| In the process of sepsis,various pathways can lead to platelet activation.Activated platelets play an important role in the occurrence and development of sepsis thrombosis and inflammatory storm,and are the bridge connecting the coagulation system and the immune system.P2Y12 receptors play a crucial role in platelet activation and thrombosis,and P2Y12 antagonists may be potential options for the treatment of sepsis.So far,whether patients with sepsis benefit from the treatment of P2Y12 antagonists,there is still lack of sufficient prospective studies,and the conclusions are inconsistent whether the treatment of P2Y12 antagonists in animal models of sepsis is effective.On the other hand,studies have shown an increased incidence of thrombotic events in the acute phase of sepsis and shortly after recovery,suggesting that patients with sepsis may benefit from antiplatelet therapy.The incidence of high ontreatment platelet reactivity(HTPR)in patients receiving clopidogrel during sepsis was significantly increased compared with the recovery period,suggesting that sepsis may reduce clopidogrel treatment response sex,but the exact mechanism is unclear.Therefore,whether the platelet P2Y12 receptor changes in sepsis patients and its mechanism,whether it affects the antiplatelet effect of P2Y12 antagonists such as clopidogrel,and what is the role of platelet P2Y12 receptor in sepsis?In order to solve the above problems,the specific research in this paper is as follows:1.Changes and mechanisms of platelet P2Y12 receptors in patients with sepsis Methods:34 patients who met the diagnostic criteria for sepsis and 17 healthy volunteers who were matched for age and sex were collected to detect the ADP-induced platelet aggregation rate,the expression of P2Y12 receptor,Pselectin,CD40L on the surface of platelets,peripheral platelet-leukocyte aggregate ratio by flow cytometry.Total P2Y12 protein,and mRNA level in platelets were also detected.The platelets of healthy volunteers were treated with LPS,ADP,TRAP-6,collagen,thrombin for 10 min in vitro to detect the changes of platelet P2Y12,P-selectin and CD40L expression.Platelets were pretreated with TLR4、P2Y12、IKK inhibitors for 10 min before stimulation with LPS,ADP and TRAP-6 to detect the expression of P2Y12,as well as the expressions of p-AKT,p-Iκb,VAMP-8 and SNAP-23 in platelets to explore the secretion mechanism of P2Y12.Results:ADP-induced platelet aggregation rate,platelet P-selectin and CD40L expression,and the ratio of platelet-monocyte aggregate and plateletneutrophil aggregate in peripheral blood were significantly increased in sepsis patients.The expression of P2Y12 receptor on the platelet surface was also significantly increased,and the degree of increase was positively correlated with the degree of platelet activation,while the total amount of P2Y12 protein and the mRNA content in platelets did not change in patients with sepsis.In vitro experiments had shown that there was a P2Y12 storage pool in platelets,and P2Y12 receptor could be further secreted to the platelet surface after activation.Different platelet stimulators had different abilities to stimulate the secretion of P2Y12,and IKK regulated the SNARE pathway to control the secretion of P2Y12.The P2Y12 receptor signaling pathway was involved in its autocrine process,and inhibition of P2Y12 could also partially inhibit the P2Y12 secretion of TRAP-6-stimulated platelets.2.Changes and effects of platelet P2Y12 receptors in sepsis ratsWe found that the expression of P2Y12 receptor on the platelet surface of patients with sepsis was elevated,independent of its transcriptional and translational levels,and related to platelet activation,which was the consequence of intracellular transfer to the cell surface.Next,we verified it in septic rats,and further explained the changes and roles of P2Y12 receptors in septic platelets by applying clopidogrel to intervene.Methods:A rat model of sepsis was established by intraperitoneal injection of 5 mg/kg LPS.The appearance and activity of the rats were observed at 4 h and 24 h,and indexes such as blood routine,coagulation,inflammation,and organ function were detected to evaluate the sepsis model.Then 4 experimental groups were set up:normal control group(intraperitoneal injection of PBS,intraperitoneal administration of vehicle),sepsis group(intraperitoneal injection of LPS,intragastric administration of vehicle),clopidogrel group(intraperitoneal injection of PBS,intragastric administration of 10 mg/kg clopidogrel)and sepsis clopidogrel treatment group(intraperitoneal injection of LPS,intragastric administration of 10 mg/kg clopidogrel).After 4 hours,the expression of P2Y12 receptor on the platelet surface of the rats in each group was detected,and platelet activation indicators included platelet aggregation,P-selectin exposure,platelet-leukocyte aggregates,and electrical stimulation was used to detect the thrombosis of the rats.Three experimental groups were set up:normal control group(intraperitoneal injection of PBS,intragastric administration of vehicle),sepsis group(intraperitoneal injection of LPS,intragastric administration of vehicle),sepsis clopidogrel treatment group(intraperitoneal injection of LPS,intragastric administration of vehicle)40 mg/kg clopidogrel).Blood was collected to separate platelets 4 hours after intervention,and proteomics techniques were used to study the changes of platelet function and the role of P2Y12 receptors in septic rats.Results:Intraperitoneal injection of LPS could lead to obvious abnormalities in the appearance,activity,and stimulation response of rats,as well as increased leukopenia,thrombocytopenia,D-dimer,APTT,inflammatory indicators IL-1β,IL-6,CRP,and ferritin.Thus,A sepsis model was successfully established.Platelet P2Y12 receptor expression was increased in sepsis rats,and the degree of platelet activation was also increased.Meanwhile,the length of the thrombus increased and the time for complete occlusion of the vessel decreased.The antiplatelet and antithrombotic effects of rats were significantly weakened.Proteomic and phosphorylation modification proteomic results showed that many functions of septic platelets were significantly altered,confirming the important role of the P2Y12 receptor in septic platelet activation and thrombosis(coagulation).In addition,P2Y12 receptor also played an important role in regulating platelet inflammatory response,regulating complement activation,leukocyte aggregation,cell-matrix adhesion and other functions or pathways.Activation of P2Y12 receptor signaling in septic rat platelets increased the activation of PKC phosphorylation,increased ROS production,and lead to activation of the NLRP3 inflammasome.P2Y12 antagonists could inhibit this pathway and inhibit the release of IL-1β,thereby participating in the regulation of platelet inflammatory responses.3.Study on the effect of a novel P2Y12 antagonist CN-218Experiments in patients and rats with sepsis showed that the expression of P2Y12 receptors on the surface of septic platelets was increased,and the degree of platelet activation was increased,resulting in significantly weakened antiplatelet activation and antithrombotic effects of clopidogrel.In addition,there are problems of drug resistance and low bioavailability during the clinical application of clopidogrel.Therefore,it is necessary to change clopidogrel to optimize its antiplatelet effect and overcome its shortcomings as much as possible.We have synthesized a series of novel clopidogrel derivatives based on clopidogrel,among which CN-218 has excellent performance in the previous pharmacokinetic study.Compared with clopidogrel,the modification of CN-218 includes selective deuteration at the methyl carboxylate position at the 7position and the introduction of a cinnamic acid side chain at the 2-position of the thiophene ring.This modification will have a huge impact on improving drug metabolism efficiency and overcoming clopidogrel resistance caused by CYP450s polymorphisms.We studied the antiplatelet and antithrombotic effects of CN-218 on healthy rats and septic rats,and investigated whether CN218 has a certain therapeutic effect on septic rats,in order to find potential antiplatelet drugs for clinical use.Methods:Set up vehicle group,clopidogrel group(10 mg/kg for rats,15 mg/kg for mice),prasugrel group(1 mg/kg for rats,1.5 mg/kg for mice),CN-218 with low,medium,high-dose group(0.5,1,2 mg/kg for rats,0.75,1.5,3 mg/kg for mice):the platelet aggregation rate,APTT and PT,bleeding time by tail docking method,and serum cAMP by Elisa method were detected 4 hours after administration.After continuous administration for 3 days,intraperitoneal injection of carrageenan was performed to establish a thrombus model to detect the antithrombotic ability.The clopidogrel group(10 mg/kg)and the CN-218 group(12 mg/kg)were set,and blood was collected at different time points before administration and after a single administration to detect the levels of plasma active metabolites.The sepsis model was established by intraperitoneal injection of LPS,and the vehicle,clopidogrel(10 mg/kg)and CN-218(2 mg/kg)were administered respectively.And platelet activation,thrombosis,and blood routine,coagulation,inflammation,and organ indexes were evaluated 4 hours later.Result:CN-218 had potent anti-platelet aggregation effects,much stronger than clopidogrel,but not as good as prasugrel.CN-218 also had a certain anticoagulant effect,which was manifested as prolonged APTT in rats.CN-218 had a similar antithrombotic effect but a lower risk of bleeding than prasugrel at the same dose.Pharmacokinetic studies of active metabolites showed that the absorption rate of CN-218 might not be as fast as that of clopidogrel,but its metabolic efficiency was much higher than that of clopidogrel.CN-218 significantly inhibited platelet activation in septic rats,and its anti-septic rat platelet aggregation and anti-thrombotic abilities were more potent than clopidogrel.CN-218 could also partially inhibit the increase of serum inflammatory indicators such as ferritin,IL-6 and IL-1β in sepsis rats,and inhibit the decline of white blood cells and platelets in sepsis rats.CN-218 had a certain inhibitory effect on the increase of D-dimer in sepsis rats.CN-218 did not show obvious protective effect on the kidney of septic rats,but had a certain protective effect on the liver and lung.Full text summary:The increased expression of P2Y12 on the platelet surface in sepsis was related to platelet activation,and was related to the significantly weakened antiplatelet effect and antithrombotic ability of clopidogrel in sepsis.In addition to its key role in platelet activation and thrombosis in sepsis,platelet P2Y12 receptors were also involved in the regulation of inflammatory responses,such as the regulation of inflammatory responses through the P2Y12/PKC/ROS/NLRP3 inflammasome/IL-1β pathway.Compared with clopidogrel,the novel P2Y12 receptor antagonist CN-218 significantly improved the metabolic efficiency and exhibited potent antiplatelet and antithrombotic effects in both healthy and septic rats.It also has a certain anti-inflammatory effect and organ protection effects on septic rats. |
