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 ◽  
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.

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

Blood ◽  
2012 ◽  
Vol 119 (26) ◽  
pp. 6317-6325 ◽  
Author(s):  
Daniel W. Bougie ◽  
Mark Rasmussen ◽  
Jieqing Zhu ◽  
Richard H. Aster
Keyword(s):  
Binding Sites ◽  
Structural Changes ◽  
Recognition Site ◽  
Platelet Inhibitors ◽  
Head Region ◽  
Drug Induced ◽  
Naturally Occurring ◽  
Β3 Integrin ◽  
Effect Of Treatment ◽  
Arginine Glycine Aspartic Acid

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.

scholarly journals Acute thrombocytopenia after treatment with tirofiban or eptifibatide is associated with antibodies specific for ligand-occupied GPIIb/IIIa

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2071-2076 ◽  
Author(s):  
Daniel W. Bougie ◽  
Peter R. Wilker ◽  
Elizabeth D. Wuitschick ◽  
Brian R. Curtis ◽  
Mohammad Malik ◽  
...  
Keyword(s):  
High Titer ◽  
Drug Binding ◽  
Prior Exposure ◽  
Severe Thrombocytopenia ◽  
Serum Samples ◽  
Igg Antibody ◽  
Drug Binding Site ◽  
Naturally Occurring ◽  
Previously Treated ◽  
Drug Dependent

Abstract Acute thrombocytopenia is a recognized complication of treatment with GPIIb/IIIa inhibitors whose cause is not yet known. We studied 9 patients who developed severe thrombocytopenia (platelets less than 25 × 109/L) within several hours of treatment with the GPIIb/IIIa inhibitors tirofiban (4 patients) and eptifibatide (5 patients). In each patient, acute-phase serum contained a high titer (range, 1:80-1:20 000) IgG antibody that reacted with the glycoprotein IIb/IIIa complex only in the presence of the drug used in treatment. Four patients had been previously treated with the same drug, but 5 had no known prior exposure. Pretreatment serum samples from 2 of the latter patients contained drug-dependent antibodies similar to those identified after treatment. No tirofiban- or eptifibatide-dependent antibodies were found in any of 100 randomly selected healthy blood donors, and only 2 of 23 patients receiving tirofiban or eptifibatide who did not experience significant thrombocytopenia had extremely weak (titer, 1:2) tirofiban-dependent antibodies. In preliminary studies, evidence was obtained that the 9 antibodies recognize multiple target epitopes on GPIIb/IIIa complexed with the inhibitor to which the patient was sensitive, indicating that they cannot all be specific for the drug-binding site. The findings indicate that acute thrombocytopenia after the administration of tirofiban or eptifibatide can be caused by drug-dependent antibodies that are “naturally occurring” or are induced by prior exposure to drug. These antibodies may be human analogs of mouse monoclonal antibodies that recognize ligand-induced binding sites (LIBS) induced in the GPIIb/IIIa heterodimer when it reacts with a ligand-mimetic drug.

scholarly journals The Use of Monoclonal Antibodies in the Treatment of Alzheimer Disease

2016 ◽  
Vol 1 (3) ◽  
pp. 59-66
Author(s):  
Hemen Moradi-Sardareh ◽  
Maryam Moradi ◽  
Elham Bordbar ◽  
Mohammadreza Malekpour ◽  
Sara Bagheri ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Monoclonal Antibodies ◽  
Alzheimer Disease ◽  
Binding Sites ◽  
Action Mechanism ◽  
Variable Binding ◽  
Human Antibodies ◽  
Delayed Onset ◽  
Antibody Structure ◽  
Phagocytosis Activity

Background and objective: Antibody molecules have similar structure but variable binding sites to antigen. Human antibodies are achieved by transgenic mouse technology or phage display. Antibodies are used for the treatment of different diseases. Alzheimer is a cow disease with a delayed onset overpresenting in adults in their 70s to 90s. This disease is the leading cause of central nervous system (CNS) degeneration during 7 to 15 years in the aging population. Several hypotheses have offered monoclonal antibodies action mechanism in relation to amyloid cleaning at Alzheimer disease. According to this mechanism, the appropriate antibody passes blood-brain barrier, enters into brain, and binds to amyloid to launch its phagocytosis activity in microglia and/or macrophage/monocytes infiltrating. The present study aimed to review antibody structure and the use of different antibodies for the treatment of Alzheimer disease.

scholarly journals Acute thrombocytopenia after treatment with tirofiban or eptifibatide is associated with antibodies specific for ligand-occupied GPIIb/IIIa

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2071-2076 ◽  
Author(s):  
Daniel W. Bougie ◽  
Peter R. Wilker ◽  
Elizabeth D. Wuitschick ◽  
Brian R. Curtis ◽  
Mohammad Malik ◽  
...  
Keyword(s):  
High Titer ◽  
Drug Binding ◽  
Prior Exposure ◽  
Severe Thrombocytopenia ◽  
Serum Samples ◽  
Igg Antibody ◽  
Drug Binding Site ◽  
Naturally Occurring ◽  
Previously Treated ◽  
Drug Dependent

Acute thrombocytopenia is a recognized complication of treatment with GPIIb/IIIa inhibitors whose cause is not yet known. We studied 9 patients who developed severe thrombocytopenia (platelets less than 25 × 109/L) within several hours of treatment with the GPIIb/IIIa inhibitors tirofiban (4 patients) and eptifibatide (5 patients). In each patient, acute-phase serum contained a high titer (range, 1:80-1:20 000) IgG antibody that reacted with the glycoprotein IIb/IIIa complex only in the presence of the drug used in treatment. Four patients had been previously treated with the same drug, but 5 had no known prior exposure. Pretreatment serum samples from 2 of the latter patients contained drug-dependent antibodies similar to those identified after treatment. No tirofiban- or eptifibatide-dependent antibodies were found in any of 100 randomly selected healthy blood donors, and only 2 of 23 patients receiving tirofiban or eptifibatide who did not experience significant thrombocytopenia had extremely weak (titer, 1:2) tirofiban-dependent antibodies. In preliminary studies, evidence was obtained that the 9 antibodies recognize multiple target epitopes on GPIIb/IIIa complexed with the inhibitor to which the patient was sensitive, indicating that they cannot all be specific for the drug-binding site. The findings indicate that acute thrombocytopenia after the administration of tirofiban or eptifibatide can be caused by drug-dependent antibodies that are “naturally occurring” or are induced by prior exposure to drug. These antibodies may be human analogs of mouse monoclonal antibodies that recognize ligand-induced binding sites (LIBS) induced in the GPIIb/IIIa heterodimer when it reacts with a ligand-mimetic drug.

An Immunoradiometric Assay for Factor VIII Related Antigen (VIIIRAg) Using Two Monoclonal Antibodies-Comparison with Polyclonal Rabbit Antibodies for Use in von Willebrand’s Disease Diagnosis

1984 ◽  
Vol 52 (03) ◽  
pp. 250-252 ◽  
Author(s):  
Y Sultan ◽  
Ph Avner ◽  
P Maisonneuve ◽  
D Arnaud ◽  
Ch Jeanneau
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Changes ◽  
Polyclonal Antibodies ◽  
Disease Diagnosis ◽  
Immunoradiometric Assay ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Structural Abnormalities ◽  
Antigenic Material ◽  
Von Willebrand

SummaryTwo monoclonal antibodies raised against FVIII/von Willebrand protein were used in an immunoradiometric assay (IRMA) to measure this antigen in normal plasma and plasma of patients with different forms of von Willebrand’s disease. The first antibody, an IgG1 was used to coat polystyrene tubes, the second one, an IgG2a, iodinated and used in the second step. Both antibodies inhibit ristocetin induced platelet agglutination and react strongly with platelets, megacaryocytes and endothelial cells. The IRMA test using these antibodies showed greater sensitivity than that using rabbit polyclonal anti VIIIRAg antibodies. A good correlation between the two tests was nevertheless found when VIIIRAg was measured in the majority of patient’s plasma. However 5 patients from 3 different families showed more antigenic material in the rabbit antibody IRMA than in the monoclonal antibody IRMA. It is suggested therefore that the monoclonal antibodies identify part of the VIIIR:Ag molecule showing structural abnormalities in these vWd patients, these structural changes remaining undetected by the polyclonal antibodies.

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

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.

THE MOLECULAR ORGANIZATION OF HUMAN ALPHA 2-MACROGLOBULIN. AN IMMUNO ELECTRON MICROSCOPIC STUDY WITH MONOCLONAL ANTIBODIES

1987 ◽  
Author(s):  
E Delain ◽  
M Barrav ◽  
J Tapon-Bretaudière ◽  
F Pochon ◽  
F Van Leuven
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Sites ◽  
Divalent Cations ◽  
The Other ◽  
Molecular Organization ◽  
Cellular Receptor ◽  
Electron Microscopic ◽  
Microscopic Method ◽  
Bait Region ◽  
Electron Microscopic Method

Electron microscopy is a very convenient method to localize the epitopes of monoclonal antibodies (mAbs) at the surface of macromolecules for studying their tree-dimensional organization.We applied this immuno-electron microscopic method to human ct2-macroglobulin (ct2M). 29 anti-α2M mAbs have been tested with the four different forms of a2M : native and chymotrypsin-transformed tetramers, and the corresponding dimers, obtained by dissociation with divalent cations. These mAbs can be classified in three types : those which are specific for 1) the H-like transformed molecules, 2) the native molecules, and 3) those which can react with both forms of α2M.1) Among the H-like α2M specific mAbs, several react with the 20 kD-domain which is recognized by the cellular receptor of transformed a2M. This domain is located at the carboxyterminal end of each monomer. One IgG binds to the end of two adjacent tips of the H-like form.The other mAbs of this type bind to the α2M tips at non-terminal positions. Intermolecular connections built polymers of alternating α2M and IgG molecules.2) Among the native a2M-specific mAbs some are able to inhibit the protease-induced transformation of the native α2M. The binding sites of these mAbs are demonstrated on the native half-molecules. One of these mAbs was also able to react with transformed dimers, in a region corresponding very likely to an inaccessible epitope in the tetrameric transformed α2M molecule.3) Among the mAbs of this type, only two were able to inhibit the protease-induced transformation of α2M. Obviously, their epitopes should be close to the bait region of α2M. The other mAbs reacting with both α2M forms did not inhibit the α2M transformation.All these mAbs can be distinguished by the structure of the immune complexes formed with all forms of α2M. The epitopes are more easily located on the dimers and on the H-like transformed α2M than on the native molecules.From these observations, we propose a new model of the tree-dimensional organization of the human α2M in its native and transformed configurations, and of its protease-induced transformation.

scholarly journals Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

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