THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255,,No. 9. Issue of May 10, pp. 4320-4327, 1980 Prrnted m U.S.A.

Crossed Immunoelectrophoresis of Human Platelet Membranes

(Received for publication, October 22, 1979)

Sabra Shulman and Simon Karpatkini From the Department of Medicine, New York University Medical School, New York, New York 10016

Human platelet membranes were prepared by the glycerol-lysis technique of Barber and Jamieson (Bar- ber, A. J., and Jamieson, G. A. (1970) J. Biol. Chem 245, 6357-6365), solubilized in 1% Triton X-100 and sub- jected to crossed immunoelectrophoresis, in 1% aga- rose, employing a rabbit anti-human platelet mem- brane antibody in the second dimension. Approxi- mately 20 immunoprecipitates (numbered 1 to 20) could be detected in normal subjects. At least 10 different antigens were observed fairly consistently with differ- ent membrane preparations. One could be identified as albumin (12A), the other as fibrinogen (1F). Four mem- brane antigens were also present in the cell sap, two of which were 12A and 1F. Absorption of the antibody with increasing concentrations of washed platelets re- vealed the disappearance of at least six different anti- gens (lF, 7-8, 10, 13, 14, and 18) at a constant rate, suggesting an external surface location. Four of these surface antigens (lF, 10, 13, and 18) reacted with con- canavalin A when this was employed as an intermedi- ate spacer gel, indicating that they are glycoproteins. At least six antigens did not react with concanavalin A. A major surface antigen, 10, was present on all preparations and had lines of identity with two other antigens, 13 and 18, which moved more cathodally. Platelets from subjects with full 10 antigen peaks had absent or diminished 13 and 18 antigen peaks, whereas platelets from subjects with absent to incomplete cath- odal arms had increased 13 and 18 antigen peaks. Fur- thermore, digestion of intact washed platelets with a- chymotrypsin resulted in a decrease in the 10 antigen peak and an increase in the 13 and 18 antigen peaks, suggesting a structural or organizational relationship among these three antigens. This is supported by stud- ies with neuraminidase digestion of intact washed platelets, which resulted in a cathodal shift of the major antigen and the disappearance of antigens 13 and 18.

Platelet membranes from three patients with Glanz- mann’s thrombasthenia (a disorder in which platelets do not aggregate with aggregating agents) revealed the absence or marked reduction of the major antigen 10, as well as of 13,18, and lF, and the exposure of a non- glycoprotein antigen, normally hidden “behind” 10 and designated loa. Platelet membranes from two patients with Bernard-Soulier syndrome, a disorder in which platelets do not adhere to injured subendothelial sur- faces, the receptor for von Willebrand factor is absent,

* This work was supported by Grant HL-13336-10 from the Na- tional Heart, Lung and Blood Institute, National Institutes of Health, and Contract DADA 17-6842-8163 from the United States Army Research and Development Command. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

and a glycoprotein, GP I (Nurden, A. T., and Caen, J. P. (1975) Nature 255,720-722) and its related soluble form, glycocalicin (GP Is), is absent (Okumura, T., Lombart, C., and Jamieson, G. A. (1976) J. Biol. Chem 251,5944- 5949) had absent or marked reduction of the major antigen 10, as well as of 13,18, and lF, the exposure of loa, the appearance of an anodal neoantigen, 21, and the absence of an antigen which reacts with a mono- specific antibody for glycocalicin, 22 Glyc. It is sug- gested that the antigenic differences noted in both groups of patients may reflect differences in endoge- nous membrane proteolysis.

Early studies on human platelet membranes, employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), revealed the presence of major surface glyco- proteins, GP I, GP 11, and GP 111, with apparent molecular weights of 155,000, 135,000, and 103,000 (1-3). More refined SDS-PAGE techniques have subsequently demonstrated the presence of at least six to seven surface glycoproteins (4). With these techniques, specific abnormalities reported in two he- reditary disorders, Glanzmann’s thrombasthenia (diminished to absent GP IIb and GPIIIa (3, 5)) and the Bernard-Soulier syndrome (diminished to absent GP I (6) and its related soluble form, glycocalicin or GP Is ( 7 ) ) .

Although crossed immunoelectrophoresis (CIE) has been employed in the analysis of serum proteins, its potential in the study of cell surface antigens and biological membranes has only recently been exploited (8-14). This technique has several advantages over SDS-PAGE.’ 1) It does not com- pletely denature the membrane proteins, so that intrinsic biologic activity can often be assayed. 2) It is 10 to 100 times more sensitive than SDS-PAGE. 3) I t is essentially quantita- tive, since the peak areas of individual immunoprecipitates are proportional to the antigen/antibody ratios. 4) It is capable of detecting at least 20 different platelet membrane antigens. 5) Comparative CIE studies of anti-membrane antiserum ab- sorbed with whole cells enable conclusions to be drawn re- garding the relative surface location of various membrane antigens. 6) Various lectins can be employed as spacer gels, thus providing an immunoafflnoelectrophoresis pattern, which provides information regarding lectin binding of surface antigens.

We have applied this technology to the detection of platelet membrane antigens. The present report provides our detailed investigation of the CIE of solubilized human platelet mem- branes.

’ The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; CIE, crossed immunoelectropho- resis; TLCK, l-chloro-3-tosylamido-7-amino-2-heptanone; Glyc, an antigen which reacts with a monospecific antibody for glycocalicin.

4320

Crossed Immunoelectrophoresis of Platelet Membranes 4321

METHODS

Preparation of Platelet Membranes-Platelet-rich plasma, ob- tained from the New York Blood Center, was centrifuged at 2,500 X g for 15 min at 4°C. The platelet pellet was washed in a human Ringer solution (15) containing 2 mM EDTA, IO mM benzamidine, and 100 pg/ml of soybean trypsin inhibitor, and the pellet was then resuspended in 1% ammonium oxalate to lyse red blood cells. The platelets were then washed twice in the same buffer mixture. A 3% (v/v) platelet suspension was then layered onto a 0 to 40% glycerol gradient dissolved in the above buffer mixture to load the cells with glycerol prior to osmotic lysis and membrane purification as described by Barber and Jamieson (16).

Platelet membranes were also prepared by four cycles of freezing (acetone-dry ice) and thawing (37OC) in the buffer mixture, followed by sonication for 15 s at 75% maximal intensity at 4"C, in a Branson sonic power sonicator, W185 (Branson Instruments Co., Plainview, N. Y.). Cellular debris was removed by centrifugation at 1,100 X g for 10 min at 4OC, and the supernatant was applied to a 30% sucrose cushion (sucrose dissolved in 0.01 M Tris buffer, pH 7.4, containing 2 mM EDTA and 0.15 M NaC1) and centrifuged for 1 h at 60,000 X g in a Beckman L2 ultracentrifuge at 4°C. The membrane layer was taken from the interface. This was then washed in the same buffer and recentrifuged at 100,OOO X g for 1 h. This gave a 3-fold greater yield. Identical patterns were obtained with both membrane preparation procedures.

Solubilization of Platelet Membranes with Triton X-100"Platelet membranes were adjusted to approximately 7.0 mg/ml of protein after extraction with 1% (v/v) Triton X-100 containing 10 mM benz- amidine and 100 pg/ml of soybean trypsin inhibitor. The supernatant was stored at -20°C until use.

Preparation of Anti-Platelet Membrane Antibody-Antiserum to purified platelet membrane was raised in a rabbit. The platelet membranes from 1 unit of blood were suspended in 0.5 ml of saline (0.9% NaCl solution) and emulsified 1:l (v/v) with Freund's complete adjuvant. This was injected into the hind foot pads of a rabbit. Subsequent booster injections, obtained from 1 unit of platelets from multiple donors, were administered subcutaneously in divided doses every 2 to 3 weeks. Sera from five consecutive bleedings were pooled, and immunoglobulin was partially purified by precipitation with 50% saturated (NH,)BO,. The precipitate was dissolved in % of its original serum volume and dialyzed against sodium acetate buffer, pH 5.0. The solution was then dialyzed against 0.1 M NaCl containing 15 mM NaN:3.

Crossed Immunoelectrophoresis-Crossed immunoelectrophoresis was performed by a modification (IO) of the Laurel1 technique (17), employing 0.02 M barbital, 0.07 M Tris buffer, pH 8.6, containing 1% (v/v) Triton X-100. Agarose gels (la, w/v) were prepared in the above buffer. The gels were cast on glass plates (50 X 50 X 0.6 mm) to give a volume-to-surface area ratio of 0.132 ml/cm'. Samples (50 pg in 20 pl) were applied to wells 4 to 7 mm in diameter and electrophoresed at 150 V for 1.5 to 3 h in a Behring diagnostic water- cooled immunoelectrophoresis cell (Behring Diagnostics, Somerville, N. J.). An agarose strip (12 X 50 mm) containing the antigens that had been subjected to electrophoresis was retained on the plate after removal of the rest of the gel, which was replaced with an adjacent gel (36 X 50 mm) containing anti-platelet membrane antibody ( 6 0 pl/ ml (2 mg/ml)). Electrophoresis in the second dimension was per- formed at 55 V for 18 h. Gels were then pressed with fiiter paper, washed 6 to 8 times in 0.1 M NaC1, air-dried, and stained with 0.25% Coomassie Brilliant Blue (dissolved in 45% methanol, 9% acetic acid) for 15 min at 22°C. The gels were destained in 45% ethanol, 9% acetic acid.

Crossed Zmmunoaffinoelectrophoresis-The initial step was iden- tical to that described above. However, after electrophoresis in the first dimension, an agarose strip (30 X 50 mm rather than 40 X 50 mm, as above) was removed and replaced by an agarose gel (30 X 50 mm) containing anti-platelet antibody. An intermediate strip of aga- rose (10 X 50 mm) located between the antibody-containing gel and the first dimension agarose strip (10 X 50 mm) was then removed and replaced with agarose containing concanavalin A. In control plates, concanavalin A was omitted from the third gel, leaving a spacer gel (10 X 50 mm) containing buffer alone.

Enzyme Digestion Experiments-Washed platelets (8 X IO8 were suspended in 6 ml of human Ringer solution, 2 mM EDTA and treated with neuraminidase (2 units/ml) for 1 h at 37°C. Sialic acid was determined by the method of Svennerbolm (18). For a-chymotrypsin digestion, 8 X 10' washed platelets were suspended in 10 ml of human

Ringer solution, 2 mM EDTA, and treated with a-chymotrypsin (1 mg/ml) for 20 min at 22°C.

Antibody Absorption Experiments-Suspensions of washed plate- lets varying from 6.7 X IO' to 1.2 X IO9 platelets were pelleted by centrifugation at 1,700 X g for 10 min at room tempelature. The supernatant was discarded and the pellet was resuspended with 0.25 ml of antibody and incubated for 30 rnin at 37°C. The platelets were removed by centrifugation at 2500 X g, and the supernatant was employed for CIE studies.

Preparation of Granulocytes and Lymphocytes-Granulocytes were produced from 30 ml of defibrinated blood (to remove platelets) by collecting blood at 22OC into a 125-ml glass Erlenmeyer flask containing glass beads. This was rotated until a clot formed. The blood was then decanted and centrifuged at 1800 X g for 20 min at 4"C, and the plasma was discarded. The blood pellet was then suspended in 22 ml of 3% Dextran-saline in a 100-ml graduated cylinder, held at a 45' angle, and allowed to sediment a t unit gravity a t 37'C for 60 min. The supernatant was centrifuged at 300 X g for 20 min at 4°C to pellet the granulocytes. These were then washed twice with 1% ammonium oxalate (to lyse red blood cells) and once with human Ringer solution, 2 mM EDTA.

Lymphocytes were prepared from defibrinated blood, as above, which was layered onto 22 ml of Ficoll-Hypaque (23.9 g of Ficoll and 50 ml of Hypaque) and centrifuged at 1,500 X g for 20 min at 4°C. The interphase was removed and centrifuged at 1,200 X g for 10 min at 4°C and washed once with 1% ammonium oxalate and twice with 0.01 M phosphate-buffered saline, pH 7.4.

MATERIALS

Agarose (Seakem HGT) was obtained from Marine Colloid Inc., Rockland, Me.). Sucrose, neuraminidase (type IX, Clostridium per- fringens), a-chymotrypsin-TLCK (type VII, bovine pancreas), soy- bean trypsin inhibitor (type 1-S), benzamidine hydrochloride, Ficoll, concanavalin A (grade IV), and insoluble concanavalin A on beaded agarose in 1 M NaCl were obtained from Sigma Chemical Co., St. Louis, Mo. Human albumin was obtained from Cutter Laboratories, Berkeley, Calif. Human fibrinogen (>95% clottable) was a gift of Dr. Margaret Karpatkin, New York University Medical Center. Freund's complete adjuvant was obtained from Miles Laboratories, Elkhart, Ind. Hypaque was obtained from Winthrop Laboratories, New York, N. Y.

RESULTS

Description of the Noma1 CIE Pattern-The first batch of anti-platelet membrane antibody (after 5 booster immuni- zations) provided the CIE pattern shown in Fig. la. At least 17 different antigens were detectable. Fig. lb demonstrates the CIE pattern from anti-platelet membrane antibody ob- tained from the same rabbit after 9 booster immunizations, and Fig. IC demonstrates the CIE pattern after 12 booster immunizations. Note the detection of new as weU as similar antigens after immunization. Fig. Id provides a drawing of 20 different antigens that are fairly consistently observed from more than 28 different normal platelet membrane prepara- tions. Letters are added to the numbers to indicate the identity or source of the antigens.

Detection of Specific Antigens-Typical patterns of im- munoprecipitate shape as well as staining intensity are consis- tently noted and reproducible on repeated determinations from the same membrane preparation. The major antigen which runs close to the anode and stains the most intensely has been numbered 10. It has lines of identity with the immunoprecipitate Peaks 13 and 18. Antigen 1F runs close to the cathode, has a typical hazy immunoprecipitate appear- ance, and has been identified as fibrinogen (Fig. 2). This has also been verified with a specific antibody for human fibrino- gen (data not shown). Antigen 12A has similarly been identi- fied as albumin (Fig. 3). Both 1F and 12A are the only antigens noted when human plasma is employed for the f i t dimension (data not shown). Antigens lF, 2CS, llCS, and 12A are also detected in the platelet cell sap (Fig. 4).

Cell Surface Location ofAntigens-The anti-platelet mem-

4322 Crossed Immunoelectrophoresis of Platelet Membranes

c m + - FIG. 1. CLE of a human platelet membrane preparation, em- which did not contain the antigens was removed and, fresh agar,

ploying an antibody obtained after 5 (a), 9 (6). and 12 (c) containing partially purified rabbit anti-human platelet membrane booster immunizations. Fifty micrograms of 1% Triton-solubilized antibody (2 mg/ml), was poured. The slide was rotated 90" and purified platelet membrane were electrophoresed on a 1% agarose electrophoresed for 18 h a t 55 V. The gel was pressed with filter slide (5 X 5 cm or 8 X 10 cm) in Tris/barbital buffer, pH 8.6, for 3.25 paper, washed, dried, and stained with 0.25% Coomassie Brilliant h a t 150 V. An agarose strip from the gel (4 X 5 cm or 4 X 10 cm) Blue. d, drawing of 20 different antigens observed fairly consistently.

FIG. 2. a, CIE of human fibrinogen (120 ng); 6, CIE of purified platelet membrane preparation (140 pg); c, co-electrophoresis of fibrinogen plus membrane preparation. Note the enhanced height of fibrinogen in c. The time interval of the fvst dimension was run for a shorter interval (1.5 h).

brane antibody was absorbed with various concentrations of washed platelets and then employed in CIE to determine which antigens could be completely adsorbed and the relative disappearance of the absorbed antibodies (Fig. 5). Fig. 5a demonstrates the control pattern before absorption in which

FIG. 3. a. CIE of human albumin (100 ng); b, CIE of purified platelet membrane preparation (50 pg); c, co-electrophoresis of albumin plus membrane' preparation. Note enhanced height of albumin in c. The time interval of the fmt dimension is 2.25 h.

the antibody was diluted with buffer instead of platelets. Fig. 5, b to f i demonstrates the rate of disappearance of these antigens following absorption of the antibody with increasing concentrations of platelets. Note that the immunoprecipitate peaks become higher as the antibody becomes weaker and

Crossed Immunoelectrophoresis of Platelet Membranes 4323

that the immunoprecipitate peaks eventually disappear. Sim- ilar results were obtained with gel-filtered platelets, which were subjected to less mechanical trauma (data not shown). Absorption of the anti-platelet membrane antibody with a comparable volume of red blood cells, granulocytes, or lym- phocytes had no effect on the CIE patterns (data not shown). The patterns of disappearance could be semiquantified by measuring the heights of the immunoprecipitates at each platelet concentration employed to absorb antibody. This was feasible with fairly strong immunoprecipitates which could readily be followed. Antigens lF, 7-8, 10, 13, 14, and 18 disappeared in a linear fashion with increasing platelet con- centration. Sufficient data could not be obtained on the dis- appearance rates of the other antigens.

Crossed Immunoaffinoelectrophoresis-Fig. 6b demon- strates the effect of concanavalin A in an intermediate spacer gel. The immunoprecipitates that react with the ligand are pulled down into the space containing the ligand. Antigens 10, 13,18, and 1F have been pulled down into the concanavalin A lectin, whereas 2CS, 3, 5 (barely visible), 7-8, and 15 are not affected.

Relationship between Antigen 10 and Antigens 13 and 18-As noted in Fig. 1, antigens 13 and 18 have lines of identity with 10. Different platelet membrane preparations exhibit different relationships between antigen 10 and anti- gens 13 and 18. When a CIE has a relatively full 10 peak, with descending cathodal arm, it has relatively absent peaks for 13 and 18 (Fig. 7a). When the descending cathodal arm is absent or incomplete, 10 appears to shift cathodally and 13 and 18 become visible (Fig. 7, band c). Similar changes were observed when a platelet membrane preparation was stored in the absence of proteolytic inhibitors for 3 weeks. Of interest were the results noted after treatment of washed platelets with a- chymotrypsin-TLCK (see below).

Digestion of Intact Washed Platelets with a-Chymotryp- sin-Fig. 8 demonstrates the CIE pattern of platelet mem- branes prepared after incubation of platelets with either buffer (Fig. 8a) or a-chymotrypsin-TLCK, 1 mg/ml for 20 min at

FIG. 6. CIE of human platelet membranes employing con- canavalin A. CIE conditions were as in Fig. 1, except for the insertion of an intermediate spacer gel (1 X 5 cm) containing concanavalin A in 1% agarose prior to electrophoresis into the second dimension. a, spacer gel contains buffer; b, spacer gel contains 250 p g / d of concan-

FIG 4. CIE of platelet cell sap. Platelet cell sap ( 5 0 p g ) was avalin A. Antigens lF, 10, 13, and 18 have been pulled down into the electrophoresed in the fmt dimension, followed by electrophoresis in spacer gel containing concanavalin A, whereas 2CS, 3, 5 (not visible the second dimension against 2 m g / d of rabbit anti-human mem- in photograph), 7-8, and 15 are not affected. (Sharp apparent peak brane antibody. IF, 2CS, IICS, and 12A can be identified. underneath 15 in 6a has been shown to be an artifact.)

FIG. 5. CIE of a human platelet membrane preparation following absorption of rabbit anti-platelet membrane antibody with increasing concentration of washed human platelets. u, 0 platelets; b, 1.25 X 10" platelets; c, 2.5 X 10" platelets; d, 6.0 X IO" platelets; e, 8.0 X 10" platelets; f, 1.0 X 10' platelets.

4324 Crossed Immunoelectrophoresis of Platelet Membranes

FIG. 7. Relationship of antigen 10 with antigens 13 and 18 on CIE of a platelet membrane preparation from normal subjects. CIE conditions were as in Fig. 1. a, antigen 10 has a complete cathodal tail with 13 and 18 not visible; b, absent cathodal tail with 13 and 18 visible; c, relatively more 13 and 18 visible, with apparent cathodal shift of 10.

FIG. 8. Effect of digestion of washed intact platelets with a- chymotrypsin-TLCK. Washed platelets (8 x lo9) were suspended in 10 ml of buffer and treated with either buffer (a) or 1 mg/ml of a- chymotrypsin-TLCK for 20 min at 20°C (b ) . Platelet membrane was then prepared for CIE employing conditions cited in the legend to Fig. 1. Note the decreased height of antigen 10 and the increased heights of antigens 13 and 18.

22°C (Fig. 86). The major antigen is diminished in height, whereas 13 and 18 are increased in height.

Digestion of Intact Washed Platelets with Neuramini- dase-Fig. 9 demonstrates the CIE pattern of platelet mem- branes prepared after incubation of platelets with either buffer (Fig. 9a) or neuraminidase, 2 units/ml for 1 h at 37°C (Fig. 96). There is a cathodal shift of antigen 10, with the disap- pearance of antigens 13 and 18, and the full extension of the cathodal tail of the major antigen 10.

CIE Patterns Obtained from Three Patients with Ghnz- mann's Thrombasthenia-The platelets of patient M. C. were employed on two different occasions for antibody absorption studies and compared to antibody absorption with a compa- rable volume of normal platelets. Fig. 10a demonstrates the CIE following absorption with 1 X lo9 washed platelets. Note the three faint immunoprecipitates which have not been com- pletely eliminated (residual 10, 14-18, and 7-8 as determined by serial absorption; data not shown). This should be com- pared to Fig. lob, where antibody has been absorbed with 1 X lo9 platelets of patient M. C. Note the retention of at least six different immunoprecipitates: lF, 10, 13, 18, and probably 15 and 16 (which were shown to be different from peaks remaining in Fig. 10a (by serial absorption)). Thus, lF, 10.13, 18, and probably 15 and 16 appear to be reduced or absent in patient M. C. with Glanzmann's thrombasthenia. (Decreased platelet fibrinogen has been reported in Glanzmann's throm- basthenia (19,20)). Identical results were obtained on a second occasion. A second patient, M. M., provided sufficient platelets to perform a CIE with her own platelets as the antigens (Fig. 11). A pattern is observed which is different from that seen

FIG. 9. Effect of digestion of washed intact platelets with neuraminidase. Washed platelets (8 X IO") were suspended in 8 ml of buffer and treated with either buffer (a) or neuraminidase (2 units/ ml) for 1 h at 37OC (b ) . Platelet membranes were then prepared for CIE employing conditions cited in the legend to Fig. 1. Note the cathodal shift of antigen 10, with the disappearance of antigens 13 and 18. Hazy peaks in background are artifacts, possibly due to fast running proteins in the fmt dimension which enter buffer and cross- react with antibody in the second dimension.

with normal individuals (see Fig. 1). Note the reduction or absence of lF, 10,13, and 18, as well as of 15 and 16 (since 15 and 16 are not always observed in normal preparations, it is difficult to draw a definite conclusion regarding their apparent absence). In the position where 10 is normally seen, an im- munoprecipitate peak is observed, which stains with a hazy pattern (Fig. loa). This hazy pattern is occasionally seen behind 10 (not fully exposed) in normal individuals (see Fig. 5, 6 and c). Similar results were obtained with platelets from a third patient, L. M. (data not shown).

CIE Pattern Obtained from Two Patients with Bernard- Soulier Syndrome-Two cousins, A. J. and T. H., with the Bernard-Soulier syndrome provided sufficient platelets for CIE employing their own platelet membranes. The CIE pat- tern obtained from A. J. (Fig. 12a) was considerably different from that noted in platelets from normal individuals. Note the decrease in lF, 10, 13, and 18, the exposure of loa, and the cathodal shift of 10. 1F is further decreased relative to the total protein applied to the slide, in light of the diminished major antigen. This should be compared to a normal prepa- ration in which the major antigen represents most of the protein. Identical results were obtained with the cousin (data not shown). The preparation was also subjected to crossed immunoaffioelectrophoresis (Fig. 126) with concanavalin A employed as an insoluble lectin. Note that 10a is not pulled down into the lectin (neither is 7-8; see Fig. 66), whereas the glycoprotein antigens lF, 10.13, and 18 are displaced into the concanavalin A lectin. The same patient was restudied 6

Crossed Immunoelectrophoresis of Platelet Membranes 4325

lo 18 14

7- 8

1 18

10- - 1F

FIG. 10. CIE of a normal platelet membrane preparation following adsorption of anti-platelet membrane antibody with platelets from a patient with Clanzmann's thrombasthenia. a, adsorption with 1 X IO' washed normal platelets; b, absorption with 1 X IOv washed platelets from the patient. Note the retention of a t least six different immunoprecipitates: IF. 10. 13. 18. and probably 15

1 100

and 16. Conditions for CIE were the same as in Fig. 1. FIG. 11. CIE of a platelet membrane preparation obtained

from a patient with Clanzmann's thrombasthenia. Note the reduction or absence of IF. 10. 13, and 18. and the exposure of loa. Conditions for CIE were the same as in Fig. 1.

4

c'?

FIG. 12. CIE of a platelet membrane preparation obtained from a patient with Bernard-Soulier syndrome, employing concanavalin A. Conditions for CIE were the same as in Fig. 1, except for the insertion of an intermediate spacer gel containing insoluble concanavalin A as in Fig. 6. a, spacer gel contains buffer; b, spacer gel contains 350 &ml of concanavalin A. Note that 10a is not pulled down into the lectin.

FIG. 13. CIE of a platelet membrane preparation obtained from a patient with Bernard-Soulier syndrome, electropho- resed on a slide (8 X 10 cm). a, platelet membrane preparation from a normal subject; b. platelet membrane preparation from patient A. J . Note diminished to absent antigens 10, 13 and 18, exposure of loa, and appearance of neoantigen 21. Conditions for CIE were the same as in Fig. 1.

FIG. 14. CIE in the presence of a monospecific antibody for glycocalicin. Conditions for CIE were the same as in Fig. 1, except for the insertion of an intermediate spacer gel containing anti-glyco- calicin antibody ( 6 0 pl/ml). a, CIE of a normal platelet preparation with buffer alone in the spacer gel; b. CIE of the same preparation with anti-glycocalicin antibody in the spacer gel (note the anodal mobility of the antigen 22 Glyc pulled down into the spacer gel); c, CIE of a platelet membrane preparation from patient A. J. with Bernard-Soulier syndrome with anti-glycocalicin antibody in the spacer gel (note the absence of 22 Glyc).

4326 Crossed Immunoelectrophoresis of PLateLet Membranes

months later with a fresh preparation on a larger slide. On this occasion, a similar pattern was noted; however, 10, 13, and 18 were considerably diminished or absent, while 10a increased in height and a neoantigen, 21, could be identified (Fig. 13b). This neoantigen did not react with concanavalin A (data not shown).

CIE Pattern Obtained with a Monospecific Antibody Against Glycocalicin-Fig, 146 demonstrates the location of an antigen which reacts with a monospecific antibody for glycocalicin (Glyc), kindly supplied by Dr. N. 0. Solum of the University of Oslo. In this preparation, the monospecific an- tibody was placed in the intermediate spacer gel. Note the considerable anodal mobility of the antigen, designated 22 Glyc, which has been pulled down into the spacer gel. Fig. 14a is the pattern observed when buffer replaces the antibody in the spacer gel. Fig. 14c is the pattern observed with a platelet membrane preparation from patient A. J. with Ber- nard-Boulier syndrome (see Fig. 13b). Note the absence of Peak 22 Glyc in the presence of anti-glycocalicin antibody in the spacer gel.

DISCUSSION

These data reveal the antigenic complexity of human plate- let membranes as determined by CIE and demonstrate the enhanced resolution of this quantitative procedure over con- ventional immunochemical procedures (21) as well as SDS- PAGE. At least 20 different antigens have been found on the platelet membrane. More than half of these are fairly consis- tently observed in every subject studied. These include lF, 4, 5, 7-8, 9, 10 (major antigen), 12A, 13, 14, and 18. The reasons for not consistently observing all 20 antigens could be related to 1) technical considerations, i.e. the optimum antigen/anti- body ratio for immunoprecipitation may vary depending upon the antigen concentration of the patient’s membrane prepa- ration, or 2) the presence or absence of specific antigens in different subjects.

Absorption studies with increasing concentrations of washed platelets resulted in the disappearance of a t least 11 antigens at different rates. This could reflect differing concen- trations or antigenicity of the various membrane antigens. It could also reflect the relative accessibility of the various antigens in the membrane, as suggested by Owen and Salton (12). The immunoprecipitates for six of these antigens, lF, 7- 8,10,13,14, and 18, disappear at a constant rate, with eventual disappearance, employing increasing concentrations of plate- lets. This is the predicted pattern of disappearance for external surface-located antigens. Furthermore, the changes noted with 10, 13, and 18 following treatment of intact platelets with neuraminidase or a-chymotrypsin, also support a surface lo- cation for these antigens. At least four of these surface anti- gens (lF, 10, 13, and 18) react with concanavalin A, indicating that they are glycoproteins. At least six antigens, 2CS, 3,5, 7- 8, loa, and 16, do not react with concanavalin A, under the conditions employed.

It is conceivable that some of the immunoprecipitates rep- resent close associations of several membrane antigens. These complexes may represent an in vivo association of membrane components due to interactions in the microenvironment (22). This may be the case with the major antigen, 10, and antigens 13 and 18, which have lines of identity with 10 and which appear to be related. For example, platelets from subjects with full 10 antigen peaks have absent or diminished 13 and 18 antigen peaks, whereas platelets from subjects with 10 antigen peaks which have absent to incomplete cathodal arms have increased 13 and 18 antigen peaks. This appears to be related to in vivo or in vitro proteolysis, since preparations stored in the absence of proteolytic inhibitors lose the cathodal

arm of the 10 antigen peak and gain the 13 and 18 antigen peaks. Of interest were the results obtained with digestion experiments employing a-chymotrypsin. In this situation, the 10 antigen peak decreased in height as 13 and 18 increased in height. A cathodal shift was not noted, as was the case with endogenous proteolysis. Studies with neuraminidase digestion of intact platelets reveal the presence of sialic acid on antigen 10 and suggest its presence on 13 and 18, since the mobility of 10 becomes more cathodal and the immunogenicity of 13 and 18 disappears. This again suggests a possible structural com- plex or organizational relationship among 10, 13, and 18. This is further supported by CIE studies on platelets from patients with Glanzmann’s thrombasthenia and Bernard-Soulier syn- drome, in which the major antigen, 10, as well as antigens 13, 18, and lF, are diminished to absent in both.

We are indebted to Drs. Hagen, Bjerrum, and Solum (23) for making a preprint of their work on CIE of human platelets available to us while our manuscript was in preparation (24).2 They employed an antibody against intact platelets and de- scribed CIE patterns noted with intact platelets as well as purified membranes as their antigen (solubilized with 1% Triton X-100). Twenty immunoprecipitates were noted with intact platelets (albumin, Factor VIII, and fibrinogen were identified with monospecific antibodies), and eight immuno- precipitates were detected with isolated membranes. Four to six surface antigens were noted, four of which were sialogly- coproteins.

Our CIE studies of the platelet membranes from two pa- tients with Bernard-Soulier syndrome and three patients with Glanzmann’s thrombasthenia, when compared to the results obtained with SDS-PAGE, point to the complexity of mem- brane abnormalities in these disorders. SDS-PAGE detects denatured proteins identified by carbohydrate staining or “H labeling of sialic acid. CIE detects a less denatured protein identified by its immunogenic capacity and antigenic proper- ties. In Bernard-Soulier syndrome, both techniques are capa- ble of detecting absent to diminished GP I. However, CIE also detects an absent to diminished major antigen, 10 (as well as 13 and 18), the exposure of a “hidden” antigen, loa, and the presence of a neoantigen, 21 (which is not detectable on normal platelets). In Glanzmann’s thrombasthenia, SDS- PAGE detects the absence or diminished presence of two glycoproteins, GP IIb and GP IIIa, whereas CIE detects the absence of the major antigen (as well as 13 and 18) and the exposure of a hidden antigen, loa.

It is conceivable that the alterations reported with SDS- PAGE partially reflect in vitro denaturation events which result in the separation of membrane proteins or complexes and that CIE (which results in less denaturation or a different type of denaturation) reveals a pattern in which the abnor- malities present in these disorders reflect themselves differ- ently. It is also conceivable that the missing membrane anti- gens in Glanzmann’s thrombasthenia and Bernard-Soulier syndrome are present but not recovered by the membrane preparation procedure. We consider this unlikely because of the presence of a neoantigen. In light of our findings on CIE, it is tempting to postulate that the antigenic differences noted in patients with Glanzmann’s thrombasthenia and Bernard- Soulier syndrome may reflect differences in endogenous mem- brane proteolysis. For example, a neoantigen, 21, which is present on platelet membranes from the Bernard-Soulier pa-

’ Presented at the Platelet Membrane Workshop of the VIIth International Congress on Thrombosis and Haemostasis, July 14, 1979, London, England, and at the 22nd Annual Meeting of the American Society of Hematology, December 4, 1979, Phoenix, Ari- zona.

Crossed Immunoelectrophoresis of Platelet Membranes 4377

tient, may reflect a proteolytic by-product (not detectable on normal platelet membranes). In this regard, it is of interest that platelets from a patient with Bernard-Soulier syndrome were noted to have numerous low molecular weight polypep- tides (absent in normal platelets) as determined by the lac- toperoxidase '"I labeling technique (25). Furthermore, a Ca"- dependent protease has been reported in platelet lysates (26).

Acknowledgments-We are indebted to the New York Blood Cen- ter for their supply of fresh platelet-rich plasma, to Dr. Margaret Johnson, Wilmington Medical Center, for making the Bernard-Soulier and Glanzmann's thrombasthenic patients available to us, and to Drs. M. R. J. Salton and Mary-Lynne Collins of New York University Medical School, Department of Microbiology, for helpful discussions and assistance.

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