A B S T R A C T Crossed immunoelectrophoresis of Triton X-100 solubilized proteins from normal and abnormal platelets was performed with rabbit antibodies raised against normnal platelets. In Bernard-Soulier platelets protein 13 was not detected, and neither the amphiphilic (probably GP Ib) nor the hydrophilic (glycocalicin) glycocalicin-related proteins were seen when monospecific antiglycocalicin antiserum was used. The most prominent precipitate, 16, and platelet fibrinogen, 24 were not detected in platelets of two patients with type I thrombasthenia, whereas in one patient with type II thrombasthenia fibrinogen was clearly detected, but the amount of protein 16 remained severely reduced. Protein 16 was heavily labeled after lactoperoxidase-catalyzed 1251 iodination ofnormal platelets, and was precipitated by IgG-L, an alloantibody from a polytransfused thrombasthenic patient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or protein 16 cut out from immunoplates showed two 125I-labeled glycoprotein bands, which migrate as GP Ilb and GP Illa. SDS-PAGE of 1251_ labeled type I thrombasthenic platelets showed no periodic acid-Schiff bands or peaks of radioactivity in the GP Ilb and GP Illa regions, whereas in the GP I region both the periodic acid-Schiff band intensity and the radiolabeling were within the normal range. Autoradiography after crossed immunoelectrophoresis of iodinated thrombasthenic platelets showed that the bulk of radioactivity was bound to protein 17. This glycoprotein, which was also present in normal and Bernard-Soulier platelets, migrates in the GP I region on SDS-PAGE. Thus, the bulk of radioactivity ob- (7), and glycocalicin (GPS or GP Is) couild not be dletected (8,9). \Ve have found a normal pattern of protein (7,10) and a normal distribution of radioactivity after SDS-PAGE of Bernard-Soulier platelets that ha(l been labele(d by lactoperoxidase-catalvzed iodination (10), whereas others have reported a grossly abnormial protein pattern and distribution of 1251-labeled proteins (11).Thrombasthenic platelets show a strongl redtuced PAS stain intensity of GP Ilb and GP lIla (GP III) comparedl to normiial (8, 12, 13) anid very little ra(lioactivity was seen in these regions after SDS-PAGE of iodinatedl, thromnibastheniic platelets (10, 13). However, SDS-PAGE is not well suiitedl for quantitative measturemenits of' proteins, anlI such an anialysis is further complicated by the facit that certaiin proteinis may comigrate in some SDS-PAGE systems (10,14). Crossed immllunoelectrophoresis in the presence of nlonlionlic detergent has proved to be a useful tool in the stuidy of imemiibrane proteins (15, 16). This two-dimensionial system permits qualitative characterization as well as a semi-quantitative estimate of the individual protein antigens. Crossed immllunoelectrophoresis of Triton X-100 extracts of normal platelets using corresponding rabbit antibodies has revealed more than 20 precipitates, andl several of these have been characterized according to their subcellular ...
Whole human platelets and platelet membranes have been solubilized in 1 % Triton X-100, and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies raised against either whole platelets or isolated membranes. 90 % of the platelet proteins were solubilized by this extraction. About twenty immunoprecipitates were observed using the extracts obtained from whole platelets, whereas normally eight immunoprecipitates were seen with extracts from isolated membranes. Albumin, factor VIII and fibrinogen were identified with monospecific antibodies.Correlation of the patterns obtained for platelets or membranes was obtained by addition experiments, by crossed-line immunoelectrophoresis and by crossed immunoelectrophoresis of a mixture of extracts from unlabeled whole platelets and membranes isolated from platelets labeled by lactoperoxidase-catalyzed "' I iodination.Four sialoglycoproteins were identified by their reduced electrophoretic migration after neuraminidase treatment, and six proteins interacted with various lectins, indicating them to be glycosylated.Seven amphiphilic proteins were identified by charge-shift crossed immunoelectrophoresis, and nine by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose.The topographical arrangement of the membrane proteins was examined with lactoperoxidasecatalyzed "'I-labeled platelets as antigens, and by antibodies absorbed with a suspension of whole platelets. Four and six radioactively labeled precipitates could be identified using the platelet and membrane extracts, respectively, indicating them to be exposed at the outer platelet surface. This was confirmed by the use of antibodies absorbed with intact platelets.Blood platelets play a crucial role in the arrest of bleeding. Adhesion to subendothelial tissue, secretion of intracellularly stored material as well as aggregate formation are essential steps in the sequence of reactions which platelets undergo during the hemostatic process [I]. There is evidence that platelet glycoproteins play important roles in these reactions, and it is thus of interest to characterize the platelet protein composition and surface structure. Lactoperoxidasecatalyzed 1251 iodination of whole platelets followed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis has demonstrated more than twenty polypeptides, and at least seven of these were radioactively labeled indicating a surface location in the intact cell [2]. However, functional studies on proteins solubilized by sodium dodecyl sulphate are difficult since this detergent tends to denature proteins [3]. A -.~ Enzyme. Neuraminidase (EC 3.2.1.18). high degree of solubilization has been achieved by extraction of membrane proteins with non-ionic detergents [3,4]. Since the antigenicity of the proteins is retained, such extracts are suitable for immunochemical examination, e.g. quantitative immunoelectrophoresis [4]. Erythrocyte membrane proteins solubilized in a non-ionicdetergent have been examined in crossed immunoelectropho...
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