Correlation of a quinidine-induced platelet-specific antibody with development of thrombocytopenia

The rapidity by which drug-dependent antiplatelet antibodies can develop is not known, since patients are only studied during or after the episode of thrombocytopenia. This report describes the development of quinidine-induced immune thrombocytopenia in a healthy volunteer during a drug study. The thrombocytopenia developed within two weeks of initiation of quinidine therapy. During the thrombocytopenic episode, but not before receiving the drug, the patient had an IgG antiplatelet antibody that bound to control platelets in the absence of the drug. This antibody was absent when the drug was discontinued and the platelet count rose. The patient's acute serum also induced the release of serotonin from control platelets, and the reaction was enhanced by quinidine. This indicates that drug-dependent antiplatelet antibodies can develop rapidly and supports the hypothesis that quinidine-induced thrombocytopenia is due to a quinidine-dependent platelet-specific IgG.     

Glycoprotein Ib/IX complex is the target in rifampicin-induced immune thrombocytopenia

Thrombocytopenia is a major adverse effect of several drug treatments. Rifampicin has been recognized as a cause of immune thrombocytopenia during intermittent high-dose therapy. We characterized the antibody of a patient who presented with purpura and thrombocytopenia during treatment of tuberculosis with rifampicin. Drug-dependent binding of the antibody to platelets was demonstrated by flow cytometry. In a glycoprotein-specific immunoassay, the binding epitope of the IgG antibody was found in the glycoprotein Ib/IX complex, using four different monoclonal antibodies (mAbs) against various epitopes on the GPIb/IX complex, as well as mAbs against GPIIb/IIIa, GPIa/IIa and GPIV. By immunoprecipitation of biotin-labelled platelets, reactivity of the antibody with GPIb/IX was found only in the presence of the drug. These findings clearly demonstrate that rifampicin induces the formation of drug-dependent antibodies capable of causing thrombocytopenia. The binding site of the rifampicin-dependent antibody, located in the GPIb/IX complex, seems to be a favoured target for antibodies induced by different drugs.     

Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpura-prone (NZW x BXSB)F1 mice

Male (NZW x BXSB)F1 (W/BF1) mice develop systemic autoimmunity involving autoantibodies, progressive thrombocytopenia, lupus nephritis, and degenerative coronary vascular disease with myocardial infarction. Platelet-associated IgG (PAIgG) on the platelet surface mediates platelet destruction by the reticuloendothelial system in the autoimmune thrombocytopenic purpura (ATP) of W/BF1 mice. Because the epitopes targeted in ATP by PAIgG have not been identifiable using serum from thrombocytopenic W/BF1 mice, we developed seven hybridomas secreting antiplatelet monoclonal antibodies (MoAbs) using splenocytes of thrombocytopenic W/BF1 mice. Epitopes recognized by three MoAbs were similar to those recognized by PAIgG, because eluted IgG from platelets of thrombocytopenic W/BF1 mice inhibited platelet binding by MoAbs in competitive micro-enzyme-linked immunosorbent assay. Hybridoma cells or purified Ig from the ascites of two clones (2A12 and 6A6), when injected into nude mice produced acute thrombocytopenia, elevated the levels of PAIgG, purpura, and megakaryocytosis. MoAbs of two clones also reacted with single-stranded DNA or double-stranded DNA, and one of these clones (4-13) bound to cardiolipin (CL) but was nonpathogenic in nude mice, suggesting that anti-CL and antiplatelet autoantibodies can be distinct. On immunoblotting analysis, antiplatelet MoAbs frequently bound a 100-Kd platelet protein. These MoAbs contribute to an understanding of the etiopathogenesis of ATP and the several antigens and autoantibodies involved.     

Idiopathic thrombocytopenic purpura

Idiopathic thrombocytopenic purpura occurs at all ages, in acute and chronic forms. Children mainly have the acute form, which usually follows a recent viral illness, occurs equally in both sexes, and generally resolves within six months. Chronic idiopathic thrombocytopenic purpura occurs more often in adults, often has an insidious onset, and shows a three:one female preponderance. Both forms are now thought to be due to an antiplatelet antibody, usually of the IgG class (platelet-associated IgG), which coats autologous platelets and leads to their phagocytosis and destruction by the reticuloendothelial system. In most patients, the spleen is the major site of the production of this platelet antibody and the destruction of the platelets. Many methods have been developed to detect this antiplatelet factor in the serum and on the platelets of patients with idiopathic thrombocytopenic purpura. Recent methods are becoming highly sensitive and may soon be simple and fast enough for routine clinical use and should significantly aid the diagnosis and management of these patients. Platelet-associated IgG levels appear significantly higher in patients with idiopathic thrombocytopenic purpura than in normal subjects, and in patients with nonimmune thrombocytopenia. Higher levels are also seen in children than in adults, and in acute cases than in chronic ones. Platelet-associated IgG levels also vary inversely with platelet count and platelet life span, can predict the disease course and response to therapy, and may predict neonatal consequences of maternal idiopathic thrombocytopenic purpura. Evidence of other alterations in immune status, as well as alterations in platelet function and HLA associations, remains controversial. Classic treatment with corticosteroids and splenectomy remains highly successful in most cases. More recent therapies include the use of immunosuppressants and alkaloid-coated platelets, plasma-exchange transfusion, and high-dose immunoglobulin. Overall, fewer than 5 percent of patients have severe hemorrhage or refractory or fatal disease.     

Thrombocytopenic purpura in narcotics addicts

Since November 1982 we have seen an association of thrombocytopenic purpura with chronic narcotic addiction in 70 patients with a mean platelet count of 53000 +/- 4000 (SE); 33 had stopped taking intravenous drugs for an average of 21.2 +/- 4.7 months; 13 of 15 had elevated antibody titers for a virus related to the acquired immunodeficiency syndrome. Platelet-bound IgG, IgM, and complement levels were 16.7-, 5.6-, and 3.1-fold greater than control values, respectively, and 2.6-, 1.9-, and 2.4-fold greater than values in 25 patients with classic autoimmune thrombocytopenic purpura studied at the same time. Thirty-three of thirty-six addicts had elevated circulating immune complexes, whereas 8 patients with autoimmune thrombocytopenia had no elevation. Eleven of eighteen addicts had positive serum platelet-reactive IgG titers, compared to 5 of 19 patients with classic autoimmune thrombocytopenia. The platelet-reactive IgG in sera of addicts was composed of 7S IgG antibody as well as high molecular weight (immune complex) IgG. Thus, chronic addicts appear to have a new immunologic platelet disorder associated with the presence of 7S IgG antiplatelet antibody, like patients with classic autoimmune thrombocytopenic purpura, and immune complex associated nonspecific platelet IgG, like male homosexual patients with thrombocytopenia.     

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a target glycoprotein in drug-induced thrombocytopenia

Drug-induced immune thrombocytopenia (DITP) is a serious complication of drug treatment. Previous studies demonstrated that most drug-dependent antibodies (DDAbs) react with the platelet membrane glycoprotein (GP) complexes IIb/IIIa and Ib/IX/V. We analyzed the sera from 5 patients who presented with DITP after intake of carbimazole. Notably, thrombocytopenia induced by carbimazole was relatively mild in comparison to patients with DITP induced by quinidine. The sera reacted with platelets in an immunoassay on addition of the drug. In immunoprecipitation experiments with biotin-labeled platelets and endothelial cells, reactivity with the platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) could be demonstrated, whereas neither GPIIb/IIIa nor GPIb/IX was precipitated in the presence of the drug. These results could be confirmed by GP-specific immunoassay (MAIPA) using monoclonal antibodies (mabs) against PECAM-1. In addition, the binding of DDAbs could be abolished by preincubation with soluble recombinant PECAM-1. Carbimazole-dependent antibodies showed similar reactivity with platelets carrying the Leu(125) and Val(125) PECAM-1 isoforms, indicating that this polymorphic structure, which is located in the first extracellular domain, is not responsible for the epitope formation. Binding studies with biotin-labeled mutants of PECAM-1 and analysis of sera with mabs against different epitopes on PECAM-1 in MAIPA assay suggested that carbimazole-dependent antibodies prominently bound to the second immunoglobulin homology domain of the molecule. Analysis of 20 sera from patients with quinidine-induced thrombocytopenia by MAIPA assay revealed evidence that DDAbs against PECAM-1 are involved in addition to anti-GPIb/IX and anti-GPIIb/IIIa. We conclude that PECAM-1 is an important target GP in DITP. (Blood. 2000;96:1409-1414)     

Cumulative experience with a simplified solid-phase radioimmunoassay for the detection of bound antiplatelet IgG, serum auto-, allo-, and drug-dependent antibodies

A simplified, sensitive, solid-phase radioimmunoassay employing 125I- staphylococcal protein A has been developed that is capable of detecting bound antiplatelet IgG as well as serum auto-, allo-, and drug-dependent antiplatelet antibodies. The simplified assay employs a ratio of test over control platelet counts per minute (cpm) for detection of positive results. All reagents are commercially available. The assay can be performed with as little as 10(6) washed platelets (10 microliters of whole blood) that have been stored for as long as 8 wk at 4 degrees C in microtiter plates. The assay time, employing stored platelets, is 4 hr. Bound platelet IgG is positive in 93% of 46 thrombocytopenic patients with autoimmune disease and correlates inversely with their platelet count, r = -0.65, p less than 0.001. The ability of this assay to detect serum antibody was studied with a rabbit anti-human platelet antibody capable of giving optimal immunoprecipitation with solubilized platelet membranes at a tier of 1:10. The present assay increases the sensitivity of antibody detection 256-fold to a titer of 1:2560. Human serum antiplatelet membrane antibody was positive in 2 of 2 patients with anti-PLA-1 antibody (titers of 1:256 and greater than 1:64); 7 of 12 multiply transfused patients who were refractory to platelet transfusion (2 had titers of greater than 1:256 and greater than 1:32); 5 of 19 patients with autoimmune thrombocytopenic purpura (2 had titers of 1:64 and 1:32); and 10 of 14 patients with clinical histories of drug-dependent antiplatelet antibody (2 had titers of 1:1280 for quinidine and 1:384 for phenazopyridine).     

Evidence for the co-existence of immunocomplexes and platelet specific antibodies in HIV infection-related thrombocytopenia in narcotic drug addicts. Experimental results and immunopathogenetic discussion.

BACKGROUND. The development of symptomatic or asymptomatic thrombocytopenia is not uncommon in HIV seropositive drug addicts. METHODS. Platelet binding immunoglobulins, as immune complexes or as platelet specific antibodies, were studied in eleven patients. Whole sera anche serum factions obtained by PEG sedimentation and gel filtration were investigated using methods able to characterize the IgG and IgM immunoglobulin classes. Immune complexes-containing fractions were used in competition assays with some monoclonal antibodies with receptor specificities. RESULTS. Immune complexes able to bind both autologous and allogenic platelets were detected in all patients, while platelet specific IgG or IgM autoantibodies were detected in most, but not in all, patients. The results obtained with the various individual tests were sometimes discrepant, owing to the low avidity binding of platelet-reacting immunoglobulins. Anti-low-affinity FcRII monoclonal antibody moderately reduced the platelet binding of IgG containing immune-complex fractions. CONCLUSIONS. In thrombocytopenic, HIV seropositive drug addicts immunecomplexes and platelet specific antibodies usually coexist and possibly have different pathogenetic potential in the development of thrombocytopenia.     

Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.     

Antibodies against glycosphingolipids in sera of patients with idiopathic thrombocytopenic purpura

Evidence is presented for the existence of antibodies against platelet glycosphingolipids in sera of patients with chronic idiopathic thrombocytopenic purpura and antiplatelet antibodies. Increased binding of IgG/IgM to neutral glycosphingolipids or gangliosides extracted from donor platelets was measured by an ELISA in 17 sera of 30 patients. Thirteen sera, five with anticardiolipin antibodies, demonstrated an increased binding to sulphatides confirmed by immuno HPTLC. Four sera showed reactivity with the platelet minor gangliosides established by immuno-HPTLC.     

Antibodies to platelet membrane glycoprotein antigens in three cases of infectious mononucleosis-induced thrombocytopenic purpura

Infectious mononucleosis may occasionally be complicated by purpura, but the mechanism of the thrombocytopenia is not known in detail. In the present study, 3 children with mononucleosis-associated purpura were found to have marked elevations of platelet-associated immunoglobulins and circulating platelet binding IgG and IgM. Employing electrophoretically (SDS-PAGE) separated normal platelet membrane proteins in an immunoblot assay, serum IgG and IgM antibodies were found to be directed to antigenic determinants situated on glycoprotein GP IIb (140 kDa) in all patients, but also on smaller proteins of molecular weights between 30 and 52 kDa. 24 control sera were negative. Positive reactions were eliminated after absorption of sera with fresh platelets. These results demonstrate autoantibodies to platelet surface membrane proteins in infectious mononucleosis-induced purpura and suggest a transient autoantibody-mediated platelet destruction as the cause of thrombocytopenia in these patients.     

Autoantibodies against platelet glycoproteins in critically ill patients with thrombocytopenia

PURPOSE: The aim of the study was to investigate immunologic causes of thrombocytopenia in critically ill patients, especially causes that were related to platelet-associated IgG antibodies. SUBJECTS AND METHODS: All patients admitted to two intensive care units between May 1 and October 30, 1997, who developed thrombocytopenia (less than 100 x 10(9) platelets/L) were studied prospectively. We measured platelet-associated IgG with a radioimmunoassay using I(125)-labeled polyclonal antihuman IgG. Characterization of platelet-associated IgG was assessed with a monoclonal antibody immobilization of platelet antigen. Circulating immune complexes were also assayed. RESULTS: Of the 61 patients with thrombocytopenia, elevated platelet-associated IgG was found in 18 (30%). Associated antiplatelet autoantibodies (glycoprotein IIb/IIIa) were detected in 4 patients, circulating autoantibodies (glycoprotein Ib/IX) were detected in sera from 2 patients, and circulating immune complexes were detected in 3 patients. The nature of the platelet-associated IgG could not be determined in 10 patients. Elevated platelet-associated IgG was associated with sepsis and previous cardiopulmonary bypass. Thrombocytopenic patients with elevated platelet-associated IgG had a lower nadir platelet count (58 +/- 27 x 10(9)/L vs 74 +/- 24 x 10(9)/L, P = 0.03). CONCLUSION: Elevated platelet-associated IgG, some of which are platelet autoantibodies, is frequent in thrombocytopenic patients with sepsis or after cardiopulmonary bypass.     

Immunochemical analysis of platelet autoantibodies in HIV-related thrombocytopenic purpura: a study of 68 patients

Serum antiplatelet IgG and platelet-associated IgG (PAIgG) were studied in 68 AIDS-free human immunodeficiency virus (HIV)-infected patients with severe immunologic thrombocytopenic purpura (ITP), for the presence of platelet autoantibodies. Serum IgG with antiplatelet activity was found in 72% of the sera. However, the presence of autoantibodies against platelet surface glycoproteins was not found in these sera by means of Western blot and immunoprecipitation procedures. Nevertheless, an immunoblot immunoassay and an indirect immunofluorescence test against semi-permeabilized platelets demonstrated the presence of antibodies in the patient sera, that reacted with intracytoplasmic platelet components, and which might participate in the elimination of platelet fragments. Direct immunofluorescence tests demonstrated an increased amount of PAIgG in 75% of the patients; the bound antibodies could be eluted with ether in 44% of the cases. These eluates were found to bind to normal platelets but not to Glanzmann type I platelets. Finally, immunoprecipitation procedures demonstrated the presence of platelet autoantibodies in six of the 35 eluates studied. These antibodies recognized GPIIb in two cases, GPIIIa in one case, and an unidentified platelet protein of 150 kDa in the three other cases. The discrepancy between sera and platelet eluates was interpreted as being due to the low titre of the antibodies and to their dilution in polyclonal hypergammaglobulinaemia. The present study provides direct evidence that isolated ITP in some HIV-positive patients is due to the presence of platelet autoantibodies. These results, however, do not exclude either direct or indirect involvement of HIV in the platelet destruction.     

Platelet autoantibody in a case of infectious mononucleosis presenting as thrombocytopenic purpura

In this unusual case acute thrombocytopenic purpura was the sole clinical manifestation of infectious mononucleosis. An antiplatelet antibody was demonstrated in the serum by two newly described technics: release of radioactive serotonin from platelets and inhibition of platelet aggregation following exposure of platelets to serum containing antiplatelet activity. The antiplatelet activity was completely inactivated by adsorption to washed platelets and was located in the immunoglobulin G (IgG) gamma globulin fraction of serum. The patient's serum also reacted against her own platelets indicating that the antiplatelet factor behaved as a true autoantibody.     

Identification of platelet proteins that bind alloantibodies and autoantibodies

We have used the techniques of radioimmunoprecipitation (RIP) and Western blot to identify the membrane proteins that bind certain alloantibodies. Anti-PlA1 sera precipitated two bands, corresponding to platelet glycoproteins IIb and III, whether or not calcium was present during the procedure. By Western blot, this antibody bound only glycoprotein III. Anti-PlA1 serum does not precipitate proteins from the platelets of a patient with Glanzmann's thrombasthenia. Two monoclonal antibodies reacting with lymphocyte HLA antigens, as well as sera from highly allosensitized patients, precipitated bands of 38,500 and 13,500 daltons. These bands correspond to the molecular weights of the two subunits of the HLA antigen, as it has been described for other cell types. The patients' sera also precipitated a protein of 72,000 daltons from some platelets. The sera of two patients with quinidine- induced thrombocytopenia precipitated a 138,000-dalton band (glycoprotein Ib-alpha) in the presence of quinidine. The purified IgG antibody from one patient did not require other plasma factors to bind to platelets in the presence of quinidine, while purified antibody from a second patient required plasma factors other than, or in addition to von Willebrand factor. Although several sera from patients with idiopathic thrombocytopenic purpura (ITP) were tested, only one precipitated membrane proteins by the RIP method; this serum identified binding proteins corresponding to glycoproteins IIb and III.     

Immunofluorescent Staining of Platelet Suspensions and Detection of Antiplatelet Antibody in Patients with Idiopathic Thrombocytopenic Purpura

Direct immunofluorescent staining of 31 specimens of platelets obtained from 13 cases of idiopathic thrombocytopenic purpura (ITP) revealed positive staining on the surface of platelets for both immunoglobulins (Igs) and human B1C globulin in 9 specimens, for only Igs in 1 specimen and for human B1C alone in 5 specimens. The pattern of the positive immunofluorescent staining was granular. Indirect immunofluorescent staining of normal platelets in serum obtained from patients with ITP was positive for antiplatelet antibody in 9 out of 31 specimens. This suggests that platelets in patients with ITP may be damaged by an antiplatelet autoantibody acting directly on the platelet surface and/or by antigen antibody complexes binding via Fc IgG receptors on the surface of the platelets.     

Two human monoclonal antiplatelet autoantibodies established from patients with chronic idiopathic thrombocytopenic purpura

Two human hybridomas secreting antiplatelet autoantibodies were established by somatic cell fusion using splenocytes from two patients with chronic idiopathic thrombocytopenic purpura (ITP). These monoclonal antibodies, HT7F and HT8C, were of the IgM isotype and reacted with autologous and allogeneic platelets fixed with paraformaldehyde (PFA). They also bound to fresh platelets. An elution study showed that eluates from allogeneic platelets reacted with autologous platelets. These results indicated that HT7F and HT8C were autoantibodies recognizing a site on the platelet surface. Both monoclonal antibodies were able to induce complement activation in vitro. HT7F was demonstrated to bind to a platelet protein having a molecular mass of 105 kDa under both nonreducing and reducing conditions. No human hybridoma synthesizing antibody against 105 kDa platelet protein has been reported to date. These antibodies may play a role in the pathogenesis of thrombocytopenia in some ITP patients.     

Detection of platelet associated IgG in immune thrombocytopenia: A new assay employing protein a and peroxidase anti-peroxidase (PROA-PAP)

Immune thrombocytopenia is frequently encountered in medical practice and is generally accepted as being caused by an IgG antibody. The capability of detecting platelet-bound IgG as a diagnostic and therapeutic modality is critical for appropriate care and management of patients with idiopathic thrombocytopenic purpura (ITP), as well as other immune thrombocytopenias We have modified our previous assay (Br J Haematol 37:265, 1977) by employing protein A and PAP as a labeled antibody. Surface bound platelet IgG was quantitated by phase contrast microscopy after incubation with PAP, graded per 100 platelets and expressed as a reactive index (RI). Controls (n=13) had RIs ranging from 0.49 to 0.72 (mean 0.63 ± 0.02 SE). The nonimmune thrombocytopenic group (n=7) had an RI ranging from 0.58 to 0.72 (mean 0.64 ± 0.01 SE). In contrast, the immune thrombocytopenic group (n=28) had RIs ranging from 1.04 to 1.75 (mean 1.43 ± 0.03 SE). Platelet-associated IgG was evaluated further by absorbing representative sera samples from each group against washed granulocytes, red cells and platelets Only when sera from the immune thrombocytopenic group were absorbed against platelets did the reactive indices of pre- and postabsorption samples change significantly. These findings suggest that our assay is clinically applicable in detecting platelet-associated IgG in immune thrombocytopenia and has the advantage of being rapid, reproducible and easy to perform in a clinical laboratory.     

Novel approaches to refractory immune thrombocytopenic purpura

Chronic immune thrombocytopenic purpura (ITP) is an organ-specific autoimmune bleeding disorder in which autoantibodies are directed against the individual's own platelets, resulting in increased Fc-mediated platelet destruction by macrophages in the reticuloendothelial system. Although ITP is primarily mediated by IgG autoantibodies, the production of these autoantibodies is regulated by the influence of T lymphocytes and antigen-presenting cells (APC). There is evidence that enhanced T-helper cell/APC interactions in patients with ITP may play an integral role in IgG antiplatelet autoantibody production. New therapies may improve platelet production, decrease platelet antibody production, and decrease monocyte function and/or B-cell and T-cell activities. Understanding these cellular immune responses in ITP may lead to the development of more specific immunoregulatory therapies for the management of this disease.     

Cell Electrophoresis for the Detection of Platelet Antibodies

Abstract. Cell electrophoresis was studied as a method for the detection of antibodies against platelets. Use was made of a Zeiss cytopherometer. After replacement of the original copper-copper sulfate electrodes by newly designed platinum electrodes, a good reproducibility of the method was obtained.
Antiplatelet heteroantibodies and usually also antiplatelet isoantibodies were found to reduce the electrophoretic mobility of platelets. The influence of drug-induced antiplatelet antibodies on the electrophoretic mobility appeared to be dependent on the presence of complement in the incubation mixture, as was the case with some anti-red cell antibodies.
From studies with sera from patients recovered or suffering from idiopathic thrombocytopenic purpura and with platelets from patients with thrombocytopenia it was concluded that cell electrophoresis is unsuitable for the demonstration of the thrombocytopenic factor.