Mechanism of quinine-dependent monoclonal antibody binding to platelet glycoprotein IIb/IIIa.

Drug-dependent antibodies (DDAbs) that cause acute thrombocytopenia upon drug exposure are nonreactive in the absence of the drug but bind tightly to a platelet membrane glycoprotein, usually α(IIb)/ß3 integrin (GPIIb/IIIa) when the drug is present. How a drug promotes binding of antibody to its target is unknown and is difficult to study with human DDAbs, which are poly-specific and in limited supply. We addressed this question using quinine-dependent murine monoclonal antibodies (mAbs), which, in vitro and in vivo, closely mimic antibodies that cause thrombocytopenia in patients sensitive to quinine. Using surface plasmon resonance (SPR) analysis, we found that quinine binds with very high affinity (K(D) ≈ 10⁻9 mol/L) to these mAbs at a molar ratio of ≈ 2:1 but does not bind detectably to an irrelevant mAb. Also using SPR analysis, GPIIb/IIIa was found to bind monovalently to immobilized mAb with low affinity in the absence of quinine and with fivefold greater affinity (K(D) ≈ 2.2 × 10⁻6) when quinine was present. Measurements of quinine-dependent binding of intact mAb and fragment antigen-binding (Fab) fragments to platelets showed that affinity is increased 10 000- to 100 000-fold by bivalent interaction between antibody and its target. Together, the findings indicate that the first step in drug-dependent binding of a DDAb is the interaction of the drug with antibody, rather than with antigen, as has been widely thought, where it induces structural changes that enhance the affinity/specificity of antibody for its target epitope. Bivalent binding may be essential for a DDAb to cause thrombocytopenia.

Structural basis for quinine-dependent antibody binding to platelet integrin αIIbß3.

Drug-induced immune thrombocytopenia (DITP) is caused by antibodies that react with specific platelet-membrane glycoproteins when the provoking drug is present. More than 100 drugs have been implicated as triggers for this condition, quinine being one of the most common. The cause of DITP in most cases appears to be a drug-induced antibody that binds to a platelet membrane glycoprotein only when the drug is present. How a soluble drug promotes binding of an otherwise nonreactive immunoglobulin to its target, leading to platelet destruction, is uncertain, in part because of the difficulties of working with polyclonal human antibodies usually available only in small quantities. Recently, quinine-dependent murine monoclonal antibodies were developed that recognize a defined epitope on the ß-propeller domain of the platelet integrin αIIb subunit (GPIIb) only when the drug is present and closely mimic the behavior of antibodies found in human patients with quinine-induced thrombocytopenia in vitro and in vivo. Here, we demonstrate specific, high-affinity binding of quinine to the complementarity-determining regions (CDRs) of these antibodies and define in crystal structures the changes induced in the CDR by this interaction. Because no detectable binding of quinine to the target integrin could be demonstrated in previous studies, the findings indicate that a hybrid paratope consisting of quinine and reconfigured antibody CDR plays a critical role in recognition of its target epitope by an antibody and suggest that, in this type of drug-induced immunologic injury, the primary reaction involves binding of the drug to antibody CDRs, causing it to acquire specificity for a site on a platelet integrin.

Bioluminescent Antibodies for Point-of-Care Diagnostics.

We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔRmax >500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera.

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

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.

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

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 beta 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.

The Role of Quinine-Responsive Taste Receptor Family 2 in Airway Immune Defense and Chronic Rhinosinusitis.

Background: Bitter (T2R) and sweet (T1R) taste receptors in the airway are important in innate immune defense, and variations in taste receptor functionality in one T2R (T2R38) correlate with disease status and disease severity in chronic rhinosinusitis (CRS). Quinine is a bitter compound that is an agonist for several T2Rs also expressed on sinonasal cells, but not for T2R38. Because of this property, quinine may stimulate innate immune defense mechanisms in the airway, and functional differences in quinine perception may be reflective of disease status in CRS. Methods: Demographic and taste intensity data were collected prospectively from CRS patients and non-CRS control subjects. Sinonasal tissue from patients undergoing rhinologic surgery was also collected and grown at an air-liquid interface (ALI). Nitric oxide (NO) production and dynamic regulation of ciliary beat frequency in response to quinine stimulation were assessed in vitro. Results: Quinine reliably increased ciliary beat frequency and NO production in ALI cultures in a manner consistent with T2R activation (p < 0.01). Quinine taste intensity rating was performed in 328 CRS patients and 287 control subjects demonstrating that CRS with nasal polyps (CRSwNP) patients rated quinine as significantly less intense than did control subjects. Conclusion: Quinine stimulates airway innate immune defenses by increasing ciliary beat frequency and stimulating NO production in a manner fitting with T2R activation. Patient variability in quinine sensitivity is observed in taste intensity ratings, and gustatory quinine "insensitivity" is associated with CRSwNP status. Thus, taste tests for quinine may be a biomarker for CRSwNP, and topical quinine has therapeutic potential as a stimulant of innate defenses.

Study of the factors that cause specific transformation in cultures of lymphocytes from patients with quinine- and quinidine-induced immune thrombocytopenia.

Quinine- or quinidine-induced thrombocytopenic purpura is caused by synthesis of an immunoglobulin (Ig)G antibody, which caused platelet damage in the presence of the offending drug. The nature of the antigenic stimulus has been examined by measuring incorporation of [3H]thymidine into DNA during lymphocyte transformation to blast cells in the presence of the drug. Although patients' lymphocytes responded normally to the nonspecific mitogen, phytohemagglutinin P, they did not respond to either drug or platelets alone. However, significant transformation occurred when patients' lymphocytes were cultured for 7 d with homologous or autologous platelets in the presence of therapeutic concentrations of the drugs (0.39-39 microM). Platelet membranes were more active than intact platelets on the basis of protein content, whereas platelets from a patient with Bernard-Soulier syndrome were inactive. Washed platelets pretreated with the drugs were inactive when cultured with lymphocytes in the absence of the drugs, whereas platelets pretreated similarly in plasma caused transformation. Control lymphocytes from 20 normal patients and 6 patients with nondrug-induced thrombocytopenia were not transformed by drugs and platelets in the presence of normal serum or serum containing drug-dependent antibody, showing that the observed response was specific for presensitized lymphocytes. Thus lymphocytes of patients with drug-induced thrombocytopenia are transformed by an antigen that forms after interaction of plasma, specific platelet membrane components and the drug.

Drug-antibody-platelet interaction in quinine- and quinidine-induced thrombocytopenia.

Binding of quinine- and quinidine-dependent antibodies to platelets was studied using an electroimmunoassay to measure platelet-bound IgG. Antibodies from four patients with drug-induced thrombocytopenia differed significantly in their interaction with platelets: association constants for binding to platelets at high drug concentrations ranged from 0.29 to 2.6 x 10(7) M(-1), the maximum number of antibody molecules bound ranged from 36,000 to 161,000/platelet, the amount of drug necessary to achieve half-maximum binding of antibodies to platelets ranged from 2 to 60 muM, and only one of the antibodies cross-reacted with the stereoisomer of the drug to which the patient was sensitized. Binding of the antibodies to platelets was enhanced at the highest achievable molar ratio of drug:antibody, 10,000:1, rather than being inhibited, as would be expected in a conventional, hapten-dependent reaction. The drug-antibody-platelet reaction was unaffected by Factor VIII/von Willebrand protein, nonspecifically aggregated IgG, or heat-labile complement components. After pretreatment with tritiated quinine, platelets retained several hundred thousand molecules of drug each, but failed to bind detectable amounts of antibody. However, platelets treated simultaneously with quinine-dependent antibody and tritiated quinine retained significantly more drug after repeated washes than platelets treated with drug and normal serum. These findings support the proposition that in quinine- and quinidine-induced thrombocytopenia, drug and antibody combine first in the soluble phase to form a complex, which then binds with high affinity to a receptor on the platelet surface (innocent bystander reaction), and demonstrate that these antibodies are heterogeneous in respect to the amount of drug required to promote their binding to platelets, the number of platelet receptors they recognize, and their binding affinities.

Fab-mediated binding of drug-dependent antibodies to platelets in quinidine- and quinine-induced thrombocytopenia.

Platelets coated with quinine- or quinidine-induced antibodies form rosettes around protein A-Sepharose beads and normal platelets form rosettes about protein A-Sepharose beads coated with these antibodies. These reactions occurred only in the presence of sensitizing drug. Platelets also formed rosettes about protein A-Sepharose beads coated with an anti-PIA1 antibody, but drug was not required. Formation of rosettes between antibody-coated platelets and protein A-Sepharose was inhibited by F(ab')2 fragments of goat antibody specific for the Fc portion of human IgG, while rosette formation between antibody-coated protein A-Sepharose and platelets was inhibited by F(ab')2 fragments directed against the F(ab')2 portion of the IgG molecule. Since binding of IgG to protein A is known to occur via the Fc region, these findings suggest that binding of drug-induced antibodies to platelets occurs at the Fab domains of the IgG molecule.

Quinine-induced thrombocytopenia following intravenous use of heroin.

Profound thrombocytopenia developed in a 22-year-old man after intravenous use of heroin. A high-titer, quinine-dependent, platelet-specific antibody was detected in his serum using lysis of normal platelets labeled with chromium 51 and an electroimmunoassay for measurement of platelet-associated IgG. The antibody was specific for quinine and failed to react with platelets in the presence of quinidine hydrochloride or two structural analogues of heroin. Quinine, a common adulterant found in heroin, was detected in the patient's blood and urine. On the basis of these observations, the patient was judged to have quinine-induced immunologic thrombocytopenia. To our knowledge, this report is the first to confirm that quinine used as an adulterant can induce immunologic thrombocytopenia following an injection of heroin.

Quinine-induced immune thrombocytopenic purpura followed by hemolytic uremic syndrome.

Immune thrombocytopenic purpura (ITP) mediated by quinine-dependent platelet reactive antibodies is well recognized. More recently there have been a number of reports of quinine-induced hemolytic-uremic syndrome (HUS). We describe a patient with quinine-induced immune thrombocytopenia who subsequently developed HUS after re-exposure to a single dose of this drug. To our knowledge, this is the first such case reported. Multiple quinine-dependent antibodies have been characterized in the patient's serum. Initially, quinine-dependent antibodies were directed solely against the platelet glycoprotein complex GPIb/IX. After rechallenge with quinine, there was broadening of quinine-dependent antibody specificities, which were now also directed against the platelet glycoprotein complexes GPIb/IX and GPIIb/IIIa, endothelial cells, and leukocytes. We have shown quinine-dependent antibody-mediated endothelial cell activation, which supports an immunopathogenic role for quinine-dependent antibodies in the causation of this disease.

Quinine-induced disseminated intravascular coagulation: case report and review of the literature.

OBJECTIVE: To describe the clinical course of quinine-induced disseminated intravascular coagulation (DIC) and review all previous cases reported in the medical literature. DESIGN: Case report/literature review. SETTING: University teaching hospital medical ICU. PATIENTS: One patient in whom thrombocytopenia, coagulopathy, intravascular hemolysis, DIC, and acute renal failure temporally followed the ingestion of quinine. DATA SOURCES: We conducted a computerized free-text MEDLINE database search from 1969 to 2000 using the keywords quinine and thrombocytopenia, quinine and hemolytic-uremic syndrome, and quinine and disseminated intravascular coagulation. STUDY SELECTION: All reported cases and reviews of quinine-induced thrombocytopenia, hemolytic-uremic syndrome (HUS), and DIC were reviewed. DIC was distinguished from quinine-induced thrombocytopenia or quinine-induced HUS based on the presence of abnormal clotting times, elevated fibrin degradation products, and/or elevated D-dimer levels. DATA SYNTHESIS: Fifteen previous patients were found to meet the criteria for DIC temporally related to the recent ingestion of quinine. The clinical course and laboratory abnormalities documented for each case are reviewed. CONCLUSIONS: Quinine-induced DIC is a distinct clinical entity, which may present as unexplained thrombocytopenia, coagulopathy, or renal failure. In susceptible patients, the immune response to quinine may result in the production of not only anti-platelet antibodies but also antibodies against leukocytes, erythrocytes, and endothelial cells. Furthermore, the varying patterns and specificities of antibody production in an individual patient may result in a spectrum of clinical disease from mild, transient thrombocytopenia to overt intravascular hemolysis, renal failure, coagulopathy, and DIC. Early recognition of quinine-induced DIC is paramount, as this diagnosis affords a better prognosis than other adult forms of HUS or DIC.

Inhibition of mitogen-induced lymphocyte proliferative responses by quinine.

In vitro mitogen-induced proliferative responses of human lymphocytes were inhibited by concentrations of quinine normally attainable during therapy of malaria infections. The clinical implications of these findings are discussed.

Monoclonal antibodies to quinine.

Monoclonal antibodies (MAbs) to quinine conjugated to a carrier protein were produced. Quinine was converted into a hemisuccinate prior to covalently linked to bovine serum albumin (BSA) by reacting with N,N'-disuccinimidyl carbonate (DSC). Coupling ratio of quinine-BSA was 13:1 calculated by spectrophotometry and 14:1 by calculation from quinine standard curve. This immunogen was used for both monoclonal antibody production and for screening test, indirect ELISA. The specificity of quinine-BSA MAbs was examined by checking the cross reactivity with BSA and the structurally related antimalarial drug, mefloquine. Six MAbs belonging to IgG1 were obtained. These MAbs slightly reacted with mefloquine-BSA because of closely related structure of mefloquine to quinine and similar conjugate preparation procedure used for conjugation. One selected MAb against quinine-BSA, showed higher reactivity with blood samples from patients previously treated with quinine when compared to normal blood. This preliminary test indicated that MAbs obtained may be useful to be used as the probe for detection of quinine in biological fluids.

[Immuno-allergic cytopenias]. / Cytopénies sanguines immuno-allergiques.

Drug-induced cytopenias are sometimes related to the so-called immuno-allergic mechanism which involves an unusual and unpredictable (i.e.: allergic) immune mediated reaction against the drug, leading to the cell-lysis of either peripheral blood granulocytes, thrombocytes; or erythrocytes. Agranulocytosis is typically induced by amidopyrine, whereas thrombocytopenia and hemolytic anemia are observed with drugs like quinine-quinidine, betalactams antibiotics, sulfonamides, rifampicin etc. In vitro methods for identification of the offending drug are cumbersome and poorly suitable for routine use. Therefore, definite diagnosis in these cases is first based on clinical and hematological grounds. These accidents recover spontaneously. However, potentially severe complications (infection, bleeding) deserve to prevent the risk of recurrence by a lifetime prohibition of the identified or suspected drug.

Drug-induced immune thrombocytopenia.

Thrombocytopenia is caused by immune reactions elicited by diverse drugs in clinical practice. The activity of the drug-dependent antibodies produces a marked decrease in blood platelets and a risk of serious bleeding. Understanding of the cellular mechanisms that drive drug-induced thrombocytopenia has advanced recently but there is still a need for improved laboratory tests and treatment options. This article provides an overview of the different types of drug-induced thrombocytopenia, discusses potential pathologic mechanisms, and considers diagnostic methods and treatment options.