scholarly journals Drug-dependent clearance of human platelets in the NOD/scid mouse by antibodies from patients with drug-induced immune thrombocytopenia

Blood ◽  
2010 ◽  
Vol 116 (16) ◽  
pp. 3033-3038 ◽  
Author(s):  
Daniel W. Bougie ◽  
Dhirendra Nayak ◽  
Brian Boylan ◽  
Peter J. Newman ◽  
Richard H. Aster
Keyword(s):  
Scid Mouse ◽  
Immune Thrombocytopenia ◽  
Small Animal ◽  
Study Drug ◽  
Human Platelets ◽  
Antibody Detection ◽  
Human Antibody ◽  
Drug Induced ◽  
Drug Dependent

Abstract Drug-induced immune thrombocytopenia (DITP) is a relatively common and sometimes life-threatening condition caused by antibodies that bind avidly to platelets only when drug is present. How drug-dependent antibodies (DDAbs) are induced and how drugs promote their interaction with platelets are poorly understood, and methods for detecting DDAbs are suboptimal. A small animal model of DITP could provide a new tool for addressing these and other questions concerning pathogenesis and diagnosis. We examined whether the nonobese diabetic/severe combined immunodeficient (NOD/scid) mouse, which lacks xenoantibodies and therefore allows infused human platelets to circulate, can be used to study drug-dependent clearance of platelets by DDAbs in vivo. In this report, we show that the NOD/scid model is suitable for this purpose and describe studies to optimize its sensitivity for drug-dependent human antibody detection. We further show that the mouse can produce metabolites of acetaminophen and naproxen for which certain drug-dependent antibodies are specific in quantities sufficient to enable these antibodies to cause platelet destruction. The findings indicate that the NOD/scid mouse can provide a unique tool for studying DITP pathogenesis and may be particularly valuable for identifying metabolite-specific antibodies capable of causing immune thrombocytopenia or hemolytic anemia.

Drug-induced thrombocytopenia: development of a novel NOD/SCID mouse model to evaluate clearance of circulating platelets by drug-dependent antibodies and the efficacy of IVIG

Blood ◽  
2010 ◽  
Vol 116 (11) ◽  
pp. 1958-1960 ◽  
Author(s):  
Simon X. Liang ◽  
Mykola Pinkevych ◽  
Levon M. Khachigian ◽  
Christopher R. Parish ◽  
Miles P. Davenport ◽  
...  
Keyword(s):  
Mouse Model ◽  
Scid Mouse ◽  
Immune Thrombocytopenia ◽  
Human Platelets ◽  
Ivig Treatment ◽  
Drug Induced ◽  
Beneficial Effects ◽  
Nonobese Diabetic ◽  
Scid Mouse Model ◽  
Drug Dependent

Abstract Drug-induced immune thrombocytopenia (DITP) is an adverse drug effect mediated by drug-dependent antibodies. Intravenous immunoglobulin (IVIG) is frequently used to treat DITP and primary immune thrombocytopenia (ITP). Despite IVIG's proven beneficial effects in ITP, its efficacy in DITP is unclear. We have established a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of DITP in which human platelets survive for more than 24 hours, allowing platelet clearance by DITP/ITP antibodies to be studied. Rapid human platelet clearance was uniformly observed with all quinine-induced thrombocytopenia (QITP) patient sera studied (mean platelet lifespans: QITP 1.5 ± 0.3 hours vs controls 16.5 ± 4.3 hours), consistent with the clinical presentation of DITP. In contrast, clearance rates with ITP antibodies were more variable. IVIG treatment partially prevented platelet clearance by DITP and ITP antibodies. Our results suggest that the NOD/SCID mouse model is useful for investigating the efficacy of current and future DITP therapies, an area in which there is little experimental evidence to guide treatment.

Drug-Dependent Murine Monoclonal Antibodies (mAbs) Mimic Antibodies Found in Patients with Drug-Induced Immune Thrombocytopenia (TP).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 731-731
Author(s):  
Daniel W. Bougie ◽  
Jessica Birenbaum ◽  
Scott Ahl ◽  
Richard H. Aster
Keyword(s):  
Molecular Basis ◽  
Immune Thrombocytopenia ◽  
Tight Binding ◽  
Human Platelets ◽  
Soluble Form ◽  
Membrane Glycoproteins ◽  
Drug Induced ◽  
Life Threatening ◽  
Drug Dependent ◽  
Covalently Linked

Abstract Drug-induced immune thrombocytopenia (DITP) is a serious and sometimes life-threatening complication of treatment with many drugs. In most instances (excluding heparin-induced TP), platelet (plt) destruction is caused by antibodies (abs) that recognize distinct epitopes on platelet membrane glycoproteins (GP) only when the sensitizing drug is present in soluble form. How drug at pharmacological levels promotes tight binding of antibody to a specific target is unknown, in part because only polyclonal human abs have been available for study. We sought to produce monoclonal abs (mAbs) that mimic the behavior of human drug-dependent abs to create tools that can be used to study the molecular basis for this interaction. Mice were immunized with GPIIb/IIIa isolated from human platelets together with soluble quinine (Qn), tirofiban (Tf), or eptifibatide (Ef), three drugs that commonly cause DITP. Hybridomas were prepared from splenic B cells using a standard protocol and approximately 550 supernatants from each cultured hybrid line were screened for DDAbs by flow cytometry using normal platelets as targets. To date, 11 Abs that react with GPIIb/IIIa only in the presence of the immunizing drug (2 Qn-, 3 Tf- and 6 Ef-specific) have been identified. Preliminary studies show that the Qn-specific abs bind reversibly to GPIIb/IIIa at 50 nM Qn, a concentration much lower than is achieved pharmacologically, and are not inhibited by Qn at 5 mM, the limit of solubility. Quinidine (Qd) the diastereoisomer of Qn, supports only weak ab binding at a concentration of 50 uM. The tirofiban and eptifibatide-dependent abs recognize GPIIb/IIIa occupied by these RGD ligand-mimetic GPIIb/IIIa inhibitors. In each of these respects, reaction patterns of the three groups of mAbs closely resemble those of abs from patients experiencing TP after treatment with one of these drugs. These findings show that mAbs mimicking the behavior of human drug-dependent abs can be produced by immunizing mice with GP and soluble drug to produce probes suitable for characterizing the molecular basis of ab-drug-target interactions leading to platelet destruction in DITP. It is noteworthy that these mAbs were induced using soluble drug and protein for immunization because this suggests that the immune response leading to DITP does not require the sensitizing drug to be covalently linked to a protein, i.e, does not require the drug to act as a classical hapten.

Immune thrombocytopenia resulting from sensitivity to metabolites of naproxen and acetaminophen

Blood ◽  
2001 ◽  
Vol 97 (12) ◽  
pp. 3846-3850 ◽  
Author(s):  
Daniel Bougie ◽  
Richard Aster
Keyword(s):  
Clinical Picture ◽  
Immune Thrombocytopenia ◽  
Drug Induced ◽  
Inflammatory Drug ◽  
Drug Metabolites ◽  
Suspect Drug ◽  
Negative Results ◽  
Responsible Agent ◽  
Drug Dependent

In patients suspected of having drug-induced immune thrombocytopenia, antibodies reactive with normal platelets in the presence of the suspect drug can sometimes be identified, but negative results are often obtained. One reason for this is that drug metabolites, formed in vivo, can be the sensitizing agents, but very little is known about the specific metabolites that can cause this complication. Five patients were studied who developed thrombocytopenia after taking the nonsteroidal anti-inflammatory drug naproxen (3 cases) or acetaminophen (2 cases) but in whom drug-dependent antibodies could not be detected by means of the unmodified drugs. In each case, antibodies that reacted with normal target platelets in the presence of a known drug metabolite (naproxen glucuronide or acetaminophen sulfate) were identified. Four of the antibodies were specific for the glycoprotein (GP) IIb/IIIa complex, but one acetaminophen sulfate–dependent antibody reacted preferentially with GPIb/IX/V. In patients with a clinical picture suggestive of drug-induced immune thrombocytopenia, tests for metabolite-dependent antibodies can be helpful in identifying the responsible agent.

scholarly journals Pathophysiology and Diagnosis of Drug-Induced Immune Thrombocytopenia

2020 ◽  
Vol 9 (7) ◽  
pp. 2212 ◽  
Author(s):  
Caroline Vayne ◽  
Eve-Anne Guéry ◽  
Jérôme Rollin ◽  
Tatiana Baglo ◽  
Rachel Petermann ◽  
...  
Keyword(s):  
Immune Thrombocytopenia ◽  
Clinical Syndrome ◽  
Severe Thrombocytopenia ◽  
Drug Induced ◽  
Platelet Factor ◽  
Life Threatening ◽  
Thrombotic Complications ◽  
Drug Dependent

Drug-induced immune thrombocytopenia (DITP) is a life-threatening clinical syndrome that is under-recognized and difficult to diagnose. Many drugs can cause immune-mediated thrombocytopenia, but the most commonly implicated are abciximab, carbamazepine, ceftriaxone, eptifibatide, heparin, ibuprofen, mirtazapine, oxaliplatin, penicillin, quinine, quinidine, rifampicin, suramin, tirofiban, trimethoprim-sulfamethoxazole, and vancomycin. Several different mechanisms have been identified in typical DITP, which is most commonly characterized by severe thrombocytopenia due to clearance and/or destruction of platelets sensitized by a drug-dependent antibody. Patients with typical DITP usually bleed when symptomatic, and biological confirmation of the diagnosis is often difficult because detection of drug-dependent antibodies (DDabs) in the patient’s serum or plasma is frequently not possible. This is in contrast to heparin-induced thrombocytopenia (HIT), which is a particular DITP caused in most cases by heparin-dependent antibodies specific for platelet factor 4, which can strongly activate platelets in vitro and in vivo, explaining why affected patients usually have thrombotic complications but do not bleed. In addition, laboratory tests are readily available to diagnose HIT, unlike the methods used to detect DDabs associated with other DITP that are mostly reserved for laboratories specialized in platelet immunology.

Drug Metabolites Formed in Vivo Promote the Clearance of Human Platelets by Metabolite-Dependent Antibodies in NOD/SCID Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1083-1083
Author(s):  
Daniel W. Bougie ◽  
Dhirendra Nayak ◽  
Richard H. Aster
Keyword(s):  
Scid Mouse ◽  
Conflicts Of Interest ◽  
Human Platelets ◽  
Serum Samples ◽  
Drug Induced ◽  
Drug Metabolites ◽  
Multiple Drugs ◽  
Test Serum

Abstract Abstract 1083 Drug induced immune thrombocytopenia (DITP) is a relatively common and often serious complication of treatment with many drugs. It can be extremely helpful clinically to identify the responsible agent, especially in patients taking multiple drugs. It is sometimes possible to demonstrate a drug-dependent antibody (DDAb) that binds to platelets only when drug is present but results are often negative even in patients with a history that strongly favors DITP. An important reason for this is that a drug metabolite rather than the parent drug can be the sensitizing agent. Because drugs often have many metabolites, most of which are not readily available, identification of the metabolite-specific, DDAbs can be very difficult. Animal models for studying immune clearance of human cells have not been available because of naturally occurring xenoantibodies. However, recent studies have shown that destruction of human platelets by human antibodies can be studied in the NOD/SCID mouse, which lacks xenoantibodies (Boylan et al 2009). Metabolism of many drugs is similar in mice and humans. We have shown that mice injected with acetaminophen and naproxen produce metabolites that promote in vivo destruction of human platelets by DDAbs specific for metabolites of these drugs (Bougie et al 2010). These findings suggested that the NOD/SCID mouse might be a useful screening tool to identify putative DDAbs specific for metabolites of other drugs in patients suspected of DITP. To detect metabolite-specific antibodies, mice are transfused with human platelets suspended in test serum. After collecting a baseline blood sample to define the relative number of human platelets in each mouse, the implicated drug is injected (intraperitoneal). Drug metabolites made by the mouse diffuse into the circulation and shorten the survival of human platelets if a metabolite-specific, platelet-reactive antibody is present in the test serum. For a result to be considered “positive” for DDAbs, human platelet survival should be unaltered in mice given test serum alone and normal serum plus drug and should be significantly shortened in mice given test serum plus drug (Figure). Findings made to date with 94 archived serum samples from DITP suspects exposed to 5 widely used medications are summarized in Table 1. No platelet-reactive antibodies were detected in vitro using unmodified drug. However, 13 patient samples caused accelerated clearance of human platelets when mice were injected with the implicated drug (2 furosemide, 6 clopidogrel, 2 pantoprazole, 3 ibuprofen). These findings suggest that DITP resulting from sensitization to drug metabolites may be much more common than has been thought and indicate that the NOD/SCID mouse model provides a highly efficient tool to screen candidate sera for metabolite-specific, platelet-reactive antibodies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals On the mechanisms of sensitization and attachment of antibodies to RBC in drug-induced immune hemolytic anemia

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1006-1010 ◽  
Author(s):  
A Salama ◽  
C Mueller-Eckhardt
Keyword(s):  
Specific Binding ◽  
Drug Induced ◽  
Cell Complexes ◽  
Large Panels ◽  
Plot Analysis ◽  
Number Of Patients ◽  
Immune Hemolytic Anemia ◽  
Drug Dependent ◽  
Reaction Patterns

Abstract The mechanisms of sensitization and attachment of drug-dependent antibodies to RBC in drug-induced immune hemolytic anemias are largely speculative. Nomifensine has been incriminated in causing immune hemolysis in a large number of patients. The hemolysis was usually of the so-called immune complex type, less commonly of the autoimmune type, and more surprisingly, few patients had developed both types of hemolysis. To determine whether nomifensine (metabolite)-dependent antibodies (ndab) exhibit specificity for antigenic structures of RBC membranes, 30 ndab were tested against large panels of RBC with common and rare antigens. We found that only 14 out of 30 ndab were invariably reactive with all cells tested. Nine antibodies were, similar to the majority of idiopathic or drug-induced autoantibodies, not or only weakly reactive with Rhnull RBC. Three antibodies did not react with cord RBC and could be inhibited by soluble I antigen. The remaining four antibodies gave inhomogeneous reaction patterns or were even negative with selected RBC; their specificity could not be identified. On a Scatchard plot analysis of one ndab, a maximum of 173,000 drug- dependent antibodies of the IgG class can specifically bind per RBC in the presence of the drug. Although nomifensine and its metabolites do not attach tightly onto RBC, our results clearly indicate that RBC do not act as “innocent bystanders,” but rather serve as a surface for a loose attachment of drugs that possibly cause a subtle structural change in the cell antigens and, by this means, allow in vivo sensitization; and a specific binding of the resultant antibodies. This concept would explain why these antibodies can be directed against drug- cell complexes, against cell antigens alone (autoantibodies), or against both in the same patient.

scholarly journals Patients with quinine-induced immune thrombocytopenia have both “drug-dependent” and “drug-specific” antibodies

Blood ◽  
2006 ◽  
Vol 108 (3) ◽  
pp. 922-927 ◽  
Author(s):  
Daniel W. Bougie ◽  
Peter R. Wilker ◽  
Richard H. Aster
Keyword(s):  
Immune Thrombocytopenia ◽  
Drug Sensitivity ◽  
Soluble Form ◽  
Severe Thrombocytopenia ◽  
Membrane Glycoproteins ◽  
Drug Induced ◽  
Specific Antibodies ◽  
Cellular Targets ◽  
Surrogate Measure ◽  
Drug Dependent

AbstractImmune thrombocytopenia induced by quinine and many other drugs is caused by antibodies that bind to platelet membrane glycoproteins (GPs) only when the sensitizing drug is present in soluble form. In this disorder, drug promotes antibody binding to its target without linking covalently to either of the reacting macro-molecules by a mechanism that has not yet been defined. How drug provides the stimulus for production of such antibodies is also unknown. We studied 7 patients who experienced severe thrombocytopenia after ingestion of quinine. As expected, drug-dependent, platelet-reactive antibodies specific for GPIIb/IIIa or GPIb/IX were identified in each case. Unexpectedly, each of 6 patients with GPIIb/IIIa-specific antibodies was found to have a second antibody specific for drug alone that was not platelet reactive. Despite recognizing different targets, the 2 types of antibody were identical in requiring quinine or desmethoxy-quinine (cinchonidine) for reactivity and in failing to react with other structural analogues of quinine. On the basis of these findings and previous observations, a model is proposed to explain drug-dependent binding of antibodies to cellular targets. In addition to having implications for pathogenesis, drug-specific antibodies may provide a surrogate measure of drug sensitivity in patients with drug-induced immune cytopenia.

scholarly journals Quinine-dependent, platelet-reactive monoclonals mimic antibodies found in patients with quinine-induced immune thrombocytopenia

Blood ◽  
2009 ◽  
Vol 113 (5) ◽  
pp. 1105-1111 ◽  
Author(s):  
Daniel W. Bougie ◽  
Jessica Birenbaum ◽  
Mark Rasmussen ◽  
Mortimer Poncz ◽  
Richard H. Aster
Keyword(s):  
Molecular Basis ◽  
Immune Thrombocytopenia ◽  
Antibody Binding ◽  
Membrane Glycoproteins ◽  
Binding Studies ◽  
Platelet Destruction ◽  
Drug Induced ◽  
N Terminus ◽  
Sequencing Studies ◽  
Drug Dependent

Abstract Drug-induced immune thrombocytopenia (DITP) is caused by drug-dependent antibodies (DDAbs) that are nonreactive in themselves but bind tightly to specific platelet membrane glycoproteins (GP) when soluble drug is present at pharmacologic concentrations. This reaction takes place without covalent linkage of drug to the target, indicating that drug does not function as a classical hapten to promote antibody binding. Studies to define other mechanism(s) responsible for this interaction have been frustrated by the polyclonal nature of human DDAbs and limited quantities of antibody usually available. We produced 2 monoclonal antibodies (mAbs), 314.1 and 314.3, from a mouse immunized with purified human GPIIb/IIIa and quinine that recognize the N terminus of the GPIIb β propeller domain only when soluble quinine is present. Both monoclonals closely mimic the behavior of antibodies from patients with quinine-induced immune thrombo-cytopenia in their reactions at various concentrations of quinine and quinine congeners. Sequencing studies showed that the 2 mAbs are closely related structurally and that mAb 314.3 probably evolved from mAb 314.1 in the course of the immune response. These monoclonal reagents are the first of their kind and should facilitate studies to define the molecular basis for drug-dependent antibody binding and platelet destruction in DITP.

scholarly journals Binding of Quinine-Dependent Monoclonal Antibodies to Gpiib/Iiia Appears to Involve Drug-Induced Modulation of Antibody Affinity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2246-2246
Author(s):  
Daniel W. Bougie ◽  
Julie A. Peterson ◽  
Mark Rasmussen ◽  
Richard H. Aster
Keyword(s):  
Monoclonal Antibodies ◽  
Immune Thrombocytopenia ◽  
Patent Application ◽  
Soluble Form ◽  
Docking Site ◽  
Fab Fragment ◽  
Drug Induced ◽  
Short Supply ◽  
Unique Type ◽  
Drug Dependent

Abstract A major type of drug-induced immune thrombocytopenia (DITP), characterized by an acute, sometimes life-threatening drop in platelets following drug exposure, is caused by a unique type of antibody that recognizes its target on a platelet membrane glycoprotein, usually αIIb/β3 integrin [glycoprotein (GP) IIb/IIIa], only when the sensitizing drug is present in soluble form. Quinine (Qn) is the prototypic drug that causes this complication, but many other drugs have been implicated. It is widely thought that drug-dependent, platelet-reactive antibodies (DDAbs) characteristic of DITP recognize drug-induced structural modifications of platelet glycoproteins (GP), but this has not been confirmed experimentally. The mechanism responsible for DDAb binding is difficult to study with human DDAbs, which are often poly-specific and in short supply. We used newly-developed, Qn-dependent monoclonal antibodies (IgG1 mAbs 314.1, 314.3) that recognize the N-terminus of GPIIb and closely mimic the serologic behavior of antibodies from patients with Qn-induced immune thrombocytopenia (Blood 2009; 113;1105-11) as an alternative tool for studying the molecular basis of drug-dependent antibody binding. Previous studies failed to demonstrate a docking site for Qn in domains of GPIIb/IIIa that are known targets for the "314" mAbs and for human Qn-dependent antibodies. Therefore we examined an alternative possibility - that binding of drug to antibody might be the first step in DDAb binding. For this purpose, Qn was perfused over the "314" mAbs immobilized on Biacore chips and surface plasmon resonance (SPR) signals were recorded. Findings showed that Qn binds specifically to both mAbs with high affinity (Kd about 30 nM) and with 2:1 stoichiometry (Qn to mAb), consistent with recognition of Qn by complementarity determining regions (CDR) of the mAbs. To characterize monovalent binding of mAb to GPIIb/IIIa, purified integrin in 0.1% triton X-100 was perfused over immobilized mAb 314.1 and SPR signals recorded. Weak, but specific binding was observed in the absence of Qn (Kd 11 uM) that was enhanced 5-fold (Kd 2.2 uM) when Qn was present. Kds for Qn-dependent binding of mAb 314.1 (bivalent interaction) and its Fab fragment (monovalent interaction) to GPIIb/IIIa were determined by flow cytometry using labeled antibody and Fab under conditions that did not require washing prior to direct measurement of platelet bound IgG and Fab. Weak Fab binding was observed in the presence of Qn (≈19 uM) but with intact IgG the effective Kd was reduced to 0.15 nM, reflecting a 100,000-fold increase in avidity. Together with studies that have failed to demonstrate any docking site for Qn on GPIIb/IIIa, the findings support a model in which DDAb-GPIIb/IIIa interaction starts with binding of drug to the antibody CDR, leading to a structural change that markedly increases the avidity of antibody for a weak autoantigen. A requirement for bivalent antibody-target interaction to achieve tight binding could explain why DDAbs almost invariably recognize GPIIb/IIIa or GPIb/IX, the most highly expressed platelet glycoproteins. How this type of DDAb is induced by drug remains uncertain but the findings are consistent with a model in which sensitization starts with drug-induced modification of a B cell receptor that increases its affinity for a weak autoantigen. Disclosures Aster: BLOODCENTER OF WISCONSIN: Patents & Royalties: A patent application has been filed based partly on these findings (Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591).

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