Proteolytic degradation of the RGD-binding and non-RGD-binding conformers of human platelet integrin glycoprotein IIb/IIIa: clues for identification of regions involved in the receptor's activation.
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ABSTRACT:
SUBMITTER: Calvete JJ
PROVIDER: S-EPMC1137975 | biostudies-other | 1994 Feb
REPOSITORIES: biostudies-other
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Project description:The major platelet integrin, glycoprotein IIb-IIIa, binds soluble fibrinogen only after platelet activation. To investigate the mechanism by which platelets convert glycoprotein IIb-IIIa into a functional fibrinogen receptor, we characterized the opening and closing of fibrinogen-binding sites in isolated platelet membranes and compared the regulatory properties of membrane-bound glycoprotein IIb-IIIa with those of the detergent-solubilized receptor. Basal fibrinogen binding to the membranes possessed many of the properties of fibrinogen binding to activated platelets; however, less than 10% of glycoprotein IIb-IIIa in the membranes was capable of binding fibrinogen. Preincubating the membranes with either an activating glycoprotein IIb-IIIa antibody or alpha-chymotrypsin increased fibrinogen binding. In contrast, agents that require intracellular mediators, such as platelet agonists, guanine-nucleotide-binding-protein activators and purified protein kinase C, did not stimulate fibrinogen binding to the membranes, suggesting that cytosolic factor(s) may be required for activation of the receptor in platelets. Occupancy of glycoprotein IIb-IIIa in the membranes with RGD (Arg-Gly-Asp)-containing peptides reversibly exposed neoantigenic epitopes and fibrinogen-binding sites in the receptor. These conformational changes required membrane fixation to be maintained following peptide removal. Similar results were obtained with purified glycoprotein IIb-IIIa incorporated into phospholipid vesicles, indicating that the resting state of the receptor is favoured in these environments. In contrast, when the conformation of detergent-solubilized glycoprotein IIb-IIIa was altered by exposure to RGD-containing peptides, the receptor remained active even after incorporation into phospholipid vesicles. These results demonstrate that platelet membranes are a useful model in which to study the regulation of glycoprotein IIb-IIIa and suggest that the environment surrounding the receptor may have a profound influence on this process.
| S-EPMC1134177 | biostudies-other
Project description:Arginine-glycine-aspartic acid (RGD)-mimetic platelet inhibitors act by occupying the RGD recognition site of ?(IIb)/?(3) integrin (GPIIb/IIIa), thereby preventing the activated integrin from reacting with fibrinogen. Thrombocytopenia is a well-known side effect of treatment with this class of drugs and is caused by Abs, often naturally occurring, that recognize ?(IIb)/?(3) in a complex with the drug being administered. RGD peptide and RGD-mimetic drugs are known to induce epitopes (ligand-induced binding sites [LIBS]) in ?(IIb)/?(3) that are recognized by certain mAbs. It has been speculated, but not shown experimentally, that Abs from patients who develop thrombocytopenia when treated with an RGD-mimetic inhibitor similarly recognize LIBS determinants. We addressed this question by comparing the reactions of patient Abs and LIBS-specific mAbs against ?(IIb)/?(3) in a complex with RGD and RGD-mimetic drugs, and by examining the ability of selected non-LIBS mAbs to block binding of patient Abs to the liganded integrin. Findings made provide evidence that the patient Abs recognize subtle, drug-induced structural changes in the integrin head region that are clustered about the RGD recognition site. The target epitopes differ from classic LIBS determinants, however, both in their location and by virtue of being largely drug-specific.
| S-EPMC3383197 | biostudies-literature
Project description:Binding sites on glycoprotein (GP) IIb/IIIa exposed by 0.5 unit/ml alpha-thrombin are insensitive to prostaglandin I2 (PGI2), in contrast with sites exposed by ADP or platelet-activating factor. Here we show that the thrombin receptor agonist peptide (TRAP) (SFLLRN; 15 microM) opens almost the same number of GPIIb/IIIa molecules as 0.5 unit/ml alpha-thrombin (64840 +/- 8920 compared with 81050 +/- 6030 molecules of fibronectin bound/platelet), but these sites rapidly close on addition of PGI2. To investigate whether alpha-thrombin and TRAP initiate different signalling pathways, we measured phospholipase C (PLC)-mediated control of GPIIb/IIIa and its sensitivity to cyclic AMP. Optimal concentrations of alpha-thrombin and TRAP activated PLC maximally, but TRAP induced only about 50% protein kinase C PKC) activation after 10 min stimulation compared with alpha-thrombin. These concentrations also suppressed PGI2-induced cyclic AMP accumulation, with alpha-thrombin inducing complete inhibition and TRAP about 10% less. Direct activation of PKC by phorbol 12-myristate 13-acetate confirmed earlier observations that PGI2-induced cyclic AMP accumulation is partly inhibited via PKC. Applying different concentration of alpha-thrombin, TRAP or a combination of alpha-thrombin and the thrombin receptor inhibitory peptide (TRIP) (Mpr-F-Cha-Cha-RKPNDK-NH2; 800 microM) (Mpr, 3-mercaptopropionic acid; Cha, cyclohexylalanine), we show that the different means of stimulating the thrombin receptor all suppressed PGI2-induced cyclic AMP accumulation via (i) activation of PKC and (ii) activation of the heterotrimeric G-protein, Gi. We conclude that complete inhibition of cyclic AMP accumulation requires activation of both PKC and Gi, as observed with 0.5 unit/ml alpha-thrombin. Although TRAP almost fully exposes GPIIb/IIIa, its activation of PKC is incomplete, enabling PGI2 to raise cyclic AMP concentration from 1.4 +/- 0.7 to 4.1 +/- 1.3 nmol/10(11) platelets (P < 0.005) which is sufficient to close exposed GPIIb/IIIa molecules.
| S-EPMC1135775 | biostudies-other
Project description:Binding of soluble fibrinogen to the activated conformation of the integrin ?IIb?3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (?C-12) sequence of the fibrinogen ? chain. However, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen A? chain inhibit soluble fibrinogen binding to ?IIb?3 and make substantial contributions to ?IIb?3 binding when fibrinogen is immobilized and when it is converted to fibrin. Here, we employed optical trap-based nanomechanical measurements and computational molecular modeling to determine the kinetics, energetics, and structural details of cyclic RGDFK (cRGDFK) and ?C-12 binding to ?IIb?3. Docking analysis revealed that NMR-determined solution structures of cRGDFK and ?C-12 bind to both the open and closed ?IIb?3 conformers at the interface between the ?IIb ?-propeller domain and the ?3 ?I domain. The nanomechanical measurements revealed that cRGDFK binds to ?IIb?3 at least as tightly as ?C-12. A subsequent analysis of molecular force profiles and the number of peptide-?IIb?3 binding contacts revealed that both peptides form stable bimolecular complexes with ?IIb?3 that dissociate in the 60-120 pN range. The Gibbs free energy profiles of the ?IIb?3-peptide complexes revealed that the overall stability of the ?IIb?3-cRGDFK complex was comparable with that of the ?IIb?3-?C-12 complex. Thus, these results provide a mechanistic explanation for previous observations that RGD- and AGDV-containing peptides are both potent inhibitors of the ?IIb?3-fibrinogen interactions and are consistent with the observation that RGD motifs, in addition to AGDV, support interaction of ?IIb?3 with immobilized fibrinogen and fibrin.
| S-EPMC5536102 | biostudies-literature
Project description:This study was to compare the efficacy and safety of combined glycoprotein IIb/IIIa inhibitor (GPI) and ticagrelor versus ticagrelor in patients with acute coronary syndrome (ACS). An observational study was conducted using the Improving Care for Cardiovascular Disease in China-ACS project. Totally, 13,264 patients with ACS and received combination therapy or ticagrelor therapy were analyzed. The primary outcome was the composite of major cardiovascular events (MACE: all-cause mortality, myocardial infarction [MI], stent thrombosis, cardiogenic shock, and ischemic stroke), and secondary outcomes included all-cause mortality, MI, stent thrombosis, cardiogenic shock, and ischemic stroke. The multivariable adjusted analysis indicated that combination therapy was associated with an increased risk of major cardiovascular events (MACE) (P = 0.001), any bleeding (P<0.001), and major bleeding (P = 0.005). Moreover, the multivariable adjusted for propensity score-matched (PSM) analysis suggested that combination therapy produced additional risk of MACE (P = 0.014), any bleeding (P<0.001), and major bleeding (P = 0.005). Moreover, PSM analysis suggested that combination therapy was associated with greater risk of stent thrombosis (P = 0.012) and intracranial bleeding (P = 0.020). Combined GPI and ticagrelor therapies did not have any beneficial effects on MACE, stent thrombosis, intracranial bleeding, any bleeding, or major bleeding.
| S-EPMC7853481 | biostudies-literature
Project description:The present study was designed to examine the interaction of the purified platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa or integrin alpha IIb beta 3) and the individual subunits of the complex with immobilized fibrinogen. Although 125I-GP IIb-IIIa binding to fibrinogen immobilized on Sepharose was specific, this interaction exhibited properties distinct from those of reversible fibrinogen binding to platelets: 125I-GP IIb-IIIa binding appeared irreversible, but non-covalent, Ca(2+)-independent, and was inhibited only weakly, or not at all, by the anti-(GP IIb-IIIa) monoclonal antibodies 10E5 and 7E3 and synthetic peptides from known platelet-binding domains of fibrinogen. Reversibly dissociated GP IIb or GP IIIa subunits inhibited 125I-GP IIb-IIIa binding to immobilized fibrinogen and bound directly to the fibrinogen. However, these subunits did not bind to peptides derived from known platelet-binding domains within the fibrinogen alpha- and gamma-chains, although the GP IIb-IIIa complex did. These results show that the complexed form of full-length GP IIb and GP IIIa is required for binding to these synthetic peptides, but not necessarily for binding to immobilized fibrinogen. Thus GP IIb-IIIa can bind to immobilized fibrinogen by a distinct mechanism that appears to involve novel binding sites on each subunit of the GP IIb-IIIa complex and on fibrinogen.
| S-EPMC1132188 | biostudies-other
Project description:BackgroundWe previously described a mouse model in which platelet immunization between selected strains leads to production of alloantibodies and severe autoimmune thrombocytopenia and mimics the human condition posttransfusion purpura (PTP). This report describes studies defining epitopes recognized by these alloantibodies.Study designHybridomas were produced from spleen cells of immunized mice. Glycoprotein (GP) targets of resulting monoclonal antibodies were characterized by immunoprecipitation using platelets from the immunizing strains. Antigens defined by single amino acid (AA) polymorphisms recognized by monoclonal antibodies were identified by mutagenizing target glycoproteins expressed in Chinese hamster ovary cells and observing the effects on antibody binding.ResultsThree monoclonal antibodies (417.1, 417.3, 425.1) were produced that recognized GPIIb on immunizing platelets. Monoclonal antibodies 417.1 and 417.3 both required G111 and 425.1 required V37, located on the beta propeller domain of GPIIb, for binding to platelets from the immunizing strains C57 and PWK, respectively. Injection of 417.3 and 425.1 into mice caused platelet destruction only in mice with GPIIb containing the targeted AAs.ConclusionsFindings made provide evidence that alloantibodies produced by mice experiencing thrombocytopenia in a mouse model of PTP are specific for single AA polymorphisms that differ in GPIIb/IIIa integrin of the immunizing and immunized strains and therefore closely resemble the potent alloantibodies found in patients with PTP. The observations show that naturally occurring single AA differences in GPIIb/IIIa integrin of various mouse strains are highly immunogenic in the mouse strains studied and readily induce antibodies comparable to human platelet antigen-specific antibodies found in transfused and pregnant humans.
| S-EPMC8035281 | biostudies-literature
Project description:BackgroundIt is apparent that the interaction between platelets and eosinophils plays a critical role in the activation of allergic inflammation. We investigated whether blocking of the glycoprotein (GP) IIb/IIIa receptor can attenuate allergic inflammation and airway hyperresponsiveness through inhibition of platelet-eosinophil aggregation (PEA) in asthma.MethodsBALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 0 and 14, followed by 3 nebulized OVA challenges on days 28-30. On each challenge day, 5 mg/kg tirofiban was administered intraperitoneally 30 min before the challenge. Mice were assessed for airway hyperresponsiveness (AHR), airway inflammation, and the degree of PEA. Finally, the activation levels of platelets and eosinophils were evaluated.ResultsTirofiban treatment decreased AHR and eosinophilic inflammation in Bronchoalveolar Lavage (BAL) fluid. This treatment also reduced the levels of interleukin (IL)-4, IL-5, and IL-13 in BAL fluid and airway inflammatory cell infiltration in histological evaluation. Interestingly, the blocking of the GP IIb/IIIa receptor more reduced PEA in both blood and lung tissue of tirofiban-treated mice than in those of the positive control mice, and both eosinophilic and platelet activations were attenuated in tirofiban-treated mice.ConclusionsThe blocking of GP IIb/IIIa receptor with tirofiban can attenuate AHR and airway inflammation through the inhibition of PEA and activation.
| S-EPMC8275907 | biostudies-literature
Project description:1. The antithrombotic effect of the glycoprotein IIb/IIIa receptor antagonist, CRL42796, was examined in canine models of carotid and coronary artery thrombosis. 2. In the carotid artery thrombosis model, occlusion occurred in all control vessels (time to thrombosis 47.6+/-8.9 min). After treatment with low dose CRL42796 (15 microg kg(-1) loading dose +0.31 microg kg(-1) min(-1) i.v.), two of five vessels occluded. Time to thrombosis increased significantly to 155.2+/-23.1 min. When the drug infusion was increased (0.69 microg kg(-1) min(-1)), each of five vessels remained patent. 3. Ex vivo platelet aggregation in response to arachidonic acid (AA) and ADP was examined in platelet rich plasma (PRP) prepared from citrate or heparin anticoagulated blood. CRL42796 reduced platelet reactivity at low and high doses in PRP from citrate anticoagulated blood. However, in PRP from heparin anticoagulated blood, only the higher infusion dose produced a significant reduction in ex vivo platelet responses. 4. A combination of oral aspirin (4.6 mg kg(-1) -41, -17 h) and the low infusion dose of CRL42796 did not produce an additional benefit beyond that provided by CRL42796 alone. 5. Coronary artery thrombosis was inhibited in four of five vessels treated with the lower infusion dose of CRL42796 and in five of five vessels treated with the higher infusion. Time to thrombosis increased with both doses (Control, 90.8+/-10.4 min; low dose, 165.8+/-14.2 min; high dose, >180.0+/-0 min). 6. The results indicate that CRL42796 is an effective in vivo antithrombotic agent against experimentally-induced carotid and coronary artery thrombosis.
| S-EPMC1573408 | biostudies-literature
Project description:Ultrasound molecular imaging (UMI) of glycoprotein (GP) IIb/IIIa receptor on activated platelets offers a unique means of identifying high-risk atherosclerosis. We hypothesized that contrast-enhanced ultrasound with microbubbles (MBs) targeted to GP IIb/IIIa could be used to detect and quantify activated platelets on the surface of advanced plaques.A mouse model of advanced atherosclerosis was generated by maintaining apolipoprotein E-deficient (ApoE(-/-)) mice on a hypercholesterolemic diet (HCD). The three other experimental groups consisted of ApoE(-/-) and wild-type (C57BL/6) mice fed a normal chow diet and C57BL/6 mice on an HCD diet. Plaque formation was confirmed by histological and immunohistochemical methods using light, fluorescence, and electron microscopy. Mice were injected with a lipid MB-conjugated cyclic Arg-Gly-Asp peptide or nonspecific control peptide, and the abdominal aorta was examined by UMI. The accumulation of GP IIb/IIIa and activated platelets on the surface of atherosclerotic plaques was highest in the ApoE(-/-)+HCD group, followed by ApoE(-/-)+chow, C57BL/6+HCD, and C57BL/6+chow groups (P<0.05). Notably, GP IIb/IIIa expression was associated with the vulnerability index and necrotic center/fiber cap ratio (P<0.05), and contrast video intensity from adhered cyclic Arg-Gly-Asp-modified MBs (MB-cRGDs) was correlated with GP IIb/IIIa expression on the plaque surface (P<0.05).GP IIb/IIIa of activated platelets on the atherosclerotic endothelium is a biomarker for high-risk plaques that can be quantified by UMI using MB-cRGDs, providing a noninvasive means for detecting high-risk plaques and preventing acute cardiovascular events.
| S-EPMC4329504 | biostudies-literature