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R. Vollenberg et al.
A
B
C
0.6 0.4 0.2 0.0
1.0
P<0.001 0.8
Figure 1. Detection of anti-GPV autoantibodies (autoabs) by surface plasmon resonance (SPR). (A) SPR analysis was performed on a protein interaction array system. Recombinant histidine (His)-tagged GP V and GP IV (CD36) were immo- bilized onto HTE sensor chips. Representative curves for the interaction of mon- oclonal antibodies with the respective proteins are shown. (B) Representative response curves from n=6 different IgG fractions obtained from immune throm- bocytopenia (ITP) patients. (Top) Reactivity of IgG fractions from n=3 sera from patients with free autoabs that were also detectable by standard serology (mon- oclonal antibody immobilization of platelet antigens, MAIPA). (Bottom) Reactivity of IgG fractions from n=3 sera without detectable anti-GP V by standard serology (MAIPA). Note the difference in the maximum response units (y-axis) and the dif- ferent behavior of antibodies with regard to association and dissociation char- acteristics. (C) Comparison of the avidity of MAIPA positive (n=29) versus MAIPA negative (n=59) ITP sera detected by SPR in a box-and-whisker plot with median, interquartile range, and highest/lowest value per group. Avidity was calculated as the R700/R350 rate, where R350 indicates the maximum anti-GP V antibody binding after 350 seconds (s) of association, and R700 indicates the remaining antibody binding after additional 350 s of dissociation.
removing platelets from the murine circulation than human alloantibodies (anti-HPA-1a as present in the WHO standard). These data demonstrate that anti-GP V antibodies of high or low avidity are capable of removing circulating platelets and thus represent a functionally rele- vant specificity of autoantibodies in ITP. To further sub- stantiate the hypothesis that the observed effects are mediated by anti-GP V IgG, we directly compared the median human platelet survival after injection of IgG frac- tions prepared from one ITP serum containing anti-GP V autoantibodies only (Figure 3C), either after the absorp- tion with recombinant glycoprotein V (rGPV; dashed line) or without (full line). The median platelet survival at t=1440 min after absorption was 48.5% (range, 44-53%) versus 18% (range, 11-20%) without absorption (P=0.028). This experiment supports our conclusion that anti-GP V IgG is capable of removing human platelets from the murine circulation.
Discussion
In this study, we demonstrate that GP V is a frequent immune target in ITP patients. Anti-GP V autoantibodies are detectable with the same frequency as those against GP IIb/IIIa and GP Ib/IX. Anti-GP V autoantibodies have the ability to induce a modest level of phagocytosis and to eliminate human platelets in a murine model.
Figure 2. Phagocytosis of platelets by splenic macrophages. Healthy donor platelets were opsonized with human immune thrombocytopenia (ITP) sera containing low-avidity (group A, red bars) or high-avidity (group B, blue bars) anti-GP V autoantibodies from four different patients (n=4 each). Serum from one anti-GPIIb/IIIa-positive ITP patient was used as a positive control (n=3) and healthy donor sera [normal human sera (NHS), n=4] or phosphate buffered saline (PBS) [non-opsonized (non-ops), n=4] were used as negative controls. Human ITP splenic macrophages were isolated by CD14 positive selection from frozen adult ITP splenic single-cell suspensions and were incubated with opsonized human platelets for 40 minutes at 37°C. Phagocytosis was deter- mined by spinning disc confocal microscopy and outside (non-phagocytosed) platelets were distinguished using an anti-GPIX antibody stain following macrophage fixation. Phagocytic index was calculated as (engulfed platelets counted / splenic macrophages counted) x 100. Error bars=Standard Deviation. Statistical significance was calculated by one-way ANOVA against NHS unless specified. NS: not significant; *P<0.05; **P<0.01; ****P<0.0001.
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