Altered Heterogeneity Of Platelet Activation In Arterial Thrombosis Under Hyperlipidemic Conditions

Cardo-cerebro-vascular disease is a serious threat to human health.As a common disease,its morbidity and mortality ranks top among all kinds of disease.The treatment for CCVD is still limited.With the improvement of economic status,hyperlipidemia emerges as one of the most important causes of atherosclerosis,thrombosis and cerebrovascular diseases.Plasma low-density lipoprotein(low density lipoprotein,LDL)is the canonical content of plasma lipids in human,and is responsible for carrying fatty acid molecules for cell metabolism.LDL can be oxidized by reactive oxygen(Reactive oxygen species,ROS)to Ox-LDL(Oxidized low density lipoprotein,ox-LDL)and both combine to platelet surface receptors.The activation of platelets may promote intracellular signaling pathways and subsequent platelet activation.Although it has been reported that substantially increased LDL level was accompanied by increased platelet reactivity and thrombotic risk,the mechanism underlying thrombus formation in hyperlipidemia is still unclear.Objective:To investigate the heterogeneity of platelet activation under hyperlipidemic condition and to analyze the dynamic process of LDL involvement in thrombosis by using the intravital microscopy and cremaster thrombosis model induced by laser damage or mechanical injuryMethods:1.C57BL/6J Wild type(WT)mice and apolipoprotein E(ApoE)knockout mice(ApoE-/-mice)were fed with high-fat feed(high-fat diet,HFD)to establish hyperlipidemia model.WT mice fed with normal diet(Chew diet,CD)were included as the control group.2.C57BL/6J WT and ApoE-/-mice on HFD for 2-3 weeks were subjected to laser induced cremaster artery thrombosis.Thrombus volume,platelet activation under hyperlipidemic condition were determined using intravital microscopy.3.Venous blood from WT mice or ApoE-/-mice on HFD for 1 week,2 weeks,3 weeks and 4 weeks were drain from the orbital venous plexus to allow analysis of platelet counts and lipid profiles.4.Abdominal aorta thrombosis in WT mice was induced by mechanical damage.Immunological staining of LDL and Ox-LDL contents in thrombus was performed.5.The red fluorescent probe labeled LDL(Dil-LDL)was used to analyze the dynamic process of LDL incorporation into the forming thrombosis induced by laser damage in the cremaster artery of WT mice.Results:1.The volume of thrombus and platelet reactivity in ApoE-/-mice on HFD were significantly larger than in those fed with CD.2.The proportion of developing thrombus tail in ApoE-/-mice on HFD was increased by 1.5 times compared to ApoE-/-mice fed with CD.3.Platelet counts in ApoE-/-mice fed with HFD for 3 weeks were decreased by 40%compared to the ApoE-/-mice on CD.4.The LDL levels in ApoE-/-mice fed with HFD for 3 weeks were significantly increased by 1.5 times compared to those on CD.5.Heterogeneity of platelet activation was observed in mouse aortic thrombosis.6.LDL and ox-LDL were incorporated into forming thrombus.LDL was found to colocalize with slightly activated platelets in thrombus.Conclusions:Our results showed that blood clots in mice with hyperlipidemia were significantly increased,and both 'core" and 'shell' of the thrombus were enlarged,accompanied by decreased thrombus abscission.Platelet reactivity was increased along with elevated LDL and ox-LDL incorporation in thrombus,when LDL was found to bind very slight activated platelets.To our knowledge,this is the first report about heterogeneity in platelet activation within large arterial thrombosis.The study further elucidated the association between hyperlipidemia and thrombosis,which may indicate a novel target in anti-thrombotic therapies.