Importance Of Cholesterol Homeostasis For The Reactivity Of Platelets

Hypercholesterolemia is a risk factor for atherothrombotic disease such as myocardial infarction.Platelets from patients with hyperlipidemia show the increased responses to various agonists.In-vitro cholesterol loading also induces the activation of human platelets.Cholesterol is an important component of lipid rafts,and lipid rafts are involved in the signal transduction of platelets for the activation and aggregation.Thus,hypercholesterolemia might affect the function of platelets by modulating the content of lipid rafts.However,the importance of cholesterol homeostasis for the content of lipid rafts in platelets are still not clear.Also the effects of the basal levels of lipid rafts on the activation and aggregation of platelets are still need to be determined.Reverse cholesterol transport?RCT?is the only way to remove excess cholesterol from peripheral tissues.As an important mediator of RCT,ATP-binding cassette transporter A1?ABCA1?promotes the cholesterol efflux to lipid-poor apolipoprotein AI?apoAI?.ABCA1 deficiency increased the content of lipid rafts in macrophages.The loss-of-function mutations of ABCA1 in human result in the hypocoagulability.Also ABCA1-deficient mice show a bleeding diathesis.However,both the reduction of plasma cholesterol levels and the absence of HDL in the ABCA1-deficient mice can affect the reactivity of platelets.Thus,the direct and specific effects of ABCA1deficiency on the content of cholesterol and lipid rafts as well as the reactivity of platelets warrant further investigation.Aims:To investigate the effects of cholesterol homeostasis in platelets on the content of lipid rafts and their reactivity.Methods:In-vitro cholesterol depletion was performed by incubation of platelets with M?CD,while a 2-week high cholesterol/high fat Western-type diet feeding to wild type and low-density lipoprotein receptor knockout?LDLr KO?mice was the way for in-vivo cholesterol loading on platelets.The Cre/Loxp system was employed to generate platelet–specific ABCA1 KO mice,and they were backcrossed to the LDLr KO background(ABCA1^Pf4-/Pf4-LDLr^-/-).The deficiency of ABCA1 in platelet was validated by PCR and Q-PCR.Lipid rafts were stained with cholera toxin B subunit?CTB?.Flow cytometry is used to determine the content of lipid raft,the expression of P-selectin and activated??b?3,and the externalization of phosphatidylserine?PS?on platelets.The platelet aggregation transmittance and tail bleeding time were performed to evaluate the in-vitro and in-vivo reactivity of platelets.Results:In-vitro cholesterol depletion increased the content of lipid rafts?1.4-fold,p<0.001?on platelets,while in-vivo cholesterol loading reduced the amount of lipid rafts?0.7-fold,p<0.01?.The expression of ABCA1 in platelets of ABCA1^Pf4-/Pf4-LDLr^-/-mice is only 19%?p<0.001?of control platelets.The platelet-specific ABCA1deficiency significantly decreases the content of lipid rafts?0.83-fold,p<0.05?.Strikingly,the induction of lipid rafts by cholesterol depletion suppressed the activation of platelets in response to various agonists?0.1-0.8-fold,p<0.01?.Conversely,the reduction of lipid rafts by cholesterol loading enhanced the activation of platelets after stimulation?1.1-4-fold,p<0.01?.Accordingly,the reduction of lipid rafts on platelets enhanced their in-vitro aggregation induced by thrombin and collagen?thrombin1.3-fold,p<0.01;collagen 1.5-fold,p<0.05?,and shortened the tail bleeding time?0.64-fold,p<0.05?of LDLr KO mice.The cholesterol loading induced differences in the content of lipid rafts and the reactivity of platelets were diminished by cholesterol removing.This indicates that the hyperreactivity from animals with hypercholesterolemia might be due to the reduction of lipid rafts by cholesterol loading.Despite the reduction of lipid rafts,the platelet-specific ABCA1 deficiency does not increase the activation and aggregation of platelets.Instead,ABCA1^Pf4-/Pf4-LDLr^-/-mice showed a trend in the decrease of the platelet aggregation and the increase of the bleeding time.The suppression of ABCA1 deficiency on the activation of platelets?0.79-0.93-fold,p<0.05?was identified by cholesterol removing.Conclusion:Cholesterol homeostasis is essential for the content of lipid rafts on platelets.Similar to cholesterol loading,the platelet-specific ABCA1 deficiency decreases the content of lipid rafts.However,in addition to the cholesterol efflux,other functions of ABCA1 favors the maintenance of the platelet reactivity.Further investigation on the importance of cholesterol homeostasis for the reactivity of platelets will provide more novel therapeutic targets and strategies for cardiovascular diseases.