Dual Role for CIB1 in Thrombopoiesis: CIB1 Suppresses Megakaryocyte Quantities but Supports Adhesion, Migration, and Proplatelet Formation

Abstract 2384

Megakaryocytes (MKs) are large polyploid cells that produce platelets through a process known as thrombopoiesis. Thrombopoietin (Tpo) is the major cytokine that regulates a variety of steps in this process, including hematopoietic stem cell (HSC) differentiation to MKs, proplatelet formation, and platelet release into the circulation. However, the molecular mechanism of thrombopoiesis is poorly understood. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in thrombopoiesis, we utilized a Cib1^−/− mouse model. We observed that Cib1^−/− mice have a slightly elevated number of platelets and bone marrow (BM)-derived MKs than wild-type (WT) controls (p<0.05). Rate of platelet clearance was comparable in Cib1^−/− and WT mice, suggesting that the defective clearance is not the cause of the observed elevated platelet number. In order to determine if the HSC differentiation is dysregulated by the ablation of Cib1, we analyzed MK-colony forming unit production, which revealed an increase in the colony forming cells with Cib1 deletion compared to WT (p<0.05). Additionally, BM from Cib1^−/− mice, cultured with Tpo for 24 hours, produced more highly polyploid MKs than WT BM (p<0.05). These results suggest that Cib1 may negatively regulate initial steps of megakaryopoiesis. Subsequent analysis of Tpo signaling revealed that activation of FAK, a known suppresser of Tpo signaling, is attenuated, as indicated by reduced FAK^Y925 phosphorylation in Cib1^−/− BM-derived MKs treated with Tpo. Consequently, Akt and ERK1/2 activation downstream of Tpo was enhanced. These results suggested that Cib1 inhibits Tpo signaling by augmenting FAK activation. Interestingly, platelet recovery in Cib1^−/− mice following platelet depletion by experimental immunothrombocytopenia was attenuated compared to WT (p<0.05). This could be due to impaired adhesion and migration of MKs on the extracellular matrix. Consistent with this notion, adhesion to fibrinogen and fibronectin and migration towards an SDF-1α gradient were significantly reduced in Cib1^−/− MKs compared to WT (p<0.05). Additionally, Cib1^−/− MKs formed fewer proplatelets compared to WT (p<0.05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in thrombopoiesis, initially by negatively regulating Tpo signaling, and later by supporting MK migration and proplatelet production.