Plasma autoantibodies against platelet glycoprotein IIb/IIIa from patients with autoimmune thrombocytopenic purpura may recognize different antigenic determinants.

Autoantibodies against platelet glycoprotein (GP) GPIIb/IIIa have been demonstrated in patients with autoimmune thrombocytopenic purpura. Recently, it has been shown that plasma autoantibodies from some patients bind to the cytoplasmic domain of GPIIIa. Our aim was to evaluate further the binding specificity of these plasma autoantibodies. From 7 patients with detectable plasma antibodies against intact GPIIb/IIIa, 1 showed strong antibody binding to a synthetic C-terminal peptide of GPIIIa. Ig class analysis of affinity purified anti-GPIIb/IIIa autoantibodies from this patient revealed an IgM antibody that reacted with intact GPIIb/IIIa as well as with recombinant GPIIb/IIIa lacking the C-terminal domains, and an IgG antibody that bound to intact GPIIb/IIIa but not to GPIIb/IIIa lacking the C-terminal region. These data indicate that this patient has at least 2 autoantibodies, an IgG directed against the cytoplasmic domain of GPIIIa and an IgM reacting with the extracellular part of GPIIIa. This may support the hypothesis that plasma IgG antibodies directed against the C-terminal domain of GPIIIa may be due to the exposition of cytoplasmic epitopes of GPIIIa as a result of increased cell lysis by IgM autoantibodies.

Blocking platelet aggregation inhibits thromboxane A2 formation by low dose agonists but does not inhibit phosphorylation and activation of cytosolic phospholipase A2.

Inhibition of aggregation by Ro 44-9883, a potent and selective non-peptide GPIIb/IIIa antagonist, resulted in inhibition of serotonin secretion induced by weak agonists such as ADP or low doses of either thrombin receptor agonist peptide (TRAP) or collagen. In contrast, alpha granule secretion was inhibited to different extents dependent on donor, averaging 60% inhibition. Inhibition of serotonin secretion correlated with an inhibition of thromboxane A2 (TxA2) formation, both of which were overcome by higher doses of TRAP or collagen. Ro 44-9883 had no effect on the already reduced serotonin secretion and TxA2 formation in Glanzmann's thrombasthenic platelets. Restoration of serotonin secretion in the absence of aggregation requires both TxA2 and lysophosphatidic acid. In addition, Ro 44-9883 inhibition of TxA2 formation was not due to a lack of phospholipase A2 (PLA2) phosphorylation and activation as assayed in vitro. These results suggest that aggregation is required for weak or low dose agonist induced in vivo activity of PLA2, possibly by either regulating phospholipid substrate availability or interaction of PLA2 with platelet membranes.

Stimulation of platelet glycoprotein IIb-IIIa (alpha IIb beta 3-integrin) functional activity by a monoclonal antibody to the N-terminal region of glycoprotein IIIa.

Platelet glycoprotein (GP) IIb-IIIa complex (alpha IIb beta 3-integrin) changes its conformation upon platelet activation that results in binding of RGD-containing ligands and expression of ligand-induced binding site (LIBS) neoepitopes. Anti-GIIb-IIIa monoclonal antibody (monAB) CRC54 bound to < or = 10% of GPIIb-IIIa on resting platelets but binding was enhanced by the occupation of GPIIb-IIIa with RGDS peptide and by platelet activation indicating that CRC54 is directed against LIBS epitope. The epitope was located within the first 100 N-terminal residues of GPIIIa and differed from other LIBS epitopes. CRC54 as well as its Fab fragments were able to induce platelet aggregation. CRC54 also stimulated interaction of GPIIb-IIIa with its ligands (fibrinogen and fibronectin) and conformation-dependent antibodies. The results indicated that changes of GPIIb-IIIa conformation, binding of ligands and platelet aggregation could be stimulated via interaction of anti-LIBS antibody with the N-terminal part of GPIIIa.

Determination of kinetic constants for the interaction between the platelet glycoprotein IIb-IIIa and fibrinogen by means of surface plasmon resonance.

The binding reaction between purified human platelet glycoprotein IIb-IIIa and fibrinogen was investigated by real-time measurements using the surface-plasmon-resonance sensor technology. In these experiments, either glycoprotein IIb-IIIa or fibrinogen was immobilized on a sensor surface. The time-dependent change in surface coverage that occurred immediately upon contact with a solution of the complementary protein was then detected. The ability to record this dynamic event from its initiation allowed the collection of kinetic and thermodynamic data over an extended time period. These data indicated that initially, in fast reaction, a reversible low-affinity complex with an equilibrium dissociation constant, Kd, of 155-180 nM was formed. In a subsequent slower reaction this complex was transformed into a more stable high-affinity complex with a Kd of 20-70 nM. Efficient dissociation of the high-affinity complex could only be induced in the presence of a competitive inhibitor such as RGDV. These data demonstrate that the binding between glycoprotein IIb-IIIa and fibrinogen is not a single monophasic reaction, but is composed of at least two consecutive processes both with their own kinetics.

Tetrafibricin has a high selectivity for GPIIb/IIIa: comparison of the effects of tetrafibricin and RGDS on GPIIb/IIIa and the vitronectin receptor.

The specificity of tetrafibricin was examined by comparing its activities on GPIIb/IIIa and on the vitronectin receptor (alpha v beta 3) with those of Arg-Gly-Asp-Ser (RGDS) on the same receptors. Tetrafibricin, which inhibited fibrinogen-GPIIb/IIIa binding 10 times more potently than RGDS, was three orders of magnitude less potent compared to RGDS on the inhibition of fibrinogen binding to alpha v beta 3. Furthermore, tetrafibricin potently inhibited platelet adhesion to both fibrinogen and von Willebrand factor. Whereas, there was no significant inhibition observed in the GPIIb/IIIa-independent cellular adhesions. These results suggest that tetrafibricin is highly selective for GPIIb/IIIa.

Tetrafibricin, a novel non-peptide fibrinogen receptor antagonist, induces conformational changes in glycoprotein IIb/IIIa.

Arg-Gly-Asp (RGD) is an amino acid sequence in fibrinogen recognized by platelet glycoprotein (GP) IIb/IIIa. Recently, it was found that RGD peptide binding to GPIIb/IIIa leads to conformational changes in the complex that are associated with the acquisition of high-affinity fibrinogen-binding function. In this study, we found that tetrafibricin, a novel non-peptidic GPIIb/IIIa antagonist, induced similar conformational changes in GPIIb/IIIa as did RGD peptides. Tetrafibricin increased the binding of purified inactive GPIIb/IIIa to immobilized pl-80, a monoclonal antibody that preferentially recognizes ligand-occupied GPIIb/IIIa. Exposure of the pl-80 epitope by tetrafibricin was also observed on resting human platelets by flow cytometry. On intact platelets, the conformational changes transformed GPIIb/IIIa into a high-affinity receptor for fibrinogen and triggered subsequent platelet aggregation. Tetrafibricin is the first non-peptidic GPIIb/IIIa antagonist reported that has the capacity to induce conformational changes in GPIIb/IIIa.

Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia.

One proposed ligand binding site on platelet integrin alpha IIb beta 3 is the region of the beta 3 subunit encompassing amino acids 211-221. However, we recently showed that synthetic peptides corresponding to amino acids 211-221 inhibit fibrinogen binding to alpha IIb beta 3 by binding to alpha IIb beta 3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214-221 of beta 3, bound to immobilized active alpha IIb beta 3 but did not inhibit fibrinogen binding to the complex. We then determined whether nonfunctional alpha IIb beta 3 on platelets with a beta 3 Arg-214-->Trp mutation (Strasbourg I variant of Glanzmann's thrombasthenia or GTV) could be induced to aggregate after treatment with dithiothreitol (DTT). DTT has been shown to expose the fibrinogen receptor on normal platelets. DTT treatment of GTV platelets did result in the formation of the fibrinogen binding site as indicated by the binding of pI-55, an MoAb that only binds to the activated form of alpha IIb beta 3. Furthermore, DTT-treated GTV platelets aggregated in the presence of fibrinogen and divalent cations. This aggregation was inhibited by EDTA, RGDS, and the selective alpha IIb beta 3 antagonist, Ro 43-5054. These data show that Arg-214 of beta 3 is not required for fibrinogen binding or for platelet aggregation. However, this amino acid appears to be critical for the formation and for the maintenance of the correct tertiary structure of the fibrinogen binding site on alpha IIb beta 3.

The integrin alpha IIb-beta 3, platelet glycoprotein IIb-IIIa, can form a functionally active heterodimer complex without the cysteine-rich repeats of the beta 3 subunit.

Integrin alpha IIb-beta 3 binds fibrinogen via the recognition sequence Arg-Gly-Asp-Ser (RGDS). We have used the baculovirus/insect cell expression system to study the structural requirements for the formation of a functionally active fragment of alpha IIb-beta 3. A tandem baculovirus transfer vector was constructed containing the cDNA coding for the heavy chain of human alpha IIb (alpha IIbH, amino acids 1-874) and the cDNA coding for a truncated form of human beta 3 (t beta 3; amino acids 1-469). Sf9 insect cells were infected with the corresponding baculovirus, and the produced soluble recombinant proteins were purified using an RGD-like affinity column. The bound receptor fragments were specifically eluted with RGDS and existed as a heterodimeric complex (rec alpha IIbH-t beta 3) with an apparent M(r) of 160,000. In an immunocapture assay, the monoclonal antibody pl-55, which only recognizes the functionally active form of alpha IIb-beta 3, bound to the purified complex. Rec alpha IIbH-t beta 3 specifically bound 125I-fibrinogen with an affinity comparable with that of purified platelet alpha IIb-beta 3. Electron micrographs of rotary-shadowed rec alpha IIbH-t beta 3 showed that the complex had the characteristic globular head, but the two rodlike tails were 4-6 nm shorter than those found in intact alpha IIb-beta 3. Thus, the cysteine-rich repeats of beta 3 are not required for assembly, stability, and functional activity of this integrin.

Peptides derived from a sequence within beta 3 integrin bind to platelet alpha IIb beta 3 (GPIIb-IIIa) and inhibit ligand binding.

Peptides derived from a sequence within the loop structure of human platelet glycoprotein (GP) IIIa (integrin beta 3) were previously shown to inhibit fibrinogen binding to purified GPIIb-IIIa. In this study a series of peptides based on the GPIIIa sequence 211-221 (SVSRNRDAPEG) was synthesized. The most active peptide was determined to be RNRDA, and its inhibitory potency was 4-fold greater (IC50 = 4.8 microM) than that of SVSRNRDAPEG. These GPIIIa peptides also inhibited the binding of two monoclonal antibodies, pl-55 and PAC-1, which are directed against the activated conformer of GPIIb-IIIa. To determine whether these peptides bound directly to GPIIb-IIIa, an affinity matrix was prepared by coupling RNRDAPEGC to Sepharose. Fibrinogen or purified GPIIb-IIIa was applied to the affinity column. Only GPIIb-IIIa was retained on the column, and it could be specifically eluted by GPIIIa peptide or RGDV but not by an irrelevant peptide. Additionally, we observed that the binding of GPIIIa peptides to purified GPIIb-IIIa induced exposure of a neoepitope on GPIIb that was recognized by the monoclonal antibody pl-80. These data suggest that sequences within the loop structure of GPIIIa can interact with the ligand binding domain of GPIIb-IIIa. Thus, this GPIIIa domain may be involved in regulating the accessibility of ligands to GPIIb-IIIa following platelet activation.

Reversible conformational changes induced in glycoprotein IIb-IIIa by a potent and selective peptidomimetic inhibitor.

Platelet glycoprotein (GP) IIb-IIIa inhibitors may become useful antithrombotic agents. Ro 43-5054 is a low molecular weight, noncyclic, peptidomimetic inhibitor that is three orders of magnitude more potent than RGDS in inhibiting fibrinogen binding to purified GPIIb-IIIa and in preventing platelet aggregation. Comparisons of RGDS and Ro 43-5054 in cell adhesion assays showed that, in contrast to RGDS, Ro 43-5054 was highly selective GPIIb-IIIa inhibitor. Effects of RGDV and Ro 43-5054 on the conformation and activation state of GPIIb-IIIa were also examined. RGDV and Ro 43-5054 induced conformational changes in purified inactive GPIIb-IIIa as determined by binding of the monoclonal antibody D3GP3 (D3). These conformational alterations were not reversed after inhibitor removal, as indicated by the continued exposure of the D3 epitope and a newly acquired ability to bind fibrinogen. Similarly, RGDV and Ro 43-5054 induced conformational changes in GPIIb-IIIa on the intact platelet. However, after removal of the inhibitors, exposure of the D3 epitope was fully reversed and the platelets did not aggregate in the absence of agonist. Thus, while RGD(X) peptides and Ro 43-5054 transformed purified inactive GPIIb-IIIa into an irreversibly activated conformer, the effects of these inhibitors were reversible on the intact platelet. This suggests that factors present in the platelet membrane or cytoplasm may regulate in part the ability of the complex to shift between active and inactive conformers.

Conformational modulation of purified glycoprotein (GP) IIb-IIIa allows proteolytic generation of active fragments from either active or inactive GPIIb-IIIa.

This study characterized conformational states of platelet glycoprotein IIb-IIIa (GPIIb-IIIa) and regions of the molecule required for fibrinogen binding. Platelet lysates were passed sequentially over concanavalin A and aminoethylglycine (Aeg)RGDS affinity columns. Approximately 10% of the total GPIIb-IIIa bound to the Aeg-RGDS column. The non-binding GPIIb-IIIa was further purified by S300 gel filtration. Only GPIIb-IIIa which recognized immobilized RGDS bound fibrinogen. The functional difference between the Aeg-RGDS binding GPIIb-IIIa (active) and the S300-purified complex (inactive) suggested that the two populations existed in different conformations. This was confirmed immunochemically and in an assay utilizing endoproteinase Arg-C. Active GPIIb-IIIa was heavily degraded by Arg-C, whereas inactive GPIIb-IIIa was highly resistant to degradation. Receptor occupancy by RGDV or peptidomimetic inhibitors prevented degradation of regions of the active complex and stimulated hydrolysis of the inactive receptor such that the two populations yielded fragments of identical electrophoretic mobility. Induction of hydrolysis of inactive GPIIb-IIIa required 15-fold higher concentrations of RGDV than protection of the active complex. Upon removal of inhibitor, fragments generated from either active or inactive GPIIb-IIIa bound fibrinogen. The ability of carboxypeptidase Y to digest inhibitor-protected GPIIb-IIIa was also examined. GPIIb was cleaved to a 58-kDa NH2-terminal fragment, whereas GPIIIa remained essentially intact. The complexed fragments bound fibrinogen with similar affinity as intact GPIIb-IIIa. This binding was inhibited by both RGDV and HHLGGAKQAGDV peptides. These data suggest that: 1) purified active and inactive GPIIb-IIIa exist in different conformations and have different affinities for RGDV; 2) certain peptidomimetic inhibitors (Ro 42-1499 and Ro 43-5054) alter the conformation of inactive GPIIb-IIIa; 3) GPIIIa and a 58-kDa NH2-terminal fragment of GPIIb alpha form a high affinity fibrinogen binding complex.

Further characterization of the loop structure of platelet glycoprotein IIIa: partial mapping of functionally significant glycoprotein IIIa epitopes.

Glycoprotein (GP) IIb-IIIa serves as the platelet fibrinogen receptor. Studies of the tertiary structure of GPIIIa have shown that the protein has a large loop structure of at least 325 amino acids in length. To further characterize this loop structure, intact platelets were digested with alpha-chymotrypsin. Digestion products were examined using the anti-GPIIIa monoclonal antibodies (MoAbs) AP3, D3GP3, and C5GP3, as well as the human alloantibody, anti-PLA1. AP3 recognized GPIIIa digestion products of 109, 95, and 68 Kd. D3GP3 and C5GP3 recognized an additional band of 51 Kd. Time course digestions demonstrated that the 51-Kd fragment was generated by proteolysis of the 68-Kd peptide. Sequence analysis of the reduced 51-Kd peptide showed that this fragment began at amino acid 422. The nonreduced 51-Kd peptide was reactive with antibodies directed against the first 13 amino acids of GPIIIa, demonstrating the presence of a covalently attached N-terminal peptide. These data suggest that: (1) the minimum length of the loop structure is at least 384 amino acids; (2) the AP3 epitope is formed at least in part by a determinant contained within residues 348 to 421; and (3) the D3GP3 and C5GP3 epitopes are contained within amino acids 422 to 692 of GPIIIa, a region that may be flexible and involved in conformational changes that occur after ligand binding.

Activation-independent exposure of the GPIIb-IIIa fibrinogen receptor.

Following platelet activation, surface receptors for fibrinogen are exposed. On the activated platelet, glycoprotein IIb-IIIa (GPIIb-IIIa) serves as the receptor for fibrinogen. However, the molecular mechanisms which regulate GPIIb-IIIa fibrinogen receptor exposure are unknown. D3GP3 is an IgG1, kappa monoclonal antibody which is specific for glycoprotein IIIa (GPIIIa). The binding of D3GP3 to GPIIIa, in intact GPIIb-IIIa complexes, induces fibrinogen binding and platelet aggregation. To determine if D3GP3 binding to GPIIIa directly caused the exposure of fibrinogen receptors or, secondarily, due to stimulus response coupling, platelet activation parameters were monitored following the addition of D3GP3 to platelets suspensions. D3GP3 binding did not induce detectable Ca++ mobilization, protein phosphorylation or activation of the pertussis toxin sensitive G-protein subunit alpha-41. Further, D3GP3-induced aggregation was not blocked by PGE1, aspirin, apyrase or the combination of all three reagents. Scanning electron microscopy of D3GP3-induced aggregates demonstrated that the aggregates were composed of discoid platelets. These data suggest that the binding of D3GP3 to GPIIIa induced a conformational change in GPIIb-IIIa such that the fibrinogen receptor was exposed in an activation-independent fashion. This provides evidence that conformational changes in the GPIIb-IIIa complex can result in the transformation of the complex to the high affinity binding competent state.

The effect of glycoprotein IIb-IIIa receptor occupancy on the cytoskeleton of resting and activated platelets.

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), serves as the receptor for fibrinogen. This study examined what effect GPIIb-IIIa receptor occupancy had on the cytoskeleton of resting and activated platelets. Triton X-100-insoluble residues (cytoskeletons) were isolated from resting washed platelets incubated with either 500 microM RGDS or 500 microM RGES and examined for protein content. RGDS did not increase the amount of GPIIb-IIIa associated with the cytoskeletal residues which sedimented at either 15,800 x g or 100,000 x g. To determine the effect of receptor occupancy on the formation of the activated platelet cytoskeleton, stirred and nonstirred RGDS-treated platelets in plasma were activated with ADP. Triton X-100-insoluble residues were isolated and examined for both protein content and retention of GPIIb-IIIa. Further, morphological studies were performed on the RGDS-ADP-stimulated platelets. The results of this study suggest that 1) RGDS peptide receptor occupancy does not lead to GPIIb-IIIa linkage to the cytoskeleton, 2) ADP-stimulated platelet shape change, polymerization of actin, and association of myosin with the cytoskeleton are unaffected by RGDS peptide receptor occupancy. 3) RGDS inhibits an aggregation-dependent incorporation of ABP, alpha-actinin, talin, and GPIIb-IIIa into the Triton-insoluble residue.

Interaction of two GPIIb/IIIa monoclonal antibodies with platelet Fc receptor (Fc gamma RII).

We have previously used the IV-3 monoclonal antibody specific for Fc gamma RII to demonstrate that platelet activation by CD9 monoclonal antibodies such as ALB-6 is mediated by the Fc gamma RII. Here, we show that platelet activation following addition of a monoclonal antibody directed against GPIIb/IIIa, P256 is completely blocked by IV-3, as monitored by serotonin release, calcium and pH modifications. However, aggregation was only partially inhibited. D3GP3 is another monoclonal antibody directed against GPIIIa which has been shown to induce platelet aggregation by exposure of the fibrinogen binding site. The present study demonstrates that this phenomenon is not accompanied by calcium flux or pH modification, nor is it blocked by pretreatment of platelet by IV-3. Despite its apparent independence from the Fc gamma RII activation pathway, D3GP3, but not its Fab fragment, was able to inhibit ALB-6 induced activation, including serotonin release, calcium flux and pH modifications. Binding studies demonstrated that D3GP3 (20 micrograms/ml, 0.13 microM) does not block ALB-6 binding to CD9 antigen but completely blocks IV-3 binding to the Fc receptor for concentrations of IV-3 ranging from 0 to 15 nM. Together, these results suggest an interaction between GPIIb/IIIa, Fc gamma RII and GPIIb/IIIa monoclonal antibodies which in some cases can result in activation of platelets through Fc gamma RII.

A conformation-dependent epitope of human platelet glycoprotein IIIa.

This study explores conformational states of human platelet glycoprotein IIIa (GP IIIa) and possible mechanisms of fibrinogen receptor exposure. D3GP3 is an IgG1, kappa monoclonal antibody generated against purified GP IIIa and found to be specific for GP IIIa by immunoprecipitation and Western blot analysis. The binding of D3GP3 to resting platelets caused fibrinogen binding (approximately 5,000 molecules/platelet) and platelet aggregation but not secretion. Platelets express 40,000-50,000 GP IIb-IIIa molecules in their surface membranes. However, resting platelets only bound approximately 5,000 D3GP3 molecules/platelet. D3GP3 binding to platelets could be increased 2-3-fold by dissociation of the GP IIb-IIIa complex with 5 mM EDTA or by occupying the fibrinogen receptor with either RGDS peptides or fibrinogen. Platelet stimulation with ADP in the absence of fibrinogen did not cause increased D3GP3 binding above control levels. These data suggest that 1) GP IIb-IIIa can exist in multiple conformations in the platelet membrane, 2) D3GP3 binding to GP IIIa can expose the fibrinogen receptor, 3) the binding of either RGDS peptides or fibrinogen causes exposure of the D3GP3 epitope, and 4) platelet activation in the absence of ligand does not induce the same conformational changes in GP IIb-IIIa as does receptor occupancy by RGDS peptides or fibrinogen.

The activation of human platelets mediated by anti-human platelet p24/CD9 monoclonal antibodies.

Anti-human platelet p24/CD9 (p24/monoclonal antibody 7) causes the activation of platelets and in the presence of calcium induces platelet aggregation. Our studies suggest that platelet response to this antibody is mediated at least in part by the pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) that stimulate phosphoinositide hydrolysis and inhibit adenylate cyclase. Prior exposure of saponin-treated platelets to anti-p24/CD9 inhibited the [32P] ADP-ribosylation of the alpha 41 protein by pertussis toxin. Platelet aggregation induced by this antibody is preceded by and/or accompanied by accelerated phosphatidylinositol turnover, the generation of inositol phosphates and diacylglycerol (DAG), calcium mobilization, and protein phosphorylation. The production of inositol phosphate(s) was measurable within 15 s of either anti-p24/CD9 or thrombin addition. Within 10 s of antibody addition (10 micrograms/ml), the level of DAG was 200% over that of the control and similar to that observed with 2 units/ml thrombin (201% over that of the control). Therefore, as it appears to be true for thrombin, platelet response upon binding of anti-p24/CD9 is primarily mediated by the activation of phospholipase C. When platelets pretreated with aspirin (200 microM) and apyrase (1 mg/ml) were subsequently exposed to anti-p24/CD9, aggregation still occurred. This indicates that neither secreted ADP nor thromboxane generation is required for this aggregation response. Using indo-1 and ratio cytofluorometry, we observed that an increase in platelet cytosolic calcium is a relatively early event and occurs in either the presence or absence of calcium in the external media. Phosphorylation studies of platelet proteins showed that anti-p24/CD9 binding to platelets caused increased phosphorylation of four proteins with apparent molecular masses of 50,000, 47,000, 36,000, and 20,000 daltons. These studies suggest that platelet activation mediated by the surface protein p24/CD9 is mainly through the stimulation of a phospholipase C, the activation of which is responsible for the generation of second messengers inositol trisphosphate and DAG.