Immunochemical Evidence for Protein Abnormalities in Platelets from Patients with Glanzmann's Thrombasthenia and Bernard-Soulier Syndrome

Abstract

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 ...

Glycoproteins of Platelet Membranes from Glanzmann's Thrombasthenia. A Comparison with Normal Using Carbohydrate-Specific or Protein-Specific Labelling Techniques and High-Resolution Two-Dimensional Gel Electrophoresis

“…A high molecular weight glycoprotein is present in normals but absent in Glanzmann's thrombasthenia platelets. There is a possibility that this glycoprotein represents a coinplex of IlbAl and IIIaAl which in the presence of nonionic detergents are coprecipitated by antisera in crossed immunoelectrophoresis [13]. However this glycoprotein is The absence or severe reduction of two major membrane glycoproteins (IIbA1 and IIIaA1) in Glanzmann's thrombasthenia platelets seems to be the consequence or the cause of a more general perturbation of the membrane surface as viewed by different surface-labelling techniques.…”

“…When immunoprecipitation line 16 [13] was cut out of the agarose and electrophoresed on an sodium dodecyl sulphate/polyacrylamide gel, in addition to glycoproteins IIb and IIIa (one-dimensional nomenclature), a third band with a molecular weight of 48000 5000 could be seen (Kunicki, T., personal communication) which is approximately the same size as glycoprotein VII.…”

“…Both PLA1 and a-actinin have been found to be greatly reduced or absent in Glanzmann's thrombasthenia platelets [11,12]. The absence of, or the severe reduction in, glycoproteins IIb and IIIa in Glanzmann's thrombasthenia platelets has been confirmed using a crossed-immunoelectrophoretic technique with a rabbit antiserum directed against normal whole platelets [13]. In addition to these two glycoprotein defects a number of recent findings indicate that in Glanzmann's thrombasthenia platelets a general perturbation of the membrane surface may be present.…”

Glanzmann's Thrombasthenia Type II in a Saudi Child: A Case Report

“…2 In type I, glycoproteins IIa and IIIb are absent, and therefore platelets do not bind fibrinogen. In type II, the platelets are reduced to 10 to 20% of normal 11 and are capable of binding subnormal amounts of fibrinogen. 12,13 The platelets also fail to aggregate in response to various aggregating agents.…”

A unique combination of inhibitory and partially activating mutations in β3 of a patient with variant-type Glanzmann thrombasthenia

“…Historically, this patient was one of the first GT patients to be characterized and possessed of the order of 10% residual IIbb3 able to bind fibrinogen (Fg) when platelets were incubated with ADP [7][8][9][10][11]. The effects of the L196P and C598Y substitutions on b3 function have both been previously examined by site-directed mutagenesis followed by transfection in CHO cells [4,12].…”