Objective To investigate programmed death including necroptosis,apoptosis,autophagy,ferroptosis,and pyroptosis in bone marrow megakaryocytes of mice during sepsis and its impact on platelet production capacity and coagulation function in mice.Methods C57BL/6J mice were randomly divided into a sham operation group(sham group)and a sepsis model group(CLP group).Peripheral blood platelets and coagulation function were measured by abdominal aortic blood sampling at 24 h postoperatively in both sham and CLP groups.After the mice were sacrificed,long bones of both lower limbs were taken,and bone marrow megakaryocytes were extracted using megakaryocyte separation solution and immunomagnetic bead separation.Laser confocal microscopy was used to observe the activation of programmed death-related marker molecules in mouse bone marrow megakaryocytes.Flow cytometry was used to detect programmed death rate,platelet production phenotype,and platelet surface markers(CD41,CD42b,CD61)of megakaryocytes.Western blotting was used to detect the expression of programmed death-related proteins in megakaryocytes.Results Compared with the sham group,the CLP group showed significant decreases in the number of platelets during acute sepsis(24 h)(P<0.000 1),significant increases in platelet distri-bution width(PDW)and mean platelet volume(MPV)(P<0.01),significant prolonging of thrombin time(TT),prothrombin time(PT),and activated partial thromboplastin time(APTT)(P<0.000 1,P<0.001,P<0.01),and significant reduction in fibrinogen(Fib)(P<0.000 1).Compared with the Con/sham group,the LPS/CLP group exhibited significant increases in the platelet production phenotype of megakaryocyte,the number of PLP in the supernatant,and the expression levels of platelet surface markers(CD41,CD42b,CD61).The rates of megakaryocyte necroptosis/apoptosis,pyroptosis,and ferroptosis were significantly elevated at 24 h post-CLP surgery.Laser confo-cal microscopy showed significant activation of LC3,P-MLKL,Caspase-1,and Fe2+in megakaryocytes of mice after CLP surgery.Western blotting results revealed that the CLP group exhibited a significant increase in the activa-tion rate of necroptosis-related protein P-MLKL(P<0.001),a significant increase in the cleavage of pyroptosis-related proteins GSDMD and GSDMD-N(P<0.01,P<0.001,respectively),a significant increase in the expres-sion of ferroptosis-related protein ACSL4(P<0.01),and a significant decrease in the expression of GPX4(P<0.01)compared to the sham group.Additionally,the CLP group demonstrated significant increases in the expression of apoptosis-related protein Bax,the cleavage of autophagy-related protein LC3B-Ⅱ,and the expression of P62(P<0.05,P<0.001,P<0.001,respectively).Inhibition of apoptosis with programmed cell death inhibitors decreased platelet production function of megakaryocyte,while inhibition of necroptosis and pyroptosis had limited effects on platelet production function of megakaryocyte.Inhibition of ferroptosis and autophagy enhanced platelet production function of megakaryocyte.Conclusion Significant programmed death of megakaryocytes was observed during the acute phase of sepsis(24 h).Among those megakaryocytes,apoptosis is an important mechanism for the differentia-tion of platelet production phenotype and increased platelet production capacity of megakaryocyte.Overactive autophagy and ferroptosis in megakaryocytes lead to megakaryocyte dysfunction,which is an important mechanism for coagulation abnormalities in sepsis.

Objective To investigate programmed death including necroptosis,apoptosis,autophagy,ferroptosis,and pyroptosis in bone marrow megakaryocytes of mice during sepsis and its impact on platelet production capacity and coagulation function in mice.Methods C57BL/6J mice were randomly divided into a sham operation group(sham group)and a sepsis model group(CLP group).Peripheral blood platelets and coagulation function were measured by abdominal aortic blood sampling at 24 h postoperatively in both sham and CLP groups.After the mice were sacrificed,long bones of both lower limbs were taken,and bone marrow megakaryocytes were extracted using megakaryocyte separation solution and immunomagnetic bead separation.Laser confocal microscopy was used to observe the activation of programmed death-related marker molecules in mouse bone marrow megakaryocytes.Flow cytometry was used to detect programmed death rate,platelet production phenotype,and platelet surface markers(CD41,CD42b,CD61)of megakaryocytes.Western blotting was used to detect the expression of programmed death-related proteins in megakaryocytes.Results Compared with the sham group,the CLP group showed significant decreases in the number of platelets during acute sepsis(24 h)(P<0.000 1),significant increases in platelet distri-bution width(PDW)and mean platelet volume(MPV)(P<0.01),significant prolonging of thrombin time(TT),prothrombin time(PT),and activated partial thromboplastin time(APTT)(P<0.000 1,P<0.001,P<0.01),and significant reduction in fibrinogen(Fib)(P<0.000 1).Compared with the Con/sham group,the LPS/CLP group exhibited significant increases in the platelet production phenotype of megakaryocyte,the number of PLP in the supernatant,and the expression levels of platelet surface markers(CD41,CD42b,CD61).The rates of megakaryocyte necroptosis/apoptosis,pyroptosis,and ferroptosis were significantly elevated at 24 h post-CLP surgery.Laser confo-cal microscopy showed significant activation of LC3,P-MLKL,Caspase-1,and Fe2+in megakaryocytes of mice after CLP surgery.Western blotting results revealed that the CLP group exhibited a significant increase in the activa-tion rate of necroptosis-related protein P-MLKL(P<0.001),a significant increase in the cleavage of pyroptosis-related proteins GSDMD and GSDMD-N(P<0.01,P<0.001,respectively),a significant increase in the expres-sion of ferroptosis-related protein ACSL4(P<0.01),and a significant decrease in the expression of GPX4(P<0.01)compared to the sham group.Additionally,the CLP group demonstrated significant increases in the expression of apoptosis-related protein Bax,the cleavage of autophagy-related protein LC3B-Ⅱ,and the expression of P62(P<0.05,P<0.001,P<0.001,respectively).Inhibition of apoptosis with programmed cell death inhibitors decreased platelet production function of megakaryocyte,while inhibition of necroptosis and pyroptosis had limited effects on platelet production function of megakaryocyte.Inhibition of ferroptosis and autophagy enhanced platelet production function of megakaryocyte.Conclusion Significant programmed death of megakaryocytes was observed during the acute phase of sepsis(24 h).Among those megakaryocytes,apoptosis is an important mechanism for the differentia-tion of platelet production phenotype and increased platelet production capacity of megakaryocyte.Overactive autophagy and ferroptosis in megakaryocytes lead to megakaryocyte dysfunction,which is an important mechanism for coagulation abnormalities in sepsis.