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Glycoprotein V in ITP
Autoantibodies against platelets are considered to be the major factors for platelet clearance in ITP.25 GP IIb/IIIa and GP Ibα are generally reported as the most common anti- genic targets.26,27 We were able to analyze the amount of platelet-bound, glycoprotein-specific autoantibodies in 343 patients in parallel. Whereas anti-GP V antibodies as the sole autoantibody entity were less often detected (2.9%) than the other specificities (20.7% and 8.9%, respectively), two-thirds of all ITP patients reacted with GP V in combination with other specificities. Anti-GP V was more often seen in association with anti-GP Ib/IX (61 out of 91, 67%) than with anti-GP IIb/IIIa (10 out of 8, 12%), but all entities were clearly separable.
In contrast to platelet-bound autoantibodies, free autoantibodies in patient plasma are only rarely detectable.28 Still, in our cohort, free anti-GP V was not less often detected than the other specificities, again, most fre- quently in association with other autoantibodies. Adding anti-GP V detection to the standard laboratory test would only mildly increase the overall test sensitivity (from 29.2% to 30.1% for platelet-bound glycoprotein specific autoantibodies and from 3.4% to 3.9% for free autoanti- bodies). In contrast to conventional testing by MAIPA, SPR technology significantly raised the test sensitivity, with no loss of specificity. Interestingly, we observed a clear difference in autoantibody avidity between those autoantibodies detected by standard serology plus SPR
and those detected by SPR only. To our knowledge, autoantibodies against platelets have not been investigat- ed for their avidity before. However, we previously demonstrated that anti-HPA-1a alloantibodies against platelets may be of low avidity and escape detection by MAIPA.23 These antibodies had a comparable profile to the SPR-only autoantibodies detected in this study: a slow binding during the association and fast detachment during the dissociation phase. This suggests that these antibodies may become washed away in conventional test methods, whereas no-wash detection by SPR increases sensitivity. Low-avidity anti-GP V autoantibodies were able to induce platelet destruction in vitro and in vivo. This finding indi- cates that these antibodies, which are not detectable using conventional methods, are of clinical relevance. This observation demonstrates that low sensitivity could, in fact, be an important drawback of autoantibody testing in the laboratory. Further development of methods might be useful to increase the clinical utility of platelet autoanti- body testing.29
Anti-GP V autoantibodies were efficient in removing platelets, regardless of their avidity; indicating that platelets loaded with anti-GP V undergo the same fate as platelets loaded with other autoantibodies.23 The presence of anti-GP V might affect the clinical picture of ITP patients in two ways: 1) by a more efficient platelet removal because of an increased overall IgG load; or 2) by
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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 circulat- ing human platelets (100%). Subsequently, antibodies were injected, and further blood sam- ples 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 pos- itive 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 inde- pendent 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 plas- mon 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 anti- bodies, 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 elimi- nation 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 out- lined above. Data are given as Mean±Standard Deviation for three independent experiments.
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