Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. I. Quantal activation of platelet subpopulations varies with adenosine diphosphate concentration.

We have previously reported that maximal platelet activation with adenosine diphosphate (100 microM ADP) causes rapid expression of all GPIIb-IIIa receptors for fibrinogen (FgR) (< 1-3 s), measured with FITC-labeled PAC1 by flow cytometry. We have extended these studies to examine the effects of ADP concentration on the graded expression and Fg occupancy of GPIIb-IIIa receptors. Human citrated platelet-rich plasma, diluted 10-fold with Walsh-albumin-Mg+2 (2 mM), was treated with ADP (0.1-100 microM). The rates of GPIIb-IIIa receptor expression or Fg binding were measured in unstirred samples by flow cytometry, using FITC-labeled monoclonal antibodies (mAb) PAC1 and 9F9, respectively, from on-rates, using increasing times between mAb and ADP additions. Fibrinogen receptors were all expressed rapidly at low (1 microM) or high (100 microM) ADP (few seconds), whereas Fg occupancy was 50% of maximal by about 2 min. The maximal extent of GPIIb-IIIa receptor expression and Fg occupancy was determined from maximal binding (Flmax) at 30 min incubation with PAC1 or 9F9. On-rates and maximal extents of binding for either PAC1 or 9F9 probes showed identical [ADP]-response profiles ("KD" approximately 1.4 +/- 0.1 microM). However, Flmax studies showed bimodal histograms consisting of "resting" (Po) and maximally "activated" (P*) platelets for both PAC1 and 9F9 binding, with the fraction of "activated" platelets increasing with ADP concentration. The data best fit a model where platelet subpopulations are "quantally" transformed from Po to P*, expressing all GPIIb-IIIa receptors, rapidly filled by Fg, but "triggered" at critical ADP concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Dynamic measurements of the platelet membrane glycoprotein IIb-IIIa receptor for fibrinogen by flow cytometry. II. Platelet size-dependent subpopulations.

Platelet aggregation has previously been shown to occur within 1 s of activation with 100 microM adenosine diphosphate (ADP) for both large (L) and small (S) platelet subpopulations, but L platelets were about twofold more sensitive and more rapidly recruited into microaggregates than were S platelets after correcting for differences in platelet surface area. Because platelet aggregation normally requires fibrinogen binding to glycoprotein IIb-IIIa receptors (FbR) expressed on the activated platelet surface, we wished to compare the kinetics and nature of FbR expression induced by ADP for L versus S platelets, and to measure size-dependent differences in FbR expression for platelets maximally activated with phorbol myristate acetate (PMA). We presented the theory and methodology in Part I (Frojmovic, M., T. Wong, and T. van de Ven. 1991. Biophys. J. 59:815-827) for measuring the rate of FbR expression (k1) and both the rate (k2) and efficiency (alpha) of binding of PAC1 to FbR as a function of activation conditions from the initial on-rate of FITC-PAC1 to FbR (V) and the maximal number of FbR expressed: these are measured, respectively, from the initial rate of increase in platelet-bound fluorescence (v) and the maximal increase in mean fluorescence (Flmax). We extended these analyses to L and S platelets, selected by electronic gating of forward scatter profiles (FSC), with corresponding fluorescence (Fl) histograms retrieved analytically. Platelet size (V) and surface area (SA), determined directly for cells separated with a cell sorter, were highly correlated with FSC, allowing v and Flmax values to be expressed per unit area of membrane for L:S comparisons. Surprisingly, ADP activation appeared to express all FbR within 1-3 s of ADP activation for both L and S platelets, whereas k1 was similar for PMA activation. In addition, L platelets maximally expressed two and three times more FbR per unit area than did S platelets when maximally stimulated, respectively, with ADP or PMA. Whereas k2 was independent of platelet size for a given activator, the efficiency of PAC1 binding (alpha), per unit area of membrane, was two times greater for L than for S platelets, for either ADP or PMA activation. Our data suggest that the FbR structure, its microenvironment, or its surface organization may vary with platelet size or activator type. Major reorganization of FbR and/or its environment appears to occur after approximately 5 min of ADP activation equally for both L and S platelets. A model is presented to account for size-dependent differences in FbR expression with implications for regulation of platelet aggregation.     

Interaction of fibrinogen with its platelet receptor. Differential effects of alpha and gamma chain fibrinogen peptides on the glycoprotein IIb-IIIa complex

The platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) recognizes peptides containing the amino acid sequence Arg-Gly-Asp, a sequence present at two locations in the alpha chain of fibrinogen. GPIIb-IIIa also interacts with peptides containing the carboxyl-terminal 10-15 residues of the fibrinogen gamma chain. We found that the alpha chain tetrapeptide, Arg-Gly-Asp-Ser (RGDS), and the gamma chain peptide, Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (LGGAKQAG-DV), each inhibited fibrinogen binding to ADP-stimulated platelets with Ki values of 15.6 +/- 2.7 and 46.2 +/- 8.2 microM, respectively. Furthermore, the inhibitory effect of the peptides was additive, indicating that they interact with GPIIb-IIIa in a mutually exclusive manner. Mutually exclusive binding suggests that either the alpha and gamma chain peptides bind to identical or overlapping sites on the GPIIb-IIIa complex or that one peptide induces a change in the complex that excludes the other. To differentiate between these possibilities, we compared the ability of RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and PAC-1, to ADP-stimulated platelets. A2A9 and PAC-1 appear to bind to different sites on GPIIb-IIIa because A2A9 binds to both stimulated and unstimulated platelets while PAC-1 only binds to stimulated platelets. RGDS specifically inhibited fibrinogen and PAC-1 binding with nearly identical Ki values of 15.6 +/- 2.7 and 20.2 +/- 3.5 microM, respectively. In contrast, LGGAKQAGDV had a differential effect on fibrinogen and PAC-1 binding, inhibiting PAC-1 binding with a Ki of 116.1 +/- 12.9 microM and fibrinogen binding with a Ki of 46.2 +/- 8.2 microM (p less than 0.005). Furthermore, while RGDS had no effect on the binding of the monoclonal antibody A2A9, LGGAKQAGDV was a partial inhibitor of A2A9 binding to activated platelets. These results suggest that the bindings sites for RGDS and LGGAKQAGDV are spatially distinct. They also suggest that ligand-induced changes in GPIIb-IIIa conformation are likely to be responsible for the mutually exclusive nature of alpha and gamma chain peptide binding.     

Ligands to the platelet fibrinogen receptor glycoprotein IIb-IIIa do not affect agonist-induced second messengers Ca2+ or cyclic AMP.

Previous studies have suggested that the platelet glycoprotein complex GPIIb-IIIa, which is the putative fibrinogen receptor, regulates Ca2+ influx into platelets, possibly operating as a Ca2+ channel. We have used RGD-peptides (peptides containing the sequence Arg-Gly-Asp; disintegrins), isolated from snake venoms, that have a high affinity and specificity for the fibrinogen-binding site of GPIIb-IIIa to address the question of whether blocking this site inhibits Ca2+ movement from the extracellular medium to the cytosol. Using fura-2-loaded human platelets, we found that neither disintegrins nor a monoclonal antibody (M148) to the GPIIb-IIIa complex altered the level of cytosolic Ca2+ obtained when the cells were stimulated with various agonists in the presence of either nominal or 1 mM extracellular Ca2+. In the presence of Mn2+, an ion that quenches fura-2 fluorescence, fura-2-loaded platelets were stimulated with thrombin or ADP. Neither disintegrins nor the monoclonal antibody altered the kinetics or the amount of quenching of fura-2 fluorescence by Mn2+. These data indicate that the binding of ligands to the fibrinogen receptor is not associated with an inhibition of Ca2+ movement through a receptor-operated channel. Furthermore, the disintegrins have no effect on platelet cyclic AMP metabolism in either the presence or the absence of phosphodiesterase inhibitors.     

Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen.

Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.     

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.     

Isolation and characterization of a chymotryptic fragment of platelet glycoprotein IIb-IIIa retaining Arg-Gly-Asp binding activity.

Platelet membrane glycoprotein (GP)IIb-IIIa exists as a divalent cation-dependent heterodimer which recognizes the Arg-Gly-Asp (RGD) sequence of adhesive proteins. To isolate the RGD binding domain of GPIIb-IIIa we performed proteolysis of GPIIb-IIIa with alpha-chymotrypsin. GPIIb-IIIa was bound to an affinity matrix of GRGDSPK-coupled Sepharose 4B and was then treated with chymotrypsin. After washing the unbound fragments, two discrete polypeptides of 55 and 85 kDa remained bound to the RGD affinity matrix and were specifically eluted by soluble HHLGGAKQAGDV (H12) or by GRGDSP, but not by GRGESP. Immunoblotting with subunit-specific polyclonal antibodies showed that the 55- and 85-kDa fragments were derived from GPIIb and GPIIIa, respectively. Amino-terminal sequencing and immunoblotting using site-specific antibodies indicated that these fragments contained the amino termini of their parent molecules. In the presence of 1 mM Ca2+ and 1 mM Mg2+, these two fragments were maintained as a heterodimer inasmuch as both fragments were immunoprecipitated by the polyclonal anti-GPIIIa antibodies. In contrast, chelating the divalent cations with 5 mM EDTA resulted in the lack of co-immunoprecipitation of the 55-kDa GPIIb fragment. After removal of the H12 peptide, the 55/85-kDa heterodimer bound to immobilized fibrinogen in an enzyme-linked immunosorbent assay by an RGD-dependent mechanism. These findings suggest that the RGD binding domain and structures required for heterodimer maintenance are present within the 55/85-kDa chymotryptic fragment of GPIIb-IIIa.     

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.     

Studies on the interaction of platelet glycoprotein IIb-IIIa and glycoprotein IV with fibrinogen and thrombospondin: a new immunochemical approach

We have designed a new binding assay based on crossed immunoelectrophoresis that allowed us to test for the relative capacities of platelet membrane glycoprotein IIb-IIIa (GP IIb-IIIa), and glycoprotein IV (GP IV) to bind purified Arg-Gly-Asp (RGD)-containing adhesive proteins. Preformed immune complexes were made by reacting a platelet lysate with murine monoclonal antibodies to GP IV (OKM5 and FA6-152) or to GP IIb-IIIa (AP-2). Upon two-dimensional electrophoretic separation in agarose gels and immunoprecipitation by a polyclonal antibody to mouse IgG, the immobilized complexes containing the desired antigen were further probed with purified 125I-labeled TSP or fibrinogen. Under these conditions, immobilized GP IV was found to specifically bind TSP, whereas it was unreactive with fibrinogen. By contrast, immobilized GP IIb-IIIa demonstrated fibrinogen binding capacity but did not demonstrate any reactivity toward TSP. These observations suggest that the overall structure of the adhesive protein may determine the accessibility of the RGD sequence to its binding site on GP IIb-IIIa.     

Dynamic measurements of the platelet membrane glycoprotein IIb-IIIa receptor for fibrinogen by flow cytometry. I. Methodology, theory and results for two distinct activators.

Platelet aggregation, which occurs within seconds of activation, is generally considered to be mediated by fibrinogen binding to glycoprotein IIb-IIIa which becomes expressed as a fibrinogen receptor (FbR) on the activated platelet surface. This receptor expression has, however, only been measured to date at relatively long activation times (greater than 15 min). We have therefore developed a theoretical and experimental approach for determining FbR expression within seconds of platelet activation using flow cytometry. The fluorescently labeled IgM monoclonal antibody FITC-PAC1, was used to report on the GPIIb-IIIa receptor for Fb (FbR). Human citrated platelet-rich plasma (PRP; diluted 1:10) was incubated with adenosine diphosphate (ADP) or phorbol myristate acetate (PMA) for varying times (tau = 0-10 s, out to 60 min), followed by incubation with fluorescein isothiocyanate (FITC)-PAC1 antibody at saturating concentrations. The time course of FITC-PAC1 binding was then measured for these variously preactivated samples (different tau) from the mean platelet-bound fluorescence (Fl), determined for greater than or equal to 5 s of PAC1 addition by dilution quenching and determination of fluorescence intensity histograms with the FACSTAR or FACSCAN (Becton-Dickinson Canada, Mississauga, Ontario) flow cytometers. Both rapid, initial rate of increase in Fl (nu) (related to PAC1 on-rates) and maximal extent of increase (Flmax) were thus determined for different tau values. These measurements yield the rate of formation of FbR (k1), and both the rate (k2) and efficiency (alpha) of binding of PAC1 to FbR as a function of activator type and time of action. We have found that ADP appears to cause rapid, maximal expression of FbR within 1-3 s (k1 greater than 20 min-1), whereas PMA expresses FbR in a slow, biphasic manner (k1 - 0.01 and 0.2 min-1). However, k2 and alpha for maximal PMA activation are about two and three times greater, respectively, than for maximal ADP-activation. Moreover, k2 decreases with post ADP activation time. These differences are discussed in terms of altered FbR organization and accessibility. This kinetic approach can be widely used to analyze the dynamics and organization of molecules on cell surfaces by flow cytometry, including studies of size-dependent subpopulations (see Part II, Frojmovic, M., and T. Wong. 1991. Biophys. J. 59:828-837).     

Trigramin. A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb-IIIa complex

Trigramin, a highly specific inhibitor of fibrinogen binding to platelet receptors, was purified to homogeneity from Trimeresurus gramineus snake venom. Trigramin is a single chain (approximately 9 kDa) cysteine-rich peptide with the Glu-Ala-Gly-Glu-Asp-Cys-Asp-Cys-Gly-Ser-Pro-Ala NH2-terminal sequence. Chymotryptic fragmentation showed the Arg-Gly-Asp sequence in trigramin. Trigramin inhibited fibrinogen-induced aggregation of platelets stimulated by ADP (IC50 = 1.3 X 10(-7)M) and aggregation of chymotrypsin-treated platelets. It did not affect the platelet secretion. Trigramin was a competitive inhibitor of the 125I-fibrinogen binding to ADP-stimulated platelets (Ki = 2 X 10(-8) M). 125I-Trigramin bound to resting platelets (Kd = 1.7 X 10(-7) M; n = 16,500), to ADP-stimulated platelets (Kd = 2.1 X 10(-8) M; n = 17,600), and to chymotrypsin-treated platelets (Kd = 8.8 X 10(-8) M; n = 13,800) in a saturable manner. The number of 125I-trigramin binding sites on thrombasthenic platelets amounted to 2.7-5.4% of control values obtained for normal platelets and correlated with the reduced number of GPIIb-GPIIIa molecules on the platelet surface. EDTA, monoclonal antibodies directed against the GPIIb-GPIIIa complex, and synthetic peptides (Arg-Gly-Asp-Ser and Tyr-Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val) blocked both 125I-fibrinogen binding and 125I-trigramin binding to platelets. Fibrinogen binding was more readily inhibited by these compounds than was trigramin binding. Monoclonal antibodies directed either against GPIIb or GPIIIa molecules did not block the interaction of either ligand with platelets. Reduced, S-pyridylethyl, trigramin did not inhibit platelet aggregation and fibrinogen binding to platelets and it did not bind to platelets, suggesting that the secondary structure of this molecule is critical for expression of its biological activity.     

Mean platelet volume: Interrelation with platelet aggregation activity and glycoprotein IIb-IIIa and Ib expression levels

Increased mean platelet volume (MPV) is an independent risk factor of thrombotic events in patients with cardiovascular diseases. Interactions of MPV with platelet aggregation activity and contents of glycoprotein (GP) IIb-IIIa (αIIb/β3 integrin, fibrinogen receptor) and GP Ib (von Willebrand factor receptor) have been investigated in this study. The study was performed in a group of healthy volunteers (n = 38) and a group of patients with acute coronary syndrome (ACS, n = 116). Patient’s blood was collected at days 1, 3–5 and 8–12 after ACS development. All patients received acetylsalicylic acid (ASA, inhibitor of thromboxane A2 synthesis) as the antiaggregant therapy and most of them also received clopidogrel (ADP receptor antagonist), except 44 patients who had not taken clopidogrel at day 1 before first blood collection. Aggregation of volunteers’ platelets was stimulated by 1.25, 2.5, 5 and 20 μM ADP, while aggregation of patients’ platelets was stimulated by 5 and 20 μM ADP. GP IIb-IIIa and GP Ib content on the platelet surface was measured using ¹²⁵I-labelled monoclonal antibodies. GP IIb-IIIa and GP Ib genetic polymorphisms were determined in ACS patients. In healthy donors significant correlations between MPV and aggregation levels have been recognized at 1.25 μM and 2.5 μM ADP (correlation coefficient (r) values of 0.396 and 0.373, p < 0.05), while at 5 μM and 20 μM ADP these interactions did not reach the level of statistical significance (r values of 0.279 and 0.205, p > 0.05). Correlations between MPV and aggregation levels were observed at day 1 of ACS in a subgroup of patients receiving ASA but before the beginning of clopidogrel treatment (r values of 0.526, p < 0.001 and 0.368, p < 0.05 for 5 and 20 μM ADP, respectively). Correlations between these parameters were not found during combined treatment of patients with ASA and clopidogrel. Strong direct correlations between MPV and GP IIb-IIIa and GP Ib contents were detected in both healthy donors and ACS patients (at all time points): the r values ranged from 0.439 to 0.647 (p ≤ 0.001 for all correlations). Genetic polymorphisms of GP IIb-IIIa (GP IIIa Leu33Pro) and GP Ib ((?5)T/C (Kozak) and Thr145Met) identified in ACS patients did not affect expression levels of corresponding glycoproteins. The data obtained indicate that increased MPV values correlate with increased platelet aggregation activity and enhanced GP IIb-IIIa and GP Ib expression.     

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.     

A mitogenic fibrinogen receptor that differs from glycoprotein IIb-IIIa. Identification by affinity chromatography and by covalent cross-linking

The mitogenic effect of human fibrinogen on the hemopoietic cell lines Raji and JM is mediated by a specific receptor with biochemical and functional properties different from those of the platelet fibrinogen receptor, the glycoprotein complex IIb-IIIa (Levesque, J.P., Hatzfeld, A., and Hatzfeld, J. (1986) Proc. Natl. Acad. Sci. U.S. A. 83, 6494-6498; Levesque, J.P., Hatzfeld, A., Hudry-Clergeon, G., Wilner, G.D., and Hatzfeld, J. (1987) J. Cell. Physiol. 232, 303-310). This work describes the identification of the mitogenic fibrinogen receptor (MFR) by two independent methods, affinity chromatography and covalent cross-linking. Affinity chromatography of surface-labeled cell extracts on fibrinogen-Sepharose revealed a 94 +/- 2-kDa membrane protein that bound specifically to fibrinogen-Sepharose only on cells that expressed the MFR. Its molecular mass was not modified after reduction. This was confirmed by cross-linking fibrinogen to surface-labeled Raji cells using the cleavable cross-linkers, ethyleneglycobis(succinimidyl succinate) and dithiobis(succinimidyl propionate). Complexes between fibrinogen and iodinated cell membrane proteins were immunoprecipitated by anti-fibrinogen antibodies. The biochemical cleavage of these immunoprecipitated conjugates gave rise to a 92 +/- 3-kDa membrane protein whose molecular mass was not modified after reduction. We conclude that fibrinogen binds specifically to a 92-94-kDa MFR which does not belong to the integrin family.     

Evidence for two functionally different fibrinogen receptors on hemopoietic cells: the glycoprotein IIb-IIIa and the mitogenic fibrinogen receptor

We have previously established that the mitogenic effect of fibrinogen on hemopoietic cell lines Raji and JM is mediated via a specific receptor (Levesque, J.-P. et al.: Proc. Natl. Acad. Sci. USA 83:6494-6498, 1986). In this study, we have further characterized the fibrinogen domain involved in the binding to the mitogenic receptor. This binding was not inhibited either by a monoclonal antibody against the C-terminal sequence of the fibrinogen gamma chains or by synthetic peptides containing the Arg-Gly-Asp sequence. Such inhibition is specific of the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. Fragments containing the fibrinogen D domain were the only plasmin degradation products of fibrinogen which were mitogenic. These fragments acted via direct binding on the mitogenic receptor with a Kd of 2.24 X 10(-6) M. This value was similar to the KI value of unlabeled fragments D (2.47 X 10(-6) M). Our results suggest the presence of two different functional types of fibrinogen receptors: the glycoprotein IIb-IIIa receptor responsible both for platelet aggregation and leukocyte adhesion and killing, and the mitogenic receptor involved in proliferation control of hemopoietic cells.     

Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro.

Glycoprotein IIb-IIIa (GPIIb-IIIa) is the fibrinogen receptor on activated platelets. GPIIIa is phosphorylated in resting platelets and the incorporation of 32Pi increases with platelet activation. To address the functional significance of this modification, the stoichiometry of GPIIIa phosphorylation was determined in resting and activated platelets by estimating the specific activity of metabolic [gamma-32P]ATP from the specific activity of phosphatidic acid. Approximately 0.01 mol of P/mol of GPIIIa was phosphorylated in resting platelets and 0.03 mol of P/mol of GPIIIa was phosphorylated in thrombin-, phorbol ester-, or U46619-treated platelets. Myosin light chain (MLC) phosphorylation served as a positive control for this method (1.2 mol of P/mol of MLC). Phosphorylation of purified GPIIb-IIIa by human platelet protein kinase C (PKC) resulted in levels of GPIIIa phosphorylation similar to that in platelets (0.05 mol of P/mol of GPIIIa). However, while GPIIIa in platelets was phosphorylated primarily on threonine, purified GPIIIa treated with PKC was phosphorylated primarily on serine. These results suggest that PKC may not directly phosphorylate GPIIIa in intact platelets. Ca2+/calmodulin-dependent kinase II phosphorylated purified GPIIIa to higher levels (0.5 mol of P/mol of GPIIIa) with phosphorylation on both threonine and serine. The limited phosphorylation of GPIIIa in intact platelets suggests that this event is unlikely to affect functions involving large populations of GPIIb-IIIa, such as its conversion to a fibrinogen receptor. However, these results may suggest the existence of a more readily phosphorylated subpopulation of GPIIb-IIIa with potentially distinct structural or functional properties.     

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.     

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.