Effects Of Cigarette Smoking Extract On Thrombomdulin&Inlfuence Of Varenicline On The Antiplatelet Action Of Clopidogrel

Cigarette smoking is independent risk factor of cardiovscular disease. Numerousstudies have shown that smoking cause signigicant endothelial dysfunction,inflammatory state, triggering of coronary thrombosis, coronary arteryspasm.Accordingly, smoking is associated with significantly increased rates of acutecoronary syndrome. However, the mechanism by which smoking cause arterialthrombosis remains unknown. Thrombomodulin (TM) is constitutively expressed onthe endothelial cell surface.The major function of TM is to form a high affinity1:1complex with thrombin.The complex of TM/Thrombin has several important rolesincluding activation of protein C and thrombin activatable fibrinolysis inhibitor,aswell as inhibition of inflammation.Thus,TM has a critical effect on anticoagulationand anti-inflammation.Functional deficiency of TM could enhance thrombosisformation. The combination of TM and thrombin depends not only on the amount ofexpression of TM but also on the single-molecule interactional force between TMand thrombin. Several studies show that the effects of cigarette smoking on TMexpression are inconsistent. Moreover, the single-molecule interactional forcebetween TM and thrombin is not reported. However, it is not clear the way leading tointravascular thrombosis whether cigarette smoking affect the expression of TM oraffect the interaction with thrombin.A research showed that continued smoking after successful percutaneouscoronary intervention (PCI) is associated with an increased risk of restenosis.However, smoking cessation can make a36%reduction in crude relative risk (RR) ofmortality for patients with CHD. Hence current management guidelines nowadvocate smoking cessation, in addition to controlling hypertension and dyslipidemia,as part of an overall cardiovascular risk reduction strategy. Varenicline is a novelselective nicotinic acetylcholine receptor partial agonist that has been approved in over70countries worldwide as an aid to smoking cessation. Clopidogrel is widelyused by patients with coronary artery disease undergoing PCI. The relationshipbetween smoking and cardiovascular disease increases the prospect of patientsreceiving smoking cessation therapy and Clopidogrel concomitantly in clinicalpractice. Plasma protein binding of Varenicline is low(≤20%) and independent of ageor renal function. The major route of clearance for varenicline is renal excretion.Clopidogrel, a prodrug, is metabolized by2consecutive cytochrome P450-dependentsteps to its active metabolite, which binds irreversibly to the platelet P2Y12receptor.The likelihood of a clinically relevant drug-drug interaction between varenicline andClopidogrel was considered to be low; nevertheless, the possibility of an interactionbetween these2drugs is lack of clinical evidences.Part Ⅰ: Effect of cigarette smoke extract on the expression of Thrombomodulinin human endothelial cellsObjective To observe the expression of thrombomodulin in HUVECs underdifferent cigarette smoke extract conditions.Methods CSE was prepared by a modification of Nakamura's method. HUVECwere isolated by0.1%type I collagenase digestion for15-20min at37℃, andcultured in low serum endothelial cell medium. After passage cultivation, we usedthe factor VIII related antibody to identify endothelial cell. The third generation ofHUVECs were incubated respectively with0,0.5%,1%,2.5%and5%CSE for6hours or exposed to5%CSE for0,6,10,12,24hours to determine the expressionchanges of TM protein and mRNA expression in HUVECs. RT-PCR and Flowcytometric analysis techniques were used for detecting TM mRNA and protein.Results After6-hour exposure to CSE, the protein level of of endothelial TM indifferent concentrations (0.5%,1%,2.5%,5%) reduced significantly (62.06±3.87,56.22±3.93,55.81±3.32,53.70±3.95) compared to control group (70.78±6.55)(P<0.01). and the mRNA level of endothelial TM also decreased significantly(0.1859±0.0139,0.1776±0.0156,0.0853±0.0136,0.0571±0.0123) compared tocontrol group (0.2550±0.0345)(P<0.01). After stimulation with5%CSE for0,6,10, 12,24hours, the levels of TM mRNA and protein decreased over time and reachedthe peak at12hours (0.0348±0.0148,30.51±0.61), which were significantly lowerthan that of control group (0.2241±0.0222,68.98±3.56)(P <0.001).Conclusion CSE significantly decreased the expression of TM in aconcentration-dependent fashion in Human Umbilical Vein Endothelial Cell. Thatsuggests cigarette smoking maybe according this way to lead to intravascularthrombosis.Part Ⅱ Study of the effect of cigarette smoking extract on the interaction betweenThrombomodulin and thrombin by live-cell single-molecule force spectroscopyObjective To study the effect of CSE on the single-molecule interactional forcebetween thrombomodulin and thrombin by live-cell single-molecule forcespectroscopy.Methods1. CSE was prepared by a modification of Nakamura's method.Total RNA was extracted from HUVECs and construct the plasmid of TM-GFP. COS-7cells were transfected with the recombinant plasmid TM-GFP and the expression of TM-GFP was detected by fluorescence microscopy and laserscanning confocal microscopy. The transfected COS-7cells were grouped (1)GFP-thrombin group (2) TM-thrombin group (3) CSE-GFP-thrombin group(4) CSE-TM-thrombin-1h group (5) CSE-TM-thrombin-6h group. Force measurements with the thrombin modified AFM tips on the living cell surface werecarried out on PicoSPM II with a Pico-Scan3000controller and a larger scanner. The force curves measured in living cells were recorded by PicoScan5software and analyzed by MATLAB R2009aMetlab. Drawn affinity between tip and cell of the Gaussian probability distribution and bonding probility wereanalyzed by Origin7.0software.2. Flow cytometry was used to detect5%CSE on the effects of the expressionTM-GFP on COS-7cells at1hours and6hours.Results1. The single-molecule binding force of Thrombomodulin and thrombin(TM-Thr) was determined60.90311±0.81723pN. The binding probability forTM-Thr was about22.58±3.95%. Antibody blocking binding probability for TM-Thr was2.58±2.0%.2. Compared to TM-THr group, the binding probability for GFP-Thr group,CSE-TM-Thr1h group,CSE-TM-Thr6h group,CSE-GFP-Thr group significantlydecreased (P<0.0001).3. After1-hour and6-hour exposure to5%CSE, there was no significantdifference between5%CSE group and control group in the expression of TM-GFP.Conclusion CSE significantly decreased the binding probability for TM-Thr.Together with the results of PartⅠ, our findings indicate that cigarette smokingdecreased the expression of TM and reduced the binding probability for TM andthrombin to lead to intravascular thrombosis.Part Ⅲ: Influence of varenicline on the antiplatelet action of clopidogrel: therandomized, open-label VACL (Varenicline Clopidogrel) studyObjective This study investigated the effect of varenicline on the antiplateletaction of clopidogrel in patients with coronary artery disease and evaluated the safetyand tolerability of concurrent administration of clopidogrel and varenicline.Methods Eligible smoking patients in hospital, with coronary arterydisease(CAD) undergoing PCI were consecutively included. All patients receivedaspirin (100mg/day) and clopidogrel (loading dose300mg, followed by75mg/day).On3day after received clopidogrel75mg/day, patients will be randomized to receiveeither associated varenicline (varenicline were titrated as follows:0.5mg/d for days1to3,0.5mg twice per day for days4to7, then1mg twice per day) or withoutvarenicline for14days. Clopidogrel effect was tested before associated varenicline,and on7,14day after associated varenicline in both groups by measuring plateletphosphorylated-VASP expressed as a platelet reactivity index (PRI) and plateletaggregation. All patients were documented adverse events and serious adverseevents.Results1.136patients were included. Conventional therapy group was68andvarenicline group was68.11of conventional therapy group failed to return on Day14for blood sampling.7of varenicline group failed to return on Day14for blood sampling.3of varenicline group did not take varenicline accoding to protocol towithdraw. Baseline characteristics were similar in the2groups.2. Changes in PRI and platelet aggregation at baseline, before taking varenicline,on day7, and on day14between the2groups.(1) In the conventional therapy group, PRI was80.08±5.60%,55.21±14.87%,41.87±17.05%and43.01±13.59%respectively at the four time. In vareniclinegroup, PRI was79.94±5.18%,53.74±14.47%,40.16±15.81%,42.18±11.27%respectively at the four time. There was no significant difference between the twogroups.(2) In the conventional therapy group, platelet aggregation was44.13±7.89%,26.53±11.27%,21.42±10.56%and23.61±10.09%respectively at the four time. Invarenicline group, platelet aggregation was43.67±7.31%,24.92±10.75%,20.45±9.75%,23.34±10.64%respectively at the four time. No significant difference wasobserved between the two groups.3. The safety and tolerability of concurrent administration of clopidogrel andvarenicline(1) The status of renal function in2weeks between the2groupsThe data of eGFR were similar in the2groups.(2) The status of adverse events and serious adverse events in2weeks betweenthe2groupsThe adverse events in varenicline group were higher than in conventional therapygroup. However, there was no significant difference between the2groups.The serious adverse events were identical between the2groups.Conclusion Clopidogrel can be safely administrated with varenicline withoutthe need for dose adjustment.