OC 39.1 - Anti-integrin immune thrombocytopenia autoantibodies impair megakaryocyte migration, polarization and proplatelet formation.

Background: Immune thrombocytopenia (ITP) is caused by antiplatelet autoantibodies directed against platelet surface glycoproteins (prevalently αIIbβ3, α2β1, GPIb/IX/V) leading to increased platelet clearance. Some data however show that they also bind surface glycoproteins on megakaryocytes (MKs) possibly impairing platelet production. Defective MK adhesion, spreading and proplatelet formation (PPF) induced by anti-αIIbβ3 and anti-αIIbβ3+anti-α2β1 autoantibodies were previously reported, but no studies have explored the effect of anti-integrin autoantibodies on MK migration and polarization.

Aims: To evaluate the effects of anti-α2β1 and anti-αIIbβ3 autoantibodies from ITP patients on MK migration, polarization and PPF.

Methods: Anti-α2β1 or anti-αIIbβ3 autoantibody-positive sera (MAIPA) from ITP patients (8 each), and control sera from healthy subjects (10) were used. Human MKs were differentiated from peripheral blood-derived CD34+ cells from healthy subjects. MK migration towards SDF-1α was assessed by a transwell system. MK polarization on fibrinogen was assessed by fluorescence microscopy upon staining with anti-CD42b MoAb and Hoechst for nuclei. PPF on fibrinogen was evaluated by fluorescence microscopy upon staining with anti-β1-tubulin antibody for microtubules, rhodamine-phalloidin for actin and Hoechst for nuclei. All data are reported as fold change±SEM versus control sera.

Results: MK migration was significantly reduced upon coincubation with anti-α2β1 and anti-αIIbβ3 ITP sera compared to control sera (α2β1=0.48±0.06, αIIbβ3=0.76±0.03; p< 0.0001). MKs polarization was significantly reduced upon coincubation with anti-α2β1 and anti-αIIbβ3 ITP sera compared with control sera (α2β1=0.42±0.06, αIIbβ3=0.43±0.04; p< 0.0005). Moreover, anti-α2β1 and -αIIbβ3 autoantibodies significantly reduced PPF on fibrinogen compared to control sera (α2β1=0.40±0.08, αIIbβ3=0.66±0.04; p< 0.0005).

Conclusion(s): Anti-α2β1 and -αIIbβ3 autoantibodies prevent MK polarization and migration and impair PPF formation by MKs, crucial steps for the release of platelets in the blood stream. Our results therefore suggest that ectopic platelet release in the bone marrow, and thus ineffective thrombopoiesis, may contribute to thrombocytopenia in a subgroup of ITP patients, besides increased peripheral destruction.