Antibodies causing thrombocytopenia in patients treated with RGD-mimetic platelet inhibitors recognize ligand-specific conformers of αIIb/β3 integrin

Arginine-glycine-aspartic acid (RGD)–mimetic platelet inhibitors act by occupying the RGD recognition site of αIIb/β3 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 αIIb/β3 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 αIIb/β3 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 αIIb/β3 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.

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Antibodies Causing Thrombocytopenia in Patients Given RGD-Mimetic Platelet Inhibitors Recognize Drug-Induced (and drug-specific) Conformational Epitopes Adjacent to the RGD Recognition Site of Gpiib/Iiia.

Blood ◽

10.1182/blood.v114.22.889.889 ◽

2009 ◽

Vol 114 (22) ◽

pp. 889-889

Author(s):

Daniel W. Bougie ◽

Mark Rasmussen ◽

Jessica Ruben ◽

Richard H. Aster

Keyword(s):

Monoclonal Antibodies ◽

Binding Sites ◽

Structural Changes ◽

Drug Binding ◽

Recognition Site ◽

Severe Thrombocytopenia ◽

Head Region ◽

Human Antibodies ◽

Drug Dependent ◽

A Domains

Abstract Abstract 889 RGD-mimetic GPIIb/IIIa inhibitors (fibans) react with the arginine-glycine-aspartic acid (RGD) recognition site of alphaIIb/beta3 integrin (GPIIb/IIIa), thereby preventing the activated integrin from reacting with fibrinogen and participating in platelet thrombus formation. This class of drugs has been shown to inhibit significantly the incidence of adverse complications following angioplasty. However, between 0.2 and 2.0% of treated patients develop acute, sometimes severe thrombocytopenia (TP) within a few hours of treatment. Platelet destruction in these cases appears to be caused by naturally occurring antibodies that recognize GPIIb/IIIa in a complex with the drug being administered (Blood 100: 2071, 2002). It has been speculated that epitopes recognized by these antibodies are the result of structural changes induced in GPIIb/IIIa when a fiban drug binds. To test this hypothesis, we studied drug-dependent binding to GPIIb/IIIa of antibodies from patients who developed acute thromboytopenia after being treated with the RGD-mimetics eptifibatide (n = 3), tirofiban (n = 3), xemilofiban (n = 1) or orbofiban (n = 1), each of which binds to a well-defined site in the “head” region of GPIIb/IIIa at the junction of the GPIIb beta propeller and GPIIIa beta A domains. Reactions of each of the human antibodies with GPIIb/IIIa were drug-specific except for the xemilofiban- and orbofiban antibodies, which exhibited some degree of cross-reactivity. None of the human antibodies recognized GPIIb/IIIa in a complex with RGDW peptide. In preliminary studies, binding sites for a family of monoclonal antibodies (mAbs) were mapped to various domains of GPIIb/IIIa using chimeric GPIIb/IIIa constructs in which one or more structural domains were switched between human and rat. We then examined the ability of these mAbs to interfere with drug-dependent binding to GPIIb/IIIa of antibodies from patients with thrombocytopenia induced by the RGD-mimetic drugs. Two eptifibatide-dependent antibodies were inhibited by mAbs specific for the beta propeller domain of GPIIb, whereas the tirofiban- and one of the eptifibatide-specific antibodies were inhibited by mAbs that recognize the beta A domain of GPIIIa. The xemilofiban- and orbofiban-dependent antibodies were inhibited to varying degrees by mAbs to both the GPIIb beta propeller and the GPIIIa beta A domains. Monoclonal antibodies specific for more distal regions of GPIIb/IIIa had no effect on drug-dependent antibody binding. The findings indicate 1) Antibodies causing thrombocytopenia in patients treated with fibans recognize various “neoepitopes” created in domains of GPIIb/IIIa near to, but not coincident with the drug binding site; 2) Different RGD-mimetic drugs, despite their structural similarity, induce slightly different structural changes in the GPIIb/IIIa “head” region that can be differentiated by the human antibodies and 3) Epitopes recognized by the human antibodies are distinct from classical ligand-induced binding sites (LIBS) produced by RGD peptide. Why these antibodies occur naturally in some individuals is an interesting question that deserves further study. Disclosures: No relevant conflicts of interest to declare.

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Secondary Structural Model of MALAT1 Becomes Unstructured in Chronic Myeloid Leukemia and Undergoes Structural Rearrangement in Cervical Cancer

Non-Coding RNA ◽

10.3390/ncrna7010006 ◽

2021 ◽

Vol 7 (1) ◽

pp. 6

Author(s):

Matthew C. Wang ◽

Phillip J. McCown ◽

Grace E. Schiefelbein ◽

Jessica A. Brown

Keyword(s):

Cervical Cancer ◽

Chronic Myeloid Leukemia ◽

Hela Cells ◽

Binding Sites ◽

Myeloid Leukemia ◽

Structural Changes ◽

Structural Model ◽

Dimethyl Sulfate ◽

K562 Cells ◽

Cancer Types

Long noncoding RNAs (lncRNAs) influence cellular function through binding events that often depend on the lncRNA secondary structure. One such lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is upregulated in many cancer types and has a myriad of protein- and miRNA-binding sites. Recently, a secondary structural model of MALAT1 in noncancerous cells was proposed to form 194 hairpins and 13 pseudoknots. That study postulated that, in cancer cells, the MALAT1 structure likely varies, thereby influencing cancer progression. This work analyzes how that structural model is expected to change in K562 cells, which originated from a patient with chronic myeloid leukemia (CML), and in HeLa cells, which originated from a patient with cervical cancer. Dimethyl sulfate-sequencing (DMS-Seq) data from K562 cells and psoralen analysis of RNA interactions and structure (PARIS) data from HeLa cells were compared to the working structural model of MALAT1 in noncancerous cells to identify sites that likely undergo structural alterations. MALAT1 in K562 cells is predicted to become more unstructured, with almost 60% of examined hairpins in noncancerous cells losing at least half of their base pairings. Conversely, MALAT1 in HeLa cells is predicted to largely maintain its structure, undergoing 18 novel structural rearrangements. Moreover, 50 validated miRNA-binding sites are affected by putative secondary structural changes in both cancer types, such as miR-217 in K562 cells and miR-20a in HeLa cells. Structural changes unique to K562 cells and HeLa cells provide new mechanistic leads into how the structure of MALAT1 may mediate cancer in a cell-type specific manner.

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A naturally occurring Tyr143HisαIIb mutation abolishes αIIbβ3 function for soluble ligands but retains its ability for mediating cell adhesion and clot retraction: comparison with other mutations causing ligand-binding defects

Blood ◽

10.1182/blood-2002-07-2144 ◽

2003 ◽

Vol 101 (9) ◽

pp. 3485-3491 ◽

Cited By ~ 17

Author(s):

Teruo Kiyoi ◽

Yoshiaki Tomiyama ◽

Shigenori Honda ◽

Seiji Tadokoro ◽

Morio Arai ◽

...

Keyword(s):

Cell Adhesion ◽

Ligand Binding ◽

Binding Sites ◽

Clot Retraction ◽

Naturally Occurring ◽

Binding Function ◽

293 Cells ◽

Ligand Binding Sites ◽

Functional Defect ◽

And Function

The molecular basis for the interaction between a prototypic non–I-domain integrin, αIIbβ3, and its ligands remains to be determined. In this study, we have characterized a novel missense mutation (Tyr143His) in αIIb associated with a variant of Glanzmann thrombasthenia. Osaka-12 platelets expressed a substantial amount of αIIbβ3(36%-41% of control) but failed to bind soluble ligands, including a high-affinity αIIbβ3-specific peptidomimetic antagonist. Sequence analysis revealed that Osaka-12 is a compound heterozygote for a single 521T>C substitution leading to a Tyr143His substitution in αIIb and for the null expression of αIIb mRNA from the maternal allele. Given that Tyr143 is located in the W3 4-1 loop of the β-propeller domain of αIIb, we examined the effects of Tyr143His or Tyr143Ala substitution on the expression and function of αIIbβ3 and compared them with KO (Arg-Thr insertion between 160 and 161 residues of αIIb) and with the Asp163Ala mutation located in the same loop by using 293 cells. Each of them abolished the binding function of αIIbβ3 for soluble ligands without disturbing αIIbβ3 expression. Because immobilized fibrinogen and fibrin are higher affinity/avidity ligands for αIIbβ3, we performed cell adhesion and clot retraction assays. In sharp contrast to KO mutation and Asp163AlaαIIbβ3, Tyr143HisαIIbβ3-expressing cells still had some ability for cell adhesion and clot retraction. Thus, the functional defect induced by Tyr143HisαIIb is likely caused by its allosteric effect rather than by a defect in the ligand-binding site itself. These detailed structure–function analyses provide better understanding of the ligand-binding sites in integrins.

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Increased cytotoxicity of normal rabbit serum for lectin-resistant mutants of animal cells.

Journal of Experimental Medicine ◽

10.1084/jem.160.4.1241 ◽

1984 ◽

Vol 160 (4) ◽

pp. 1241-1246

Author(s):

C Jones

Keyword(s):

Structural Changes ◽

Chinese Hamster ◽

Rabbit Serum ◽

Cytotoxic Action ◽

Normal Rabbit Serum ◽

Animal Cells ◽

Naturally Occurring ◽

Alternate Complement Pathway ◽

Surface Carbohydrates ◽

Naturally Occurring Antibodies

Plant lectins are cytotoxic and can be used to select for mutants of animal cells that exhibit structural changes in cell surface carbohydrates reflecting glycosylation defects. We isolated eight lectin mutants of Chinese hamster ovary (CHO) cells that appear to represent three different phenotype classes. These lectin mutants were much more sensitive to the cytotoxic action of normal rabbit serum (NRS) than were the parental cells. This increased cytotoxicity was heat sensitive, specifically absorbed, and inhibited by simple and complex carbohydrates. No killing was observed under conditions in which only the alternate complement pathway was active. An NRS-resistant subclone that was isolated from one lectin mutant was shown to have also regained wild type behavior when tested with the lectins. The possibility that naturally occurring antibodies in rabbit serum are reacting with incomplete carbohydrate chains on the surface of the lectin mutants is discussed.

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Design of Protein–Protein Binding Sites Suggests a Rationale for Naturally Occurring Contact Areas

Journal of Chemical Theory and Computation ◽

10.1021/acs.jctc.8b00667 ◽

2018 ◽

Vol 15 (2) ◽

pp. 1383-1392 ◽

Cited By ~ 1

Author(s):

Francesca Nerattini ◽

Luca Tubiana ◽

Chiara Cardelli ◽

Valentino Bianco ◽

Christoph Dellago ◽

...

Keyword(s):

Protein Binding ◽

Binding Sites ◽

Protein Binding Sites ◽

Contact Areas ◽

Naturally Occurring

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Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity

Thrombosis and Haemostasis ◽

10.1160/th13-08-0634 ◽

2014 ◽

Vol 112 (07) ◽

pp. 53-64 ◽

Cited By ~ 42

Author(s):

Sven Brandt ◽

Krystin Krauel ◽

Kay E. Gottschalk ◽

Thomas Renné ◽

Christiane A. Helm ◽

...

Keyword(s):

Conformational Changes ◽

Structural Changes ◽

Cd Spectroscopy ◽

Platelet Factor 4 ◽

Drug Induced ◽

Platelet Factor ◽

Preclinical Drug Development ◽

Varying Length ◽

Endogenous Proteins

SummaryHeparin-induced thrombocytopenia (HIT) is the most frequent drug-induced immune reaction affecting blood cells. Its antigen is formed when the chemokine platelet factor 4 (PF4) complexes with polyanions. By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD)-spectroscopy, we show that PF4 structural changes resulting in antiparallel β-sheet content >30% make PF4/polyanion complexes antigenic. Further, we found that polyphosphates (polyP-55) induce antigenic changes on PF4, whereas fondaparinux does not. We provide a model suggesting that conformational changes exposing antigens on PF4/polyanion complexes occur in the hairpin involving AA 32–38, which form together with C-terminal AA (66–70) of the adjacent PF4 monomer a continuous patch on the PF4 tetramer surface, explaining why only tetrameric PF4 molecules express “HIT antigens”. The correlation of antibody binding in immunoassays with PF4 structural changes provides the intriguing possibility that CD-spectroscopy could become the first antibody-independent, in vitro method to predict potential immunogenicity of drugs. CD-spectroscopy could identify compounds during preclinical drug development that induce PF4 structural changes correlated with antigenicity. The clinical relevance can then be specifically addressed during clinical trials. Whether these findings can be transferred to other endogenous proteins requires further studies.

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1P001 Cancer-related Mutations in BRCA1-BRCT Cause Structural Changes in Protein-Protein Binding Sites : A Molecular Dynamics Study

Seibutsu Butsuri ◽

10.2142/biophys.45.s32_1 ◽

2005 ◽

Vol 45 (supplement) ◽

pp. S32

Author(s):

Craig Gough ◽

T. Gojobori ◽

T. Imanishi

Keyword(s):

Molecular Dynamics ◽

Protein Binding ◽

Binding Sites ◽

Structural Changes ◽

Protein Binding Sites

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Drug-induced lung disease

Oxford Textbook of Medicine ◽

10.1093/med/9780198746690.003.0433 ◽

2020 ◽

pp. 4272-4281

Author(s):

S. J. Bourke

Keyword(s):

Lung Disease ◽

Enzyme Inhibitors ◽

Oral Contraceptive Pill ◽

Drug Effects ◽

Angiotensin Converting Enzyme Inhibitors ◽

Vascular Involvement ◽

Drug Induced ◽

Converting Enzyme Inhibitors ◽

Effect Of Treatment ◽

Respiratory Conditions

Drug-induced lung disease is common and needs to be considered in the differential diagnosis of many respiratory conditions. The nature and timing of events often provide important clues and are sometimes sufficiently characteristic for drug-induced lung disease to be diagnosed with confidence, with resolution of symptoms on drug cessation providing further supportive evidence. Direct drug effects may arise through toxic, pharmacological, allergic, or idiosyncratic mechanisms (there may also be indirect effects). From a clinical perspective, adverse effects may be classified according to the induced disorder and the site of involvement. Asthma is the most common airway disorder to be induced or exacerbated by drugs. Cough is a well-recognized side effect of treatment with angiotensin-converting enzyme inhibitors. Pulmonary vascular involvement includes venous thromboembolism (e.g. oral contraceptive pill), and pulmonary hypertension (e.g. aminorex, now withdrawn), dasatinib, and interferons. Pleural effusions and thickening may result from drugs (e.g. dantrolene, bromocriptine, methysergide, and dasatinib).

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Atrial Flutter Associated with Carboplatin Administration

Annals of Pharmacotherapy ◽

10.1345/aph.1q376 ◽

2011 ◽

Vol 45 (11) ◽

pp. 1451-1451 ◽

Cited By ~ 2

Author(s):

Sammy Zakaria ◽

Kit Yu Lu ◽

Veronique Nussenblatt ◽

Ilene Browner

Keyword(s):

Atrial Flutter ◽

Causal Link ◽

Cardiac Monitoring ◽

Cardiac Complications ◽

Medication Regimen ◽

Cardiac Events ◽

Drug Induced ◽

Arrhythmogenic Effect ◽

Adverse Cardiac Events ◽

Effect Of Treatment

Objective: To report a novel case of atrial flutter associated with carboplatin administration and review chemotherapy-related cardiac toxicities, focusing on platinum-containing compounds. Case Report: A 69-year-old man with extensive small cell lung cancer and asymptomatic cardiovascular and cerebrovascular disease was inconsistently adherent to his medication regimen. While undergoing carboplatin infusion, he developed atrial flutter. He had no other immediate arrhythmogenic causes of atrial flutter and the arrhythmia spontaneously reverted to sinus rhythm after 24 hours. His condition remained stable until he died 8 days later. The cause of death was unknown and the family declined postmortem examination. Discussion: Although this patient's cardiac history and nonadherence to his medications may have increased his susceptibility to develop atrial arrhythmias, the Naranjo probability scale reveals a possible relationship between atrial flutter and Infusion of carboplatin. A literature search revealed other adverse cardiac events due to platinum compounds; however, to our knowledge, this case is the first to describe an association with atrial flutter. A definitive causal link cannot be determined, but this may have been the result of a direct arrhythmogenic effect of treatment or to a novel hypersensitivity reaction. Given the potential deleterious impact of drug-induced arrhythmias, we have reported this case to the Food and Drug Administration as a new adverse effect of carboplatin. Conclusions: Providers should consider cardiac monitoring during carboplatin infusion in patients with known cardiac disease or at high risk of cardiac complications.

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