Characterization of Human Platelet Proteins Solubilized with Triton X‐100 and Examined by Crossed Immunoelectrophoresis

Abstract

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...

Characterization of an anti‐thrombospondin monoclonal antibody (P8) that inhibits human blood platelet functions

“…After 30 rnin incubation at 4°C the solubilized platelet extract was centrifuged in an Eppendorf microfuge for 5 min and the supernatant was carefully removed and recentrifuged in the microfuge for 5 min before being stored at -70 "C. Crossed immunoelectrophoresis of platelet extracts or purified human platelet thrombospondin was carried out as described by Hagen et al [39]. Approximately 80 pg solubilized platelets (or 4.5 pg isolated thrombospondin) were separated in a first-dimension agarose (1%) gel in Triton X-lOO/Tris/glycine (pH 8.7) at 10 V/cm, for 60 min at a temperature of 10 "C. The second-dimension separation was performed, on a GelBond film (FMC Corp., Rockland, ME, USA) at 2 V/cm, for 18 h at a temperature of 10°C.…”

Surface Components of Platelets and Their Changes during Storage

“…References Two-dimensional immunoelectrophoresis has recently been used for the detection of platelet membrane antigens in normal and pathological conditions [25,26]. This tech nique may prove valuable in evaluating sur face changes during storage.…”

Protein and Glycoprotein Abnormalities in an Unusual Subtype of Glanzmann’s Thrombasthenia

“…In this way we identified albumin, fibrinogen, and factor VUI-related antigen. According to Hagen et al [20], and Kunicki et al [21], the most prominent precipitate was identified as GP Ilb-IIIa complex, previously designated '16' by Hagen et al [20]. This complex is the major antigen of platelet membranes [19,22], and of the a-granules [23],…”