STUDIES

ON HUMAN AND BOVINE PLATELET MEMBRANE GLYCOPROTEINS

JOHN F. MCMASTER and MILTON E. NOELKEN Department of Biochemistry. University of Kansas Medical Center. Kansas

City.

KS 66103. U.S.A. (Receired 18 October 1978)

1. We have re-evaluated the number of platelet membrane glycoproteins with use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two highly sensitive detection techniques. 2. The first involves incorporation of the fluorophore dansyl hydrazine into the carbohydrate moiety after post-electrophoresis periodate oxidation. 3. The second involves tritium labelling of galactosyl residues in the intact platelet, clectrophoresis of membrane proteins. and autoradiography. 4. In each case more glycoproteins were detected with these procedures than with periodate-Schiffs base stain. 5. The human platelet membrane contains about 16 resolvable glycoprotein components in the 35.000-300.000 molecular weight range and the bovine platelet membrane contains about 17.

Abstract-

INTRODUCTION The platelet membrane contains several glycoproteins that are thought to be involved in hemostasis because the) are located on the outer surface, which permits interaction with potentiators of aggregation as well as adhesion to vascular tissue (Michaeli & Orloff. 1976). Supportive evidence for this is the greatly reduced levels of certain glycoproteins in human platelet deficiency syndromes such as Glanzmann’s thrombasthenia (Nurden & Caen, 1976), a defect in adhesion. Okumura & Jamieson (1976a. b) have found that a water-soluble glycoprotein, glycocalicin. can inhibit thrombin-induced human platelet aggregation. Phillips & Agin (1977) have reported that a second glycoprotein, of smaller size, is hydrolyzed by thrombin under physiological aggregation-promoting conditions and hence may also be involved in the process. In addition to participation in specialized functions of the platelet it would be expected that some‘ of them would be involved in other functions such as ipn transport and cell surface antigenicity. A complete understanding of platelet membrane structure and function thus requires characterization of the glycoproteins. In view of the demonstration by Gahmberg that the human erythrocyte membrane contains glycoproteins that are not readily detectable by periodic acid-Schiff’s base (PAS) staining (Gahmberg & Hakamori, 1973a) we decided to re-determine the number of platelet membrane glycoproteins by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for resolution and two highly sensitive techniques with different chemical bases to detect them. One of them, the autoradiography technique described by Gahmberg, involves treatment of intact platelets with neuraminidase and galactose oxidase, followed by incorporation of tritium by reduction with sodium borotritiide. This is a highly sensitive procedure but has the potential disadvantage of altering the glycoproteins and perhaps cell function. Gahmberg & Andersson (1977), have I,
300 260 195-210* I52* 140* 118* I05 99 90*

25&300 212* I60*

150* l25* 103s 92*

Laemmli (1970) buffer; 7.5” 0 _eels

Wcber-Osborne (1969) buffer: 5”,, gels

Autoradiograph)

Periodate-Schiff

2 bands > 300 19c-250*

105* 89’

85 7x* 67 60 54 51 43* 38 35

59 51 49 46*

46*

200* 160*

I46* 135* 121 115* 96*

80 62 50;

453

35 25 * An asterisk

indicates

a relatively

intense

band.

so we used those systems in most of our experiments. Photographs of representative dansyl hydrazinestained gels are presented in Figs I and 2 and autoradiograph patterns in Fig. 3. We roughly indicated the relative intensities of carbohydrate-detecting stain or tritium incorporated in each electrophoretic band by assigning single asterisks to relatively intense bands and none to fairly faint bands. A scan of a dansyl hydrazine-stained gel containing human platelet membrane protein is shown in Fig. 4. In addition to the glycoprotein components detected by

method of detection used, at least two membrane preparations were used and from 2 to 6 gels for each preparation. The only exception was that of bovine platelet membranes electrophoresed in the WeberOsborne buffer system and stained with PAS where only one preparation was used. Initial results indicated that the Fairbanks et al. (1971) and Laemmli (1970) buffer systems afforded better resolution of platelet membrane proteins by SDS-PAGE than did the Weber-Osborne system (1969) in terms of number of detectable components and narrowness of bands. Table 2. Glycoprotein components Fairbanks rr al.

Fairbanks er ul.

(1971) buffer. 5% and 7.5”/, gels Dansyl hydrazine

buffer with 8 M urea 37, and So;, gels Dansyl hydrazine

of bovine platelet membranes

Laemmli (1970) buffer. 7.5”,, gels Dansyl hydrazine 3 bands > 300 300

220

240 215

Autoradiography 2 bands

Laemmli (1970) buffer; 7.5”,, gels Periodate--SchilT

> 300

250 215 190

175

170 146: 132* 119* 101* 88*

225

Laemmli (1970) buffer; 7.5”,, gels

156 145* 128* 115* 99* 79*

160

74

72 66

135* 125’ 108* 90* 82* 76 66

42’

42*

42*

25

25

* An asterisk indicates a re!atively intense band.

137* 117* 102* 92* X0* 74 62 52 41 36

134* 125* 104 89

JOHN F.

454

MCMASTIR and

scannmg there were scvcral faint bands that could be detected by the eye. The relative intensities of the peaks shown in Fig. 4 agreed with our visual observations. The weaker bands were always verified by an independent observer. The results for the human platelet membrane are summa&cd in Table 1 BJhcre WC have listed the apparent moiccular weights of the glycoproteins detected under diirerent conditions. A statistical analysis of the reprodtlcibility of the mobilities of the standard proteins used in determination of opparent molecular weights indicated ;I standard deviation of S’,, in the range above 91.500: J- 5”,# from 92,500 to 66,000: and 4”,, in the range below 66.000. These values also appl) to the results for bovine platelet membrane glycoprotcins. listed in Table 1. All of the components hstcd in Tables I and 2. could be stained with Coomassie blue. as would bc cxpectcd for glycoproteins. Representative gels. stained for protein by Coomassie blue. are shown in Fig. 5. The Laemmli buffer system proved to be superior to the Fairbanks C*Itrt. system for resolving platelet membrane proteins. cspec~ally those of apparent molecular &eight greater than I50.000. This is in great part duo to the increased ability of larger chains to penetrate the ~1. For example. rabbit muscle myosin with a subuntt molecular weight of 211.000 has a mobility relative to the tracking dye of only 0.03 in a 7.5” o gel when the Fairbanks 6~ trl. system is used. but it is 0.1X i 0.01 with the Laimmli system. It is important to use a system that yields maximum rcsolution because it has been observed in other studies that this factor can have a dramatic elect on the number of components observed. Taylor & Crawford (1976) for example, were able to detect four pig piatclet membrane g~~~~~proteins b!: SDS-PAGE using a ~oIltinllous Tris~ borate buffer system and PAS staining while Cierncwski c*t LZ~. (1976) found only thrco when ;I continuous phosphate system was

J

30

20

5

IO

Molecular

-

Weight

(xIO-~I

Fig. 4. Stun or a fluorescent gel containing X75/~g of hum;m membrane protein which had been stained with dansyl h)drazine. Eicc(rophoresis conditions were the same as utcd

in the legend

to Fig. 1.

MILTOX E. NOILKLN

There is generally good agreement between the number of components as revealed by dansyl hydrazine staining and autoradiography after tritium incorporation. For example. in the human system (Table 1. columns 3 and 4) there arc five rather intense bands in the 79.00% 140.000 range which must contain substantial percentages of carbohydrate or else be present in relatively large amounts in the inembran~, Four of these five can also bc detected readi b! PAS staining. In addition there are other bands which could not be dctocted by PAS unless inordinateI> kq?c amounts of platelets were used, In spite of the generalI? good agreement of the results obtained nith autoradIography and dansyl hydrarine staining there are some differences th:lt should bc noted. A 52.000 apparent molecular bvcight component was routinely observed by autoradiography but ncvcr with dansyl hydrazine. Similarly. two bands of 160.000 and 190,000 apparent molecular weight appear upon autoradiography whereas a single band of 175.000 apparent molecular weight IUS found b\ dans~t hydrazinc staining. Furthermore. a 36.OCK)band was seen only with ~~~lt~~radiograpily. These differences must derive from the respective chemistries of the two procedures. In autoradiography. ;1s used in this study. aidehyde residues are generated bb oxidation of thr: number 6 carbon of galactosql rc&ues. Tritium is then incorporated by reduction with sodium borotritiide. On the other hand. the dansql hydrazine method involves oxidation of vicinal hydroxyl groups of carbohydrate moieties with pcriodate to yield aldehyde groups. These are then coupled to dansyl hydrazine via SchitT’s base formation. Thus there is no a priori reason to expect that ever> membrane glycoprotein should be detectable by both procedures. We were able to rule out the possibility that these differences in bands detected were artifacts due to either noncovalent binding of dansyl hydrazine or incorporation of tritium into reducible non-aldehyde functions because wt‘ routinely ran control gels in which the oxidizing steps were omitted. Control ,g,els routinely showed negligible incorporation of trmum. Dansyl hqdrazine was found to bind just as well to a few platelet membrane proteins before oxidation as after. hut we did not include thcsc proteins in our results. An interesting observation was made concerning the effects of neuraminidasc on the ciectrophoretic mobilities of SDS-solubilized platelet membrane gllcoprotein. In the ~iutor~~di~~~ra~~h~procedure neuraminidase is used to remove &lic acid residues and thus expose additional galactosyl residues to galactosc oxidase and enhance the sensitivity of the method. In Fig. 6 are shown the SDS-PAGE patterns of two portions of a sample of human platelet membranes. One had been treated with neuraminidase and the other was not. After olectrophoresis both gels were stained with dansyl hqdrazine. It was found that the sialic acid removal decreased the mobilitics of three of the major glycoprotein components, with apparent molecular weights of about 115.000~125.000. 99.00&108,000. and 79,000 90.000. respectively. by about 3 to 4”,,. While this difference is small and detectable only bq direct comparison of ~~ppropriate gels it is signi~cant because it corresponds to an artifactual increase in apparent molecular weight of about 5000 for these species when in fact the real molecular

A Fig. 3. Autoradiographs of human and bovine tritiated platelet membrane glycoproteins. The samples are. left to right: A. 441 pg human protein obtained from membranes of neuraminidase-. galactose oxidase-. and Na-‘BH,-treated platelets, exposure time 186 hr; B. 148 pg bovine protein, other conditions same as A; C. control for A, 735 pg protein platelets treated the same except that the neuraminidase and galactose oxidase steps were deleted and the exposure time was 289 hr; D, control for B. 225 pg protein conditions were the same as in C except that the exposure time was 456 hr. Relatively long exposure times were used with the samples in order to show the weaker bands.

455

C

Fig. 6 Increased apparent molecular weights of human platelet membrane glycoproteins with Ivzuraminidase. galactose oxidasc and No’BH,. The second gel from the left contair from platelets which had been treated with the two enzymes and Na’BH,. The gel a con ltrol using the same conditions except for dclction of the enrqmcs. The t\co I are si milar to the other two except that the platelets had hccn treated with thromb in the : text; the right-hand gel is the sample and the other is the control. The arro glycol -nateins which arc affected by treatment with ncuraminidasc. galactose oxidase

457

treatment coproteins the left is hand pals described ldicate the Na’BH,.

Studies

on human and bovine

height has decreased. This further complicates the use of SDS-PAGE to determine the molecular weights of glycoproteins. which have already been shown to deviate from simple proteins. used as molecular weight standards, in their electrophoretic behavior (Segrest & Jackson. 1972). The simplest explanation for the lowered mobility is the loss of sialic acid residues. which have negatively charged carboxylate groups. upon ncuraminidase treatment. This would outweigh the tendency towards an increased mobility caused by the reduced molecular weight. Similar observations were made by Gahmberg & .Andersson (1977) in their studies with the selective incorporation of a radioactive label in crythrocyte surface glycoproteins. Neuraminidase and galactose oxidase treatmcnt resulted in lower mobilities than when aldehyde groups were generated by periodate oxidation. The latter caused cleavage limited to the bonds between carbon 7 and carbon 8 or carbon 8 and carbon 9 and did not cause removal of carboxyl groups. Masher ct trl. (1977) and Phillips & Agin (1977) have used the same autoradiograph~ methods for human platelet membrane glycoprotems as we have in this study. Mosher t’t ai. reported ten tritium labelfed bands with the following apparent molecular wetghts: I (2~,t~), II (185.000). III (i~,~). IV (140,000). v (I 22,000). VI (I 12.000). VII (80.000). VIII (68.000). IX (52,000) and X (38.000). Bands IV, V, VI and VIII were the most heavily labelled and lie in the same apparent molecular weight range where we obxerved maximal incorporation of tritium. We found a tkttal of 16 bands; three of the “extra” bands could perhaps bc related to enhanced resolution in the 92.(K)&36.000 region. as we found seven bands there. It should be noted that we found several of the lowest apparent molecular weight bands to be rather weak (Table 1). In addition the two bands of greater than 300.000 apparent molecular weight were weak and diflicult to resolve. I-Iowcver. the 250.000 apparent molecular weight component which we observed was readily detectable (Fig. 3). Phillips & Agin (1977) listed ten glycoprotein bands with apparent molecular weights lying in the range 160,000-44,000 but inspection of F&. 1 of their papers shows four bands above this r~nge~includin~ one at the top of the gel. Our results for bovine membranes are presented in Table 2. In column 4 are the apparent molecular weights of the giycoprotein components detected by using a S’,, gel. I”,, 2-mercaptoethanol, the continuous phosphate buffer system and PAS staining. We found components of 200,000, 160.000. 105,000 and 89,000. Cierllewski t’f iii. (1976) used very similar conditions and reported only three components of apparent molecular weight 230,000. 155,000 and lOl.GQO. However. in Fig. 3 of their paper. in which a spectrophotometric scan of the gel is represented. there is a small shoulder on the low molecular weight side of their 101,000 peak which could correspond to our 89.000 component. In our studies though, a definite 89,000 peak was obtained which was clearly separated from the 105.000 peak. In columns 2 and 3 are the results obtained with the Laemmli buffer system under reducing conditions and using either dansyl hydrazine staining or autoradiography to detect glycoproteins. AS with human platelets there are several glycoproteins which can be readily detected by these two

platelet membrane

glyooproteins

359

methods which would be difficult to find by PAS staining unless very large quantities of platelets were used. Essentially identical results for the bovine system were obtained with the Ruorescencc and autoradiography methods. An exception to this is that the 67,000 and 38,000 components were not detected by the former method, but were by the latter. The Fairbanks r’t trl. buffer system (column I) used with 3 and Y,, gels and 8 M urea allowed detection of fewer components \vith dansll hydrazine staining than did use of the Laemmli skstem with 7.5”,, gcis. further indicating the superior& of the latter system. The 160.000 apparent molecular weight bovine 4) stained quite glycoprotein (Table 2. column intensely with the periodate-Schiff reagent but only poorly with Coomassie blue. while strong protein staining components comigr~~te with the other glycoproteins. This behaviour has been noted for a glycoprotein of similar apparent molecular weight found in platelet membranes of other mammalian species. including Ho,uo srrpic,rl.>, when the Wcber- Osborne continuous phosphate buffer system was used (Nurden & Caen, 1977). We observed this disparity in staining for the 13?.000 apparent molecular weight human glycoprotein (Table 1. column 4) \%hen the Laemmli buffer system was used. Each of these two glycoproteins corresponds to glycoprotein I in the nomenclature system used by Nurden & Caen (1977). in terms of relative staining intensity with protein and carbohydrate stains and position relative to the two nearby intensely staining glycopr~~teins of lower apparent molecular weight. It is possible that bovine glycoprotein I is heterogeneous since use of either the buffer svstem of Fairbanks YI trl. (1971) or that of Laemmii yields an additional component of similar apparent molecular weight. In addition to the glycoprotein components listed in Tables I and 2 we noted material which barely penetrated the polyacrylamide gels. In both the bovine and human membranes it stained with Coomassie blue, dansyl hydrazine. and could be labelled with tritium. To allow further comparison of the membranes we digested intact platelets with bovine thrombin because it has a rather limited proteolytic spccificity. directed to the pcptide bond on the carboxyftermmal side of arginyl residues (Lundblad PI ul., 1976). We found that most of the tritium label was lost from this material after thrombin treatment. indicating a similarity in structure of part of this fraction in the two types of platefets. The conditions used were much more drastic than required for platelets to undergo thronlbin-induced aggregation. i.e. we used thrombin levels of 10 NIH units;ml of platelet suspension (lo9 platelets/ml) for 1 hr at 37 C, or hlternalively 5 units/ml for 2 hr. whereas platelet aggregation is induced in a matter of seconds with 1 unit/ml. Thus. these results do not necessarily support a role for the very high molecular weight fraction in thrombin-induced aggregation but. on the other hand, does suggest a common structure in the two membranes. In addition, by use of autoradiography we noted the disappearance of a human glycoprotein band corresponding to an apparent moleular weight of 66,000. This is in accord with the finding of Mosher et a/. (1977) who reported that a 68.000 molecular weight

Jot!~ 1-. MCMASTFK and

460

glycoprotein disappeared after 20 min incubation of human platelets with thrombin (I UrnI). Since some of the products of hydrolysis in our stud! migrated in the 90.00& 135.ooO molecular weight region we did not obtain any evidence that woutd hear on the report of Phillips & Agin (1977) that an 89.000 apparent molecular weight glycoprotein disappeared after thrombin action. In the case of bovine plutelcts the only detectable change in the glycoprotein distribution was the decrease in the amount of material at the top of the get. DISCUSSION

We have re-evaluated the number of human and bovine platelet membrane glycoprotcins with use of sodium dodecyl sulfatepolyacrylamidc gel electrophoresis and two highly sensitive detection techniques. The first involves incorpor~~tion of the fluorophore dansyl hydrazine into the carbohydrate moiety after periodate oxidation. The second involves labelling of exposed galactosyl residues of intact platelets through oxidation catalyzed by galactoso oxidase. followed by reduction with tritiated sodium horohydridc and subsequent ~~utor~di[~graphy involt-ing exposure of x-ray films to eiectrophoresis gels. The number of giycoprotein chains present in these membranes is substantially more than were found in other studies primarily because we used more sensitive detection techniques and also because of our USC of several buffer systems. We found that human piatelet membranes contain about 16 glycoprotcil~ c~~mponents in the molecular weight range 35.C!O@300,~ which can be resolved from each other by SDS-PAGE using the Laemmli buffer system and can be detected by at least one of our two techniques. Five of thcsc. in the molecular weight range 80.000 137,000. can be stained intensely with dansyl hydrazine and;or labelled heavily with tritium indicating that they contain large percentages of carbohydrate or. aiternatively, are present in large amounts in the membrane. Similarly, we found 17 resolvable glycoprotein components in the 35.000-300,000 molecular weight range in bovine platelet membranes. five of which are in the 78,00&152,000 apparent molecular weight range and are easily detected by one or both of our methods. However, comparison of gels containing human and bovine glycoproteins (Figs I and 2) clearly indicates substantial differences between glycoproteins of equal clectrophoretic mobility in the quantity of label which can be added. Species variations in the carbohydrates of platelet membrane giycoproteins have been reported by Nurden & Caen (1977) who found differences among 13 mammalian species. including man. in the number and electrophoretic mobility of acidic glycopeptides released by tryptic action. Using SDS-PAGE with the relatively insensitive periodate-SchitYs base staining method and a continuous phosphate buffer system that was less effective at resolving glycoprotejns than the two we used most freyuently. they found three major glycoproteins of apparent molecular weight 14X,000- 170.000. I25.00@ 138,000 and 9X.000- 108.000 in most of the species studied. However. the relative amounts of these was distinctive for a given species and the higher tnoiecular weight component even

MILTOK E. NOELKLN

appeared to be missing in four of the 13 species. Ciernewski et (II. (1976) have reported such differences in the periodate-Schiff base staining intensity between bovine. porcine. and human platelets. The results of our comparative study of human and bovine indicate that the species variation is still apparent when differcnt buffer systems and more sensitive detection techniqucs are used. However. it is noteworthy that in each of these systems we were able to resolve and detect about 16-l 7 giycoprotein components in the molecular weight range 35,000 to about 3~,~. Each of the bands corresponding to a human glycoprotein appears to have its bovine counterpart in terms of electrophoretic mobility. even though there are important quantitative differences in dansyl hydrazine staining intensity or in tritium labeiling. These results suggest a strong quantitative similarity between the two membranes. It would seem possible that some of the glycoprotein chains of essentially identical mobility in bovine and human platelet membranes would also have the same function. Platelets of various species have been found to differ in fundamental properties such as size (Lewis. 1975; ProstDvojakovic of uf., 1975) and some of them even have special distinguishing ultrastructural characteristics (Lewis. 1975). They do, however, have common biological functions which generally vary in a quantitative rather than qualitative way (Lewis, 1975; Macmillan & Sim. 1970: Sinakos & Caen, 1967). The species variations could derive from differences in levels of functional entities as well as from differences in amino acid sequence and carbohydrate composition. Tritium label& by the method used in this study isspecificfor externally located carbohydrate moieties whereas dansyl hydrazine was used to detect carbohydrate following separation, solubilization, and electrophoresis of membrane proteins. Since the results obtained by these two methods were very similar we conclude that essentially all of the carbohydrate of the platelet membrane is exposed to the exterior, unless of course. certain glycoproteins would contain both internal and external carbohydrate moieties. However. this would be unlikely as it has not been demonstrated in any other type of ceil. Thus. the dansyl hydrazine staining technique should be very useful in future studies of platelet membranes because it reAccts accurately the number of giycoproteins. is relatively rapid, and is quite sensitive. The autoradiography procedure is capable of greater sensitivity but is inherently a slower process, as the intensity on x-ray film corresponding to a tritiated glycoprotein depends on the length of exposure of the film to the gel and typically requires several days. Acknomlrdyern~nts-This research was supported by American Heart Association-Kansas Affiliate grants KK76-I5 and 77-55-22. We wish to thank Dr Billy G. Hudson for valuable suggestions and criticism. REFERENCES

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12.