Human and bovine endothelial cells synthesize membrane proteins similar to human platelet glycoproteins IIb and IIIa

Human umbilical vein endothelial (HUVE) and bovine aortic endothelial (BAE) cells in culture were examined to determine whether membrane proteins similar to human platelet glycoproteins (GP) IIb and IIIa were present. The HUVE and BAE cells were either 125I-surface labeled or metabolically labeled. Triton X-100 lysates of labeled cells were immunoprecipitated with polyclonal antibodies prepared against purified human platelet GP IIb-IIIa complex. Two membrane proteins were detected on both HUVE (Mr = 130,000 and 110,000) and BAE (Mr = 135,000 and 105,000) cells, which were similar to human platelet GP IIb (Mr = 125,000) and GP IIIa (Mr = 108,000). The two membrane proteins from HUVE cells and the two from BAE cells cosedimented in sucrose gradients, indicating that they exist as a complex. Unlike the human platelet GP IIb-IIIa complex, the HUVE and BAE membrane protein complexes were not dissociated by chelation of Ca2+. Platelet GP IIb and GP IIIa and the related membrane proteins on both HUVE and BAE cells showed similar changes in electrophoretic mobility upon disulfide reduction. These data demonstrate that human and bovine endothelial cells synthesize membrane proteins that have properties similar to the platelet membrane GP IIb-IIIa complex.     

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ($\text{p}\text{I}$) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.     

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.     

Quantitation and characterization of human platelet glycoprotein IIIa by radioimmunoassay

Glycoprotein IIIa was quantitated in human platelets by radioimmunoassay using antisera specific to platelet membranes and purified glycoprotein IIIa. Glycoprotein IIIa and glycoprotein IIb were isolated from washed platelets by Triton X-114 extraction followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioiodinated glycoprotein IIIa was further purified by affinity chromatography on Lentil lectin-Sepharose 4B. Purified glycoprotein IIb showed little crossreactivity with 125I-labeled glycoprotein IIIa using the anti-platelet membrane or anti-glycoprotein IIIa antisera on a competition inhibition radioimmunoassay. The expression of glycoprotein IIIa epitopes were the same for the purified glycoprotein IIIa and glycoprotein IIIa in Triton X-100 solubilized platelets. A 66 kDa protein derived from glycoprotein IIIa by limited proteolysis of platelet membranes also expressed the same epitopes as intact glycoprotein IIIa. Solubilized platelets contained approximately 16 micrograms of total glycoprotein IIIa antigen per 10(9) cells. The level of glycoprotein IIIa determined by radioimmunoassay in one patient with Glanzmann's thrombasthenia amounted to 6.7% of normal and it was close to the values obtained by other methods.     

Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb-IIIa using monoclonal antibodies

Platelet membrane glycoprotein IIb-IIIa exists as a calcium-dependent complex of two large peptides (designated IIb and IIIa) in Triton X-100 solutions, but it remains unknown if these peptides are subunits of one glycoprotein or are actually two individual glycoproteins in the intact platelet membrane. We used crossed immunoelectrophoresis to define the epitopes of two monoclonal antibodies to IIb-IIIa, then used these antibodies to study the structural and functional organization of IIb and IIIa in the platelet membrane. Human platelets solubilized in Triton X-100 were electrophoresed through an intermediate gel containing 125I-monoclonal IgG, then into an upper gel containing rabbit anti-human platelet antibodies. Our previously characterized antibody. Tab, and a new monoclonal antibody, T10, both bound to the immunoprecipitate corresponding to the IIb-IIIa complex. When platelets were electrophoresed after solubilization in 5 mM EDTA, 125I-Tab bound to the dissociated IIb polypeptide, but not to IIIa. In contrast, 125-I-T10 did not react with either IIb or IIIa. Thus, Tab recognizes a determinant on IIb, while T10 recognizes a determinant created only after the association of IIb and IIIa. Gel-filtered platelets from six normal donors bound 50,600 +/- 5,600 125I-T10 molecules/platelet and 47,800 +/- 11,200 125I-Tab molecules/platelet, consistent with IIb-IIIa being a heterodimer. 125I-T10 binding was identical in unactivated platelets and platelets stimulated with 10 microM ADP. However, platelets did not aggregate or bind 125I-fibrinogen until ADP was added. T10, but not Tab or nonimmune mouse antibody, inhibited ADP-induced platelet aggregation and 125I-fibrinogen binding. Our findings suggest that IIb and IIIa exist as subunits of a single membrane glycoprotein in unstimulated platelets. Fibrinogen binding appears to require not only the interaction of IIb and IIIa, but also some additional change occurring after platelet activation.     

A method for purifying the platelet membrane glycoprotein IIb-IIIa complex

A method has been developed for the rapid isolation of platelet membrane glycoproteins (GP) IIb and IIIa. This method produces an excellent yield and does not require the prior isolation of platelet membranes. Outdated platelets were washed and solubilized in Triton X-100. Concanavalin A affinity chromatography was used to purify a platelet glycoprotein fraction. The concanavalin A-retained glycoproteins were eluted and adsorbed with a heparin-Sepharose column to remove a major contaminant, thrombospondin. Sephacryl S-300 gel filtration was used as the final purification step to remove most fibrinogen and low-molecular-weight contaminants. Wheat germ agglutinin affinity chromatography was used to completely remove trace amounts of fibrinogen. The purified GP IIb and GP IIIa were analyzed by sucrose gradient sedimentation and found to consist of heterodimer complexes.     

Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography

The major platelet membrane glycoproteins have been solubilized in 1.0% sodium deoxycholate and subjected to affinity chromatography on the lectins from Lens culinaris, wheat germ and Abrus precatorius. Polyacrylamide gel electrophoresis in the presence and absence of a reducing agent together with the differential binding of the lectins to the glycoproteins permitted the distinction of at least seven separate glycoprotein entities. A new nomenclature for the glycoproteins is proposed to accomodate the additional data.Using combinations of lectin columns, glycoproteins Ia and Ib could be prepared in a pure state and IIb and IIIa could be greatly purified. The binding of lectins to glycoprotein Ib has been strongly implicated as a necessary step in the aggregation response of platelets to lectins.     

Comparative study of the major glycoproteins of the plasma membrane and secretory granule membranes of porcine platelets

The major glycoproteins of porcine platelet plasma membranes and alpha-granule membranes were compared. Significant cross-contamination of the two preparations was ruled out by surface labeling and proteolysis experiments. At least four of the major glycoproteins of the alpha-granule membrane had counterparts in the plasma membranes with identical molecular weight and lectin-binding properties. Two of these (mol. wts 110,000 and 125,000) were further analysed by one-dimensional peptide mapping. The results confirmed that there are two distinct pools of identical glycoproteins: one on the surface membrane and the other on the alpha-granular membrane. The 110,000 and 125,000 mol. wt glycoproteins are probably equivalent to glycoproteins IIb and IIIa of the human platelet and may therefore be involved in fibrinogen binding.     

Role of tumor cell glycoproteins immunologically related to glycoproteins Ib and IIb/IIIa in tumor cell-platelet and tumor cell-matrix interactions

A panel of monoclonal and polyclonal antibodies raised against human platelet GpIb or the GpIIb/IIIa complex were used to detect immunologically related molecules on two cell lines derived from human solid tumors. Human cervical carcinoma (MS751) and human colon carcinoma (clone A) expressed molecules immunologically related to platelet GpIb and GpIIb/IIIa complex. These molecules were localized to their plasma membranes by immunofluorescence and immunocytochemistry. The immunologically related GpIb was evenly distributed on the tumor cell membrane with occasional areas of aggregates, whereas the immunologically related GpIIb/IIIa had a pronounced punctate distribution of aggregates in prefixed cells. When MS751 or clone A cells were pretreated with antibodies against platelet GpIb and/or the GpIIb/IIIa complex, their ability to induce platelet aggregation was significantly inhibited. In addition, when tumor cells were pretreated with antibodies against the platelet IIb/IIIa complex, adherence to fibronectin-coated plates was also significantly inhibited. These results suggest a role for these immunologically related tumor cell glycoproteins in tumor cell-host cell (i.e., platelet, endothelial cells) interactions, tumor cell interactions with components of the subendothelial matrix, and subsequent tumor metastasis.     

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.     

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.     

Platelet membrane glycoprotein IIb-IIIa complex incorporated into phospholipid vesicles. Preparation and morphology

Platelet membrane glycoproteins (GP) IIb and IIIa have been identified as platelet aggregation sites. These glycoproteins form a heterodimer complex (GP IIb-IIIa) in the presence of Ca2+. To study the morphology of this glycoprotein complex in membranes, we incorporated GP IIb-IIIa into artificial phospholipid vesicles using a detergent (octyl glucoside) dialysis procedure. Phosphatidylserine-enriched vesicles (70% phosphatidylserine, 30% phosphatidylcholine) incorporated approximately 90% of the GP IIb-IIIa as determined by sucrose flotation. Glycoprotein IIb-IIIa incorporation into the vesicles was unaffected by ionic strength, suggesting a hydrophobic interaction between the glycoprotein and the phospholipid. In both intact platelets or phospholipid vesicles, GP IIb was susceptible to neuraminidase hydrolysis, indicating that most of the glycoprotein complexes were oriented toward the outside of the platelets or vesicles. The morphology of GP IIb-IIIa in the phospholipid vesicles was observed by negative staining electron microscopy. Individual GP IIb-IIIa complexes appeared as spikes protruding as much as 20 nm from the vesicle surface. Each spike consisted of a GP IIb "head," which was distal to the vesicle and was supported by the GP IIIa "tails." The GP IIb-IIIa complex appeared to be attached to the vesicle membrane by the tips of the GP IIIa tails. Treatment of vesicles with EGTA dissociated the GP IIb-IIIa complex. The dissociated glycoproteins remained attached to the phospholipid vesicles, indicating that both GP IIb and GP IIIa contain membrane-attachment sites. These data suggest a possible structural arrangement of the GP IIb-IIIa complex in whole platelets.     

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.     

pH and magnesium alter 45calcium binding to platelets at sites other than glycoproteins I or IIb/IIIa

Calcium is a cofactor of human platelet aggregation. Moreover a direct correlation between the ability of platelets to bind this divalent cation and to aggregate has been demonstrated. Since magnesium can substitute for calcium in supporting aggregation, especially in the presence of low calcium concentrations, and platelet aggregation is inhibited at low pH, the present study was designed to examine the effects of magnesium and low pH on 45calcium binding to human platelets, and to determine whether such effects might be associated with calcium binding to glycoproteins I (GPI) or IIb/IIIa (GPIIb/IIIa), the putative fibrinogen receptor. 45Calcium binding to aspirin-treated platelets that had been depleted of surface-associated calcium by brief exposure to EDTA was evaluated. Magnesium (5-10 mM) or a change in hydrogen ion concentration to decrease the pH from 7.5 to 6.0 was found to inhibit the binding of 45calcium to platelets from healthy donors by 34 +/- 6 and 32 +/- 8% (mean +/- SD, n = 13), respectively. Similar results were obtained with platelets incubated with chymotrypsin to selectively remove GPI or platelets from a patient with the Bernard Soulier Syndrome, congenitally deficient in GPI. In contrast, calcium binding to platelets from two patients with thrombasthenia, lacking GPIIb/IIIa, was reduced 49 +/- 6% and 42 +/- 8% (n = 4) by magnesium and hydrogen ions, respectively. This apparently increased inhibition was attributed to the combined effects of an overall decrease (approximately 50%) in calcium binding to thrombasthenic platelets compared with that in control platelets, and a similar absolute reduction in calcium binding in the presence of magnesium and/or hydrogen ions. No additional inhibition of 45calcium binding was noted in the presence of magnesium and at low pH, indicating that magnesium and hydrogen ions may affect the same platelet membrane binding sites. The data suggest that although modulation of platelet aggregation by magnesium and pH is accompanied by changes in platelet-associated calcium, calcium binding to the three major platelet membrane glycoproteins, GPI, IIb, and IIIa is unaffected.     

N-terminal sequence of human leukocyte glycoprotein Mo1: conservation across species and homology to platelet IIb/IIIa

Mo1 and gp160-gp93 are two surface membrane glycoprotein heterodimers present on granulocytes and monocytes derived from humans and guinea pigs, respectively. We purified both antigens and found that their alpha subunits had identical N-termini which were significantly homologous to the alpha subunit of the human adhesion platelet glycoprotein IIb/IIIa.     

Ca2+ transport across the platelet plasma membrane. A role for membrane glycoproteins IIB and IIIA

Human platelets maintain a low cytosolic free Ca2+ concentration in part by controlling plasma membrane Ca2+ transport. The present studies examine the role in this process of two well-characterized membrane proteins: glycoproteins IIb and IIIa. These glycoproteins form a Ca2+-dependent complex which serves as both the platelet fibrinogen receptor and the principle site for high affinity Ca2+ binding on the platelet surface. The kinetics of plasma membrane Ca2+ exchange were compared in normal platelets and in thrombasthenic platelets, which lack the IIb X IIIa complex. Under steady-state conditions, the maximum rate of plasma membrane Ca2+ exchange in the thrombasthenic platelets was half the rate observed in normal platelets. The size of the cytosolic exchangeable Ca2+ pool and the cytosolic free Ca2+ concentration, however, were normal. A quantitatively similar decrease in plasma membrane Ca2+ exchange was seen in normal platelets after incubation with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 37 degrees C, conditions that dissociate the IIb X IIIa complex. This decrease in the Ca2+ exchange rate in normal platelets could be prevented by preincubating platelets with a complex-specific anti-IIb X IIIa monoclonal antibody, but not by preincubating platelets with an anti-IIIa monoclonal antibody. In order to determine whether loss of the IIb X IIIa complex primarily affects Ca2+ influx or Ca2+ efflux, both processes were also examined under nonsteady-state conditions. An immediate decrease in the 45Ca2+ influx rate was seen when Ca2+ was added back to platelets preincubated with EGTA at 37 degrees C. The 45Ca2+ efflux rate, on the other hand, was not immediately affected. These data suggest, therefore, that an intact IIb X IIIa complex is necessary for normal Ca2+ homeostasis in platelets.     

Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation

Platelet activation is accompanied by the appearance on the platelet surface of approximately 45,000 receptor sites for fibrinogen. The binding of fibrinogen to these receptors is required for platelet aggregation. Although it is established that the fibrinogen receptor is localized to a heterodimer complex of the membrane glycoproteins, IIb and IIIa, little is known about the changes in this complex during platelet activation that result in the expression of the receptor. In the present studies, we have developed and characterized a murine monoclonal anti-platelet antibody, designated PAC-1, that binds to activated platelets, but not to unstimulated platelets. PAC-1 is a pentameric IgM that binds to agonist-stimulated platelets with an apparent Kd of 5 nM. Binding to platelets is dependent on extracellular Ca2+ (KCa = 0.4 microM) but is not dependent on platelet secretion. Platelets stimulated with ADP or epinephrine bind 10,000-15,000 125I-PAC-1 molecules/platelet while platelets stimulated with thrombin bind 20,000-25,000 molecules/platelet. Several lines of evidence indicate that PAC-1 is specific for the glycoprotein IIb.IIIa complex. First, PAC-1 binds specifically to the IIb.IIIa complex on Western blots. Second, PAC-1 does not bind to thrombasthenic platelets or to platelets preincubated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 37 degrees C, both of which lack the intact IIb.IIIa complex. Third, PAC-1 competitively inhibits the binding of 125I-A2A9, and IgG monoclonal antibody that is specific for the IIb.IIIa complex. Fourth, the antibody inhibits fibrinogen-mediated platelet aggregation. These data demonstrate that PAC-1 recognizes an epitope on the IIb.IIIa complex that is located near the platelet fibrinogen receptor. Platelet activation appears to cause a Ca2+-dependent change involving the glycoprotein IIb.IIIa complex that exposes the fibrinogen receptor and, at the same time, the epitope for PAC-1.     

Involvement of divalent cations in the complex between the platelet glycoproteins IIb and IIIa

The major immunoprecipitate (No. 16) seen on crossed immunoelectrophoresis of Triton X-100-solubilized platelet proteins against whole platelet antibodies represents a complex containing the membrane glycoproteins IIb and IIIa. When EDTA is present during the solubilization, immunoprecipitate 16 as such is not observed, and two new arcs, termed 16a and 16b, appear. As with 16 these immunoprecipitates become radioactively labelled on lactoperoxidase-catalyzed iodination of platelets. Immunoprecipitate 16a showed partial immunochemical identity with 16, and was precipitated by an antibody raised against immunoprecipitate 16. The areas covered by immunoprecipitates 16, 16a and 16b were strongly reduced compared to normal with platelets from a patient with Glanzmann's thrombasthenia type II. Such platelets are known to contain reduced amounts of glycoproteins IIb and IIIa. The new arcs appearing when divalent cations are chelated by EDTA thus represent proteins derived from the immunoprecipitate 16 proteins, and divalent cations seem to be necessary to preserve the protein complex containing glycoprotein IIb and IIIa. The different complex formations between the components of immunoprecipitate 16 may reflect biochemical alterations of functional importance.     

Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex

Triflavin, an antiplatelet peptide containing Arg-Gly-Asp, purified from Trimeresurus flavoviridis venom, inhibits aggregation of human platelets stimulated by a variety of agonists. It blocks aggregation through interference with fibrinogen binding to its specific receptor on the platelet surface membrane in a competitive manner, but it has no apparent effect on intracellular events, such as thromboxane B2 formation, phosphoinositides breakdown and intracellular Ca2+ mobilization of thrombin-activated platelets. In this study, we determined the complete sequence of triflavin, which is composed of a single polypeptide chain of 70 amino acids. Its sequence is rich in cysteine and contains Arg-Gly-Asp at residues 49-51 in the carboxy-terminal domain. Triflavin shows about 68% identity of amino acid sequence with trigramin, which is a specific antagonist of the fibrinogen receptor associated with glycoprotein IIb/IIIa complex. [125I]Triflavin binds to unstimulated and ADP-stimulated platelets in a saturable manner and its Kd values are estimated to be 76 and 74 nM, respectively; the corresponding numbers of binding sites are 31,029 and 34,863 per platelet, respectively. [125I]Triflavin binding is blocked by Gly-Arg-Gly-Asp-Ser in a competitive manner. EDTA, the Arg-Gly-Asp-containing peptides (including naturally occurring polypeptides, trigramin and rhodostomin), and monoclonal antibody, 7E3, raised against GP IIb/IIIa complex, inhibit [125I]triflavin binding to unstimulated and ADP-stimulated human platelets. In conclusion, triflavin specifically binds to fibrinogen receptor associated with GP IIb/IIIa complex and its binding site is located at or near GP IIb/IIIa complex, overlapping with those of 7E3 and another Arg-Gly-Asp-containing polypeptide, rhodostomin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy

The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes and examined for glycoprotein stoichiometry and morphology. To determine the ratio of glycoproteins in the complex, the isolated glycoproteins were solubilized with sodium dodecyl sulfate and separated by high-performance liquid chromatography. Quantitative amino acid analysis of individual glycoproteins showed that the ratio of GP IIb to GP IIIa in the Ca2+-dependent complex was 0.93:1. Morphology was determined by electron microscopy of rotary-shadowed and negatively stained specimens. Individual complexes consisted of two domains: an oblong head of approximately 8 X 10 nm with two rodlike tails extending approximately 14-17 nm from one side of the head. Treatment of the isolated complex with EDTA resulted in the appearance of a mixture of oblong and filamentous structures, which could be separated by a sucrose gradient sedimentation in Triton X-100. As seen by rotary and unidirectional shadowing, GP IIb was a compact structure, approximately 8 X 10 nm in size. Isolated GP IIIa was more heterogeneous but was most often observed in an elongated form, varying in length from 20 to 30 nm and in width from 2 to 3 nm. By comparing these structures to that of the heterodimer complex, it was determined that the oblong domain was GP IIb and the rodlike tails were GP IIIa. Each milligram of isolated GP IIb-IIIa complex bound 0.30 mg of [3H]Triton X-100, indicating that the glycoprotein complex contained limited hydrophobic domains. Upon removal of detergent, GP IIb-IIIa complexes formed aggregates that sedimented in sucrose gradients as a diffuse peak ranging from 14 to 32 s. Examination of these aggregates by electron microscopy showed that they were composed of clusters or "rosettes" of 2 to 20 or more of the GP IIb-IIIa complexes. The orientation of these rosettes was such that the tails were joined in the center, with the head portions directed away from the interacting tails. It thus appears that the primary hydrophobic domains of the GP IIb-IIIa complex exist at the tips of the GP IIIa tails. Because the GP IIb-IIIa complex is an intrinsic membrane glycoprotein, these findings indicate a potential membrane attachment site for the GP IIb-IIIa complexes.