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.     

Effect of calcium on the stability of the platelet membrane glycoprotein IIb-IIIa complex

Platelet membrane glycoproteins IIb and IIIa form a Ca2+-dependent heterodimer complex that contains binding sites for fibrinogen, von Willebrand factor, and fibronectin following platelet stimulation. We have studied the effect of Ca2+ on the stability of the IIb-IIIa complex using a IIb-IIIa complex-specific monoclonal antibody A2A9 to detect the presence of the complexes. Soluble IIb and IIIa interacted with A2A9-Sepharose only in the presence of Ca2+ with 50% IIb-IIIa binding requiring 0.4 microM Ca2+. In contrast, at 25 degrees C 125I-A2A9 binding to intact unstimulated platelets suspended in buffers containing EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was independent of the presence of Ca2+. However, the effect of Ca2+ chelators on 125I-A2A9 binding varied with temperature. At 37 degrees C, 125I-A2A9 binding to intact platelets became Ca2+-dependent with 50% binding requiring 0.4 microM Ca2+. This effect of temperature was not due to a change in platelet membrane fluidity because enrichment or depletion of platelet membrane cholesterol did not influence antibody binding. But, 125I-A2A9 binding to intact platelets at 25 degrees C did become Ca2+-dependent when the pH was increased above 7.4. Thus, at 1 nM Ca2+ and 25 degrees C, 50% antibody binding occurred at pH 9.0. Our studies demonstrate that Ca2+-dependent IIb-IIIa complexes are present on unstimulated platelets and that the Ca2+ binding sites responsible for the stability of these complexes are located on the external platelet surface. Our experiments also suggest that changes in platelet cytosolic Ca2+ do not regulate the formation of IIb-IIIa complexes.     

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.     

The IIb-IIIa glycoprotein complex that mediates platelet aggregation is directly implicated in leukocyte adhesion

Evidence is presented that the IIb-IIIa glycoprotein complex, which functions as the receptor for fibrinogen on platelets and is central to platelet aggregation, is expressed on the surface of leukocytes where it may function as a receptor for fibronectin. F(ab')2 fragments of a monoclonal antibody, 25E11, raised against activated large granular lymphocytes, inhibited killing by natural killer cells, blocked the binding of fibronectin-coated particles by monocytes, and stimulated neutrophils to exhibit increased antibody-dependent killing. Immunoprecipitation studies of leukocytes and platelets, and the ability of 25E11 to inhibit platelet aggregation, identified the antigen as an epitope on the IIb-IIIa complex. This glycoprotein thus constitutes the first example of a receptor mediating both platelet aggregation and leukocyte adhesion.     

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.     

Modulation of an RGDS binding site on the platelet membrane glycoprotein IIb-IIIa complex

Fibronectin, von Willebrand factor, and fibrinogen each bind to the glycoprotein IIb-IIIa complex on activated platelets via an arg-gly-asp-ser (RGDS) sequence present within the adhesive proteins. Both the IIb and IIIa polypeptides of the IIb-IIIa complex on thrombin activated platelets are specifically and extensively labeled by a radiolabeled, photoactivatable arylazide derivative of the RGDS sequence when the labeling is performed in the presence of concentrations of Ca++ or Mg++ approaching 0.5 mM. In contrast, labeling of unactivated platelets, ADP activated platelets, or thrombin activated platelets in the presence of low concentrations of divalent cations resulted in restriction of labeling to the IIb polypeptide of the complex.     

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.     

Characterization of a novel membrane glycoprotein involved in platelet activation

When platelets bind certain specific ligands they are induced to secrete the contents of their cytoplasmic granules and to aggregate. Studies of the molecular events accompanying this vital physiological response have led to a greater understanding of cell activation in general since the pathways involved are common to a number of cell types. By contrast most of the information about the cell surface molecules that initiate signal transduction has emerged from work on T lymphocyte activation, a process essential to the initiation of the immune response. We have described an activation antigen on T lymphocytes that is involved in the differentiation of these cells. In the present report it is demonstrated that the antigen is expressed on the platelet membrane with about 1,200 copies/platelet. A monoclonal antibody detecting this antigen stimulates platelet secretion and aggregation with a half-maximal response at approximately 10(-8) M. Characterization of the antigen, termed PTA1, reveals a glycoprotein of Mr 67,000 showing extensive N-linked carbohydrate, much of which appears to be heavily sialated. The amino-terminal sequence of PTA1, EEVLWHTSVPFAEXMSLEXVYPSM, indicates that the protein has not previously been characterized. Preliminary investigation of the mechanism by which PTA1 mediates platelet activation suggests involvement of protein kinase C and the 47-kDa protein of platelets is rapidly phosphorylated upon antibody-mediated activation. During this process PTA1 is also phosphorylated, as it is following platelet activation by the other agonists, collagen, thrombin, and 12-O-tetradecanoylphorbol 13-acetate. These results provide the first example of a cell surface glycoprotein that is directly involved in both platelet and T lymphocyte activation.     

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)     

Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIIa and extracellular calcium.

The biochemical responses of intact human platelets to the monoclonal antibody (mAb) AG-1 were investigated. AG-1 is a murine IgG mAb that recognizes a series of platelet membrane glycoproteins (Gp) from M(r) 21,000 to 29,000, one of which is the M(r) 24,000 (p24) receptor for anti-CD9 mAbs. AG-1 causes platelet aggregation and secretion. Platelets binding AG-1 demonstrate a dose- and time-dependent breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), production of diacylglycerol, and generation of phosphatidic acid (PA). These events are associated with the activation of protein kinase C (PKC), an increase in intracellular calcium, and fibrinogen binding. Platelet PA generation and PKC activation in response to AG-1 are inhibited by mAbs to platelet GpIIb-IIIa or by extracellular EGTA, but not by a mAb to platelet GpIb or by inhibiting platelet Na+/H+ exchange with 5-(N-ethyl-N-isopropyl)amiloride. Platelet cytoplasmic free calcium ([Ca2+]i) is elevated in response to AG-1, and this elevation is inhibited by mAbs to GpIIb-IIIa, an RGDS peptide or by chelating extracellular calcium. These results suggest that AG-1 binding to a unique platelet-surface glycoprotein initiates platelet responses through the activation of PIP2-specific phospholipase C, and that this occurs through a signal pathway that is dependent on GpIIb-IIIa and extracellular calcium.     

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.     

Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins

Treatment of platelets with thrombin was shown previously to induce rapid changes in tyrosine phosphorylation of several platelet proteins. In this report, we demonstrate that a variety of agonists which induce platelet aggregation also stimulate tyrosine phosphorylation of three proteins with apparent molecular masses of 84, 95, and 97 kD. Since platelet aggregation requires the agonist-induced activation of an integrin receptor (GP IIb-IIIa) as well as the binding of fibrinogen to this receptor, we examined the relationship between tyrosine phosphorylation and the function of GP IIb-IIIa. When platelets were examined under conditions that either precluded the activation of GP IIb-IIIa (prior disruption of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (addition of RGDS or an inhibitory mAb), tyrosine phosphorylation of the 84-, 95-, and 97-kD proteins was not observed. However, although both GP IIb-IIIa activation and fibrinogen binding were necessary for tyrosine phosphorylation, they were not sufficient since phosphorylation was observed only under conditions in which the activated platelets were stirred and allowed to aggregate. In contrast, tyrosine phosphorylation was not dependent on another major platelet response, dense granule secretion. Furthermore, granule secretion did not require tyrosine phosphorylation of this set of proteins. These experiments demonstrate that agonist-induced tyrosine phosphorylation is linked to the process of GP IIb-IIIa-mediated platelet aggregation. Thus, tyrosine phosphorylation may be required for events associated with platelet aggregation or for events that follow aggregation.     

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.     

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.     

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.     

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.     

A receptor-induced binding site in fibrinogen elicited by its interaction with platelet membrane glycoprotein IIb-IIIa.

The interaction of fibrinogen with membrane glycoprotein GPIIb-IIIa regulates platelet aggregation. This ligand:integrin receptor interaction elicits conformational changes in GPIIb-IIIa as evidenced by the induction of ligand-induced binding sites which are recognized by antibodies that react selectively with the occupied receptor. The dynamic nature of these conformational changes is now demonstrated by the identification and characterization of a receptor-induced binding site (RIBS) elicited in fibrinogen bound to GPIIb-IIIa. A monoclonal antibody to fibrinogen, anti-Fg-RIBS-I, failed to bind to nonstimulated platelets in the presence or absence of fibrinogen. However, when platelets were stimulated with an agonist, the antibody reacted with platelet-bound fibrinogen even in the presence of a marked excess of unbound fibrinogen. A key element of the RIBS epitope has been precisely localized to residues 373-385 of the gamma chain of fibrinogen. Conformational elements also are important in defining the epitope. Fab fragments of the antibody inhibited platelet aggregation. As these fragments also inhibited fibrin polymerization, a commonality between these two diverse functions of fibrinogen in thrombus formation is indicated. In general, antibodies to RIBS and ligand-induced binding site provide unique probes for characterizing ligand:receptor interactions.     

Examination of the platelet membrane glycoprotein IIb-IIIa complex and its interaction with fibrinogen and other ligands by electron microscopy.

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), is a calcium-dependent heterodimer that binds fibrinogen, von Willebrand factor, and fibronectin after platelet activation. We examined GPIIb-IIIa alone and bound to these ligands by electron microscopy after rotary shadowing with platinum/tungsten. We found, as observed previously, that in the presence of detergent and 2 mM Ca2+, GPIIb-IIIa consists of an 8 x 12-nm globular head with two 18-nm flexible tails extending from one side. We also found that in the presence of EDTA, GPIIb-IIIa dissociates into two similar comma-shaped subunits, each containing a portion of the globular head and a single tail. Using monoclonal antibodies to GPIIb, GPIIIa, and the GPIIb-IIIa heterodimer, we found that the tails contained the carboxyl termini of each subunit, while the nodular head was composed of amino-terminal segments of both subunits. Electron microscopy of GPIIb-IIIa bound to fibrinogen revealed a highly specific interaction of the nodular head of GPIIb-IIIa with the distal end of the trinodular fibrinogen molecule and with the tails of GPIIb-IIIa extended laterally at an angle of approximately 98 degrees with respect to the long axis of fibrinogen. When a GPIIb-IIIa was bound to each end of a single fibrinogen, the tails were oriented to opposite sides of fibrinogen, enabling fibrinogen to bridge two adjacent platelets. Electron microscopy of GPIIb-IIIa bound to fibronectin revealed GPIIb/IIIa-binding sites approximately two-thirds of the distance from the amino terminus of each end of the fibronectin molecule, while GPIIb-IIIa was found to bind to von Willebrand factor protomers along a rod-like region near the central nodule of the molecule.     

Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. II. Quantal activation parallels platelet capture in stir-associated microaggregation.

There is broad agreement that platelet aggregation is generally dependent on fibrinogen (Fg) binding to the glycoprotein (GP) IIb-IIIa receptor expressed on the activated platelet surface. We therefore compared rates and extents of aggregation and of fibrinogen receptor expression and specific Fg binding to activated platelets, as a function of ADP concentration. Human citrated platelet-rich plasma (diluted 10-fold) was stirred with adenosine diphosphate (ADP) for 10 s or 2 min to measure rates and extent of aggregation, respectively, determined from the decrease in the total number of particles. The number of fibrinogen receptors and bound Fg were measured from mean fluorescence values obtained with FITC-labeled IgM monoclonal antibody PAC1 and the IgG monoclonal antibody, 9F9, respectively, using flow cytometry as presented in part I (Frojmovic et al., 1994). Because flow cytometric and aggregation measurements were routinely determined at room temperature and 37 degrees C, respectively, we also compared and found temperature-independent initial rates of aggregation. The fraction of platelets with fluorescence values above one critical threshold value, corresponding to maximally "activated" platelets (P*), increased with increasing activator concentration and correlated linearly with the fraction of platelets recruited into aggregates for ADP (r > 0.9). Aggregation was not rate-limited by fibrinogen receptor expression or by Fg binding. It appears that each platelet expresses its maximal Fg receptors at a critical ADP concentration, i.e., occupancy of ADP receptors. This, in turn, leads to rapid Fg occupancy and capture of such "quantally activated" platelets into aggregates.