Recombinant Mouse Anti-GPIIb Antibody (314.1) (CAT#: PABX-066)

Figure 1 Monoclonals 314.1 and 314.3 reacted with normal human platelets.

Histograms show reaction in the presence (solid histograms) but not the absence (dashed histograms) of soluble quinine. Mean fluorescent intensity (MFI) values for each histogram are shown.

Bougie, D. W., Birenbaum, J., Rasmussen, M., Poncz, M., & Aster, R. H. (2009). Quinine-dependent, platelet-reactive monoclonals mimic antibodies found in patients with quinine-induced immune thrombocytopenia. Blood, The Journal of the American Society of Hematology, 113(5), 1105-1111.

Figure 2 Drug-dependent reactions of monoclonal 314.1 with intact platelets were similar to those of 2 human quinine-dependent antibodies.

All studies were done with flow cytometry. (A) Reactions of mAb 314.1 (0.2, 1, and 5 μg/mL) at various concentrations of quinine. (B) Reactions of 2 human quinine-dependent antibodies at various concentrations of quinine. (C,D) Reactions of mAb 314.1 (1.0 μg/mL; C) and 2 human quinine-dependent antibodies (D) at various concentrations of quinine and quinine congeners. Drug concentrations are shown on the abscissa. Reactions of mAb 314.3 (not shown) were essentially identical to those of mAb 314.1.

Bougie, D. W., Birenbaum, J., Rasmussen, M., Poncz, M., & Aster, R. H. (2009). Quinine-dependent, platelet-reactive monoclonals mimic antibodies found in patients with quinine-induced immune thrombocytopenia. Blood, The Journal of the American Society of Hematology, 113(5), 1105-1111.

Figure 3 Monoclonals 314.1 and 314.3 reacted with isolated GPIIb/IIIa in the presence, but not in the absence of quinine.

Platelet GPIIb/IIIa was captured from a platelet lysate by monoclonal AP2 (anti-GPIIb/IIIa complex) immobilized in microtiter wells. Biotinylated mAbs 314.1 and 314.3 were incubated with this target in the presence (■) and absence (□) of 0.4 mM quinine. Bound mAb was detected with alkaline-phosphatase-labeled streptavidin. Brackets show the mean plus or minus 1 standard deviation of triplicate determinations.

Bougie, D. W., Birenbaum, J., Rasmussen, M., Poncz, M., & Aster, R. H. (2009). Quinine-dependent, platelet-reactive monoclonals mimic antibodies found in patients with quinine-induced immune thrombocytopenia. Blood, The Journal of the American Society of Hematology, 113(5), 1105-1111.

Figure 4 Reactions of mAb 314.1 with CHO cells expressing mixed (human/rat) GPIIb/IIIa integrins.

In the presence of quinine (solid histograms) mAb 314.1 reacted with cells expressing human GPIIb paired with either human (H/H) or rat (H/R) GPIIIa but failed to react with cells expressing rat GPIIb paired with either human (R/H) or rat (R/R) GPIIIa. No reactions were obtained in the absence of quinine (dashed histograms).

Bougie, D. W., Birenbaum, J., Rasmussen, M., Poncz, M., & Aster, R. H. (2009). Quinine-dependent, platelet-reactive monoclonals mimic antibodies found in patients with quinine-induced immune thrombocytopenia. Blood, The Journal of the American Society of Hematology, 113(5), 1105-1111.

Figure 5 Quinine-dependent reactions of mAb 314.1 with CHO cells expressing human/rat chimeric or wild-type human GPIIb paired with human GPIIIa.

(A) mAb 314.1 reacted with wild-type GPIIb (H, solid histogram) and with a GPIIb containing human sequence in the N-terminal β propeller domain (H1-452R, dotted histogram) but not with the inverse construct containing rat sequence in the β propeller domain (R1-452H, dashed histogram). (B) mAb 314.1 binding was lost when the first 2 (R1-144H, dotted histogram) or 3 (R1-246H, dashed histogram) N-terminal repeats (blades) of the human β propeller domain were replaced by a rat sequence. All reactions contained quinine 0.4 mM. There were no significant reactions in the absence of quinine (not shown). (C,D) Reactions of mAb AP2 (specific for the GPIIb/IIIa complex) with the same CHO cell lines showed that all constructs were satisfactorily expressed.

Bougie, D. W., Birenbaum, J., Rasmussen, M., Poncz, M., & Aster, R. H. (2009). Quinine-dependent, platelet-reactive monoclonals mimic antibodies found in patients with quinine-induced immune thrombocytopenia. Blood, The Journal of the American Society of Hematology, 113(5), 1105-1111.

Figure 6 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).

Bougie, D. W., Nayak, D., Boylan, B., Newman, P. J., & Aster, R. H. (2010). Drug-dependent clearance of human platelets in the NOD/scid mouse by antibodies from patients with drug-induced immune thrombocytopenia. Blood, The Journal of the American Society of Hematology, 116(16), 3033-3038.