Glycoprotein IIb Alpha Research Articles

Antibodies to GPIIb alpha (300-312) inhibit Fg binding, clot retraction, and platelet adhesion to multiple ligands.

We previously reported that a peptide with the sequence Gly-Ala-Pro-Leu (GAPL) found as residues 309-312 in glycoprotein IIb alpha (GPIIb) comprises at least part of a Fg binding site on GPIIb (1). Subsequent studies demonstrated that a peptide corresponding to residues 300-312 of GPIIb alpha can bind to Fg and Vn, and is a potent inhibitor of platelet aggregation and the adhesion of activated platelets to at least four adhesive ligands: Fg, Fn, Vn, and vWf (2). Here, the production and initial characterization of polyclonal antibodies against this peptide are described. ELISAs and dot-blot assays reveal the specificity of the antibodies for the peptide immunogen. In immunoblots, the antibodies recognize GPIIb under reducing conditions. The binding of Fg to immobilized GPIIb/IIIa, the rate of clot retraction and the adhesion of stimulated platelets to Fg, fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWf) were inhibited by these antibodies, but not by a control IgG. These results together with our earlier published data indicate that GPIIb alpha (300-312) comprises at least part of a common ligand binding site within the integrin alpha IIb subunit.

Calcium binding to human platelet integrin GPIIb/IIIa and to its constituent glycoproteins. Effects of lipids and temperature

Platelet plasma membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form a Ca(2+)-dependent heterodimer. GPIIb/IIIa, which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. Using equilibrium dialysis measurements, it was established that both GPIIb and GPIIIa in solution have low-affinity Ca(2-)-binding sites (Kd0.2-0.3 mM), five in GPIIb and two in GPIIIa, and it was confirmed that only the alpha-chain of GPIIb (GPIIb alpha) binds Ca2+. Furthermore, Ca2+ binding was found with two CNBr fragments of GPIIb, GPIIb alpha-(1-285) and GPIIb alpha-(314-489), which carry three out of the four putative Ca(2+)-binding sites. GPIIb/IIIa in solution has a single high-affinity Ca(2+)-binding site (Kd1 80 +/- 30 nM at 21 degrees C), whose degree of saturation regulates the state of association of GPIIb and GPIIIa in the GPIIb/IIIa heterodimer at room temperature, and 3-4 medium-affinity Ca(2+)-binding sites (Kd2 40 +/- 15 microM at 21 degrees C). When GPIIb/IIIa was incorporated into liposomes, Kd1 decreased by an order of magnitude (9 +/- 3 nM at 21 degrees C) and reached the dissociation constant estimated for the high-affinity Ca(2+)-binding sites at the platelet surface [Brass & Shattil (1982) J. Biol. Chem. 257, 1400-1405], whereas Kd2 remained unchanged. The high-affinity Ca(2+)-binding site of GPIIb/IIIa in solution at 4 degrees C has almost the same affinity (Kd1 65 +/- 20 nM) as at 21 degrees C; however, at 37 degrees C, either its affinity decreases enough so as to become experimentally indistinguishable from the medium-affinity Ca(2+)-binding sites determined at this temperature (number of binding sites 3.9 +/- 1.2 mol of Ca2+/mol of GP, Kd 25 +/- 11 microM), or vanishes altogether. Studies on Ca(2+)-dependent dissociation of GPIIIb/IIIa at 37 degrees C in solution seem to support the former interpretation. Further work will be necessary to decide whether the dissociation of GPIIb/IIIa in the platelet membrane at 37 degrees C is regulated by the degree of saturation of the high-affinity Ca(2+)-binding site, as occurs in solution. It is suggested that the high-affinity Ca(2+)-binding site could be related to the putative GPIIIa-binding region in GPIIb (residues 558-747 of the alpha chain).

Human platelets and megakaryocytes contain alternately spliced glycoprotein IIb mRNAs.

The earliest marker of the megakaryocyte lineage in human bone marrow is a heterodimeric complex of glycoproteins IIb (GPIIb) and IIIa (GPIIIa). GPIIb-IIIa is a member of the integrin family and serves as a receptor for fibrinogen and other adhesive glycoproteins on the surface of activated blood platelets. Mature platelet GPIIb is processed from a single precursor peptide into a disulfide-linked GPIIb alpha and GPIIb beta chain. To study possible mechanisms which could determine ligand specificity of the GPIIb-IIIa complex we investigated whether there are multiple forms of GPIIb mRNA by screening an unamplified lambda gt10 human erythroleukemia cell cDNA library. Four GPIIb clones were isolated and sequenced. Two contained a 102-nucleotide in-frame deletion corresponding to 34 amino acids in the extracellular domain of GPIIb beta. Polymerase chain reaction amplification of human erythroleukemia cell cDNA confirmed the presence of two different transcripts. Normal genomic DNA sequence was shown to contain consensus splice-donor and splice-acceptor sites indicating a 102-base pair exon that is spliced in or out. Using primers specific for either transcript, both forms of GPIIb mRNA were identified by the polymerase chain reaction in normal human platelets and megakaryocytes, but not in reticulocytes or two white blood cell lines. These data provide evidence for two forms of GPIIb in platelets and megakaryocytes and suggest a mechanism for variations in receptor ligand affinity or localization.

Interchain and intrachain disulphide bonds in human platelet glycoprotein IIb. Localization of the epitopes for several monoclonal antibodies

The single interchain disulphide bond in platelet glycoprotein IIb (GPIIb) is accessible to extracellular reductants, and selective cleavage does not liberate GPIIb alpha from platelet plasma membrane, confirming that non-covalent interactions contribute to maintaining attachment of this subunit to the membrane. Eosin-maleimide labelling of isolated GPIIb after selective cleavage of this interchain disulphide bond, followed by full reduction and alkylation, CNBr cleavage, and analysis of the cleavage products allowed us to establish that this interchain disulphide bridge is formed between GPIIb beta (GPIIb beta-subunit) Cys-9 and GPIIb alpha Cys-826, and this conclusion was confirmed by independent routes. The other two cysteines of GPIIb beta (Cys-14 and Cys-19) form the single intrachain disulphide bond in this subunit. Last, the intrachain disulphides in GPIIb alpha (GPIIb alpha-subunit) are distributed in four main peptide domains which are not disulphide-bonded among themselves. The linear epitope for monoclonal antibody M1 is localized between Pro-4 and Met-24 (or Met-31) of GPIIb beta. The linear epitope for M3 is situated between Cys-826 and the C-terminus of GPIIb alpha. The M4 epitope is also linear and localized somewhere between residues 115 and 285 of GPIIb alpha. Finally, the epitopes for M5 and M6 are somewhere between Cys-608 and Met-704, within a 35 kDa membrane-bound chymotryptic product of digestion of GPIIb in whole platelets. The N-terminal amino acid sequences determined for eight different cleavage products of GPIIb alpha and GPIIb beta agree with the corresponding amino acid sequences predicted by cDNA sequence for human-erythroleukaemic-cell GPIIb [Poncz, Eisman, Heindenreich, Silver, Vilaire, Surrey, Schwartz & Bennett (1987) J. Biol. Chem. 262, 8476-8482].

Complete localization of the intrachain disulphide bonds and the N-glycosylation points in the alpha-subunit of human platelet glycoprotein IIb.

Glycoprotein IIb (GPIIb), one of the two molecular components of the inducible receptor for fibrinogen on the platelet surface, is formed from two subunits, GPIIb alpha (114 kDa) and GPIIb beta (22.5 kDa), joined by a single disulphide bond. CNBr cleavage of GPIIb, together with tryptic or endoproteinase Lys-C digestion of some of the isolated CNBr peptides, followed by amino acid and N-terminal sequence analysis of the isolated fragments, allowed us to locate unambiguously all the unknown disulphide bonds and the N-glycosylation points in platelet GPIIb. It could be established that each cysteine residue in GPIIb, beginning at alpha-Cys-56, is disulphide-bonded to its nearest neighbour in the amino acid sequence. Given the extensive structural similarity among the two-chain alpha-subunits of Arg-Gly-Asp adhesion receptors and the conservative positions of cysteine residues in their amino acid sequences, the intrachain and interchain disulphide-bond pattern found here in GPIIb will most probably be conserved in all two-chain alpha-subunits of these receptors. The N-linked glycosylation points found here in platelet GPIIb are the same as the five N-glycosylated asparagine residues suggested after cDNA sequencing of human erythroleukaemic-cell GPIIb [Poncz, Eisman, Heindenreich, Silver, Vilaire, Surrey, Schwartz & Bennett (1987) J. Biol. Chem. 262, 8476-8482]. Some of the general features of the structure of GPIIb, such as the existence of distinct domains in the alpha- and beta-subunits, as well as the identification of well-defined points in its external topography, are discussed.

Palmitylation of the Glycoprotein IIb-IIIa Complex in Human Blood Platelets

The presence of covalently bound palmitic acid in fibrinogen receptors, glycoproteins (GP) IIb and IIIa, has been explored in human blood platelets. Membrane fractions were isolated from fresh blood platelets labeled with [9,10-3H]palmitic acid and then analyzed for radioactive proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were visualized by staining with Coomassie Brilliant Blue, excised, and counted in a liquid scintillation counter. The results indicate that membrane proteins with electrophoretic mobility corresponding to glycoproteins IIb and IIIa incorporate [9,10-3H]palmitic acid. The palmitylated glycoproteins IIb and IIIa were immunoprecipitated by specific anti-GP IIb and GP IIIa antisera. It is interesting to note that the palmitylation of these glycoproteins occurred rapidly in platelets activated with 0.5 unit of thrombin or 30 microM ADP. At the concentration used (100 micrograms/ml), cycloheximide did not inhibit incorporation of [3H]palmitate into the glycoproteins showing that this process is not dependent upon protein synthesis. The acyl moiety was resistant to denaturating detergents, delipidation with organic solvents, and hydrolyzable with hydroxylamine. In the case of membrane protein with the electrophoretic mobility of GP IIb, the radioactive label was significantly decreased after reduction with 2-mercaptoethanol. Final identification of GP IIIa as an acylated product in human platelets incubated with [9,10-3H]palmitic acid was provided by two-dimensional polyacrylamide gel electrophoresis. In contrast to GP IIb alpha, GP IIIa isolated by this method showed the presence of attached radioactive palmitic acid residues. Analysis by high performance liquid chromatography after methanolysis of the [3H]palmitate-labeled glycoproteins confirmed the fatty acid nature of the label. Palmitylation is a newly identified post-translational modification of the fibrinogen receptor which may play an important role in its interaction with the membrane and/or its biological function.

Immunochemical characterization of an autoantigen on platelet glycoprotein IIb in chronic ITP: comparison with the Baka alloantigen

Employing an immunoblotting procedure, we have identified and characterized an autoantigen carried on glycoprotein (GP) IIb in a patient with chronic idiopathic thrombocytopenic purpura (ITP), and have compared the location of the autoantigen with that of the platelet-specific alloantigen Baka. Immunoblots, using the partially purified GP IIb/IIIa complex as the target antigen, indicated that GP IIb alpha carried both the ITP autoantigen and the Baka alloantigen. The ITP plasma contained another antibody against a 100 kD protein (P100), a trace contaminant in the GP IIb/IIIa sample, which is probably a proteolytic fragment of an internal 124 kD protein. After chymotrypsin treatment, the auto- and alloantigen were found to be located on 65 kD fragments detectable under reducing conditions. In addition, immunoblots made after two-dimensional nonreduced-reduced SDS-polyacrylamide gel electrophoresis (SDS-PAGE) directly demonstrated that both 65 kD fragments had a molecular weight of 80 kD under nonreducing conditions; this provides evidence that these fragments were one and the same, and were derived from GP IIb alpha. Immunoblots of platelets digested in situ with chymotrypsin indicated that the 65 kD fragment of GP IIb alpha was retained by the platelet membrane. We conclude, therefore, that a 65 kD fragment, which represents the membrane side of the chymotrypsin cleavage site on GP IIb alpha, carries a clinically important determinant(s) recognized not only by the anti-Baka alloantibody, but also by the ITP autoantibody.

Identification of Bakb, a new platelet-specific antigen associated with posttransfusion purpura

Baka is a platelet alloantigen whose putative allele, Bakb, has not been identified previously. By using a serum, “Har,” obtained from a patient with posttransfusion purpura, we describe the platelet alloantigen Bakb. The Har serum reacted with an NP-40-extractable platelet membrane protein of 142 kd with mobility similar to platelet glycoprotein IIb alpha. We found that the antigen recognized by the Har serum is inherited in an autosomal dominant mode with an apparent gene frequency of .39. Chi-square analysis of observed and expected phenotype frequencies indicated that serum Har recognizes Bakb, the anticipated allele of Baka. Our findings provide new evidence for polymorphism of glycoprotein IIb and for the association of posttransfusion purpura with alloimmunization to determinants on this glycoprotein.

Identification of Bakb, a New Platelet-Specific Antigen Associated With Posttransfusion Purpura

Baka is a platelet alloantigen whose putative allele, Bakb, has not been identified previously. By using a serum, "Har," obtained from a patient with posttransfusion purpura, we describe the platelet alloantigen Bakb. The Har serum reacted with an NP-40-extractable platelet membrane protein of 142 kd with mobility similar to platelet glycoprotein IIb alpha. We found that the antigen recognized by the Har serum is inherited in an autosomal dominant mode with an apparent gene frequency of .39. Chi-square analysis of observed and expected phenotype frequencies indicated that serum Har recognizes Bakb, the anticipated allele of Baka. Our findings provide new evidence for polymorphism of glycoprotein IIb and for the association of posttransfusion purpura with alloimmunization to determinants on this glycoprotein.

Isolation and characterization of the receptor on human neutrophils that mediates cellular adherence.

The receptor on human neutrophils (polymorphonuclear leukocytes or PMN) that mediates cellular adherence has been purified from the peripheral blood PMN obtained from an individual with chronic myelogenous leukemia (CML). This receptor consists of two noncovalently associated subunits, designated alpha M (Mac-1 alpha, CD11b) (Mr = 170,000) and beta (Mac-1 beta, CDw18) (Mr = 100,000), respectively, which are identical on normal and CML PMN. The subunits were purified by monoclonal antibody 60.1-Sepharose (anti-alpha M) affinity chromatography and separated in 5-nmol quantities by high pressure liquid chromatography on a TSK-4000 gel filtration column. Subunits were characterized by amino acid composition, NH2-terminal amino acid sequence, and carbohydrate content. The NH2-terminal sequence of the human PMN alpha M subunit contains regions of homology with the human platelet glycoprotein IIb alpha. We conclude that nanomole amounts of individual alpha M and beta subunits of the receptor on human PMN that mediates cellular adherence can be isolated and separated using CML PMN.

Molecular characterization of human platelet glycoproteins IIIa and IIb and the subunits of the latter.

Sedimentation equilibrium and low-angle laser-light scattering were used to determine the molar mass of the glycoprotein moieties in the complexes of sodium dodecyl sulphate with the human platelet membrane glycoproteins IIb (GPIIb), IIIa (GPIIIa), and the alpha (GPIIb alpha) and beta (GPIIb beta) subunits of GPIIb. The values obtained by both procedures, except those for GPIIb, agree within experimental error with those calculated from their chemical composition: GPIIb alpha (114,000 g mol-1), GPIIb beta (22,200 g mol-1), and GPIIIa (91,500 g mol-1). The molar mass of GPIIb determined by light scattering (142,000 g mol-1 and sedimentation equilibrium at different solvent densities (134,000 g mol-1) also agree, within experimental error, with the values calculated either from its chemical composition (136,500 g mol-1) or from the sum of the molar masses of its subunits. However the molar mass determined by sedimentation equilibrium at constant solvent density, is consistently underestimated (116,000 g mol-1). High-performance size-exclusion chromatography in sodium dodecyl sulphate solutions overestimates the molar mass of these glycoproteins and their Stokes radii, and therefore the maximal frictional ratios derived from them.

Isolation and biochemical characterization of the alpha- and beta-subunits of glycoprotein IIb of human platelet plasma membrane.

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.

Platelet glycoproteins IIb and IIIa: evidence for a family of immunologically and structurally related glycoproteins in mammalian cells.

Human and bovine cultured cell lines and circulating leukocytes were examined for the presence of surface proteins similar to platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human endothelial cells, smooth muscle cells, and MG-63 fibroblast-like cells were found to have surface proteins that cross-reacted with platelet GPIIb and GPIIIa antibodies, existed as complexes, and had molecular weights similar to those of the corresponding platelet glycoproteins. Bovine endothelial cells and smooth muscle cells also expressed GPIIb- and GPIIIa-like surface proteins. Metabolic labeling studies with [35S]methionine demonstrated that the cultured cells synthesized these glycoproteins. The GPIIIa-like protein in human endothelial and smooth muscle cells had the same isoelectric point as platelet GPIIIa, whereas their GPIIb alpha-like protein was slightly more acidic than platelet GPIIb alpha (pI = 5.2-5.3 versus 5.5). Platelet and endothelial cell GPIIb alpha (but not GPIIIa) showed an increased electrophoretic mobility in Ca2+ -containing versus EDTA-containing gels, implying a Ca2+ -GPIIb alpha interaction. The amino acid sequence of the amino termini of platelet GPIIb alpha and GPIIb beta and of the alpha chains of the leukocyte LFA-1 and Mac-1 glycoprotein complexes had significant sequence homology. These data indicate that glycoproteins that have either immunological cross-reactivity or amino-terminal sequence homology with the platelet GPIIb-IIIa complex are widely distributed in human and non-human adherent cells and circulating leukocytes and suggest that these proteins may be the products of a large gene family whose expression is cell specific.

Acquired Thrombasthenia Due to GPIIb/IIIa-Specific Platelet Autoantibodies

An otherwise healthy woman developed a hemorrhagic diathesis with fluctuating clinical symptoms and laboratory findings, but without thrombocytopenia, over 8 years. In periods of bad clinical condition, a platelet defect, characteristic of thrombasthenia, was found. In contrast to classic thrombasthenia, electrophoresis of the patient's platelet membranes revealed normal amounts of glycoproteins IIb alpha, IIb beta, and IIIa in the normal positions. Monoclonal antibodies, specific for GPIIIa and GPIIb/IIIa, respectively, bound normally to the P1A1-positive platelets from the patient. Although no antibody and no platelet function inhibitor were evident in the autologous plasma, an IgG1 antibody that was bound to the patient's platelets and was directed against GPIIb/IIIa could be demonstrated. After elution from the patient's platelets, this antibody immunoprecipitated GPIIb (both subunits), IIIa, and a 200-kilodalton (kd) band (probably undissociated GPIIb/IIIa complex) from solubilized normal platelets, but did not react with thrombasthenic platelets. Adding the eluate from the patient's platelets to normal platelet-rich plasma immediately caused concentration-dependent inhibition of adenosine diphosphate (ADP)-induced and collagen-induced aggregation and also strong inhibition of ADP-stimulated fibrinogen binding. Because it was very unlikely from the patient's medical history that the antibody was caused by alloimmunization, the hemorrhagic diathesis must be interpreted as acquired thrombasthenia due to an anti-GPIIb/IIIa autoantibody.

Acquired thrombasthenia due to GPIIb/IIIa-specific platelet autoantibodies

An otherwise healthy woman developed a hemorrhagic diathesis with fluctuating clinical symptoms and laboratory findings, but without thrombocytopenia, over 8 years. In periods of bad clinical condition, a platelet defect, characteristic of thrombasthenia, was found. In contrast to classic thrombasthenia, electrophoresis of the patient's platelet membranes revealed normal amounts of glycoproteins IIb alpha, IIb beta, and IIIa in the normal positions. Monoclonal antibodies, specific for GPIIIa and GPIIb/IIIa, respectively, bound normally to the P1A1-positive platelets from the patient. Although no antibody and no platelet function inhibitor were evident in the autologous plasma, an IgG1 antibody that was bound to the patient's platelets and was directed against GPIIb/IIIa could be demonstrated. After elution from the patient's platelets, this antibody immunoprecipitated GPIIb (both subunits), IIIa, and a 200-kilodalton (kd) band (probably undissociated GPIIb/IIIa complex) from solubilized normal platelets, but did not react with thrombasthenic platelets. Adding the eluate from the patient's platelets to normal platelet-rich plasma immediately caused concentration-dependent inhibition of adenosine diphosphate (ADP)- induced and collagen-induced aggregation and also strong inhibition of ADP-stimulated fibrinogen binding. Because it was very unlikely from the patient's medical history that the antibody was caused by alloimmunization, the hemorrhagic diathesis must be interpreted as acquired thrombasthenia due to an anti-GPIIb/IIIa autoantibody.

Platelet-collagen adhesion: inhibition by a monoclonal antibody that binds glycoprotein IIb.

To identify platelet surface structures involved in adhesion to collagen, the effect of 16 murine antiplatelet membrane hybridoma antibodies were tested in a defined, in vitro assay. Four of these antibodies inhibited platelet-collagen adhesion and reacted with a polypeptide with Mr approximately 125,000, as determined by immunoblots after gel electrophoresis under reducing conditions. Through detailed studies with one of these antibodies, the monoclonal antibody PMI-1, the relevant antigen was identified as platelet glycoprotein IIb alpha, based upon (a) co-migration with this glycoprotein in two-dimensional gel electrophoresis and (b) co-purification by immunoaffinity chromatography with a protein with apparent Mr identical to that of glycoprotein III, under conditions in which glycoproteins IIb and III form a complex. Univalent antibody fragments prepared from monoclonal antibody PMI-1 inhibited greater than 80% of platelet-collagen adhesion, and inhibition was completely blocked by the immunopurified antigen. These results indicate that glycoprotein IIb participates in some aspect of platelet-collagen adhesion. In contrast, the purified antigen only partially neutralized a polyclonal antiserum that blocked platelet-collagen adhesion, to a maximum of approximately 25%, at saturating antigen concentrations. Thus, by these immunological criteria, glycoprotein IIb is not the only molecule involved in this process.