Mechanism Of Immune Thrombocytopenia Research Articles

Association between megakaryocyte abnormalities on bone marrow smear and response to thrombopoietin receptor agonists in adult patients with primary immune thrombocytopenia

Impaired platelet production is a mechanism of immune thrombocytopenia (ITP). Morphological abnormalities of megakaryocytes (MKs) are sometimes observed in this disease. Two studies have suggested an association between MK abnormalities and response to corticosteroids in primary ITP, but none have investigated this association for thrombopoietin-receptor agonists (TPO-RAs). This was the aim of this study. The source of population was the French CARMEN registry with prospective follow-up of adult patients with incident ITP. We included patients with primary ITP, treated by TPO-RA and with a bone marrow smear before initiating TPO-RA. MK abnormalities were categorized by the presence of dysplasia and by the stage of maturation. Among 451 patients screened, 38 were included in the analysis. There was no difference in the median percentage of dysplastic MKs between responders to TPO-RA (4.0%, 95% confidence interval – CI: 2.3–6.4) and non-responders (4.5%, 95% CI: 0.7–7.1). There was a slightly higher proportion of granular MKs (4.5%, 95% CI: 3–6) and basophilic MKs (30.1%, 95% CI: 21.9–39.1) in non-responders compared to responders (granular: 2.0%, 95% CI: 0–4.1; basophilic: 21.3%, 95% CI: 11.4–40.7). In conclusion, MK abnormalities were not associated with response achievement in ITP patients treated with TPO-RA in this series of 38 patients.

ATRA Could Correct the Defective S1P-Mediated Cytoskeletal Reorganization in Proplatelet Formation of ITP

Introduction Sphingosine-1-phosphate (S1P) is now emerging as a vital lipid mediator. Activation of sphingosine kinase (SphK) produces intracellular S1P, which in turn can be secreted out of the cell and act extracellularly by binding to S1P receptors (S1PR). Recent studies suggest that the "inside-out" signaling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). PPF requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases which can be activated by S1P, including Rac1 and Cdc42, have been shown to be master regulators of cytoskeletal rearrangements. The pathogenesis mechanisms of immune thrombocytopenia (ITP) are not entirely understood. Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that S1P-mediated microtubule reorganization is defective in PPF of ITP. All-trans retinoic acid (ATRA), which has demonstrated to be a promising option for ITP patients in our previous study (Lancet Haematology, 2017), could correct the altered microtubule reorganization and promote PPF. Methods Thirty consecutive patients with primary ITP and 20 healthy donors were enrolled in our study. MKs were isolated from bone marrow samples, and they were collected again after ITP patients received ATRA therapy. MK mRNA sequencing by microarray was used to assess the difference of gene expression between ITP and controls. Microtubule regrowth assay was performed to observe microtubule dynamic behavior. In this assay nocodazole was first used to induce complete depolymerization of microtubule network, followed by drug washout to allow microtubule regrowth over time. ATRA was added to the culture medium of MKs to determine the mechanism of ATRA in correcting impaired PPF. Additionally, ITP mice model was established to observe the therapeutic effects of ATRA in PPF. Pf4-Cre/loxP system was used to specifically knock down gene of MKs. Results S1P concentration in bone marrow from ITP patients was lower compared to healthy donors. MKs mRNA sequencing demonstrated that S1P synthetase SphK2 and S1P receptor S1PR1 gene were downregulated while S1P lyase (SPL) gene was upregulated in ITP patients, which caused abnormal S1P signaling. Furthermore, we observed that PPF capacity of MKs in patients with ITP was reduced. Pharmacological disruption of S1PR1 blocked PPF, exogenous S1P corrected impaired PPF. Collectively, deregulation of S1P signaling was associated with impaired PPF in ITP. To verify the downstream role of S1P in regulating PPF, the Rho GTPases detection of MKs revealed a decrease in Cdc42 and Rac1 levels from ITP patients. Immunofluorescence of the differentiated MKs showed that the expression and distribution of β1 tubulin were abnormal from ITP patients. Early PPs from MKs of healthy donors displayed a well-organized tubulin bundles resembling bunches of grapes. In contrast, in MKs from ITP patients, tubulin was disorganized in thick bundles. In addition, TEM analysis of the MKs showed an irregular distribution of granules, tortuous membranes and impaired proplatelet structure. In microtubule regrowth assay, MKs from ITP patients had significantly lower microtubule regrowth at 10 min post-nocodazole washout compared with controls. Together, microtubule alteration resulted in impaired PPF in ITP. We tested whether S1P pathway were required for microtubule reorganization, both SphK2-/- and S1PR1-/- mice displayed significantly reduced S1P, Cdc42 and Rac1, altered microtubule architecture and defective PPF. Taken together, abnormal S1P pathway accounted for impaired microtubule reorganization in ITP. Next, we explored the effect of ATRA on microtubules reorganization in ITP patients, our data showed that in vitro treatment with ATRA restored microtubules structure by upregulating S1P and activating Rho GTPases. In vivo studies showed that ARTA could rescue the impaired PPF in both patients and mice model with ITP. Conclusions The MKs of ITP patients displayed defective cytoskeletal reorganization regulated by S1P pathway. ATRA restored cytoskeletal structure and corrected impaired PPF by upregulating S1P and activating Rho GTPases. It sheds light on a novel mechanism of ITP pathogenesis and provides a basis for the therapeutic potential of ARTA in ITP patients. Disclosures No relevant conflicts of interest to declare.

Glycosylation of autoantibodies: Insights into the mechanisms of immune thrombocytopenia

Immune thrombocytopenia (ITP) is a bleeding disorder caused by IgG autoantibodies (AAbs) directed against platelets (PLTs). IgG effector functions depend on their Fc-constant region which undergoes posttranslational glycosylation. We investigated the role of Asn279-linked N-glycan of AAbs in vitro and in vivo. AAbs were purified from ITP patients (n=15) and N-glycans were enzymatically cleaved by endoglycosidase F. The effects of native AAbs and deglycosylated AAbs were compared in vitro on enhancement of phagocytosis of platelets by monocytes and complement fixation and activation applying flow cytometry, laser scanning microscopy, and a complement consumption assay. AAb-induced platelet phagocytosis was inhibited by N-glycan cleavage (median phagocytic activity: 8% vs 0.8%, p=0.004). Seven out of 15 native AAbs bound C1q and activated complement. N-glycan cleavage significantly reduced both effects. In vivo survival of human PLTs was assessed after co-transfusion with native or N-glycan cleaved AAbs in a NOD/SCID mouse model. Injection of AAbs resulted in rapid clearance of human platelets compared to control (platelet clearance after 5h (CL(5h))75% vs 30%, p<0.001). AAbs that were able to activate complement induced more pronounced platelet clearance in the presence of complement compared to the clearance in the absence of complement (CL(5h) 82% vs 62%, p=0.003). AAbs lost their ability to destroy platelets in vivo after deglycosylation (CL(5h) 42%, p<0.001). N-glycosylation of human ITP AAbs appears to be required for platelet phagocytosis and complement activation, reducing platelet survival in vivo. Posttranslational modification of AAbs may constitute an important determinant for the clinical manifestation of ITP.