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.     

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.     

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.     

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.     

Characterization of the proteins of isolated human platelet alpha-granules. Evidence for a separate alpha-granule-pool of the glycoproteins IIb and IIIa

The protein composition of a well-defined alpha-granule preparation isolated from human platelets has been studied. Crossed immunoelectrophoresis against polyspecific platelet antibodies revealed more than 20 immunoprecipitates. The glycoprotein IIb-IIIa complex represented a major antigen in the Triton X-100-solubilized alpha-granule preparation and cross-reacted with the corresponding platelet membrane antigen. Furthermore, after lactoperoxidase-catalyzed 125I-iodination of whole platelets it was not labelled, in contrast to its membrane-located counterpart. This indicates an intracellular location of glycoproteins IIb and IIIa, probably as constituents of the alpha-granules. Fibrinogen, platelet factor 4, albumin, factor VIII-related antigen and the main granule glycoprotein (thrombinsensitive protein, thrombospondin) were identified in the alpha-granule preparation by the crossed immunoelectrophoresis technique. Crossed affinity immunoelectrophoresis using lectins revealed the presence of at least seven glycoproteins, and six sialoglycoproteins were identified by their altered electrophoretic mobility after neuraminidase treatment. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of reduced samples of the alpha-granules revealed at least 15 Coomassie Brilliant Blue-staining polypeptide bands, one of which comigrated with myosin heavy chain. No prominent band was observed in the actin region. Five glycopolypeptide bands were observed after periodic acid-Schiff staining. The dominant three represented the main granule glycoprotein, glycoprotein IIb and glycoprotein IIIa, respectively. More glycoproteins seem to be present in the alpha-granules than was previously recognized.     

Ca(2+)-dependent structural transitions of the platelet glycoprotein IIb-IIIa complex. Preparation of stable glycoprotein IIb and IIIa monomers

The platelet membrane glycoprotein (GP) IIb-IIIa complex is the receptor for adhesive proteins on activated platelets that mediates platelet aggregation. In the present study, factors affecting the structural stability of the purified GP IIb-IIIa complex and the dissociated subunits were investigated. Purified GP IIb-IIIa was incubated in various Ca2+ concentrations, and the percentage of dissociated subunits was quantitated by sucrose gradient sedimentation. Two Ca(2+)-dependent transitions were observed, one at about 60 microM Ca2+, where half of the complexes became dissociated, and the other at 0.1 microM Ca2+, where half of the dissociated subunits became incapable of reforming heterodimer complexes when higher Ca2+ concentrations were readded. This loss in ability to reform heterodimer complexes was caused primarily by a Ca(2+)-dependent transition in GP IIIa, leading to an apparent unfolding of this subunit, followed by the formation of high molecular weight aggregates. The formation of these aggregates was time- and temperature-dependent and could not be reversed by added Ca2+. Although Mg2+ prevented dissociation of GP IIb-IIIa, it failed to promote reassociation of the dissociated subunits. Based on these findings, conditions were developed for the preparation of dissociated GP IIb and GP IIIa such that 70% of the subunits remained functional in that they retained the ability to reform heterodimer complexes.     

Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb-IIIa complex

Platelet activation converts the membrane GP IIb-IIIa complex into a functional receptor for fibrinogen, but the mechanism is poorly understood. We asked whether induction of receptor competency coincides with a conformational change affecting the spatial arrangement of exoplasmic domains of the IIb and IIIa subunits. Epitopes on these subunits were labeled with monoclonal antibodies conjugated to either a donor fluorescein (FITC) or an acceptor tetramethylrhodamine (TR) chromophore. Then, fluorescence resonance energy transfer (RET) between platelet-bound FITC and TR was measured by flow cytometry. In unstimulated platelets, 6-8% RET efficiency was detected between antibody B1B5, bound to GP IIb, and antibody SSA6, bound to GP IIIa, regardless of which antibody served as RET donor. RET was also observed between these antibodies and A2A9, an antibody specific for the GP IIb-IIIa complex. Cell stimulation by thrombin, ADP plus epinephrine or phorbol-ester caused up to a 2-fold increase in RET between chromophore-labeled, platelet-bound B1B5, SSA6, and A2A9 (p less than or equal to 0.05), suggesting a change in the separation or orientation of these epitopes within the GP IIb-IIIa complex. The activation-related conformational change detected by the increase in RET between antibody B1B5 and SSA6 was independent of receptor occupancy since it was unaffected by the addition of fibrinogen or by the inhibition of fibrinogen binding by the antibody, A2A9, or the peptide, RGDS. In contrast to these results with antibodies bound to different epitopes within GP IIb-IIIa, no RET was observed between FITC-A2A9 and TR-A2A9 bound to different GP IIb-IIIa complexes or between a TR-labeled GP Ib antibody and FITC-labeled GP IIb-IIIa antibodies. These studies demonstrate that platelet activation causes a change in the spatial separation or orientation of exoplasmic domains within GP IIb and IIIa, which may serve to convert this integrin into a functional adhesion receptor.     

Immunochemical and amino-terminal sequence comparison of two cytoadhesins indicates they contain similar or identical beta subunits and distinct alpha subunits

Platelet membrane glycoprotein (GP) IIb-IIIa is functionally and antigenically related to proteins present on many cell types, suggesting that it is a member of the proposed cytoadhesin family of membrane proteins. We have compared the purified tissue vitronectin receptor (VnR) with GP IIb-IIIa. Anti-VnR immunoprecipitated GP IIb-IIIa and a related endothelial cell protein. In immunoblots, GP IIIa reacted with anti-VnR and the beta subunit of the VnR reacted with poly and monoclonal anti-GP IIIa. In contrast, the alpha subunit of the VnR failed to react either with a polyclonal anti-GP IIb or with monoclonal anti-GP IIb. Furthermore, the amino-terminal sequence of GP IIIa and the beta subunit of VnR were identical at determined residues while the alpha subunit and the GP IIb were different, but showed 33% identity. These data indicate the identity or close homology of GP IIIa and the beta subunit of the VnR. In contrast, the alpha subunit and GP IIb are distinct polypeptides which may be homologous. Since GP IIb-IIIa and the VnR differ in ligand recognition specificity, the data also suggest that this specificity may be governed by the alpha subunit of cytoadhesins.     

Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. Identity with platelet glycoprotein IIIa and similarity to "integrin"

Platelet membrane glycoprotein (GP) IIIa forms a Ca2+-dependent heterodimer complex with GP IIb. The GP IIb-IIIa complex constitutes the fibrinogen and fibronectin receptor on stimulated platelets. A biochemically and immunologically similar membrane glycoprotein complex is present on endothelial cells. A human umbilical vein endothelial cell cDNA library was screened using oligonucleotide probes designed from peptide sequences obtained from platelet GP IIIa. A cDNA clone was sequenced and found to encode a protein of 84.5 kDa. The translated endothelial cDNA contained five sequences that corresponded to peptide sequences in platelet GP IIIa, including the amino-terminal 19 residues. Thus, the endothelial and platelet forms of GP IIIa are apparently identical. Glycoprotein IIIa consists of a long amino-terminal extracellular domain with several potential N-linked glycosylation sites and four cysteine-rich tandem repeats, a 29-residue hydrophobic transmembrane segment, and a short carboxyl-terminal cytoplasmic domain. Glycoprotein IIIa has a 47% amino acid sequence homology to "integrin," a fibronectin receptor from chicken embryo fibroblasts. This homology suggests that GP IIIa is a member of a family of cell-surface adhesion receptors.     

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.     

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.     

Competition for related but nonidentical binding sites on the glycoprotein IIb-IIIa complex by peptides derived from platelet adhesive proteins

Two distinct sequences of amino acids, RGDS and HHLGGAKQAGDV, each inhibit the binding of fibrinogen, fibronectin, and von Willebrand factor to the platelet membrane glycoprotein IIb-IIIa complex. We have employed radiolabeled, photoactivatable aryl azide derivatives of the two sequences to explore the relationship between the binding sites for these peptides on the glycoprotein IIb-IIIa complex. Each probe specifically labeled only the glycoprotein IIb-IIIa complex of intact platelets. Since each peptide inhibited labeling of the receptor complex by the other, the peptides compete for binding sites on the receptor complex. However, the binding sites do not appear to be identical. Whereas the RGDS probe specifically labeled both glycoproteins IIb and IIIa, the HHLGGAKQA-GDV probe specifically labeled only glycoprotein IIb.     

Dissociation of the glycoprotein IIb-IIIa complex in isolated human platelet membranes. Dependence of pH divalent cations

The platelet membrane glycoproteins IIb and IIIa normally exist as a complex which forms a predominant immunoprecipitate after crossed immunoelectrophoresis of Triton-X-100-solubilized platelets. Dissociation of the complex occurs by solubilization in the presence of EDTA or EGTA at pH 8.7 and is readily verified by crossed immunoelectrophoresis. Incubations of isolated membranes with EDTA or EGTA at various pH levels were performed. Removal of the chelators and solubilization showed no dissociation of the glycoprotein IIb-IIIa complex in membranes incubated at pH below 8.0. At pH above 8.0 a dissociation which increased with increasing pH was seen. Under these conditions, dissociation appears to take place already in the intact membranes. The tendency of the glycoprotein IIb-IIIa complex to become dissociated with EDTA or EGTA at increasing pH seems to be due to increased chelating capacity of the chelators concomitant with a decreased chelating capacity of glycoprotein IIb and IIIa. The divalent cations Ca2+ and Mg2+, but not Cu2+, Zn2+, Mn2+ or Sr2+, in molar concentrations below that of EGTA were able to prevent the dissociation of the glycoprotein IIb-IIIa complex by the chelator at pH 9.0, indicating that Ca2+ as well as Mg2+ can be used to keep the complex together. In some experiments it was possible to reverse the dissociation in the membranes after removal of EDTA. At pH 7.5 reassociation occurred within 15 min whether divalent cations were added or not. At pH 9.0. reassociation occurred within 2 h provided Ca2+ was present. The tendency of glycoprotein IIb and IIIa to form a complex thus appeared to be most pronounced over the physiological pH range and to be a rapid process in platelet membranes under such conditions.     

Inhibition of fibrinogen binding to GP IIb-IIIa by a GP IIIa peptide.

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) mediates platelet aggregation and is a member of the cytoadhesin family of receptors that bind adhesive proteins such as fibrinogen, fibronectin, and von Willebrand factor. Despite the wide range of cell-substrate interactions mediated by these receptors, ligand binding domains have not yet been identified on any of the integrins. The present study was designed to determine potential fibrinogen binding domain(s) on the GP IIb-IIIa complex. Synthetic peptides derived from residues 1-288 of the amino-terminal portion of GP IIIa were tested for their abilities to block the binding of fibrinogen to purified GP IIb-IIIa in a solid-phase microtiter assay. Two overlapping peptides encompassing residues 204-229 of GP IIIa were identified which blocked fibrinogen binding in this assay. Polyclonal antibodies to these peptides blocked fibrinogen binding to purified GP IIb-IIIa as well as platelet aggregation. The overlapping residues of these two peptides GP IIIa (211-222), SVSRNRDAPEGG-NH2, blocked the binding of fibronectin, von Willebrand factor, and vitronectin to purified GP IIb-IIIa. Finally, direct binding of GP IIIa (204-229) to fibrinogen and fibronectin was demonstrated by enzyme-linked immunosorbent assay. We conclude from these studies that the amino acid sequence 211-222 of GP IIIa is critically involved in adhesive protein binding, and may represent an important portion of the GP IIb-IIIa ligand binding domain.     

Reconstitution of the purified platelet fibrinogen receptor. Fibrinogen binding properties of the glycoprotein IIb-IIIa complex

Several lines of evidence indicate that the platelet membrane glycoprotein IIb-IIIa complex (GP IIb-IIIa) is necessary for the expression of platelet fibrinogen receptors. The purpose of the present study was to determine whether purified GP IIb-IIIa retains the properties of the fibrinogen receptor on platelets. Glycoprotein IIb-IIIa was incorporated by detergent dialysis into phospholipid vesicles composed of 30% phosphatidylcholine and 70% phosphatidylserine. 125I-Fibrinogen binding to the GP IIb-IIIa vesicles, as measured by filtration, had many of the characteristics of 125I-fibrinogen binding to whole platelets or isolated platelet plasma membranes: binding was specific, saturable, reversible, time dependent, and Ca2+ dependent. The apparent dissociation constant for 125I-fibrinogen binding to GP IIb-IIIa vesicles was 15 nM, and the maximal binding capacity was 0.1 mol of 125I-fibrinogen/mol of GP IIb-IIIa. 125I-Fibrinogen binding was inhibited by amino sugars, the GP IIb and/or IIIa monoclonal antibody 10E5, and the decapeptide from the carboxyl terminus of the fibrinogen gamma chain. Furthermore, little or no 125I-fibrinogen bound to phospholipid vesicles lacking protein or containing proteins other than GP IIb-IIIa (i.e. bacteriorhodopsin, apolipoprotein A-I, or glycophorin). Also, other 125I-labeled plasma proteins (transferrin, orosomucoid) did not bind to the GP IIb-IIIa vesicles. These results demonstrate that GP IIb-IIIa contains the platelet fibrinogen receptor.     

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.     

Interaction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets

A murine monoclonal antibody, designated AP-2, reacts specifically with the complex formed by human platelet membrane glycoproteins IIb and IIIa, but does not react at all with the individual glycoproteins. Purified AP-2 covalently coupled to Sepharose CL4B was used as an immunoadsorbent column to purify the IIb-IIIa complex from a preparation of Triton X-100-solubilized human platelet proteins. Radioiodinated AP-2 was shown to bind to a single class of sites, with 57,400 +/- 9,700 molecules bound per cell (mean +/- S.D.) at saturation and a dissociation constant (Kd) of 0.64 +/- 0.15 nM (mean +/- S.D.). Binding could not be readily reversed even after a 1-h incubation with a 100-fold excess of cold antibody. AP-2 inhibits ADP-induced binding of radiolabeled fibrinogen to gel-filtered platelets in a noncompetitive fashion, consistent with the previous observation that AP-2 also inhibits the aggregation of platelets in plasma induced by a number of physiologic agonists, including adenosine diphosphate, epinephrine, collagen, thrombin, and arachidonic acid. Using AP-2, we have obtained evidence that the IIb-IIIa complex exists in the membrane of intact nonstimulated platelets and that complex integrity is not affected by external calcium ion concentration.     

Ligand inhibition of the platelet glycoprotein IIb-IIIa complex function as a calcium channel in liposomes

Platelet glycoproteins IIb and IIIa function as a fibrinogen receptor on the activated platelet. We have shown that these glycoproteins can be incorporated onto the surface of phosphatidylcholine vesicles with retention of fibrinogen and antibody binding properties and can permit Ca2+ transit across the phospholipid bilayer. In the current study we demonstrate that this apparent Ca2+ channel function is specifically inhibited by the synthetic analogue of the fibrinogen gamma COOH-terminal peptide, His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (His-12-Val), but not by the adhesive protein sequence Arg-Gly-Asp-Ser (RGDS). Prior incubation of IIb-IIIa liposomes with RGDS prevented Ca2+ transit inhibition by 25 microM His-12-Val, analogous to RGDS inhibition of His-12-Val binding to platelets. His-12-Val inhibited a minor component of transmembrane Ca2+ influx into ADP and thrombin-activated human platelets but had no effect on steady-state platelet 45Ca flux. These data indicate that ligand binding may exert a regulatory influence on transmembrane Ca2+ influx into activated platelets. The difference in inhibitory potency of the peptides studied may be related to differences in conformational changes in the glycoprotein IIb-IIIa complex induced by His-12-Val and RGDS, steric considerations, or differences in interactions with glycoprotein IIb Ca2+ binding domains.     

Synthetic peptides derived from fibrinogen and fibronectin change the conformation of purified platelet glycoprotein IIb-IIIa

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a platelet cell-surface receptor for fibrinogen and fibronectin. A carboxyl-terminal decapeptide of the fibrinogen gamma-chain (Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val LGGAKQAGDV] and a tetrapeptide (Arg-Gly-Asp-Ser (RGDS] from the fibrinogen alpha-chain and the fibronectin cell-binding domain appear to mediate the binding of these ligands to GP IIb-IIIa. The present study was designed to examine the effects of these and related peptides on the structure of purified platelet GP IIb-IIIa. Treatment of GP IIb-IIIa with various synthetic peptides affected the glycoprotein so that GP IIb alpha became a substrate for hydrolysis by thrombin. The order of potency of these peptides was as follows: RGDS greater than LGGAKQAGDV greater than KGDS greater than RGES. This is the same order of potency in which these peptides inhibit fibrinogen binding to platelets. This effect was time-, temperature-, and concentration-dependent; RGDS induced a half-maximal effect at approximately 60 microM. In addition, RGDS, but not RGES, decreased the intensity of the intrinsic protein fluorescence of GP IIb-IIIa. Finally, the decapeptide or RGDS decreased the sedimentation coefficient of GP IIb-IIIa from 8.5 to 7.7 or 7.4 S, respectively, whereas RGES had a minimal effect. This decrease was accompanied by an increase in the Stoke's radius from 74 to 82 A with RGDS or 85 A with the decapeptide, indicating a peptide-induced unfolding of the GP IIb-IIIa complex. This change in conformation may be related to changes in the distribution and function of GP IIb-IIIa on the platelet surface that occur when adhesive proteins or peptides from the GP IIb-IIIa binding domains of these proteins bind to GP IIb-IIIa.     

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.