High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes

The proteins and glycoproteins of human blood platelets and platelet membranes in both the reduced and the unreduced states have been analysed by isoelectric focusing and sodium dodecyl sulphate-discontinuous polyacrylamide gel electrophoresis in a two-dimensional technique. Gels which had been stained with periodic acid-Schiff's reagent could be counter-stained with Coomassie Brilliant Blue, simplifying the recognition of components which stain with both reagents. The major glycoproteins and some of the proteins have been identified and the characteristics of the membrane and of the whole platelet components established in this system.     

Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes.

By using highly purified surface and intracellular membrane fractions prepared from human platelets by free-flow electrophoresis, the polypeptide and glycopeptides of these membranes have been characterized by high-resolution gel electrophoresis under reducing and non-reducing conditions. Silver staining and a variety of glycoprotein-staining procedures have been applied to identify the major components. The principal finding was the clear disparity between the distribution patterns for these two membrane fractions. There are proportionately more low-Mr acidic components present in the intracellular membrane than in the surface-derived membrane. Of the major platelet surface glycoproteins GPIb, IIb, IIIa and IIIb (or IV) well expressed in the surface membrane only, GPIIb and IIIa appear as trace components in the intracellular membrane. The cytoskeleton proteins, actin, myosin, tropomyosin, actin-binding protein and alpha-actinin are prominent features of the surface membrane and essentially absent from the intracellular membrane. Neuraminidase treatment at the whole-cell level, before homogenization, which is an essential requirement for good resolution of the two membrane subfractions, modifies a number of the glycoprotein subunits with respect to their pI characteristics, suggesting much molecular micro-heterogeneity with respect to sialic acid content. A comparison of the staining characteristics of the major glycoproteins with periodic acid/Schiff's reagent and concanavalin A/peroxidase detection and a combined procedure revealed significant differences in associated carbohydrate structures, and the major concanavalin A-binding component was shown to be GPIIIa. These observations are discussed in the context of functional activities of both membrane systems in the physiological behaviour of the platelet.     

Inherited abnormalities of platelet glycoproteins

Glanzmann's thrombasthenia and the Bernard-Soulier syndrome are inherited blood disorders characterized by abnormalities in different aspects of platelet function during haemostasis. Platelets from patients with thrombasthenia do not aggregate in response to the normal physiological platelet aggregation inducing stimuli, while Bernard-Soulier platelet have a reduced capacity to adhere to exposed subendothelium. Deficiencies of different membrane glycoproteins have been located in the platelets of both disorders and suggest specific roles for membrane glycoproteins in different aspects of platelet function.     

Platelet membrane glycoproteins: role in primary hemostasis and component antigens

The biochemical details of the platelet surface as they relate to normal platelet function have been elucidated through study of labeled membranes from both normal platelets and those with congenitially defective function. Several cytoadhesive glycoprotein complexes which are integral components of the platelet membrane have been demonstrated to act as important receptors for extracellular matrix macromolecules. Glycoproteins Ia/IIa (collagen receptor), Ic/IIa (fibronectin receptor), and IIb/IIIa (fibrinogen receptor) belong to a family of cytoadhesive complexes called the integrins, while glycoprotein Ib/IX, the major von Willebrand receptor, has different features. These same major glycoproteins comprise all of the alloantigens and most of the autoantigens that have been characterized. Glycoprotein IIb/IIIa contains the alloantigens, PlA (Zw), Bak (Lek), and Pen (Yuk), as well as the most frequent target antigenic sites for anti-platelet autoantibodies. Because a number of platelet alloantigens were discovered independently by more than one group, nomenclature is confusing at present, although a system analogous to that used for histocompatibility antigens has been proposed. Precise identification of the antigenic epitopes has not yet been accomplished for all of the platelet antigens. Current research efforts include characterization of antigenic epitopes, elucidation of mechanisms by which platelet immunization occurs, and determination of the clinical implications of the presence of various platelet antibodies.     

Purification of glycoproteins IIb and III from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein IIb-III complex

Human platelet membrane glycoproteins IIb and III are two major integral membrane components that have been identified as sites mediating thrombin-induced aggregation. For purposes of our study, glycoproteins IIb and III were solubilized by extracting platelet plasma membranes with a buffer containing 0.1% Triton X-100 and were separated by gel filtration chromatography on Sephacryl S-300, employing Triton X-100-containing column buffers with or without urea or guanidine hydrochloride. The physical properties of the purified glycoproteins were: for glycoprotein IIb, Rs = 61 A, s20.w = 4.7, f/f0 = 1.7, Mr = 125,000 (hydrodynamic values), Mr = 136,000 (sodium dodecyl sulfate gels); for glycoprotein III, Rs = 67 A, s20,w = 3.2 f/f0 = 2.1, Mr = 93,000 (hydrodynamic values), Mr = 95,000 (sodium dodecyl sulfate gels). Although the amino acid compositions of the two glycoproteins were similar, antibodies raised against glycoprotein IIb did not crossreact with glycoprotein III. If divalent cations were not chelated in the Triton extract, glycoproteins IIb and III coeluted during gel filtration chromatography (apparent Stokes radius of 71 A) and co-sedimented on sucrose gradients (apparent s20.w of 8.6), from which Mr = 265,000 was calculated. Glycoproteins IIb and III were coprecipitated by an antibody monospecific for glycoprotein IIb. The two glycoproteins dissociated into monomers when EDTA was added to Triton lysates. Readdition of Ca2+ caused them to reassociate into a complex with physical properties similar to those of the complex in the original Triton lysate. The data show that glycoproteins IIb and III are a heterodimer complex, that complex formation depends upon the presence of Ca2+, and that chelation of Ca2+ causes dissociation into monomeric glycoproteins.     

Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes

Platelets have previously been shown to contain a membrane skeleton that is composed of actin filaments, actin-binding protein, and three membrane glycoproteins (GP), GP Ib, GP Ia, and a minor glycoprotein of Mr = 250,000. The present study was designed to determine how the membrane glycoproteins were linked to actin filaments. Unstimulated platelets were lysed with Triton X-100, and the membrane skeleton was isolated on sucrose density gradients or by high-speed centrifugation. The association of the membrane glycoproteins with the actin filaments was disrupted when actin-binding protein was hydrolyzed by activity of the Ca2+-dependent protease, which was active in platelet lysates upon addition of Ca2+ in the absence of leupeptin. Similarly, activation of the Ca2+-dependent protease in intact platelets by the addition of a platelet agonist also caused the membrane glycoproteins to dissociate from the membrane skeleton. Affinity-purified actin-binding protein antibodies immunoprecipitated the membrane glycoproteins from platelet lysates in which actin filaments had been removed by DNase I-induced depolymerization and high-speed centrifugation. These results demonstrate that actin-binding protein links actin filaments of the platelet membrane skeleton to three plasma membrane glycoproteins and that filaments are released from their attachment site when actin-binding protein is hydrolyzed by the Ca2+-dependent protease within intact platelets during platelet activation.     

Loss of yolk platelets and yolk glycoproteins during larval development of the sea urchin embryo

The fate of the yolk platelets and their constituent yolk glycoproteins was studied in Strongylocentrotus purpuratus eggs and embryos cultured through the larval stage. Previous studies have shown that the yolk glycoproteins undergo limited proteolysis during early embryonic development. We present evidence that the yolk glycoproteins stored in the yolk platelets exist as large, disulfide-linked complexes that are maintained even after limited proteolysis have occurred. We provide additional evidence that acidification of the yolk platelet may activate a latent thiol protease in the yolk platelet that is capable of correctly processing the major yolk glycoprotein into the smaller yolk glycoproteins. Because we previously showed that these yolk glycoproteins are not catabolized during early embryonic development, it was of interest to study their fate during larval development. Using a specific polyclonal antibody to a yolk glycoprotein, we found that both yolk glycoproteins and the yolk platelets disappeared in feeding, Day 7, larval stage embryos, but that starvation did not significantly affect the levels of the yolk glycoproteins. We also found that the yolk glycoproteins reappeared in 30-day-old premetamorphosis larvae.     

Effects of diamide and iodoacetamide on the expression of the glycoprotein IIb/IIIa complex on blood platelets

We have examined the effects of two agents that alter platelet thiol-disulphide status on platelet aggregation and on the ability of platelets to bind a monoclonal antibody (M148) that is directed toward an epitope on the glycoprotein IIb/IIIa complex. The immediate effect of both diamide and iodoacetamide is to enhance aggregation but after further incubation diamide, but not iodoacetamide, inhibits platelet aggregation. Incubation of platelets with diamide, but not iodoacetamide, is accompanied by a marked increase in the amount of M148 that binds to platelets. This is presumably a reflection of an altered distribution of glycoproteins on the platelet surface. It is known that diamide, but not iodoacetamide, leads to polymerisation of cytoskeletal proteins in platelets. Thus evidence is provided that agents that interact with the cytoskeleton inhibit platelet behaviour via an effect on surface glycoproteins.     

Inhibition of human tumor cell induced platelet aggregation by antibodies to platelet glycoproteins Ib and IIb/IIIa

Tumor cell induced platelet aggregation was shown to be inhibited in a dose dependent manner by preincubation of human platelets with antibodies to platelet glycoprotein Ib and the IIb/IIIa complex. Combination of antibody to Ib and antibody to the IIb/IIIa complex at concentrations which produced half maximal inhibition of platelet aggregation alone caused complete inhibition of tumor cell induced platelet aggregation. Antibodies to platelet glycoproteins Ib and the IIb/IIIa complex also inhibited platelet synthesis of thromboxane A2, but not synthesis of 12-hydroxyeicosatrienoic acid. Inhibition of tumor cell induced platelet aggregation with antibodies against platelet glycoproteins suggests a role for these glycoproteins in tumor cell-platelet interactions and possibly platelet facilitated tumor cell metastasis.     

Distribution of platelet glycoproteins and phosphoproteins in hydrophobic and hydrophilic phases in triton Triton-114 phase partition

Platelets, either unlabelled, surface-labelled by the periodate NaB³H₄ method or metabolically labelled with ³²P were solubilized in Triton X-114 and partitioned into aqueous and detergent phases. The phases were analysed by two-dimensional polyacrylamide gel electrophoresis followed by silver-staining, fluorography or indirect autoradiography. Each of the phases contains a distinct set of proteins. The surface-labelled glycoproteins partition into the hydrophobic phase with the notable exceptions of glycoproteins Ib and GP₁₇^5.8–6.5 and minor amounts of a few others. The phosphoproteins which undergo increased phosphorylation on platelet activation in general separate in the hydrophobic phase, while higher molecular weight phosphoproteins were principally in the hydrophilic phase. This method might be used as a first step in purifying many platelet components.     

Concanavalin A induces interactions between surface glycoproteins and the platelet cytoskeleton

We have measured the association of platelet surface membrane proteins with Triton X-100 (Triton)-insoluble residues in platelets surface labeled with 125I. In both concanavalin A (Con A)-stimulated and resting platelets, this fraction is composed largely of polypeptides with apparent molecular weights of 45,000, 200,000, and 250,000 which comigrate with authentic actin, myosin heavy chain, and actin binding protein, respectively, as judged by PAGE in SDS. Less than 10% of the two major 125I-labeled surface glycoproteins, GPiib and GPIII, were associated with the Triton residue in resting platelets. Within 45 s after Con A addition, 80-95% of these two glycoproteins became associated with the Triton residue and the amount of sedimentable actin doubled. No cosedimentation of GPIIb and III with the cytoskeletal protein-containing Triton residue was seen when Con A was added to a Triton extract of resting cells, indicating that the sedimentation of GPIIb and III seen in Con A-stimulated platelets was not due to precipitation of the glycoproteins by Con A after detergent lysis. Treatment of Triton extracts of Con A-stimulated platelets with DNase I (deoxyribonucleate 5'-oligonucleotidido-hydrolase [EC 3.1.4.5]) inhibited the sedimentation of actin and the two surface glycoproteins in a dose-dependent manner. This inhibition of cosedimentation was not due to an effect of DNase I on Con A-glycoprotein interactions since these two glycoproteins could be quantitatively recovered by Con A- Sepharose affinity absorption in the presence of DNase I. When the Con A bound to the Triton residue was localized ultrastructurally, it was associated with cell-sized structures containing filamentous material. In intact cells, there was simultaneous immunofluorescent coredistribution of surface-bound Con A and myosin under conditions which induced a redistribution of platelet myosin. These data suggest that Con A can, in the intact platelet, induce physical interactions between certain surface glycoproteins and the internal cytoskeleton.     

A biochemical study on the level of lipids and glycoproteins in the serum and platelets of liver cirrhotic bleeders

Bleeding complication and abnormal platelet functions are associated with liver cirrhosis. The aim of the present investigation was to assess the functional integrity of platelets in terms of lipids like cholesterol and phospholipids, glycoproteins and membrane-bound enzymes. Liver cirrhotic patients with bleeding complications were studied. Age and sex matched normal healthy volunteers were also involved in this study as a control group. Levels of cholesterol, phospholipids, glycoproteins and adenosine triphosphatases were assessed in isolated platelet membrane fraction. The level of glycoproteins and the activity of adenosine triphosphatases were found to be decreased significantly in cirrhotic patients. The cholesterol/phospholipid ratio was found to be altered significantly, indicating an alteration in the fluidity of platelet membrane. The results of this study reveal that the functional impairment of platelets in liver cirrhotic patients which is responsible for their bleeding tendency might also be due to altered lipid and enzyme levels in platelet membrane.     

Effect of certain lectins on platelet aggregation in healthy people

A study was made of the ability of some plant lectins, which bind specifically to different carbohydrate determinants of glycoproteins, to induce the platelet aggregation in healthy humans. It has been shown that phytohemagglutinin (PHA) and wheat germ agglutinin (MGA) induce a more marked platelet aggregation than concanvalin A (Con A). Lens culinaris agglutinin (LCA) had a slight aggregate activity. It was pointed at different roles played by carbohydrate determinants of platelet glycoproteins in fulfilling their aggregation function.     

Electrophoretic Distribution of the Proteins and Glycoproteins of Influenza Virus and Sendai Virus

The proteins of influenza (WSN) and Sendai virus have been separated by polyacrylamide gel electrophoresis into five components. In both cases, three of these components were shown to be glycoproteins containing fucose, galactose, and glucosamine. Two protein components of each virus were probably free from these sugar residues, including the structural unit of the viral ribonucleoprotein (molecular weight of about 60,000 daltons).     

Identification of a surface protein of the rabbit blood platelet with high affinity for collagen

Platelet membrane components adhering with high affinity to collagen fibers were studied by means of an affinity column in which fibrillar type I collagen was physically immobilized. Intact rabbit platelets in 1 mM EGTA adhered to the column but did not aggregate. Adhesion was dependent on the collagen concentration and on the number of platelets applied. Passage through the column without adhesion did not affect the potential for subsequent platelet binding. Surface-labelled whole platelets were passaged through this column, lysed in Triton and in SDS and labelled components adhering to the collagen were analysed on SDS-polyacrylamide gels. It was found that Triton lysis removed most of the major surface glycoproteins but left the cytoskeleton on the column. Subsequent SDS elution removed the cytoskeletal proteins along with the remaining major surface glycoproteins. The label left on the column could not be eluted with 8 M urea or up to 4 M NaCl. Collagenase digestion of the column collagen released a single surface glycoprotein of Mr 80,000. Limited chymotryptic digestion of the labelled platelets prior to their application to the column did not affect their binding. A radiolabelled band of the same molecular weight (MW) became bound to the collagen following passage of the chymotrypsin-treated platelets. This band was trypsin-sensitive following SDS-polyacrylamide gel electrophoresis (SDS-PAGE). These results, along with other published evidence, suggest that at least one platelet membrane component, expressed on the surface of the unstimulated platelet, binds with high affinity to fibrillar type I collagen and is probably involved in platelet collagen recognition.     

Distribution of platelet glycoproteins and phosphoproteins in hydrophobic and hydrophilic phases in Triton X-114 phase partition

Platelets, either unlabelled, surface-labelled by the periodate NaB3H4 method or metabolically labelled with 32P were solubilized in Triton X-114 and partitioned into aqueous and detergent phases. The phases were analysed by two-dimensional polyacrylamide gel electrophoresis followed by silver-staining, fluorography or indirect autoradiography. Each of the phases contains a distinct set of proteins. The surface-labelled glycoproteins partition into the hydrophobic phase with the notable exceptions of glycoproteins Ib and GP17(5.8-6.5) and minor amounts of a few others. The phosphoproteins which undergo increased phosphorylation on platelet activation in general separate in the hydrophobic phase, while higher molecular weight phosphoproteins were principally in the hydrophilic phase. This method might be used as a first step in purifying many platelet components.     

Platelet membrane glycoproteins IIb and IIIa are substrates of purified pp60c-src protein tyrosine kinase.

Human platelet glycoproteins IIb and IIIa form the receptor for fibrinogen, von Willebrand factor and fibronectin. Isolated human glycoproteins IIb-IIIa are phosphorylated by purified pp60c-src protein tyrosine kinase. Analysis of the phosphorylated proteins on SDS-PAGE showed that under reducing conditions both phosphoproteins change their relative molecular masses from 135 to 120 kDa and from 97 to 105 kDa, which are characteristic properties of glycoproteins IIb-IIIa. Phosphorylated proteins could be immunoprecipitated with an antiserum against glycoproteins IIb-IIIa but not by control serum. Some kinetic properties of the glycoprotein phosphorylations are also investigated. How the glycoprotein IIb-IIIa complex acquires its receptor activity in stimulated platelets is unknown; however, phosphorylation could be an important mechanism.     

Photoaffinity labeling of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) on intact platelets with 8-azido-[gamma-32P]ATP.

The fibrinogen receptor GPIIb-IIIa plays a crucial role in platelet aggregation. Here we show that the adenine nucleotide, 8-azido-ATP, inhibits ADP-induced conformational change of the platelet fibrinogen receptor GPIIb-IIIa (integrin alpha IIb beta 3). Photoaffinity labeling of intact platelets with 8-azido-[gamma-32P]ATP exclusively modifies two plasma-membrane glycoproteins which are identical with both subunits of GPIIb-IIIa. The presence of adenine-nucleotide-binding sites on GPIIb-IIIa implies that the platelet fibrinogen receptor is directly regulated by extracellular adenine nucleotides.     

Topological and regulatory aspects of dolichyl phosphate mediated glycosylation of proteins

From the time of their synthesis in the rough endoplasmic reticulum until they are secreted, packaged in lysosomes, or appear as membrane components at the cell surface, the polypeptide chains of N- and O-linked glycoproteins remain associated with intracellular membranes that are components of the secretory pathway. The various co-translational and post-translational modifications of the carbohydrate moieties of glycoproteins have been shown to occur within morphologically and functionally distinct regions of this complex membrane system. However, the sugar nucleotides, which serve as precursors to the oligosaccharide moieties of these glycoproteins, are synthesized almost exclusively in the cytoplasm. These findings raise a number of questions about the mechanisms involved in the transmembrane assembly of membrane and secretory glycoproteins. In this paper these questions are reviewed and recent studies directed towards providing answers to them are summarized. In addition, information related to the possible role of dolichyl phosphate in regulating the glycosylation of proteins is presented.     

Tissue specificity of chromatin glycoproteins recognized by concanavalin A

Chromatin glycoproteins recognized by Concanavalin A have been isolated from pig liver, kidney and heart by the use of immobilized lectin. Two groups of proteins differing in affinity for DNA have been analysed. Glycoproteins are mainly present in the group of proteins which are tightly bound to DNA. Mono and bidimensional electrophoretic patterns of total tightly bound proteins reveal a similarity among the three organs examined, while the corresponding patterns of the glycoproteins are typical for each organ. The tissue specificity of chromatin glycoproteins, together with their capability to interact not only with DNA but possibly also with other nuclear components, suggest a role for these proteins in the mechanism of genome expression.