Involvement of divalent cations in the complex between the platelet glycoproteins IIb and IIIa

Biochimica et Biophysica Acta, 701 (1982) 1-6 Elsevier Biomedical Press

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BBA 31041

INVOLVEMENT OF DIVALENT CATIONS IN THE COMPLEX BETWEEN THE PLATELET GLYCOPROTEINS lib AND Ilia INGER HAGEN a , , OLE JANNIK BJERRUM b GEIR GOGSTAD a RANDI KORSMO a and NILS OLAV SOLUM a

a Research Institute for Internal Medicine, Section on Hemostasis and Thrombosis, University of Oslo, Oslo 1 (Norway) and h The Protein Laboratory, University of Copenhagen, DK-2200, Copenhagen N (Denmark) (Received June 17th, 1981)

Key words: Divalent cation," Glycoprotein comples; (Platelet)

The major immunoprecipitate (No. 16) seen on crossed immunoelectrophoresis of Triton X-100-solubilized platelet proteins against whole platelet antibodies represents a complex containing the membrane glycoproreins lib and Ilia. When EDTA is present during the soluhilization, immunoprecipltate 16 as such is not observed, and two new arcs, termed 16a and 16b, appear. As with 16 these immunoprecipltates become radioactively labelled on lactoperoxidase-catalyzed iodination of platelets. Immunoprecipitate 16a showed partial immunochemical identity with 16, and was precipitated by an antibody raised against immunoprecipitate 16. The areas covered by immunoprecipitates 16, 16a and 16b were strongly reduced compared to normal with platelets from a patient with Glanzmann's thrombasthenia type II. Such platelets are known to contain reduced, amounts of glycoproteins lib and llla. The new arcs appearing when divalent cations are chelated by EDTA thus represent proteins derived from the immunoprecipitate 16 proteins, and divalent cations seem to be necessary to preserve the protein complex containing glycoprotein lib and Illa. The different complex formations between the components of immunoprecipitate 16 may reflect biochemical alterations of functional importance.

Introduction Crossed immunoelectrophoresis in the presence of a nonionic detergent has proved to be a useful method for characterization of membrane proteins (for review see Ref. 1). Analysis of Triton X-100solubilized platelet proteins has revealed that the most prominent immunoprecipitate (No. 16) contains two glycoproteins, termed IIb and Ilia [2]. These glycoproteins are not detected or are present in greatly reduced amounts in platelets from patients with Glanzmann's thrombasthenia [2-4], and are believed to play an important role in platelet aggregation. Despite the fact that glycoproteins

Abbreviation: SDS, sodium dodecyi sulphate. 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

lib and Ilia are enriched in isolated membranes compared to whole platelets [6], crossed immunoelectrophoresis of proteins solubilized from isolated membranes revealed considerable variation in the appearance of immunoprecipitate 16. In many experiments no prominent immunoprecipitate corresponding to No. 16 was seen, but new arcs appeared [5], indicating that the complex did not form, or that some sort of splitting had occurred under these conditions. The aim of the present study was to establish the conditions resulting in these changes, and to characterize the immunochemical relationship between the antigens contained in immunoprecipitate 16. Our resuits suggest that glycoproteins lib and IIIa exist as a complex stabilized by divalent cations, and that this complex can be split by EDTA.

phoresis was performed at 8-10 V/cm for 45 min, and the second-dimension overnight at 1-2 V/cm.

Materials

Chemicals and radiochemicals. Agarose, type HSA, was from Litex, Glostrup, Denmark. Trypsin and chymotrypsin were from Sigma Chemical Co., St. Louis, U.S.A. Thrombin was a gift from Dr. Frank Brosstad, Oslo. Concanavalin A was from Pharmacia Fine Chemicals, Uppsala, Sweden, and leupeptin from Sigma Chemical Co., St. Louis, U.S.A. usI, code IT.3, was from Institutt for Energiteknikk, Kjeller, Norway. Antibodies. Rabbit antibodies to whole human platelets (46 mg protein/ml) and antiglycocalicin antiserum were obtained as described in Refs. 5 and 7, respectively. Antiserum to the components of immunoprecipitate 16 was obtained by injecting rabbits with immunoprecipitate which had been cut out from the washed, unstained immunoplates. The antibody to 132 microglobulin was purchased from Dakopatts, Copenhagen, Denmark.

Results and Discussion

Methods

Blood was obtained from registered blood donors and, with informed consent, from a patient with Glanzmann's thrombasthenia, type II. The platelets were washed as described elsewhere [5]. Surface labelling of washed platelets was performed by lactoperoxidase-catalyzed iodination [8]. Protein was determined according to the method of Miller [9]. Platelet membranes were prepared either by a modified glycerol-lysis technique [10,11], or by French Pressure Cell homogenization [12]. Solubilization of platelets and isolated membranes were performed as described in Ref. 5. The final concentration of detergent was 1% (v/v). The supernatants obtained after ultracentrifugation (74000 X g, 60 rain, 4°C) were used in the subsequent immunochemical studies. Solubilization of platelets in the presence of leupeptin was done as in Ref. 13. Crossed immunoelectrophoresis and crossed affinity immunoelectrophoresis were performed on 5 X 7 cm glass plates using 1% (w/v) agarose containing 1% (v/v) Triton X-100 as described in Refs. 14 and 15, respectively. The electrophoresis buffer consisted of 0.038 M Tris, and 0.1 M glycine, pH 8.7. The first-dimension electro-

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The major immunoprecipitate seen upon crossed immunoelectrophoresis of Triton X- 100-solubilized platelet proteins (No. 16) has been shown to contain the glycoproteins lib and Ilia [2]. These glycoproteins are present in the platelet surface membranes as well as in the a-granule membranes, and the surface membrane components become radioactively labelled by lactoperoxidase-catalyzed 125Iiodination of whole platelets [2,4,16]. Crossed immunoelectrophoresis of Triton-solubilized proteins from isolated membranes revealed considerable variation in the appearance of immunoprecipitate 16 (Figs. 1A-D). In some instances No. 16 had a similar shape and appearance to that normally seen on analysis of whole platelets (Fig. 1A, termed type A pattern), whereas in other cases no traces of immunoprecipitate 16 as such could be observed (Fig. 1D, termed type D pattern). However, glycoproteins lib and IIIa were present normally in all the extracts, as evidenced by SDSpolyacrylamide gel electrophoresis (not shown). Figs. 1B and C represent typical intermediate patterns. A disappearance of the cathodic part of immunoprecipitate 16 is seen concomitant with the appearance of at least two new arcs. For reasons discussed below, these were termed 16a and 16b. Occasionally, a third immunoprecipitate, 16c, was seen (Figs. 1B and 2B). The pattern variability was seen regardless of the method used for membrane isolation. The essential factor for obtaining a type D pattern seemed to be the presence of EDTA during the solubilization [17,18]. Membranes washed in a medium without EDTA and solubilized i n 0:038 M Tris/0.1 M glycine, pH 8.5, containing 1% (v/v) Triton X-100 gave a type A pattern on crossed immunoelectrophoresis. However, if EDTA was added to the solubilization medium (1 mM), a type D pattern was obtained. Partial or complete splitting was also seen when the membranes were washed in a medium containing EDTA, followed by solubilization in the Tris/glycine/Triton buffer. This was probably due to the presence of residual EDTA from the washing fluid. At the pH used

Fig. 1. Crossed immunoelectrophoresis of Triton X-100solubilized proteins from platelet membranes (approx. 25/~g) using antibodies raised against whole platelets (750 #g/cm2). A, membranes washed in the absence of EDTA prior to solubilization; B, membranes washed in the presence of 0.6 mM EDTA prior to solubilization; C, membranes washed in the presence of 1 mM EDTA prior to solubilization; and D, membranes washed in the presence of 2 mM EDTA prior to solubilization. A pattern as in D was also obtained when membranes prepared as in A were solubilized in the presence of 1 mM EDTA. Arrows indicate partial immunochemical identity. Note the disappearance of immunoprecipitate 16 in D. The numbered arcs represent albumin (No. 6) and glycocalicinrelated protein (glycoprotein Ib, No. 13).

(pH 8.5), or at higher pH values, the splitting of immunoprecipitate 16 seemed to be particularly sensitive to small amounts of EDTA (Gogstad, unpublished results). The transition between 16 and 16a + 16b could be verified in Triton extracts of whole platelets, provided that EDTA ( ~ 1 mM) was present during solubilization (not shown).

Several lines of evidence indicate that the proteins contained in immunoprecipitates 16a and 16b are derived from those contained in 16. Firstly, the pattern variability was only observed with immunoprecipitate 16, provided that the same batch of antibodies was used. 16a and 16b were only seen when 16 was absent or present in reduced amounts compared to the type A pattern. 16a and 16b represent surface membrane components, as they become labelled by lactoperoxidase-catalyzed 125I-iodination of whole platelets (Fig. 2). Fartial immunochemical identity between 16a and 16 was seen (Figs. 1B and C, arrow), and 16a and 16 were precipitated by an antibody raised against immunoprecipitate 16 (Figs. 3A and B). Furthermore, coelectrophoresis of a mixture of two different membrane extracts, one of type A and the other of type D, showed reactions of partial identity between 16a and 16, but no reactions between 16b and 16 (not shown). A detailed analysis of the reactions of partial identity between immunoprecipitate 16 and the derived precipitates is difficult, due to the various degrees of splitting seen from one experiment to the other. Furthermore, the patterns are disturbed by the presence of several other immunoprecipitates representing different proteins. However, it is consistent that immunoprecipitate 16b in the present figures does not show obvious cross-reactions with 16, indicating that the determinants are cryptic (shielded) in the complex. The proteins contained in immunoprecipitate 16b (and 16) were present in strongly reduced amounts in the membranes from a patient with Glanzmann's thrombasthenia, type II (Figs. 4A and B). Immunochemical analysis of such platelets has previously demonstrated that immunoprecipitate 16 is present in reduced amounts (13% of normal) in these platelets [2], and that this is a specific abnormality in these platdets as observed by crossed immunodectrophoresis. Finally, the observed events do not seem to result from an unspecific aggregation of membrane proteins. This is supported by the fact that two well-defined membrane constituents, glycoprotein Ib (glycocalicin-rdated protein) and the fl2-microglobulin bearing histocompatibility antigen (HLA-ABC) [19], contained in immunopre-

Fig. 2. (left-hand figure) Autoradiography of the immunoplate obtained by crossed immunoelectrophoresis of Triton X- 100-solubilized membrane proteins isolated from t25I-iodinated whole platelets. The corresponding protein stained immunoplate is shown in Fig. IC. Fig. 3. (two right-hand figures) Crossed immunoelectrophoresis of proteins solubilized from platelet membranes using whole platelet antibodies. The intermediate gel contained antiserum raised against immunoprecipitate 16. A, membranes washed in the absence of EDTA prior to solubiliz'ation, and B, membranes washed in the presence of 2 mM EDTA prior to solubilization. Corresponding controls with no antiserum included in the intermediate gel are shown in Figs. IA and D, respectively. Note that No. 16 and 16a are retained in the intermediate gel compared to the controls, and that the antiserum also contained antibodies to albumin (No. 6).

Fig. 4. Crossed immunoelectrophoresis of platelet membranes washed in the presence of I mM EDTA and solubilized in Triton X-100 using antibodies raised against whole platelets. A, membranes isolated from normal platelets, and B, membranes isolated from thrombasthenic (type II) platelets. Arrow indicates immunoprecipitate 16a. Note that 16, 16a and 16b .are absent or present in strongly reduced amounts in the thrombasthenic membranes.

cipitates 13 and 20, respectively, are not involved in the formation of immunoprecipitates 16, 16a or 16b (Figs. 4A and 5A and B). Almost identical patterns were obtained upon repeated examination of one and the same extract. This was seen also when EDTA (1-5 mM) or divalent cations (1-5 mM of Zn 2+, Ca 2+ or Mg2 + ) were added to the extract (not shown), indicating that little or no transformation occurred after the proteins had been solubilized in Triton X-100. This suggests that the patterns of partial identity observed do not derive from interactions between the proteins during electrophoresis. The relationships between glycoproteins IIb and Ilia on the one hand and immunoprecipitates 16a, 16b and 16c on the other, is not dear. Crossed affinity immunoelectrophoresis using concanavalin A showed a strong interaction between 16a and the lectin, whereas 16b was practically unaffected (Figs. 6A and B). Interaction between 16 and concanavalin A has been demonstrated previously [5]. Kunicki and Aster [20] have shown that glyco-

Fig. 5. Crossed immunoelectrophoresis of Triton X-100solubilized platelet proteins using anti-platelet antibodies, followed by incubation of the immunoplate with 125I-labelled antibodies to flE-microglobulin. For experimental details see Ref. 19. A, protein stain, and B, autoradiography, f12" microglobulin is part of the histocompatibility antigen (HLAABC) which by this experiment is shown to correspond to immunoprecipitate 20 in our reference pattern [5].

protein IIb appears in the flow-through fraction on affinity chromatography with concanavalin A, whereas glycoprotein IIIa is retained on the column. This indicates that 16a represents glycoprotein IIIa or split products from this glycoprotein, and that 16b may represent or be derived from glycoprotein IIb. The immunoprecipitate termed 16c appears as an arc congruent with 16b. 16c

might represent the fl- or a-polypeptide chain of glycoprotein IIb, but, since the antigenicity of proteins is often lost upon reduction, this explanation seems less likely. Another possibility would be that 16c represents a complex of glycoprotein IIb (or glycoprotein IIb-derived) molecules. Shulman and Karpatkin [21] suggested that the splitting of immunoprecipitate 16 (termed 10 in their nomenclature) was due to a proteolytic event, since the splitting was seen in extracts kept in the absence of proteolytic inhibitors. Similar effects were also seen after chymotrypsin treatment of the platelets. However, we were unable to detect any effect on the appearance of immunoprecipitate 16 following treatment of the platelets with trypsin, thrombin or chymotrypsin (data not shown). Also, it seems unlikely that the splitting of immunoprecipitate 16 is a result of the action of a Ca 2+activated protease, since solubilization of the proteins in the presence of leupeptin or other inhibitors of the Ca 2+-activated protease did not reveal a type D pattern on crossed immunoelectrophoresis (not shown). It is not clear whether glycoproteins IIb and IIIa exist in the membrane as separate glycoproreins that rapidly associate upon solubilization, or whether they exist in the membrane as a complex that is stabilized by divalent cations (Ca 2+ ?). The experiments described above seem to favour the latter explanation. However, whatever the native condition may be, it is tempting to speculate that the different patterns may reflect biochemical alterations of functional importance. Platelet activation has been associated with mobilization of Ca 2+ from platelet subcellular structures, resulting in an increase of the concentration of Ca 2÷ in the cytosol [22]. Further studies on this topic are in progress.

Acknowledgements Fig. 6. Crossed affinity immunoelectrophoresis of platelet membranes washed in the presence of I mM EDTA and solubilized in Triton X-100. The antibodies were raised against isolated membranes (550 #g/cm2). A, control, no lectin incorporated in the first-dimension gel; and B, concanavalin A (100 # g / c m 2) was incorporated in the first-dimension gel and added to the sample before application. Immunoprecipitate 16 and 16a were affected by concanavalin A, whereas 16b was practically unaltered.

We thank Dr. T. Plesener, Copenhagen, for the use of radioactively labelled f l 2 " m i c r o g l ° b u l i n antibody. The work was supported by the Norwegian Council on Cardiovascular Diseases.

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