The Role And Mechanism Of Klotho In Regulating Thrombopoiesis And Platelet Function

Platelets play very important roles in physiological and pathological processes.The main function of platelets is to participate in coagulation and hemostasis.Dramatic platelet reduction in blood such as acute radiation sickness-induced thrombocytopenia will further cause severe hemorrhage and infection.On the contrary,high platelet level or platelet hyperactivation will result in thrombotic diseases.Platelets are produced by megakaryocytes that residing in the bone marrow.There are several key steps of thrombopoiesis involving commitment of hematopoietic stem cells into megakaryocytes,expansion of megakaryocyte progenitors,differentiation and maturation of megakaryocytes,polyploidization,migration of mature megakaryocytes to vascular sinus,adhesion to the endothelium,protrude proplatelet into vascular sinus and platelet production and release.Our preliminary studies find that the production of platelets by megakaryocytes is regulated by the reactive oxygen species（ROS）levels in the microenvironment.Notably,ROS is also the primary mediator of radiation injury.Ionizing radiation can rapidly induce massive production of ROS,which can bind and oxidize the cellular biological macromolecules such as DNA to damage cells,thus resulting in cell apoptosis and necrosis.The dramatic reduction of peripheral blood cells is related to the apoptosis and necrosis of hematopoietic cells in bone marrow.Thrombocytopenia is not only a main manifestation of bone marrow radiation injury,but also the major cause of hemorrhage,infection and even death.Therefore,it may be effective to prevent and mitigate ionizing radiation-induced thrombocytopenia by inhibiting ionizing radiationinduced apoptosis of megakaryocyte progenitors,thus preventing hemorrhage and infection.Klotho is an anti-aging protein that mainly expressed and secreted by renal tubular epithelial cell.As a humoral protein,Klotho can be secreted to the circulation to achieve to the tissues of the whole body,thus exerting its systematic anti-aging effect.Klotho possesses a strong ability to inhibit ROS production and scavenge redundant ROS in cells to resist aging.As reported,Klotho can effectively protect endothelial cells and renal cells from oxidative stress-induced apoptosis.As both normal thrombopoiesis and radiation injury-induced thrombocytopenia are related to oxidative stress,it deserves in-depth study to investigate whether Klotho participates in the regulation of thrombopoiesis and precaution and treatment of radiation injury-induced thrombocytopenia.On the other hand,ROS also play important roles in the process of platelet activation.Apart from the exogenous ROS,the endogenous ROS generated by platelets and the downstream signalling pathway also play important roles in platelet activation,the adhesion of platelets to vessel wall,recruitment of other platelets,platelet aggregation and thrombus formation.The hyperactivation of platelets under conditions of certain disease will increase the risk of thrombus formation,thereby further causing ischemic diseases such as myocardial infarction and stroke.However,there is still a lack of effective measures to prevent platelet activation and thrombus formation at this moment.Several studies have demonstrated that Klotho level is negatively correlated with the onset risk of thrombosisrelated disease.Moreover,chronic kidney disease（CKD）is a disease characterized by severely decreased Klotho level in circulation.However,the platelet function is often changed in CKD,suggesting that Klotho as an anti-oxidative agent may also affect the process of platelet activation.Therefore,to investigate the effects of Klotho on thrombopoiesis and platelet function and the underlying pathophysiological significance,in this study,we will firstly analyze the effect of Klotho on normal thrombosis by using human primary megakaryocytes and megakaryoblast cell line M07 e cell through cell activity analysis and flow cytometry.Then,by application of blood routine examination after total body irradiation,we will investigate the effect and the mechanism of Klotho treatment on the precaution and treatment of radiation injury-induced thrombocytopenia.Finally,by creation of a 5/6 nephrectomy CKD mouse model,we will study the counteraction effect of Klotho on CKD-induced platelet hyperactivation and the underlying mechanisms by using western blot,flow cytometry,cell immunofluorescence,laser confocal microscopy and in vivo thrombosis model.The main results and conclusions are shown as follows:1.In vitro studies show that,Klotho has no significant effect on the proliferation of megakaryocyte progenitors,differentiation and maturation of megakaryocytes,platelet production and release.2.Sublethal dose of ionizing radiation can induce ROS overproduction and apoptosis of megakaryocyte progenitors and dramatic decrease of platelet levels.However,Klotho has a distinct ability to protect megakaryocyte progenitors from ionizing radiation-induced ROS overproduction and apoptosis and to accelerate the recovery of platelet levels in mice receiving total body irradiation.These results suggest that Klotho can be used to prevent and treat ionizing radiation-induced thrombocytopenia.3.In the CKD mouse model,we find that indoxyl sulphate（IS）level significantly elevated,accompanied by dramaticly decreased serum Klotho level and abnormally enhanced platelet activity.4.IS can induce platelet hyperactivation both in vitro and in vivo,while Klotho can counteract IS-induced platelet hyperactivation.Furthermore,Klotho reduction in CKD mice can further aggravate IS-induced platelet hyperactivation and thrombus formation.5.Results from in vitro studies show that IS can induce ROS overproduction in platelets and the subsequent activation of its downstream signaling pathway,p38 MAPK,while Klotho can suppress IS-induced ROS overproduction and p38 MAPK activation in platelets.These results indicate that the ability of Klotho to counteract IS-induced platelet hyperactivation and thrombus formation is related to its strong anti-oxidative capacity.6.By using apo E^-/-mice and apo E^-/-mice with CKD,we find that IS and CKD-I nduced platelet hyperactivation can aggravate the progression of atherosclerosis,while exogenous Klotho administration can attenuate IS and CKD-induced atherosclerosis acceleration.Conclusions: In this study,we for the first time show that Klotho as an anti-oxidative protein not only can prevent ionizing radiation-induced apoptosis of megakaryocyte progenitors and dramatic platelet reduction,but also can suppress IS-induced platelet hyperactivation,thus preventing thrombosis and atherosclerosis.Our study provide new insights into the treatment of thrombocytopenia and thrombosis-related disease in clinic.