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Editorials
Is the mysterious platelet receptor GPV an unsuspected major target for platelet autoantibodies?
Paquita Nurden and Alan T Nurden
Institut Hospitalo-Universitaire LIRYC, Pessac, France E-mail: PAQUITA NURDEN - paquita.nurden@gmail.com
doi:10.3324/haematol.2018.214908
To confirm an origin of immune thrombocytopenia (ITP), serum autoantibodies are routinely tested for by enzyme-linked immunosorbent assay (ELISA), or monoclonal antibody immobilization of platelet anti- gens (MAIPA), with the integrin alpha IIb-beta 3 (αIIbb3) and glycoprotein (GP)Ib-IX receptors on platelets as major targets.1 A range of commercially available kits also allows the detection of human antibodies to these and other platelet targets. In spite of this, confirmation of an immune process for an acquired thrombocytopenia is often difficult to establish, as many potential antigenic targets occur on less well-characterized or minor surface components of platelets that are not tested for. Over the past 20 years, familial thrombocytopenia, often linked with increased platelet size, has no longer been systemat- ically considered as ITP.2 The recognition of a possible genetic origin, even in the absence of a family history, has been facilitated by the tremendous progress linked to the development of next generation sequencing procedures.3 This revolution has reinforced the need for more positive criteria to diagnose ITP and to be able to elaborate the best strategy for their treatment.
In this issue of Haematologica, Vollenberg et al.4 identify GPV, a little understood constituent of the GPIb-IX-V complex, as a frequent target for autoantibodies in ITP. Firstly, they developed methods to improve the detection of platelet-bound and free autoantibodies, and also to evaluate their pathological consequences, using a phago- cytosis assay or a NOD/SCID mouse model. In particular, they applied surface plasmon resonance (SPR) technology using recombinant His-tagged GPV. With SPR, they were able to detect lower avidity autoantibodies in the sera of patients compared to indirect MAIPA. Using a direct MAIPA, they found platelet-bound anti-GPV autoanti- bodies either alone (2.9%) or more often associated with antibodies to GPIb-IX and/or to αIIbb3 in 61.8% of 343 positive samples in a series of 1140 ITP patients. Free anti- bodies to GPV in the sera were found in 66.6% of the 45 patients found positive by indirect MAIPA, which is more frequent than those found for αIIbb3. When sera from 222 patients positive for platelet-bound GPV were tested using SPR, 88 (39.6%) now tested positively, showing a higher specificity due to the ability of the SPR approach to detect lower avidity antibodies. It should be noted, however, that low avidity antibodies to αIIbb3 and GPIb- IX were not evaluated in serum. Blocking with recombi- nant GPV confirmed the specificity for serum autoanti- bodies to GPV in MAIPA.
Vollenberg et al.4 clearly demonstrated the role of anti- GPV autoantibodies in mediating platelet clearance. First, they showed that both high- and low-avidity anti-GPV were able to mediate platelet uptake in a phagocytosis
assay using human macrophages. They then performed an in vivo experiment using a NOD/SCID mouse model with transfused human platelets. Their model was vali- dated using a murine monoclonal antibody (MoAb) SW16 against human GPV injected at two concentrations that induced comparable clearance to a murine MoAb (SZ21) specific for αIIbb3. Next, the IgG fraction of sera from patients containing exclusively anti-GPV autoanti- bodies of low- or high-avidity were administered. Interestingly, the human anti-GPV increased platelet clearance, although to a lower extent than was seen with SW16; results were similar for the high- or low-avidity IgG fractions and platelet survival was increased after adsorption of the sera with recombinant GPV. It should be noted, however, that the extent to which platelet GPV was saturated was not evaluated. In conclusion, as for classical autoantibodies in ITP, anti-GPV are able to cause thrombocytopenia.
The mechanism by which platelets are cleared for patients with anti-GPV antibodies is worthy of further investigation. Early biosynthesis and assembly of the GPIb-IX complex includes N-glycosylation of the individ- ual subunits and extensive O-glycosylation of the extra- cellular mucin-like domain of GPIbα.5 GPV (approx. 85 kDa) is non-covalently associated by way of transmem- brane interactions with GPIbα and its association with GPIb-IX is required for full expression at the platelet sur- face.6 Classically, the GPIb-IX-V complex is lacking or dysfunctional in patients with inherited biallelic Bernard- Soulier syndrome (BSS) where bleeding occurs through the adhesion defect caused by the absence of GPIbα, the platelet receptor for von Willebrand factor (VWF).7 It has 50% expression in monoallelic forms of BSS linked to macrothrombocytopenia alone.2,7 In contrast to the other subunits, the specific absence of GPV in mice (GPV-/-) did not result in BSS, platelet and MK morphology were nor- mal, and there was abundant GPIb-IX expression.8 Classically, mutations in GPV do not give rise to human BSS.7
In ITP, pathways to remove platelets from the circula- tion include Fc-receptor-mediated clearance by mononu- clear macrophages primarily, although not exclusively, located in the spleen.9 Vollenberg et al.4 clearly show that antibodies to GPV can bring about phagocytosis by macrophages. But are there other possible mechanisms? Platelet activation in platelets incubated with monoclonal antibodies (MoAbs) to GPIb (but not with antibodies to αIIbb3) or in ITP patients with anti-GPIb autoantibodies can be followed by neuraminidase translocation to the platelet surface.10 Removal of terminal sialic acid residues from the O-linked oligosaccharides of the mucin-like domain of GPIbα results in severe thrombocytopenia due
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