1985) J. Biol. Chem. 260, 1743-1749

of biological. chemistry no. 5, 4882-4890,1988 by ... · plasma membrane has been termed the...

Documents