Glycoprotein V is a relevant immune target in patients with immune thrombocytopenia

Abstract

Platelet autoantibody-induced platelet clearance represents a major pathomechanism in immune thrombocytopenia (ITP). There is growing evidence for clinical differences between anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib/IX mediated ITP. Glycoprotein V is a well characterized target antigen in Varicella-associated and drug-induced thrombocytopenia. We conducted a systematic study assessing the prevalence and functional capacity of autoantibodies against glycoprotein V. A total of 1140 patients were included. In one-third of patients, platelet-bound autoantibodies against glycoproteins Ib/IX, IIb/IIIa, or V were detected in a monoclonal antibody immobilization of platelet antigen assay; platelet-bound autoantiglycoprotein V was present in the majority of samples (222 out of 343, 64.7%). Investigation of patient sera revealed the presence of free autoantibodies against glycoprotein V in 13.5% of these patients by an indirect monoclonal antibody immobilization of platelet antigen assay, but in 39.6% by surface plasmon resonance technology. These antibodies showed significantly lower avidity (association/dissociation ratio 0.32±0.13 vs 0.73±0.14; P<0.001). High- and low-avidity antibodies induced comparable amounts of platelet uptake in a phagocytosis assay using CD14⁺ positively-selected human macrophages [mean phagocytic index, 6.81 (range, 4.75-9.86) vs 6.01 (range, 5.00-6.98); P=0.954]. In a NOD/SCID mouse model, IgG prepared from both types of anti-glycoprotein V autoantibodies eliminated human platelets with no detectable difference between the groups from the murine circulation [mean platelet survival at 300 minutes, 40% (range, 27-55) vs 35% (16-46); P=0.025]. Our data establish glycoprotein V as a relevant immune target in immune thrombocytopenia. We would suggest that further studies including glycoprotein V will be required before ITP treatment can be tailored according to platelet autoantibody specificity.

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 immobilized onto HTE sensor chips. Representative curves for the interaction of monoclonal antibodies with the respective proteins are shown. (B) Representative response curves from n=6 different IgG fractions obtained from immune thrombocytopenia (ITP) patients. (Top) Reactivity of IgG fractions from n=3 sera from patients with free autoabs that were also detectable by standard serology (monoclonal 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 different behavior of antibodies with regard to association and dissociation characteristics. (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.

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 determined 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.

Figure 3.

Platelet elimination in a NOD/SCID mouse model. NOD/SCID mice (NOD.CB17-Prkdcscid/J) were injected with freshly isolated human platelets (200 μL, 2x10⁹/mL) into the lateral tail vein. After 30 minutes (min), blood was taken to determine the baseline of circulating human platelets (100%). Subsequently, antibodies were injected, and further blood samples were taken at 60, 120, 300 and 1440 min (24 h) after baseline, as indicated. (A) Platelet elimination in the presence of monoclonal antibodies. SZ21 against GP IIIa was used as positive control. Note that SW16 against GP V eliminated human platelets with the same kinetics as SZ21 when 10 μg were injected. Significant, but less pronounced platelet elimination was observed at a lower concentration. Data are given as Mean±Standard Deviation for three independent experiments. Murine IgG was used as negative control. (B) Platelet elimination in the presence of human anti-GP V autoantibodies. Group A: anti-GP V IgG detected by surface plasmon resonance (SPR) only (low avidity antibodies; n=3); group B: anti-GP V IgG detected by SPR and monoclonal antibody immobilization of platelet antigens (MAIPA) (high avidity antibodies, n=3). Compared to human control IgG, IgG obtained from immune thrombocytopenia (ITP) sera in both groups induced significant platelet removal. No difference in platelet elimination was observed between group A and group B. As to be expected, autoantibodies were less effective in removing platelets from the murine circulation than a human control alloanti-body [anti-HPA-1a present in the World Health Organization (WHO) standard]. (C) Human ITP serum containing anti-GP V autoantibodies only was absorbed (dashed line) or not absorbed (full line) with recombinant GP V prior to IgG isolation. Platelet elimination was studied as outlined above. Data are given as Mean±Standard Deviation for three independent experiments.