The role of 12-lipoxygenase in regulating megakaryocyte maturation and platelet-like particles production

Megakaryocytes, a type of myeloid cell primarily produced in the bone marrow, are essential for releasing platelets into the bloodstream. Through platelet production, megakaryocytes transfer their own biological content to these cells, including inflammatory enzymes. Amongst these enzymes, 12-lipoxygenase (12-LO) has been shown to modulate platelet activation and is involved in several chronic inflammatory conditions. However, the role of 12-LO in megakaryocyte maturation and the subsequent release of platelets remains uninvestigated. This study demonstrates the importance of 12-LO expression in megakaryocyte maturation and functions. Flow cytometry and fluorescence microscopy were utilized to analyze DAMI cell differentiation. Inflammatory enzyme profiles were assessed through Western blot analysis and LC-MS/MS. To evaluate megakaryocyte functionality, platelet-type 12-LO knockout mice were employed. Differentiation of DAMI cells into mature megakaryocytes resulted in increased surface marker expression, enhanced platelet-like particle production, reduced cell proliferation, and elevated cell death. The arachidonic acid release from cell membranes was increased in differentiated cells as well as the expression of several inflammatory enzymes such as 12-LO, cyclooxygenase-1 and thromboxane synthase. The production of 12(S)-hydroxyeicosatetraenoic acid and thromboxane was also increased in these cells. In DAMI cells deficient in 12-LO expression (ALOX12-/-), we found no difference in CD41 expression following differentiation compared to ALOX12+/+ cells. However, 12-LO-deficient cells produced fewer proplatelets and, consequently, fewer platelet-like particles. In vivo, platelet counts in 12-LO-deficient mice were comparable to those in wild-type mice, and the number of bone marrow megakaryocytes remained consistent between the groups. Ex vivo analysis revealed that megakaryocytes lacking 12-LO produced fewer platelets. These findings indicate that 12-LO may play a regulatory role in megakaryocyte function, particularly in proplatelet formation and platelet release.