Labeling of platelet surface proteins with 125I by the Iodogen method

ANALYTICAL

BIOCHEMISTRY

130, 166-170 (1983)

Labeling of Platelet Surface Proteins with 1251by the lodogen GEORGE P. TUSZYNSKI, LINDA C. KNIGHT,

ELIZABETH

Method

KORNECKI,

AND SITA SRIVASTAVA Specialized Center for Thrombosis Research, Temple University Health Sciences Center, Philadelphia, Pennsylvania 19140 Received September 20, 1982 A procedure for the ‘251-iodination of platelet suspensions is described. The procedure utilizes Iodogen, a solid-phase oxidizing agent similar to chloramine-T. Platelets were labeled under a variety of conditions, including in the presence of 0.1% albumin, and showed between 7 and 28% incorporation of i*‘I Best labeling results were obtained at low platelet concentrations (3-5 X 10’ platelets/ml), short reaction times (15 min), and with 2-ml glass vials coated with 100 rg of Iodogen. Analysis of the labeled platelet proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed that the same major protein bands were labeled by this procedure as were labeled by the lactoperoxidase procedure. At low platelet concentrations, the Iodogen procedure gives twice the amount of iodine incorporation.

The identification of cell surface proteins by the use of radiolabeled chemical probes has greatly aided our understanding of the structure-function relationships of membrane glycoproteins. By far the most widely used labeling procedure has been the lactoperoxidase (LPO)-catalyzed iodination of cell surface proteins (1). In this report, we present a simpler alternative labeling procedure. The method is based on the solid-phase Iodogen technique which has been used in the iodination of cell membrane proteins (2-4,9) and soluble proteins (2-4,8). We have applied this technique to the labeling of platelet surface proteins under a variety of buffer conditions. The procedure is relatively easy to perform and compares favorably with the lactoperoxidase procedure. MATERIALS

AND

METHODS

was purchased from New England Nuclear, Boston, Massachusetts. Platelet suspension buffers were: HEPES = 3.8 mM Hepes’ buffered with 3.8 mM NaH2P04, pH 7.35, containing 0.137 M NaCl, 2.7 mM KCl, 1 mM MgC12, and 0.01% dextrose; HEPES + BSA = HEPES containing 1 mg/ml BSA; HEPES + EDTA = HEPES containing 1 InM EDTA; HEPES + EDTA + BSA = HEPES containing 1 mrvr EDTA and 1 mg/ml BSA. Platelet washing buffer, Tyrodes, pH 7.35 = NaHCOs (11.9 mM), NaH2P04. H20 (0.36 mM), CaC12 * 6H2O (2 mM), MgC12 * 6H2O (1 mM), glucose (O.l%), bovine serum albumin (0.35%). Apyrase was prepared from potatoes by the method of Molnar and Lorand (5). Preparation of platelets. Platelets were washed by the method of Mustard et al. (6) and resuspended in the buffers listed above. Briefly, fresh whole blood was obtained from healthy donors, collected into acid citratedextrose and containing 2500 units/liter heparin, and centrifuged at 120g to obtain plate-

Reagents. Common buffers and reagents for gel electrophoresis were purchased from Sigma Chemical Company, St. Louis, Mis’ Abbreviations used: BSA, bovine serum albumin; souri. Iodogen (1,3,4,6-tetrachloro-3a,6a-diDTT, dithiothreitol; EDTA, ethylenediaminetetraacetic phenylglycoluril) was obtained from Pierce acid; Hepes, 4-(2-hydroxyethyl)-l-piperazine-ethanesulChemical Company, Rockford, Illinois. Na1251 fonic acid; SDS, sodium dodecyl sulfate. 0003-2697/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.

166

LABELING

OF

PLATELET

SURFACE

let-rich plasma. All centrifugations were performed at 23°C. Platelets were pelleted at 1lOOg for 15 min, and approximately 0.5 ml of packed platelets resuspended in 10 ml of Tyrodes-albumin solution, pH 7.35 (see above), containing 2500 units/liter of heparin and 100 mg/liter apyrase, pelleted at 950g for 10 min, and resuspended in 10 ml of Tyrodes-albumin solution, pH 7.35, containing 100 mg/liter apyrase but no heparin. Platelets were centrifuged again for 10 min and resuspended in the platelet suspension buffers listed above. Platelet concentrations were adjusted to the desired levels. Platelet iodination. Two-milliliter septumcapped glass vials (Wheaton, Vineland, N. J.) were coated with varying amounts of Iodogen by injecting each vial with a solution of Iodogen (usually 1 mg/ml) dissolved in chloroform. The chloroform was evaporated by means of a syringe needle fitted to a vacuum source. The syringe needle was inserted into the septum-capped vial containing the Iodogen-chloroform solution and, with the vacuum on, the vial was slowly rotated on its side until all of the solution was evaporated, leaving a thin film of Iodogen deposited on the sides of the vial. One milliliter of a platelet suspension was placed into a septum-capped microcentrifuge tube and the desired amount of Na’251 activity was added. This mixture was then transferred to the Iodogen-coated vials by means of a disposable syringe and incubated at room temperature for 15 to 30 min with occasional shaking. The reaction was terminated by removing the platelet suspension by syringe and transferring it to a 1.5-ml conical tube for washing. The pellet was washed three times in HEPES + BSA by centrifugation at 10,OOOg for 2 min. To minimize release of volatile 1251activity the first centrifugation was performed in a septum-capped microcentrifuge tube. Washed platelet pellets were dissolved in 100 ~1 of SDS sample buffer containing no reducing agents. Labeling of platelets by the lactoperoxidase procedure was carried out according to the method of Phillips and Morrison (1). Briefly,

PROTEINS

WITH

IODINE

167

1 ml of platelets suspended in HEPES buffer was placed in a septum-capped microcentrifuge tube. By means of a glass Hamilton syringe, loo-220 &i of Na’251 was added, followed by 20 1 ~1 of a 1 mg/ml lactoperoxidase solution in HEPES. Immediately, 10 ~1 of 3 mM H202 solution was added and the platelets were gently mixed. This procedure was repeated four times every 5 min. After the addition of H202, 100 ~1 of a 100 mg/mlcysteine solution was added to stop the reaction, and the platelets were washed by centrifugation as described in the Iodogen procedure. SDS.-polyacrylamide-gel electrophoresis. Labeled platelet proteins were analyzed according to the procedure of Laemmli (7). Fixed and stained gels were pressed onto paper, and autoradiograms were prepared using intensifying screens (DuPont Cronex Lighting-Plus screens mounted in Spectroline Cassettes, Reliance X-ray Inc., Oreland, Pa.). Kodak XOmat-AR film was used and developed according to the instructions provided with the film. Films were exposed for 1 week at -70°C. RESULTS

Efect ofreaction time and Iodogen level on 12jI incorporation into platelets. Figure 1 shows that 100 pg of Iodogen gave highest incorporation of 125Iinto platelets after 30 min of incubation at room temperature. Higher concentrations of Iodogen gave lower incorporation, and after 30 min of incubation the ‘25I incorporation appeared to decrease slightly. The reason for the lower incorporations at longer reaction times and higher Iodogen concentrations is unknown. We speculate that extended exposure to even a mild oxidizing agent such as Iodogen may cause platelet membrane damage. Efect of platelet concentration on 12’1 incorporation into platelets. Platelet concentrations on the order of 3.0-5.4 X lo8 platelets/ ml gave highest ‘25I incorporations (Table 1). Higher platelet concentrations incorporated significantly lower amounts of ‘251.

168

TUSZYNSKI

Eflect of bufler composition on “‘I incorporation into platelets. Various commonly used platelet buffers changed the amount of ‘25I incorporated into the platelet suspensions only when 1 mg/ml BSA was present (Table 2). Reduction of the incorporation of activity into platelets was expected in the presence of albumin, which is easily radioiodinated. However, the amount incorporated into the cells was still reasonably high (7%), indicating that the procedure can be used in proteincontaining buffers. Analysis of the labeled proteins on SDS-gel electrophoresis. Figure 2 shows that basically the same labeling pattern was obtained whether or not the platelets were suspended in albuminor EDTA-containing buffers (compare lanes l-4 and lanes 6-9, Fig. 2). Comparison of the Iodogen procedure with the lactoperoxidase procedure. Figure 2 also shows that the Iodogen method and the lactoperoxidase procedure label the same major proteins (compare lane 4 with lane 5, and lane 9 with lane 10, Fig. 2). The major proteins labeled by both procedures are the glycoprotein complex (GPII,,, III) which migrate with

ET AL. TABLE

1

COMPARISON OF THE EFFECT OF PLATELET CONCENTRATION ON THE I29 INCORPORATION INTO PLATELETS BY MEANS OF THE IOLXK~EN AND LACTOPEROXIDASE(LPO) METHODS

Platelet concentration (X108/ml) 5.4 21 54

‘25I incorporated (Ki)

Percentage incorporation

Iodogen

LPO

Iodogen

LPO

35 12 8

18 13 10

28 9.6 6.4

14 10 8

a Platelets were suspended in HEPES and labeled as described under Materials and Methods. Approximately 125 &i of carrier-free Na’*sI was added to each sample. Platelet numbers were determined electronically by a Coulter cell counter.

apparent molecular weights of 130,000 and 96,000 under nonreducing conditions (compare lane 1 with lane 5, Fig. 2). The Iodogen method and lactoperoxidase procedure also show differences in the labeling patterns obtained for some of the less highly labeled proteins. For example, under reducing conditions a component of approximately M, TABLE 2 EFF-ECTOFBWFERCOMPOSITION INCORPORATION

IO

P Reaction

50

40

50

Suspension buffer”

‘*‘I incorporated (PC9

Percentage incorporation

HEPES HEPES + BSA HEPES + EDTA HEPES + EDTA + BSA

36 13 35

18 7 18

23

12

60

Time (min)

I. Effect of Iodogen concentration and reaction time on percentage i2*I incorporation into platelets. One milliliter of 3.0 X lo8 platelets in HEPES was added to three separate 2-ml vials coated with 50 pg Iodogen (+), 100 PLgIodogen (0) and 200 rg Iodogen (0). Each vial contained approximately 200 pCi of ‘r51, and at the reaction times indicated, small aliquots of platelets were withdrawn, washed, and the pellets counted for radioactivity. FIG.

ON 12s1

INTOPLATELETS

a Platelets were suspended in the various buffers listed above (see Materials and Methods for exact buffer compositions) to a final concentration of 3.0 X lOa platelets/ ml. One milliliter of each suspension was labeled for 30 min with 200 &i of carrier-free Na’*‘I in a 2-ml glass vial coated with 100 rg of Iodogen. Platelets were then transferred to microcentrifuge tubes and washed three times by centrifugation as described under Materials and Methods to remove unbound iodide. The final platelet pellet was counted for radioactivity.

LABELING

OF PLATELET

SURFACE PROTEINS

MWxlO” 200 II6 96 r 68 -

1234

5

6

7 8 9 IO

FIG. 2. SDS-gel electrophoresis of labeled platelets. One milliliter of 3.0 X lo* platelets was labeled in various buffers by the Iodogen method, and one sample was labeled by the lactoperoxidase procedure. Pellets were solubilized in SDS sample buffer, and approximately 100,000 cpm of labeled proteins run on a 10% SDS-gel for 3 h at 30 mA. After electrophoresis, gels were fixed, stained and subjected to autoradiography using intensifying screens. (1) Iodogen-labeled platelets in HEPES buffer; (2) Iodogen-labeled platelets in HEPES + BSA buffer; (3) Iodogen-labeled platelets in HEPES + EDTA buffer; (4) Iodogen-labeled platelets in HEPES + EDTA + BSA buffer; (5) Iactoperoxidase-labeled platelets in HEPES buffer. (610) Same samples run in lanes l-5, respectively, except that each sample was reduced with 3 mM DTT. Molecular weights were determined by running Bio-Rad high- and low-molecular-weight standard mix under reducing conditions in parallel with the labeled proteins.

85,000 is labeled by the lactoperoxidase procedure but not by the Iodogen procedure (compare lane 10 with lanes 6 through 9, Fig. 2). Alternatively, the Iodogen procedure labels a component of approximately h4,3 1,000 not labeled by the lactoperoxidase procedure (compare lane 6 with lane 10, Fig. 2). Ap proximately 14% of the radioactivity is incorporated by the lactoperoxidase procedure when 5.4 X lo8 platelets per ml are labeled. This value is half as much as was obtained by the Iodogen procedure under comparable conditions (Table 1). The comparison in the amount of label incorporated by the two methods is not absolute, since different oxidizing equivalents are used in both procedures.

WITH

IODINE

169

The Iodogen procedure appears to label a considerable amount of low-molecular-weight material that runs near the dye front (Fig. 2). We speculate that this material is lipid in nature since it will eventually wash out of the gel by repeated prolonged soaking of the gel in methanol-acetic acid. The higher ‘25I incorporation by the Iodogen procedure may in fact be due to the labeling of this lipid-like material. However, sufficient label is incorporated into protein to make the procedure useful as a platelet membrane protein labeling procedure. DISCUSSION

Iodogen (1,3,4,6-tetrachloro-3cy,6c+diphenylglycoluril) is a solid-phase oxidizing agent that has been used for labeling soluble proteins with “‘1 without damaging protein function. Iodogen has also been used to label isolated membranes (2-4) and the surface proteins of viruses and eucaryotic cells (9). We have used the Iodogen procedure to label platelets with 1251.Our results indicate that reasonable incorporation of label occurs under the following conditions: (1) platelet suspensions that are on the order of 5 X lo8 platelets/ml; (2) Iodogen levels of 100 pg coating the inside surface of 2-ml glass vials; (3) reaction times of 30 min at room temperature; (4) platelet suspension buffers that contain no proteins, although the presence of albumin does not adversely affect the incorporation of iodine (Table 1). The Iodogen procedure appears to label the same major protein species as does the commonly used lactoperoxidase procedure (1). Basically, the major surface glycoprotein species GPII,-GPIII complex (lo), having apparent molecular weights of 130,000 and 96,000 under nonreducing conditions (see Fig. 2, lanes l-5) and 120,000 and 116,000 under reducing conditions (see Fig. 2, lanes 6-10) are labeled to the same relative intensity by both the lactoperoxidase procedure and the

170

TUSZYNSKI

Iodogen procedure. Differences in the labeling patterns obtained by the two procedures seem to be confined to some of the less highly labeled protein species (see Results). The reasons for these differences are unknown. Therefore, the Iodogen procedure can quickly and efficiently label the major platelet surface proteins with a minimum of reagents, and platelets can also be labeled in the presence of albumin, which stabilizes platelet function (11) without much loss in the amount of label incorporated. The Iodogen procedure also allows for the elimination of the reduction step employed in other iodination procedures that employ soluble oxidizing agents such as chloramine-T ( 12). The Iodogen procedure should be useful in production of labeled antigens for the screening of platelet antibodies to specific platelet membrane glycoproteins (13). This procedure has also been found useful in the investigation of the membrane components which remain on the platelet surface following enzymatic treatment (14). We have preliminary evidence (15) that platelets labeled by the Iodogen method respond normally to such physiological platelet activators as thrombin, and form platelet cytoskeletons having protein compositions similar to those isolated from unlabeled platelets. The procedure may find use in the study of the role of membrane proteins in platelet function. ACKNOWLEDGMENTS This work was supported by Department of Health and Human Services Grants HL28149 and HL14217, Na-

ET AL. tional Research Council Service Award HLQ6356, and an American Heart Association Special Pennsylvania Chap ter Investigatorship Fellowship. The authors would like to thank Dr. Ed Kirby for his helpful suggestions. The authors are grateful to Monica Kollmann for excellent technical assistance.

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Amaout, M. Amin, Pitt, J., Cohen, H. J., Melamed, J., Rosen, F. S., and Colten, H. R. (1982) N. Engl. J. Med.

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4. Salisbury, J. G., and Graham, J. M. (198 1) B&hem. J. 194, 351-355. Molnar, J., and Lot-and, L. (1961) Arch. B&hem. Biophys.

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Mustard, J. F., Perry, D. W., Ardlie, N. G., and Packham, M. A. (1972) Brit. J. Haematol22,193-204. Laemmli, U. K. (1970) Nature (London) 227, 680685. Knight, L. C., Budzynski, A. Z., and Olexa, S. A. (1981) Thromb. Haemostas. (Stuttgart) 46, 593596. Markwell, M. A., and Fox, C. F. (1978) Biochemistry 17,4807-4817. Phillips, D. R. (1980) Thromb. Haemostas. (Stuttgart) 42, 1638-1651. Walsh, P. N. (1972) Brit. J. Haematol. 22, 205-217. McConahey, P. J., and DixoniF. J. (1966) Znt. Arch. Allergy

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13. Komecki, E., Niewiarowski, S., and Tuszynski, G. P. (1981) Blood 58, 198a. 14. Komecki, E., Tuszynski, G. P., and Niewiarowski, S. (1983) submitted for publication. 15. Rao, A. K., Tuszynski, G. P., Knight, L., Willis, J., and Beckett, C. (1981) Thromb. Haemostas. (Stuttgart) 46, 109.