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.     

Evidence that activation of platelet calpain is induced as a consequence of binding of adhesive ligand to the integrin, glycoprotein IIb-IIIa

Calpain (a Ca(2+)-dependent protease) is present in many cell types. Because it is present in the cytosol, the potential exists that it may regulate critical intracellular events by inducing crucial proteolytic cleavages. However, the concentrations of Ca2+ required to activate calpain are higher than those attained in the cytoplasm of most cells. Thus, the physiological importance of calpain and the mechanisms involved in its activation have remained elusive. In this study, we show that calpain rapidly moved to a peripheral location upon the addition of an agonist to suspensions of platelets, but it remained unactivated. We provide three lines of evidence that calpain was subsequently activated by a mechanism that required the binding of an adhesive ligand to the major platelet integrin, glycoprotein (GP) IIb- IIIa: calpain activation was prevented by RGDS, a tetrapeptide that inhibits the binding of adhesive ligand to GP IIb-IIIa; it was also prevented by monoclonal antibodies that inhibit adhesive ligand binding to GP IIb-IIIa; and its activation was markedly reduced in platelets from patients whose platelets have greatly reduced levels of functional GP IIb-IIIa. Thus, in platelets, binding of the extracellular domain of GP IIb-IIIa to its adhesive ligand can initiate a transmembrane signal that activates intracellular calpain. Because calpain is present in focal contacts of adherent cells, the interaction of integrins with adhesive ligands in the extracellular matrix may regulate activation of calpain in other cell types as well.     

Aggregation efficiency of activated normal or fixed platelets in a simple shear field: effect of shear and fibrinogen occupancy.

Shear rate can affect protein adsorption and platelet aggregation by regulating both the collision frequency and the capture efficiency (alpha). These effects were evaluated in well defined shear field in a micro-couette for shear rate G = 10 - 1000 s-1. The rate of protein binding was independent of G, shown for adsorption of albumin to latex beads and PAC1 to activated platelets. The initial aggregation rate for ADP-activated platelets in citrated platelet-rich plasma followed second order kinetics at the initial platelet concentrations between 20,000 and 60,000/microliters. alpha values, which dropped nearly fivefold for a 10-fold increase in G, were approximately proportional to G-1, contrary to a minor drop predicted by the theory that includes protein cross-bridging. Varying ADP concentration did not change alpha of maximally activated platelet subpopulations, suggesting that aggregation between unactivated and activated platelets is negligible. Directly blocking the unoccupied but activated GPIIb-IIIa receptors without affecting pre-bound Fg on "RGD"-activated, fixed platelets (AFP) by GRGDSP or Ro 43-5054 eliminated aggregation, suggesting that cross-bridging of GPIIb-IIIa on adjacent platelets by fibrinogen mediates aggregation. Alpha for AFP remained maximal (approximately 0.24) over 25-75% Fg occupancy, otherwise decreasing rapidly, with a half-maximum occurring at around 2% occupancy, suggesting that very few bound Fg were required to cause significant aggregation.     

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.     

Ca2+-mediated association of glycoprotein G (thrombinsensitive protein, thrombospondin) with human platelets

Washed human platelets suspended in buffers containing either 1.8 mM Ca2+ and 0.49 mM Mg2+ or 1 mM EDTA were treated with human alpha-thrombin to induce secretion. Glycoprotein G, a major glycoprotein in alpha-granules, was quantitatively secreted from platelets activated in the EDTA-containing buffer but remained with the platelet in the presence of Ca2+ and Mg2+. Addition of Ca2+ to the platelets that were activated in the presence of EDTA caused glycoprotein G to bind to platelets. To determine if glycoprotein G is expressed on the membrane surface of the activated platelet, platelets were rapidly labeled by a method employing lactoperoxidase-catalyzed iodination. Although glycoprotein G was barely detected on the surface of unstimulated platelets, labveling 1 min after thrombin treatment showed that glycoprotein G rapidly became one of the prominent surface proteins. These findings show that an alpha-granule protein, glycoprotein G, is one of the major glycoproteins on the membrane surface of thrombin-activated platelets and that its binding is dependent on divalent cations.     

Human platelet glycoprotein V: a surface leucine-rich glycoprotein related to adhesion

Human platelet glycoprotein V (Mr 82,000) is a surface glycoprotein and a substrate for thrombin, undergoing proteolytic cleavage by thrombin and releasing a soluble fragment, glycoprotein Vfl (Mr 69,000). It does not appear to be the receptor for thrombin's agonist effect on platelets. A congenital platelet disorder, Bernard-Soulier syndrome, is marked by a deficiency of glycoprotein V and two other surface glycoproteins, Ib-IX. The latter two, Ib-IX, constitute the platelet receptor for von Willebrand factor, mediate arterial platelet adhesion, and contain unique 24-amino acid sequences, termed "leucine-rich glycoprotein" segments. The segments relate to adhesive function and distinguish the leucine-rich glycoprotein family. Surface glycoprotein V is not physically associated with Ib-IX nor does it bind to von Willebrand factor. To date, no common denominator has been found that explains the combined deficiency of glycoproteins V and Ib-IX in Bernard-Soulier syndrome. This study describes the isolation of glycoprotein V/anti-glycoprotein V antibody and the analysis of three glycoprotein V peptides that contain "leucine-rich" sequences. Therefore, glycoprotein V shares the "leucine-rich" structure with platelet glycoproteins Ib-IX and belongs to the family of leucine-rich glycoproteins.     

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.     

Immunogold-surface replica study of ADP-induced ligand binding and fibrinogen receptor clustering in human platelets

Platelet cohesion requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins GPIIb and GPIIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad expanses of surface membranes in unstimulated and ADP-activated human platelets. We found that the gold prove was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. To ascertain whether the receptors clustered prior to ligand binding or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the secretion of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa binding domains of fibrinogen--namely, the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.     

The occupancy of glycoprotein IIb-IIIa complex modulates thrombin activation of human platelets

Platelet membrane glycoprotein (GP IIb-IIIa), besides its activity as adhesive protein receptor, displays a number of properties supporting its involvement in the mechanisms of transduction of the activation signal. Recently we have observed that GP IIb-IIIa ligands, mostly fibrinogen, inhibit Ca2+ movement and cytoskeleton reorganization caused by mild platelet activation. These findings led us to investigate the effect of GP IIb-IIIa ligands on agonist-induced platelet responses, with particular attention to the two major messenger generating systems, involving the activation of phospholipase C and the inhibition of cAMP production. In this paper we demonstrate that the occupancy of the major adhesive protein receptor on the platelet surface modulates the phosphatidylinositol cycle decreasing the amount of IP3, IP2 and IP produced after mild platelet activation as well as the pattern of protein phosphorylation. The platelet cAMP content of activated platelets was also affected and kept higher when evaluated under the same experimental conditions. Our data provide evidence for a role of fibrinogen binding in regulating the degree of activation of circulating platelets.     

In vitro activation and DNA binding affinity of human lymphoid (CEM-C7) cytoplasmic receptors labeled with the antiglucocorticoid RU 38486

The data reported here demonstrate that the synthetic steroid RU 38486 functions as an optimal antagonist in the glucocorticoid-sensitive human leukemic cell line CEM-C7. This steroid blocks the ability of the potent agonist triamcinolone acetonide (TA) to induce glutamine synthetase activity and to ultimately cause cell lysis, but when given alone does not exhibit partial agonist activity. Both [3H]RU 38486 and [3H]TA bind with high affinity and specificity to cytosolic glucocorticoid receptors in this cell line. However, under a variety of in vitro conditions (elevated temperature and presence of exogenous ATP), [3H]TA promotes receptor activation more effectively than [3H]RU 38486. This difference in the extent of activation was verified by two independent techniques: DEAE-cellulose chromatography and DNA-cellulose binding. [3H]RU 38486 and [3H]TA dissociate at the same rate from the unactivated receptors but at 25 degrees C (not 0 degree C) [3H]RU 38486 dissociates slightly more rapidly from the activated receptors. The defective receptors in the glucocorticoid-resistant subclone 3R7 appear to be "activation labile" (rapid dissociation of ligand from activated form) using either tritiated steroid. Once activated in vivo, the CEM-C7 [3H]TA- and [3H]RU 38486-receptor complexes undergo similar nuclear translocation and those activated complexes generated in vitro appear to bind to nonspecific DNA-cellulose with the same relative affinities. Thus the precise mechanism(s) by which RU 38486 exerts its potent antiglucocorticoid effect in this human cell line cannot be easily explained in terms of a defect in one of the crucial steps (specific high affinity binding, activation, translocation, DNA binding) required to elicit a physiological response. However, the data presented here do suggest that when comparing an antagonist and agonist which both bind to receptors with the same relative high affinity, the agonist may be more effective in facilitating the conformational change associated with in vitro activation.     

Thrombin-platelet interactions: an assessment of the roles of saturable and nonsaturable binding in platelet activation

Thrombin binds to platelets and induces platelet activation, but the relationship of binding to activation is not clear. To better define this relationship, we have analyzed parameters of binding and activation by alpha-thrombin and by three analogous proteases that activate platelets somewhat differently. The proteases were nitro-alpha-thrombin, a derivative with nitrated tyrosine, gamma-thrombin, a product of partial proteolysis of alpha-thrombin, and trypsin, a homologous protease. Nitro-alpha-thrombin and native alpha-thrombin activated platelets similarly, whereas gamma-thrombin and trypsin activated to a slightly lesser extent than alpha-thrombin and only after a distinctive delay. alpha-Thrombin and nitro-alpha-thrombin bound to platelets to about the same extent, but only alpha-thrombin showed evidence of saturable binding. Hirudin, a thrombin inhibitor, blocked both platelet activation and saturable binding by alpha-thrombin. With nitro-alpha-thrombin, hirudin blocked platelet activation, but it had no effect on binding. gamma-Thrombin and trypsin bound less than alpha-thrombin and with no evidence of saturable binding. There were identical relationships between the total amount bound and the extent of platelet activation for the four proteases (some show no saturable binding) but distinct differences in the relationships of total amount bound and the rate of activation; similar rates of activation required the binding of three to five times more gamma-thrombin or trypsin than alpha-thrombin. That is, without saturable binding, activation was slower. These data thus show a correlation between total amount bound and extent of activation but no correlation between amount saturably bound and the extent of platelet activation. Conversely, the rate of activation is more closely correlated with saturable binding than with total binding. We conclude that high-affinity saturable binding is not essential for thrombin-induced platelet activation but that it may accelerate the reaction.     

The physicochemical nature of 37 degrees C cytoplasmic glucocorticoid receptors in HeLa S3 cells

The physicochemical properties of size, shape and surface charge have been determined for the soluble fraction of cytoplasmic glucocorticoid receptors which are located in the HeLa S3 cell cytoplasm after incubation of whole cells with glucocorticoid at 37 degrees C. Under hypotonic buffer conditions approximately 80% of the total recovered [3H]triamcinolone acetonide receptor complexes sedimented through a 5-20% density gradients to the tube bottom, and approximately 90% eluted from a Sephacryl S-300 gel exclusion column in the void volume. Increasing the [KCl] of the buffer in the sucrose density gradients, and gel exclusion columns to 0.15 M caused a reduction in the percentage of this large aggregate to approximately 64% and approximately 75%, respectively. Further increases in the [KCl] during analysis to 0.4 M reduced the percentage of rapidly sedimenting receptors to approximately 62%, and shifted the sedimentation coefficient of the slower sedimenting receptors from approximately 5.2 S to 3.9 S. These conditions also decreased the fraction of receptor in the void volume of gel exclusion columns to 67%. Ion exchange analysis of receptor binding to DEAE cellulose, hydroxylapatite, phosphocellulose, and DNA cellulose revealed heterogenous populations of receptor species; comprising both "unactivated" and "activated" receptor forms. The ratios of unactivated/activated receptors was highly dependent on the matrix employed and differed substantially among those evaluated. For example, by the criteria of DEAE cellulose and phosphocellulose chromatography approximately 60% of the total 37 degrees C cytoplasmic receptors were in the "activated" state. A large fraction of these receptors, however, failed to bind to DNA cellulose. These results demonstrate that the glucocorticoid receptors which remain in the HeLa S3 cytoplasm at 37 degrees C do not bind to ion exchange materials, which are used as indexes of receptor "activation," in a uniform manner. We hypothesize that the diminished DNA binding capability of these receptors accounts for their cellular localization in the HeLa S3 cell cytoplasm at 37 degrees C.     

GPVI and GPIbα mediate staphylococcal superantigen-like protein 5 (SSL5) induced platelet activation and direct toward glycans as potential inhibitors

Background

Staphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to platelet glycoproteins and induce platelet activation. This study investigates further the interaction between SSL5 and platelet glycoproteins. Moreover, using a glycan discovery approach, we aim to identify potential glycans to therapeutically target this interaction and prevent SSL5-induced effects.

Methodology/Principal Findings

In addition to platelet activation experiments, flow cytometry, immunoprecipitation, surface plasmon resonance and a glycan binding array, were used to identify specific SSL5 binding regions and mediators. We independently confirm SSL5 to interact with platelets via GPIbα and identify the sulphated-tyrosine residues as an important region for SSL5 binding. We also identify the novel direct interaction between SSL5 and the platelet collagen receptor GPVI. Together, these receptors offer one mechanistic explanation for the unique functional influences SSL5 exerts on platelets. A role for specific families of platelet glycans in mediating SSL5-platelet interactions was also discovered and used to identify and demonstrate effectiveness of potential glycan based inhibitors in vitro.

Conclusions/Significance

These findings further elucidate the functional interactions between SSL5 and platelets, including the novel finding of a role for the GPVI receptor. We demonstrate efficacy of possible glycan-based approaches to inhibit the SSL5-induced platelet activation. Our data warrant further work to prove SSL5-platelet effects in vivo.     

The human fibroblast class II extracellular matrix receptor mediates platelet adhesion to collagen and is identical to the platelet glycoprotein Ia-IIa complex

A monoclonal antibody, P1H5, to the human fibroblast class II extracellular matrix receptor (ECMR II) specifically inhibits human fibroblast adhesion to collagen and immunoprecipitates a cell surface receptor containing an alpha and beta subunit of approximately 140 kilodaltons each (Wayner, E. A., and Carter, W. G. (1987) J. Cell Biol. 105, 1873-1884). We report here that P1H5 also specifically inhibits adhesion of unactivated human platelets to type I and III collagens, but not to fibronectin. Immunoprecipitation of the class II ECMR from Triton X-100 detergent lysates of platelets, after cell surface iodination, identified the platelet collagen receptor. Peptide mapping confirmed that the II alpha and II beta subunits immunoprecipitated from platelets are structurally homologous with those derived from fibroblasts. The platelet ECMR II alpha and -beta subunits comigrate with platelet membrane glycoproteins Ia and IIa, respectively, on two-dimensional nonreduced-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. These results indicate that platelet and fibroblast adhesion to collagen are both mediated by a similar receptor and that the alpha and beta subunits of this receptor are identical to platelet membrane glycoproteins Ia and IIa, respectively. Although glycoprotein Ia has been previously implicated as a collagen binding protein, our results are the first direct evidence that platelet glycoprotein Ia is associated with glycoprotein IIa in a heterodimer complex and that this complex, by mediating platelet attachment, is an actual receptor for platelet adhesion to collagen.     

Lysine binding to activated human platelets and its similarity to fibrinogen binding

Platelet surface glycoproteins IIb-IIIa are considered to function as the binding site for fibrinogen. Fibrinogen binding is essential for platelet aggregation and several amines have been shown to inhibit this binding. The present study compares the binding properties of 125I-fibrinogen and [3H]lysine with platelets activated by the Ca2+ ionophore A23187. Many lines of similarities in the binding properties are apparent; however, several differences were also found. The similarities are listed below and the differences are pointed out in parentheses. Marked enhancement by platelet activation; deficiency of binding by thrombasthenic platelets lacking the glycoproteins IIb-IIIa; saturability (fibrinogen binding approaches saturation at more than 12 microM, within 10 min; lysine binding at more than 100 mM within 1 min); Ca2+-dependence (at 1 mM Ca2+ lysine binding is minute and fibrinogen binding is half-saturated); reversibility; the binding achieved within 10 min is exchangeable; dissociation depends upon time and external ligand concentration; inhibition by the oligoamines His-Lys and Lys4; inhibition by serum from a thrombasthenic patient who developed anti-glycoproteins IIb-IIIa antibodies; specificity; alanine neither binds to activated platelets nor inhibits fibrinogen binding; it thus appears that the lysine which associates with activated platelets is mostly bound onto the surface of the cells rather than being incorporated. Moreover, the major site of lysine binding seems to be the complexed glycoproteins IIb-IIIa.     

The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.     

Specific binding of blood coagulation factor XIIIa to thrombin-stimulated platelets

We studied the binding of 125I-platelet and plasma Factor XIII (125I-Factor XIII) to human platelets. When 125I-Factor XIII was incubated with gel-filtered platelets, calcium chloride (5 mM) and thrombin (1 unit/ml) at 37 degrees C, saturable binding was observed. Half-maximal binding occurred at 1 min. Binding was inhibited 93% by a 100-fold molar excess of unlabeled ligand but not by other purified proteins. Greater than 87% of platelet-bound radioactivity migrated as thrombin-cleaved a-chains (a'-chains) in sodium dodecyl sulfate-polyacrylamide gels indicating that Factor XIIIa but not Factor XIII binds to platelets. 125I-Factor XIIIa does not bind to unstimulated platelets. When platelet secretion was blocked, binding was markedly inhibited. 125I-Factor XIIIa bound minimally to platelets stimulated with agonists other than thrombin. Thus, binding is dependent on platelet activation, as well as modification of platelets by thrombin. 125I-Factor XIIIa bound to gamma-thrombin-stimulated platelets, at concentrations which did not clot fibrinogen. Therefore, Factor XIIIa is not bound to fibrin associated with platelets. Binding was only partially reversible. Approximately 12,000 molecules of Factor XIIIa were bound per platelet. 125I-Factor XIIIa bound normally to platelets from patients with severe Glanzmann's thrombasthenia indicating that 125I-Factor XIIIa does not bind to platelet glycoproteins IIb or IIIa, or platelet-bound fibrinogen. Chymotrypsin treatment of platelets inhibited 125I-Factor XIIIa binding by 78% without inhibiting secretion. Methylamine and putrescine, Factor XIIIa substrates, and N-ethylmaleimide, an active site inhibitor, did not inhibit binding. Factor XIIIa bound to platelets was enzymatically active and catalyzed [3H]putrescine incorporation into platelet proteins. The specific binding of Factor XIIIa to platelets suggests it may play a role in physiologic reactions involving platelets.     

Exposure of binding sites for vitronectin on platelets following stimulation

Vitronectin is a glycoprotein that mediates cell adhesion and spreading in a number of cell culture systems. Liposomes containing platelet glycoproteins IIb-IIIa complex have been shown to bind vitronectin-coated surfaces through an Arg-Gly-Asp cell attachment mechanism. We examined the expression of the binding sites for vitronectin on the surface of intact, resting platelets and following stimulation. 125I-Labeled vitronectin bound specifically in a saturable manner to platelets treated with physiological concentrations of thrombin. The binding reached saturation at 100 nM concentration, and, at saturation, approximately 5000 specific binding sites were detected per platelet. The binding was divalent cation-dependent and only partially reversible after complete saturation. A synthetic hexapeptide containing the Arg-Gly-Asp sequence inhibited vitronectin binding to platelets. A monoclonal antibody against platelet glycoprotein IIb-IIIa complex also inhibited the binding of vitronectin to stimulated platelets. These data suggest that platelets possess an inducible divalent cation-dependent receptor for vitronectin and that the glycoprotein IIb-IIIa complex is involved in the expression of the vitronectin receptor.     

The "activated" hepatic glucocorticoid-receptor complex. Its generation and properties.

The glucocorticoid receptor-glucocorticoid complex of the hepatic cytosol need undergo an "activation" to enable its binding to nuclei, chromatin, or stripped DNA. The conditions of this activation have been studied using native calf thymus DNA absorbed to cellulose. At low ionic strength, activation is very slow at 0 degrees, but, takes place rapidly at 25 degrees, reaching completion at 1 hour. Addition of 10 mm CaCl2 or 150 mm NaCl increases the rate of activation of the receptor at 0 degrees. Neither magnesium nor manganese ions can replace calcium with respect to enabling activation of the steroid-receptor complex to occur at low temperatures. Isofocusing studies reveal that the major component of the unactivated steroid-receptor complex has an isoelectric point of 7.1. Incubation of the steroid-receptor complex at 25 degrees for 30 min leads to its conversion to a form with an isoelectric point of 6.1 concurrent with the development of its ability to bind to DNA-cellulose. Sucrose density gradient analysis reveals that no detectable alteration in the sedimentation coefficient of the steroid-receptor complex occurs during its activation. MnCl2 (20mm) effeciently precipitates the unactivated hormone-receptor complex and to a lesser degree, precipitates the activated hormone-receptor complex.     

Mapping the binding domains of the alpha(IIb) subunit. A study performed on the activated form of the platelet integrin alpha(IIb)beta(3).

alpha(IIb)beta(3), a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the alpha(IIb) subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of alpha(IIb) potentially involved in the platelet-aggregation event. Regions alpha(IIb) 313-332, alpha(IIb) 265-284 and alpha(IIb) 57-64 of alpha(IIb)beta(3) were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of alpha(IIb)beta(3). Furthermore, alpha(IIb) 313-332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in alpha(IIb) 313-332, 265-284 and 57-64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.