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

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.

Figure 1

Quinine-dependent mAb 314.1 promotes platelet destruction in NOD/scid mice given quinine. Human platelets were infused into NOD/scid mice followed by intraperitoneal injection of 50 μg of mAb 314.1 or control monoclonal. One hour later (time 0) and at 5 hours, buffer or quinine (74 μg), was injected intraperitoneally Platelet survival was markedly shortened in mice given mAb 314.1 and quinine (triangles) but not in mice given the mAb alone (squares) or quinine plus an irrelevant mAb (circles). Values shown are the average ± 1 SEM of triplicate experiments. ***P < .001 relative to controls.

Figure 2

Human platelets survive “normally” when infused into NOD/scid mice as a suspension in normal citrated plasma or serum containing 0.02M sodium citrate. Values shown are the average of triplicate experiments ± 1 SEM. Survival values at 24 hours of platelets suspended in normal plasma or serum was 70% ± 15% and 84% ± 11% of the baseline (time 0) values, respectively.

Figure 3

Human antibodies specific for quinine and sulfamethoxazole promote platelet destruction in NOD/scid mice given these drugs. Human platelets suspended in plasma containing DDAbs or normal plasma were infused into NOD/scid mice followed by intraperitoneal injection of quinine (3.7 mg/kg; A) or sulfamethoxazole (50 mg/kg; B) at 0 and 5 hours. Platelet survival was markedly shortened in mice given DDAb and the appropriate drug (triangles), but not in mice given DDAb alone (squares) or drug plus normal plasma (circles). Values shown are the average of triplicate experiments ± 1 SEM. ***P < .001 relative to controls.

Figure 4

Binding of quinine- and sulfamethoxazole-specific DDABs to platelets is enhanced by using drug at supratherapeutic concentrations. Reactions of quinine-specific (A) and sulfamethoxazole-specific (B) DDAbs used at dilutions of 1:5 and higher to normal platelets were enhanced by using the drugs at supratherapeutic concentrations. Binding of patient Ig to platelets was measured by flow cytometry. MFI indicates median platelet fluorescence; values on the abscissa indicate final concentration of drug in the reaction mixture. Vertical lines denote expected mean peak levels achieved after administration of a conventional dose of quinine (10μM) and sulfamethoxazole (80μM).^,

Figure 5

Drug-dependent clearance of human platelets in NOD/scid mice is enhanced by supra-therapeutic concentrations of drug. Human platelets suspended in plasma containing DDAbs at the indicated dilutions were infused into NOD/scid mice followed by intraperitoneal injection of quinine (50 mg/kg; A) or sulfamethoxazole (250 mg/kg; B) at 0 and 5 hours. Platelet survival was markedly shortened in mice given DDAbs diluted 1:15 (squares) and 1:45 (triangles) followed by injection of the appropriate drug. Values shown are the average of triplicate experiments ± 1 SEM. **P < .01, ***P < .001 relative to controls.

Figure 6

Drug metabolites produced in vivo promote accelerated clearance of platelets by metabolite-specific DDAbs. Human platelets suspended in serum containing DDAbs specific for acetaminophen glucuronide (A) or naproxen glucuronide (B) were infused into NOD/scid mice. Acetaminophen (50 mg/kg) or naproxen (75 mg/kg) were injected intraperitoneally at 0 and 5 hours. Platelet survival was significantly shortened in mice given DDAb and the unmodified primary drug (triangles) but not in mice given DDAb (squares) or drug with normal serum (circles). Values shown are the average of triplicate experiments ± 1 SEM. *P < .05, **P < .01, ***P < .001 relative to controls.