Vol.

THE JOIJRNAL OP BIOLOC~CAL CHEMISTW 352, No. 6, Issue of March 25, pp. 2121-2126, Printed in U.S.A.

1977

Platelet
EVIDENCE REDUCED

Plasma

Membrane

Glycoproteins
DISULFIDE BONDS USING NONREDUCED-

FOR THE PRESENCE TWO-DIMENSIONAL

OF NONEQUIVALENT GEL ELECTROPHORESIS

(Received

for publication,

April

30, 1976, and in revised form, September

14, 1976)

DAVID From 38101

R. PHILLIPS$ the Department

AND

PATRICIA

POH

AGIN

of Biochemistry,

St. Jude

Childrens

Research

Hospital,

Memphis,

Tennessee

Proteins on the surface of human platelets were labeled by the lactoperoxidase-iodination procedure. The polypeptides were separated by a two-dimensional sodium dodecyl sulfate electrophoretic technique in which the solubilized platelet polypeptides were electrophoresed first with disulfide bonds intact followed by electrophoresis in the second dimension after disulfide reduction. To obtain reproducible nonreduced patterns, it was essential to prevent oxidation before separation in the first dimension either by solubilizing the platelets in anaerobic solutions or by blocking native sulfhydry1 groups with N-ethylmaleimide. Some platelet disulfides required a high concentration of reducing agent (5% dithiothreitol) to achieve complete reduction. Analysis of the twodimensional separation of iodinated platelet proteins revealed that seven polypeptides with molecular weights from 90,000 to 160,000 were iodinated. The labeled components were designated Ia, Ib, Ic, IIa, IIb, III, and IV; all but Ic were stained by the periodic acid-Schiff reaction. Three of these, Ib, Ic, and IIb, were shown to be attached to nonidentical subunits by disulfide linkages. Four components, Ia, IIa, III, and the larger subunit of IIb, gave evidence for intramolecular disulfide bonds as well. Nonequivalence of these various bonds in the sodium dodecyl sulfate-denatured state was evident, since intermediate states of reduction were readily obtained. The order or reduction was Ia = Ib = IIa = IIb (intersubunit disulfide) > III (first reduction) > IIb (intrasubunit disulfide) > III (second reduction). Thus, contrary to the generalization that disulfide cross-linking is unimportant in membrane structure, many of the platelet membrane polypeptides contain disulfides as an integral part of their structure.

Platelets then aggregate, provide a surface to enhance coagulation, and they retract the ensuing clot. Membrane glycoproteins may serve as specific receptors for some of these reactions, e.g. adhesion and aggregation (3-6). It appears that a thorough understanding of these hemostatic processes depends upon a characterization of the surface glycoproteins. Recently, confusion has developed concerning the number of platelet membrane glycoproteins and their apparent molecular weights, and a consistent nomenclature has not been developed (3, 5, 7-11). Perhaps this is because the separation techniques that have been used were inadequate to resolve all the molecular species. An example comes from the study of the inherited bleeding disorder Glanzmanns thrombasthenia. Platelets from patients with this disorder had a decreased concentration of a glycoprotein which was labeled IIb (5). One of the other glycoproteins, III, also appeared to be affected, since it was much less iodinated by lactoperoxidase than in normal platelets (5). With techniques previously used, it was impossible to determine whether the decreased iodination was due to the absence of this molecule or to a genetically controlled alteration of its properties. We now report an improved method for separation of the platelet membrane glycoproteins. This method is based on the observation that several of these glycoproteins contain disulfide bonds. We have found that some of the glycoproteins, including IIb, which is missing in thrombasthenia (5), were bound to small subunits by disulfide linkages. Other glycoproteins, including III, were clearly resolved and seemed to contain intrachain disulfides.
MATERIALS AND METHODS

Downloaded from www.jbc.org by guest, on March 14, 2013

Blood platelets are involved in several events in hemostasis, that is, the arrest of blood flow (for review, see Refs. 1 and 2). In the initial event, adhesion, platelets adhere to collagen and other elements of exposed subendothelial tissue. Adhering platelets then undergo a release reaction, whereby the contents of their storage organelles are extruded outside the cell. * This work was supported by the United States Public Health HL 15616 from the National Heart and Lung Institute.
Service Grant $ Recipient of a Research Career Development from the United States Public Health Service. Award (HL-0080)

Isolation of Platelets-For most experiments, 17.2 ml of blood, collected from adult donors, was mixed with 2.8 ml of acid citrate dextrose. The acid citrate dextrose solution contained 2.5 g of Na,citrate. 2H,O, 2 g of glucose, and 1.5 g of citric acid in 100 ml of H,O (12). The blood was centrifuged at 160 x g for 10 min at room temperature to obtain platelet-rich plasma. The platelets were then separated from plasma and washed as described by Massini and Liischer (13). The medium for the first two washing steps contained 0.12 M sodium chloride, 0.0129 M Na,,-citrate, and 0.03 M glucose. For the last step it contained 0.154 M sodium chloride, 0.01 M Tris, and 0.001 M EDTA, pH 7.4. The washed platelets were resuspended in this EDTA buffer at a concentration of 1 x lo9 platelets/ml. More than 99.9% of the cells in these samples were platelets, as determined with a phase-contrast microscope. Zodination of Platelets -One millicurie of lzaI (carrier-free, Schwarz) was added to 1 ml of a stirred, washed platelet suspension

2121

2122

Disulfides

in Platelet

Membrane

Glycoproteins
(23).

containing 1 x lo platelets (7). This was followed by 10 ~1 of 0.1 M sodium phosphate, pH 7.4, containing 2.5 nmol of lactoperoxidase. The iodination reaction was started by adding five aliquots (10 ~1 of each) of freshly prepared hydrogen peroxide solution (0.001 M in the EDTA buffer) at 10-s intervals, followed by 9 ml of a buffer containing 0.154 M sodium chloride, 0.01 M Tris, and 0.001 M EDTA, pH 7.4. The platelets were sedimented by centrifugation and washed once with 10 ml of the same buffer. After complete removal of buffer from the platelet pellet, the tube was flushed with N,. The platelets were rapidly suspended in 0.2 ml of deaerated water and solubilized immediately by adding 0.1 ml of deaerated 10% SDS. This mixture was then immersed in boiling water for 3 min to effect solubilization. The solubilization step and subsequent incubations were performed in a N, atmosphere. The solubilized platelets were sealed under N, in glass ampoules. Electrophoresis -Electrophoresis was performed according to the method of Laemmli (15). Except where indicated, samples were electrophoresed in a commercial slab apparatus (Bio-Rad model 220 dual vertical slab electrophoresis cells). The resolving gel contained 7.5% acrylamide, was 9 cm high, 16 cm wide, and 0.15 cm thick, overlaid with 1 cm of a stacking gel containing 3% acrylamide. The slot for each sample was 0.8 cm wide. Gels were electrophoresed at 25 V until the tracking dye reached the bottom of the gel (approximately 18 h). When reduced and nonreduced samples were run simultaneously, it was found essential to leave at least 1 cm between each sample, preventing any possible effect of the reducing agent on a neighboring nonreduced sample. For one-dimensional electrophoresis, 10 to 20 ~1 of the solubilized platelets containing 50 pg of protein were mixed with 50 ~1 of sample buffer containing various concentrations of reducing agents, depending on the amount of reduction desired. To separate nonreduced polypeptides, samples were incubated at 100 for 3 min with an anaerobic sample buffer free of reducing agents. To separate partially reduced polypeptides, the samples were heated at 100 for 3 min with buffer which contained either 0.1% or 1% P-mercaptoethanol. To obtain complete reduction, samples were heated at 100 for 10 min with a buffer which contained either 10% P-mercaptoethanol or 20% dithiothreitol. In the latter case, complete reduction was obtained even when heating was limited to 3 min. For nonreduced-reduced two-dimensional slab gels, the method of Wang and Richards (16), with slight modification, was employed. Approximately 50 ~1 of solubilized platelets containing 200 pg of protein were mixed with 50 ~1 of the sample buffer free of reducing agents and electrophoresed on a slab gel. After electrophoresis, the lane containing the resolved platelet polypeptides was cut out and incubated in 5 ml of the reducing sample buffer (containing 20% dithiothreitol) for 15 min at 60 to reduce the disulfide bonds in the platelet polypeptides. This strip of acrylamide was then rinsed twice with electrode buffer and placed on top of the stacking gel of a second slab. This sample was then electrophoresed for 90 min at 15 V and then at 25 V until the tracking dye reached the bottom of the gel. Gels were stained for protein with Coomassie brilliant blue by the method of Fairbanks et al. (17). Autoradiography -Stained gels of labeled samples were dried under vacuum and stored next to Kodak Royal X-O-Mat RP/R-54 x-ray film. After 1 to 5 days exposure the film was processed as suggested by the manufacturer. Molecular Weight Determinations -Apparent molecular weights of platelet polypeptides were determined by comparing their electrophoretic mobility with standard proteins, as previously reported (7). Molecular weight standards were: platelet myosin (200,000); /3-galactosidase (130,000), Worthington; bovine serum albumin (68,000), ovalbumin (43,000), and cytochrome c (12,400), Sigma; chymotrypsinogen A (25,700), Mann. Other Procedures - Lactoperoxidase (a gift from Dr. M. Morrison) was isolated by the procedure of Morrison and Hultquist (20) and platelet myosin as described by Pollard et al. (21). Protein concentrations were determined by the method of Lowry et al. (22) using MoniTrol (American Hospital Supply) as the standard. Sulfhydryl con The abbreviations used are: SDS, sodium dodecyl sulfate; DTNB, 5,5-dithiobis(2-nitrobenzoic acid); PAS, periodic acid-Schiff reagent; TSP, thrombin-sensitive protein, glycoprotein described by Baenziger et al. (14). 2 All molecular weights are presented as apparent since many of the components examined (a) are glycoproteins and (b) contain disulfide bonds. These two factors lead to errors in molecular weight determinations by SDS-electrophoresis (18, 19).

centration was determined using DTNB as reported by Ellman Plasma fibrinogen was supplied by A. B. Kabi, Stockholm.
RESULTS

of Zodinated Platelets b-y Nonreduced-Reduced Analysis Two-Dimensional Gels -Initially, analysis of platelet samples by electrophoresis in the absence of reducing agents led to complications since random polymerization of the solubilized proteins occurred. Although these polymers could be converted to monomers by agents which cleaved disulfide bonds, such preparations were not useful for obtaining reliable nonreduced patterns. Two procedures were employed to prevent sulfhydryl oxidation after platelet solubilization. In the first procedure, native sulfhydryl groups were blocked with N-ethylmaleimide before platelet disruption. Titration of SDS-solubilized platelets with 5,5-dithiobis(2-nitrobenzoic) acid (DTNB) showed 100 ? 10 nmol of sulfhydryl rng- of protein. Platelets were also incubated for 30 min at 37 with N-ethylmaleimide (which permeates plasma membranes) (24) at a concentration of 1 pmol/mg of protein. This concentration of N-ethylmaleimide blocked >98% of the native sulfhydryl groups as determined by DTNB titration of the platelets after solubilization. These solubilized platelets were resistant to sulfhydryl oxidation for several weeks. The second procedure used was solubilization of the platelets in an anaerobic detergent solution. It was found that polypeptides in this preparation, when stored at -20 sealed under nitrogen, remained monomeric for 2 weeks. This preparation yielded reproducible electrophoretic patterns which were identical with those from N-ethylmaleimidetreated material and to freshly solubilized platelets and appeared to reflect the inherent state of the polypeptides. All electrophoresis experiments reported herein, however, were performed immediately on samples which were solubilized in anaerobic solutions to minimize the effects of sample storage. Platelets were radiolabeled by the lactoperoxidase iodination technique and solubilized in SDS. In a previous study (7) it was shown that only membrane components were iodinated by this procedure. The solubilized platelets were then fractionated on a nonreduced-reduced two-dimensional slab (Fig. 1). In the first step of this procedure, the solubilized platelets were separated by electrophoresis on a 5% gel in the absence of reducing agents so that the disulfides remained intact. Separation in the second dimension was also on a 5% gel and was performed after the disulfides in the resolved polypeptides were cleaved with 20% dithiothreitol. Seven prominent labeled membrane components, Ia, Ib, Ic, IIa, IIb, III, and IV, were observed. Using this two-dimensional electrophoretic procedure, any protein which did not contain a disulfide bond would be on the diagonal line since its mobility would be the same in both directions. Any shift either above the line or below it, indicates that the polypeptide contained a disulfide bond. Six of the labeled membrane components were not on the diagonal, indicating that each contained at least one disulfide bond. Comparisons of the horizontal and vertical axes indicated that Ia, IIa, and III increased in apparent molecular weight after reduction, whereas the apparent molecular weights of Ib, Ic, and IIb decreased. Fig. 1B shows a two-dimensional slab which was stained for protein with Coomassie brilliant blue. Most of the platelet polypeptides fell on the diagonal line. We have concluded that most platelet polypeptides either did not contain disulfide bonds or, if they did, were not affected in their electrophoretic mobility by disulfide cleavage. A notable exception was the glycoprotein TSP which was identified and characterized by

Downloaded from www.jbc.org by guest, on March 14, 2013

Disulfides

in Platelet

Membrane

Glycoproteins

2123

FIG. 1. Nonreduced-reduced two-dimensional electrophoresis of iodinated platelets. Washed human platelets were iodinated by the lactoperoxidase iodination procedure and solubilized anaerobically as described under Materials and Methods. The sample was then either (I) reduced with 20% dithiothreiml and electrophoresed on the 5% polyacrylamide slab or (2) subjected to two-dimensional analysis. The nonreduced sample was electrophoresed in the first dimension. The first dimension separation gel was subjected to reducing conditions and electrophoresed in the second dimension. A, autoradiogram of the dried slab; B, Coomassie blue-stained slab. The dashed areas inB refer to the labeled components inA. The precise positions of the radioactive components were determined by superimposing the developed x-ray film over the stained, dried slab. The symbols refer to: M, platelet myosin;A, platelet actin; TSP, thrombin-sensitive protein (14).

Majerus and co-workers (14). Nom-educed TSP had an apparent molecular weight greater than nonreduced fibrinogen (too high to be estimated on these gels) whereas reduced TSP had an apparent molecular weight of 185,000. The positions of the labeled membrane components which were shown in Fig. 1 have been identified in this figure by the dashed circles. The protein staining of Ia and IIa could barely be detected and did not reproduce on the photograph: Ilb and III were detectably stained for protein while Ib was stained weakly. When platelet plasma membranes were electrophoresed on this two-dimensional gel and were stained for carbohydrate with periodic acid-Schiff reagent (not shown), Components lb, IIb, III, and IV stained predominantly. Ia and IIa were stained also, but weakly. Three of the iodinated components in Fig. 1 had a lower molecular weight after reduction. One possible explanation is that these components are composed of nonidentical subunits connected by a disulfide bond. To test this possibility, platelets

were electrophoresed in the second dimension in a higher concentration of acrylamide (10%) and examined for the presence of low molecular weight polypeptides (Fig. 2). This procedure gave a curved line of polypeptides because the low molecular weight polypeptides (smaller than actin, 45,000) migrated as a cohort close to the dye front in the first dimension, but were resolved by the higher per cent acrylamide in the second dimension. Several features were observed in the nonreduced-reduced gel in Fig. 2 which were not apparent in Fig. 1. First, the (Y, p, and y subunits of fibrinogen, which were disulfide-linked and remained near the top of nonreduced gels, dissociated after reduction and were clearly distinguished. Second, several iodinated components with apparent molecular weights less than actin were resolved. Third, this technique also showed the presence of three polypeptides below the glycoprotein region of the gel. The autoradiogram of the nonreduced-reduced gel (Fig. 2A) had two radioactive spots, one directly below Ic, and the other below Ilb. When the proteins in the nonreducedreduced gel were stained with Coomassie blue (Fig. 2B) two polypeptides, the one below IIb and the one below Ib were stained. We have designated these small molecular weight polypeptides the p chains of the corresponding molecules and we will refer to them as Ib& I@, and IIbbp, respectively. We designated the corresponding larger subunits 01 chains. Comparison of Fig. 2, A and B, revealed that lh/3 was stained by Coomassie blue but was not iodinated by lactoperoxidase on intact platelets, Ilbp was stained and was also labeled, while Icp was iodinated but was not stained. No staining of these polypeptides was obtained by the PAS procedure. The molecular weights of these polypeptides, determined by re-electrophoresis in a 10% gel after extraction from a nonreduced onedimensional gel, were 22,000, 27,000, and 23,000 for Ibp, I@, and Ilbp, respectively. Fig. 2, C and D, show that when platelets were reduced before the first dimensional electrophoresis, all polypeptides and labeled glycoproteins were located on the diagonal. Comparative Rates of Reduction of Platelet Polypeptides The results in Figs. 1 and 2 indicated that some of the surface glycoproteins as well as some of the other proteins (e.g. TSP and fibrinogen) contained disulfide bonds. These data also showed that the order of migration of the polypeptides in a one-dimensional gel of solubilized platelet polypeptides with disulfides intact would be different then that obtained after reduction. In order to determine the apparent molecular weights of the various labeled components as well as their relative susceptibilities to reducing agents, SDS-solubilized platelets were treated with different concentrations of reducing agents and electrophoresed. The time of incubation at 100 (up to 20 min in the absence of reducing agent) did not affect the electrophoretic separation (data not shown). The stained gels (Fig. 3A) showed that individual polypep;ides in SDSsolubilized platelets had different susceptibilities to reduction by P-mercaptoethanol. The nonreduced-reduced relationship shown in Figs. 1 and 2 provided a means of identification of individual bands in the one-dimensional analysis. In 0.1% pmercaptoethanol, fibrinogen was almost totally reduced while TSP was unaffected. This condition was also sufficient to affect the apparent molecular weight of two other polypeptides, which were subsequently identified as IIb and III. Under these conditions, the apparent molecular weight of IIb decreased (142,000 to 128,000) while that of III increased (99,000 to 110,000). At 1% /?-mercaptoethanol, two additional changes were observed. This concentration of reducing agent was suffl-

Downloaded from www.jbc.org by guest, on March 14, 2013

2124

Disulfides

in Platelet

Membrane

Glycoproteins

A--

IST NONREDUCED,

7+% NJ

c
d--

IsT REDUCED 73%

* h,

Downloaded from www.jbc.org by guest, on March 14, 2013

FIG. 2. Two-dimensional electrophoresis of iodinated platelets. Samples were electrophoresed on a 7.5% polyacrylamide slab with disulfides intact (nonreduced) in the first dimension. The gel strip was cut out, incubated with the reducing sample buffer to cleave disulfide bonds, and electrophoresed in the second dimension on a 10% slab. The gels were then stained with Coomassie brilliant blue and dried for autoradiography. A, autoradiograph of the nonreducedreduced gel; B, nonreduced-reduced slab stained for protein with

Coomassie brilliant blue; C, autoradiograph of the reduced-reduced gel; D, reduced-reduced gel stained for protein with Coomassie brilliant blue. A and C are autoradiographs of B and D, respectively. The symbols in the figure refer to: TSP, thrombin-sensitive protein; A, platelet actin; Fib, fibrinogen. The dotted circle in A is the position of IbP and the radioactive spot, IQ, is referred to in B by a dotted circle.

cient to reduce TSP and allowed it to dissociate into its subunits (apparent molecular weight of 185,000). Also, IIba! underwent a second reduction, which resulted in an apparent molecular weight increase to 132,000. Finally, a higher concentration of the reducing agent (10% P-mercaptoethanol, Gel d) resulted in a second change to the apparent molecular weight of III, a molecular weight increase to 114,000. No additional changes were observed with either a higher concentration of P-mercaptoethanol (20%), with 20% dithiothreitol, or with longer incubation times (20 min at 100). Iodinated platelets were also subjected to various reducing conditions, as shown in Fig. 3B. The apparent molecular weight decrease of IIb and the increase of III caused by 0.1% pmercaptoethanol which were seen in the stained gels were readily observed by the change in apparent molecular weight of these radioactive components. The concentration of /3-mercaptoethanol also caused the apparent molecular weight of Ia and IIa to increase, Ia increasing from 153,000 to 167,000 and IIa from 138,000 to 157,000. In addition, the apparent molecu-

lar weight of Ib was also affected by this concentration of reducing agent. In some experiments (not shown), conditions were such that the first molecular weight change of IIb had occurred completely while III was equally divided between the molecular weights of 99,000 and 110,000. This showed that the disulfides in IIb were more easily cleaved than those in III. The effects of additional reductions of IIb and III were also observed in the iodinated samples. All these changes in apparent molecular weights were also seen when the samples were reduced with 1 mM tetramethylammonium borohydride, indicating that the changes with the sulfhydryl reagents were due to cleavage of disulfide bonds and not to any abnormal reactions of the sulfhydryl reagents.
DISCUSSION

Previous studies on the characterization of the platelet membrane have identified either three or four glycoproteins in this membrane (3, 5, 7-11). In the present study we have achieved improved resolution by taking advantage of the ob-

Disulfides

in Platelet

Membrane

Glycoproteins

2125

Downloaded from www.jbc.org by guest, on March 14, 2013

FIG. 3. Electrophoresis of nonreduced, partially reduced, and reduced iodinated platelet polypeptides. Washed platelets were iodinated, solubilized anaerobically, and electrophoresed on 7.5% slab gel either nonreduced (a) or after reduction with 0.1% (bl, 1% (cl, or 10% (cl) pmercaptoethanol. Gels were stained for protein with Coomassie brilliant blue. A, Coomassie-stained gels; B, autoradiograms of these same gels. TSP, thrombin-sensitive protein; Fib, fibrinogen.

servation that the membrane glycoproteins contain disulfides. Because of this feature, the apparent molecular weights of the glycoproteins can be altered by reducing the disulfides. Thus, the use of nonreduced-reduced two-dimensional electrophoresis affords an improved separation. Using this procedure, we showed that the platelet plasma membrane contains at least six glycoproteins between the molecular weights of 90,000 and 160,000. We termed those glycoproteins Ia, Ib, IIa, IIb, III, and IV. Summaries of the apparent molecular weights of these glycoproteins and their staining properties are presented in Table I. Both inter- and intramolecular disulfides were present in these glycoproteins. Glycoproteins Ib and IIb were disulfidelinked to small subunits. This conclusion is based on the observation that two polypeptides were released from the parent molecules after treatment with reducing agents. Furthermore, the sums of the apparent molecular weights of the reduced products approximated those of the parent molecules (see Table I). We designated the larger subunit the (Y chain and the smaller the /3. Another polypeptide, termed Ic, which was also iodinated by lactoperoxidase on intact platelets, also was composed of subunits. It is not known if this component is glycoprotein, since it did not stain with the PAS reaction as did Ib and IIb. We have also concluded that the p chains of Ic and IIb were exposed on the outer surface of the membrane, since both were iodinated by lactoperoxidase in intact cells. Four glycoproteins, Ia, IIa, IIbcu, and III, appeared to contain intrachain disulfides. This conclusion is based on the observation that these glycoproteins had increased apparent molecular weights, i.e. decreased electrophoretic mobilities, after disulfide cleavage. Several proteins with intrasubunit disulfides such as superoxide dismutase, lysozyme, and plasmin digests of fibrinogen have been shown to have increased apparent molecular weights after disulfide cleavage (18, 25, 26). An explanation for the decreased electrophoretic mobility

TABLE

Apparent molecular weights and staining properties of iodinated platelet membrane components on 7.5% Laemmli gels
Relative Glycoprotein AppFt r %zTzstaining PAS propertid Synonyms -

Ia

Ib Iba N-3 Ic Iccu I@ IIa

(nonreduced) (reduced) (nonreduced)

(nonreduced)

(nonreduced) (reduced) IIb (nonreduced) IIba (partially reduced) IIbcv (reduced) IW III (nonreduced) (partially reducedY (reduced) IV

153,000 167,000 170,000 143,000 22,000 148,000 134,000 27,000 138,000 157,000 142,000 128,000 132,000 23,000 99,000 110,000

+ + + + + ++ ++

+ ++++ ++++ ?

+ +++ +++ +++ IIb IIa (10)

+ ++++

RV,
MGPa (11)

114,000 97,000

++

(LReduced apparent molecular weights were obtained in 10% pmercaptoethanol. b Relative intensities for each st.ain on the acrylamide gel. + + + +, most intense stain; k, detection uncertain; - , no stain detected. c The molecular weight of the p chains were determined on 10% Laemmli gels. rl In Ref. 10, IIa refers to the nonreduced form of IIb. r Reduced with 0.1% P-mercaptoethanol. Reduced with 1% P-mercaptoethanol. (I MGP, major glycoprotein.

2126

Disulfides

in Platelet

Membrane

Glycoproteins

is that the effective chain length of SDS-solubilized proteins increases with cleavage of intrasubunit disulfides. However, cleavage of intrasubunit disulfides may also increase the amount of SDS bound to proteins (27). These considerations complicate the determination of molecular weights of proteins containing disulfide bonds by SDS-acrylamide gel electrophoresis (28). Nonetheless, the phenomenon of decreased mobility after disulfide cleavage seems to be a general feature of proteins containing intrasubunit disulfides. Thus, it would appear that four membrane glycoproteins contain intrasubunit disulfide bonds. Although intersubunit disulfides are present in some membrane polypeptides (e.g. IgG and histocompatibility antigens), and can be induced in others (Band 3 in the erythrocyte membrane (2911, to our knowledge this is the first example of native intrasubunit disulfides in a plasma membrane polypeptide. Thus, contrary to the generalization that disulfide cross-linking is unimportant in membrane structure (30, 31) many of the platelet membrane polypeptides contain disulfides as an integral part of their structure. The use of different concentrations of reducing agents showed that the glycoprotein disulfides could be cleaved selectively. For instance, the intrachain disulfides in glycoproteins Ia and IIa as well as the interchain disulfides joining the subunits in Ib and IIb were reduced by low concentrations of pmercaptoethanol, whereas other disulfides were cleaved only by higher concentrations of reducing agent. The order of reduction of the latter was III (first reduction) = TSP > IIbcv (second reduction of IIb) > III (second reduction). Considering that the proteins were denatured by SDS, these differences in rates of reduction were surprising. Since wide variation in oxidation-reduction potentials of disulfides seems unlikely, it appeared that the variations were due to differences in accessibility of the disulfides to the reducing agent. Indeed, accessibility and conformation have been shown to affect reduction rates (19). Our data suggested that even in the presence of SDS protein disulfides are not necessarily made equivalent in reactivity. The increased resolution afforded by the nonreduced-reduced two-dimensional gel system shows that the glycoprotein composition of the platelet membrane surface is more complicated than previously thought (3, 5, 7-11). The one-dimensional analyses used in earlier studies gave incomplete resolution for three reasons. First, some of the membrane glycoproteins had similar apparent molecular weights. Accordingly, the assumption that a single band on an acrylamide gel represented a single molecular species was erroneous. Second, some of the membrane glycoproteins were reduced only by high concentrations of reducing agents (10% P-mercaptoethanol or 5% dithiothreitol). Since these conditions were not employed in previous work, only partially reduced samples were obtained. Third, the apparent molecular weights of five of the six identified glycoproteins changed significantly after reduction. Thus, the number of glycoproteins observed and the apparent molecular weights reported in previous studies were influenced not only by the resolving capabilities of the gel but also by the extent of reduction. The increased resolution offered by

two-dimensional electrophoresis should permit a more precise evaluation of both the platelet proteolytic receptor for thrombin (6) and the membrane defects present in the genetic disorders Glanzmanns thrombasthenia (5) and Bernard-Soulier syndrome (3). Acknowledgments-We wish to thank Doctors M. Morrison and D. Kingsbury for critical reading of this manuscript.
REFERENCES A., and Zucker, M. B. (1965) 1. Marcus, Platelets, Grune and Stratton, New
The Physiology

of

BZood 22,

2. Mustard, J. F., and Packham, 97-187 3. Jenkins, C. S. P., Phillips, D. R., Clemetson,

York M. A. (1970) Pharmacol.

Reu.

Larrieu,

M.-J.,

and Liischer,

K. J., Meyer, D., E. F. (1976) J. Clin. Znuest. 57,

112-124 255, 720-722 4. Nurden, A. T., and Caen, J. P. (1975) Nature D. R.. Jenkins. C. S. P.. Liischer. E. F.. and Larrieu. 5. Phillius. M.-J. (1975) Nature 257, 599-606 6. Phillips, D. R., and Agin, P. P. (1974) Biochim. Biophys. Acta 352. 218-227 7. Phillips, D. R. (1972) Biochemistry 11, 4582-4588 8. Tanner, M. J. A.. Boxer, C. H., Gumming, J., and VerrierJones, J. (1974) Biochem. J. 14i, 909-911 9. Gates, R. E., Phillips, D. R., and Morrison, M. (1975) Biochem. J. 147. 373-376 10. George, J. N., Potterf, R. D., Lewis, P. C., and Sears, D. A.

Downloaded from www.jbc.org by guest, on March 14, 2013

(1976) J.
11. Nachman,
Chem.

Lab.

Clin. 2928-2936

Med.

88. 232-246

R. L., Hubbard,
248,

A., and Ferris,

B. (1973) J.

Biol.

12. Aster, R. H., and Jandl, J. H. (1964)J. Clin. Invest. 43, 843-855 13. Massini, P., and Liischer, E. F. (1974) Biochim. Biophys. Acta 372, 109-121 14. Baenziger, N. L., Brodie, G. N., and Majerus, P. W. (1972) J. Biol. Chem. 247, 2723-2731 15. Laemmli, U. K. (1970) Nature (Land.) 227, 680-685 16. Wang, K., and Richards, F. M. (1974) J. Biol. Chem. 249, 80058018 17. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Biochemistry 10, 2617-2624 18. Dunker, A. K., and Kenyon, A. J. (1976) Biochem. J. 153, 191-197 19. Iyer, K. W., and Klee, W. A. (1973) J. Biol. Chem., 248, 707-710 20. Morrison, M., and Hultquist, D. E. (1963) J. Biol. Chem. 238,
2847-2849

21. Pollard,
Biochem.

T. D., Thomas,
60, 258-266

S. M., and Niederman,

R. (1974)AnaZ.

22. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193. 265-275 23. Eliman; G. T. (1959) Arch. kochem. Biophys. 82, 70-77 24. Vansteveninck, J., Weed, R. I., and Rothstein, A. (1965) J. Gen.
Physiol. 48, 249, 617-632

25. Abernethy,
Chem.

J. L., Steinman,
7339-7347 Chem.

H. M., and Hill, R. L. (197415. Biol.

26. Mosesson,
J. Biol.

27. Pitt-Rivers, 825-830 28. Fish, W. W., Reynolds, J. A., and Tanford, C. (1970) J. Biol. Chem. 245, 5166-5168 29. Steck, T. L. (1972) J. Mol. Biol. 66, 295-305 Molecular Biology 30. Steck, T. L., and Fox, C. F. (1972) Membrane (Fox, C. F., and Keith, A., ed) pp. 27-75, Sinauer Associates, Inc. Models and the Formation of 31. Benson, A. A. (1968) Membrane Biological Membranes (Bolis, L., and Pethica, B. A., ed) pp. 190-202, North Holland, Amsterdam

M. W., Finlayson, J. S., and Galanakis, D. K. (1973) 248, 7913-7929 R., and Impionbato, F. S. A. (1968) Biochem. J. 109,