Patients with quinine-induced immune thrombocytopenia have both "drug-dependent" and "drug-specific" antibodies

Abstract

Immune 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.

Figure 1.

Patient samples contain drug-dependent antibodies that immunoblot GPIIIa in the presence of quinine. Patients 1, 2, 3, 5, and 6 had IgG antibodies that recognized a protein corresponding to GPIIIa in the presence but not in the absence of 0.4 mM quinine (immunoblot against whole platelet lysate). GPIIIa is marked with monoclonal AP6 specific for amino acid residues 211-221. The prominent high-molecular-weight bands correspond to naturally occurring platelet IgG.

Figure 2.

Characterization of quinine-conjugated HSA. (Left) A quinine-specific sheep antibody reacts with Qn-HSA but not unmodified HSA in an immunoblot and is inhibited by soluble quinine 0.4 mM. (Right) Reactions of HSA and Qn-HSA with the HSA-specific monoclonal HSA-11.

Figure 3.

Patient samples contain drug-specific antibodies that recognize Qn-HSA. Patients 1, 2, 3, 5, 6, and 7 had antibodies that recognized Qn-HSA in an immunoblot and were inhibited by soluble quinine 0.4 mM. None of the antibodies recognized unmodified HSA (not shown).

Figure 4.

Demonstration of 2 distinct antibody populations in serum from patient 6. (Left) An eluate prepared from platelets following incubation with antibody reacted with platelets in the presence of drug. No detectable platelet-reactive antibody remained in the absorbed serum. (Right) The absorbed serum, but not the eluate, reacted with Qn-HSA. The eluted antibody failed to react with platelets in the absence of drug and the reaction of the absorbed serum with Qn-HSA was completely inhibited by soluble quinine (not shown).

Figure 5.

Inhibition of drug-specific antibody binding to Qn-HSA by quinine analogues. Reactions of serum from patient 6 with Qn-HSA were inhibited by quinine and cinchonidine (desmethoxy-quinine) but not by quinidine, quinoline, or quinuclidine.

Figure 6.

Structural models of quinine and quinidine. The ovals delineate the carbon bridge linking the quinoline and quinuclidine components of the molecules. Stereoisometry at the C8 and C9 positions causes the 2 molecules to assume quite different conformations in 3 dimensions.

Figure 7.

A proposed model for DDAb binding to an epitope on a platelet glycoprotein. (Left) Antibodies capable of causing drug-dependent thrombocytopenia react weakly with an epitope on a glycoprotein. The K_a for this interaction is too small to allow significant numbers of antibody molecules to bind in the absence of drug. (Right) Drug contains structural elements that are complementary to charged or hydrophobic domains (H) on the GP epitope and the complementarity-determining region (CDR) of the antibody. Drug interacts with the target protein and antibody to improve the “fit” between the 2 proteins, increasing the K_a to a value that permits binding to occur at levels of antibody, antigen, and drug achieved in the circulation after ingestion of the drug.