Loss of Autophagy Leads to Megakaryocytes Differentiation Failure and Defective Platelets Function

Background

Megakaryocytes (MKs), large progenitor cells residing in the bone marrow, are the source of platelets. In 2009, Colosetti P et al. reported that PMA and SB could induce megakaryocytic differentiation of the chronic myelogenous leukemia cell line K562 by triggering autophagy. It gives us the first insight of autophagy induction in in vitro megakaryocytic differentiation. Although Feng W et al. reported autophagy also exists in human platelets, the role of autophagy in megakaryocyte-platelet commitment axis remains poorly understood. In this study, we elucidated the biological effects of autophagy deficiency on megakaryopoiesis and thrombosis using hematopoietic system conditionally atg7 knockout mice.

Methods and Materials

To evaluate the biological effects of autophagy deficiency on platelets, the following experiments were performed: (1) complete blood count of wild type and atg7^-/- mice, (2) the tail bleeding time assay of wild type and Atg7^-/- mice, (3) the effect of atg7 knockout on platelet aggregation and activation of CD62P and JON/A (αIIbβ3) were analyzed by flow cytometry. To assess whether the observed changes in platelets of atg7^-/- mice result in aberrations of megakaryopoiesis, the following experiments were performed: (1) the percentage of BM CD41⁺CD61⁺ cells was analyzed by flow cytometry, (2) the AchE activity assay of platelets and murine BM Lin^- cells cultured with murine TPO and SCF, (3) Morphology of megakaryocytes derived from BM Lin^- cells was evaluated by Wright-Giemsa staining, (4) megakaryocytic differentiation from BM Lin^- cells was evaluated by CD41/forward-scatter (FSC) dot plot. To evaluate the role of reactive oxygen species in MK differentiation, the Lin^- cells were stained with MitoTracker Green and MitoSox Red and then analyzed by flow cytometry.

Results

(1) The number of platelets in the peripheral blood of atg7^-/- mice was significantly decreased (WT: 904.2±75.5, Atg7^+/-: 942.8±136.3, Atg7^-/-: 330.5±282.2, p<0.01), while the size of platelets (MPV) was increased compared with WT mice (WT: 5.6±0.1, Atg7^+/-: 5.7±0.1, atg7^-/-: 6.8±0.5, p<0.01). (2) The bleeding time was significantly longer (WT: 51.5±14.8s, Atg7^-/-: 915.2±282.9s, p<0.01) and thrombin-induced platelet aggregation was decreased (WT: 97.5±2.5, Atg7+/-: 62.5±7.5, Atg7-/-: 7.75±7.25, p<0.05) in Atg7-/- mice than in wild-type mice. (3) The activation of CD62P (WT: 14.5±0.09, atg7^+/-: 11.17±0.06, atg7^-/-: 3.2±0.03, p<0.01) and JON/A (αIIbβ3) (WT: 48.1±0.1, atg7^+/-: 13.5±0.1, atg7^-/-: 5.9±0.2, p<0.01) was decreased in atg7^-/- platelets. These results indicated that atg7-dependent autophagy is important for thrombosis and platelet function. (4) The percentage of CD41⁺CD61⁺ cells was decreased in bone marrow of atg7^-/- mice (WT: 30.4±0.6, atg7^+/-: 27.9±1.3, atg7^-/-: 18.9±0.3, p<0.01). (5) In mice lacking autophagy, both the Lin^- cells stimulated by TPO (WT: 0.35±0.03, atg7^-/-: 0.22±0.05, p<0.01) and the platelets collected through the inferior vena cava (WT: 0.099±0.005, atg7^-/-: 0.05±0.009, p<0.01) had significantly lower AChE activity compared with WT mice. (6) Low level of CD41⁺/FSC^high cells were seen in the in vitro culture of atg7^-/- BM Lin- cells with TPO and SCF ( WT:5.6±2.2, Atg7^-/-: 0.2±0.03, p<0.01). These results reflected a significant reduction in MK differentiation from autophagy defective hematopoietic progenitors. An accumulation of mitochondria (WT: 547.3±7.0, atg7^-/-: 737.8±126.6, p<0.01) and mitochondrial superoxide (WT: 280.2±4.8, atg7^-/-: 343.8±42.4, p<0.05) was found in atg7^-/- BM Lin^- cells, which may severely disturb the progress of MK differentiation.

Conclusion

Autophagy is essential for the megakaryopoiesis and thrombosis by maintaining mitochondrial homeostasis. Elevated reactive oxygen species might be the cause of megakaryocytic differentiation blockade.