S848 allelic polymorphism of platelet glycoprotein genes gpia and gpiib as a possible predictive marker of response to therapy in patients with primary immune thrombocytopenia

Background: Inherited mutations in the transcription factors (TF) Runt-related transcription factor-1 (RUNX1), Growth factor independence 1B (GFI1B) and GATA Binding Protein1 (GATA1) cause megakaryocyte and platelet dysfunction resulting in bleeding disorders. Several platelet abnormalities overlap (e.g. platelet α-granule paucity) suggesting commonly affected pathways. Aims: The functional effects of many pathogenic mutations remain largely unknown. To further the understanding of the pathobiology of TF mutated bleeding disorders, we identified the affected pathways and proteins important during megakaryopoiesis. Methods: We identified variants in RUNX1 (TD2-6, Q154fs, G165S), GFI1B (Q287∗, T174N) and GATA1 (R216Q) by next generation sequencing of DNA from cases with thrombocytopenia/pathy and bleedings. According to AMCG guidelines all but the RUNX1 G165S and GFI1B T174N mutations were classified as pathogenic. To identify (commonly) affected biological pathways downstream of mutated TFs, we analyzed platelets from these cases and five controls with label free quantification (LFQ) mass spectrometry. Results: Proteomes from controls were highly similar and clearly distinct from all cases with bleedings. Besides, the non-pathogenic variants in RUNX1 (G165S) and GFI1B (T174N) clustered together but separate from controls and cases with pathogenic mutations. Pathogenic RUNX1, GFI1B and GATA1 proteomes were also clearly distinct from each other (P < 0.001, between 220 and 520 proteins significantly differentially expressed). For cases with pathogenic TF mutations, gene ontology (GO) pathway analysis detected significantly downregulated platelet a-granule, - degranulation and - activation pathways. Platelet α-granule proteins such as platelet factor 4V1, SERPINE2 and F5, but also the structural MYL9 protein were all significantly downregulated. In addition, all pathogenic TF patient samples had negative enrichment for coagulation proteins, compared to control samples. Remarkably, in all TF affected platelets we observed a negative enrichment for JAK-STAT signaling (AKT1, JAK2, JAK3), normally required for proper megakaryocyte differentiation. To address this finding in more detail, we determined the proteomes of normal megakaryocytes at different stages of differentiation derived from primary immature CD34 positive cells. TF mutated platelet proteomes clearly resembled immature rather than mature megakaryocytes, in line with defective megakaryocyte maturation. For example, in affected platelets an increase in oxidative phosphorylation proteins (ATP6V1A, NDUFS3, NDUFA2) was observed, that are downregulated upon normal megakaryocyte maturation. Many other affected pathways, including the ubiquitin-proteasome pathway were deregulated in affected platelets. Summary/Conclusion: Pathogenic TF mutations have dominant distinctive effects on platelet proteome composition. Platelets from cases without a proven TF mutation have similar affected proteomes distinct from controls and cases with pathogenic TF mutations. Thus, the studied TF mutations have a dramatic effect on the platelet proteome, in line with the plethora of abnormalities in megakaryocytes and platelets. Identifying which pathways and proteins act during megakaryopoiesis and which are affected by TF mutations allows identification of biological pathways previously unknown to be fundamental to platelet development.