氧化型低密度脂蛋白活化血小板机理的初步探讨
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摘要
背景：
随着社会的发展和人们生活水平的提高、胆固醇摄入的增加,高脂血症及动脉粥样硬化疾病的发病率逐年上升。作为动脉粥样硬化的主要疾病、目前冠心病已成为我国人群死亡的主要病因。已明确动脉粥样硬化斑块的形成及斑块不稳定导致的局部血栓是冠心病、急性冠脉综合征(ACS)发生的重要病理环节,研究已经肯定低密度脂蛋白及血小板在其中扮演主要角色,两者是冠心病研究的重要课题。氧化型低密度脂蛋白(oxidized low-density lipoprotein, ox-LDL)是低密度脂蛋白致动脉粥样硬化的主要活性成份,近期文献报道ox-LDL除致斑块形成外、对血小板活化也有直接的促进作用,在动脉血栓的形成过程中可能发挥重要作用,但具体机制尚缺乏深入研究。因此,研究ox-LDL对血小板活化的影响对阐明冠心病的发病机理、防治ACS临床事件的发生意义重大。
作为动脉血栓形成的核心环节,尽管还有许多有待阐明的具体环节,血小板活化的主要步骤已得到充分的研究。研究显示：血小板活化时,其胞膜上的花生四烯酸(AA)在环氧化酶1(COX-1)的催化下合成血栓烷A2(TXA2),后者可扩散到胞膜外进而活化其他血小板。血小板上有两种TXA2受体：TPα和TPβ,两者均可与蛋白Gq、G12/13偶联。在血管损伤部位,循环中的凝血酶原迅速生成凝血酶,后者可与血小板表面的蛋白酶激活受体(PAR1和PAR4)反应,激活与之偶联的Gq、G12/13和Gi蛋白,启动活化过程,并促进血小板胞浆颗粒内容物的释放。其中血小板致密颗粒内的ADP,可扩散到胞膜外激活血小板表面的P2Y1和P2Y12受体,并使偶联的Gq和Gi蛋白发生构型改变。构型改变了的Gq蛋白可激活磷脂酶C (PLC),后者可催化降解磷酸肌醇生成三磷酸肌醇(IP3)和二酰基甘油(DAG). DAG能够激活血小板内蛋白激酶C (PKC)进而引起蛋白磷酸化,IP3则促进内质网内Ca2+的释放,使胞浆中Ca2+的水平升高。Gi蛋白可抑制腺苷酸环化酶(AC)的活性并促进PI3K的活性。AC活性的下降可降低胞浆环一磷酸腺苷(cAMP)的水平,使磷酸化的血管舒张剂刺激磷蛋白(VASP)去磷酸化。PI3K活性升高可使Akt磷酸化增强,进而通过mTORs并与VASP-起活化GpⅡb/Ⅲa受体。G12/13蛋白可与Rho鸟苷酸交换因子(RhoGEFs)结合,诱发Rho因子介导的细胞骨架反应,从而导致血小板形状改变。最后促进血小板的粘附、聚集,导致血小板血栓形成。
在上述血小板活化过程中,P2Y12受体占据中心地位,因为其活化不但可以激活血小板,还可以放大和维持TXA2和凝血酶引起的血小板的活化,稳定形成的血栓。在研究高脂血症对血小板功能影响的过程中,考虑到高脂血症中最重要的致病因素是ox-LDL,而P2Y12受体在血小板活化中又占有中心地位,因而选择ox-LDL对血小板活性的影响以及ox-LDL是否可通过P2Y12受体促进血小板活化是关键的切入点,对阐明动脉血栓机制具有重要的意义。
目的：
1.进一步明确ox-LDL对不同血小板活化指标的影响；
2.初步探讨ox-LDL活化血小板的具体机制,重点明确ox-LDL是否可通过P2Y12受体活化血小板。
方法：
通过改良沉淀和硫酸铜氧化法制备ox-LDL。按文献报道,将不同浓度的ox-LDL分别与成年健康者的血小板孵育5分钟后,分别检测ADP诱导的血小板最大聚集率、纤维蛋白原诱导的铺展面积和血小板表面活化的GpⅡb/Ⅲa受体数目,从而观察ox-LDL对上述血小板活化指标的影响。然后通过检测致密颗粒内ADP的释放、P2Y12受体下游信号cAMP、VASP和Akt的水平来初步探讨ox-LDL活化血小板的机制。
1. ox-LDL制备和鉴定及血小板的准备：
1.1ox-LDL的制备和鉴定：采用改良沉淀法提取人血浆低密度脂蛋白(low density lipoprotein,LDL),然后以0.01mmol/L的硫酸铜氧化24-48小时制备ox-LDL,以硫代巴比妥酸(thiobarbituric acid, TBA)检测ox-LDL的氧化修饰程度,并以BCA法测定蛋白浓度；
1.2血小板的制备：自志愿者肘部静脉取血36m1,以1：6的右旋枸橼酸ACD抗凝,常温300g离心10分钟,分离富血小板血浆,然后900g离心10分钟,分离血小板,重悬在含0.02U/mL apyrase的苔氏液(Tyrode buffer)中,并将血小板终浓度调整至2.5×108个/毫升。
2. ox-LDL对血小板活化指标的影响
2.1以10uM的ADP刺激不同浓度ox-LDL孵育后的血小板,使用双通道血小板聚集仪以1cm/mi n的走纸速度、测定苔氏液参比下的最大聚集率,观察ox-LDL对血小板聚集率的影响；
2.2血小板铺展及面积测量。实验组血小板预先与ox-LDL共孵育5min,对照组未做处理。用20μg/mL的纤维蛋白原包被chamber后,将标本接种于chamber内,以玫瑰红-鬼笔环肽荧光染料染色,37℃孵育45分钟,4%多聚甲醛固定,染色,荧光显微镜下观察。在低倍视野下比较未铺展血小板数目,在高倍视野下观察血小板铺展面积；
2.3全血法流式细胞术检测干预前后血小板膜表面纤维蛋白原受体(PAC-1) GpⅡb/Ⅲa复合物的表达。取出30μl全血,加入缓冲液稀释至300μl；每一标本取流式管A、B,均加入上述液体30μl；加入60μg/ml的ox-LDL共孵育,在30分钟、60分钟、120分钟分别取出10μll。A管PAC-1(FITC标记)、CD42b (APC标记),B管：PAC-1同型、CD42b (APC标记)；室温避光孵育15min；最后加入1%多聚甲醛,置于4℃避光保存,1小时内流式细胞仪上检测。血小板PAC-1及CD62p含量以其阳性血小板百分率表示。使用SystemⅡ软件采集样本,参照阴性空白对照,用EXPO TM32Multicomp软件分析数据,得出PAC-1-FITC抗体荧光的百分率；
3. ox-LDL活化血小板的具体机制探讨
3.1虫荧光素-荧光素酶标记的ATP曲线测定。血小板活化时,其致密颗粒内容物(如ADP和ATP)释放,可以通过血小板聚集仪和虫荧光素-荧光素酶观察血小板胞浆内内ATP含量的变化来间接反映ADP的释放。加用ox-LDL预先孵育血小板5min,再用ADP刺激血小板时,测量虫荧光素-荧光素酶标记的ATP曲线,并与不加ox-LDL的标本对照,观察ox-LDL对血小板致密颗粒内ADP释放的影响；
3.2血小板P2Y12受体下游cAMP的测定：获得富血小板血浆,加入2μCi/mL [74kBq/mL][3H]腺嘌呤和1mM的阿司匹林,37℃孵育1小时,通过[3H]腺嘌呤标记血小板内的ATP,离心分离血小板,重悬在含apyrase0.02U/mL的苔氏液中。加入ox-LDL预先孵育5分钟,恒定搅拌速度900rpm,将血小板与ox-LDL共孵育后,加入ADP刺激,然后利用柱层析法分离ATP和cAMP,并使用液闪仪测定含[3H]标记的cAMP和ATP,最后通过公式：{[3H]cAMP/([3H]ATP+[3H]cAMP) x103},计算血小板内自ATP转化生成的cAMP浓度；
3.3血小板P2Y12受体下游磷酸化VASP的测量采用ox-LDL处理血小板,加入SDS细胞裂解液和SDS蛋白上样缓冲液液制备样品,以10%的分离胶和5%的浓缩胶分别加入80μ1预染蛋白和10μl的血小板样品,60V-110V电压聚丙烯酰胺凝胶电泳1小时分离VASP；结束后进行蛋白质全湿法电转移至硝酸纤维素膜上1.5小时,加入终止液1.5小时；然后加入1：1000的p-VASP兔抗人单克隆抗体,4℃过夜孵育；以洗涤液洗涤3次(摇床上连续摇动),再加入1：2000标有辣根过氧化物酶的山羊抗兔二抗,常温孵育2小时；洗涤3次后加1：1的发光试剂发光约5分钟；暗室曝光30秒,显像,定影。通过western-blot检测分析磷酸化的VASP水平。
3.4ox-LDL影响血小板内Akt浓度的测量：采用上述方法用ox-LDL处理血小板,血小板裂解后液制备样品,以10%的分离胶和5%的浓缩胶分别加入80μl预染蛋白和10μl的血小板样品,60-110V电压聚丙烯酰胺凝胶电泳1小时分离Akt；结束后进行蛋白质全湿法电转移至硝酸纤维素膜上1.5小时,加入终止液1.5小时；然后加入1：1000加入p-Akt兔抗人单克隆抗体,4℃过夜孵育；洗涤液洗涤3次,再加入1：2000标有辣根过氧化物酶的山羊抗兔二抗,常温孵育2小时；洗涤3次后加1：1的发光试剂发光约5分钟；暗室曝光30秒,显像,定影。通过western-blot检测分析Akt的水平；
4.统计分析。每个实验重复3～6次,文中采用的图片均为有代表性的图片。实验数据中,正态分布的计量资料录入Microsoft excel工作表,计算组内均数±标准差(mean±SE)表示,组间比较采用t检验,以p<0.05为具有统计学显著性差异标准。
结果：
1. ox-LDL制备和鉴定：人血浆分离LDL,经5%琼脂糖电泳证实为单一条带。LDL氧化程度由TBARS值(单位：nM MDA/ml)反映。实验结果显示：新鲜LDL的值为3.69±0.47,而ox-LDL的值为30.72±2.24, ox-LDL的MDA含量明显增高,且ox-LDL的TBARS值大于LDL的TBARS值5倍以上,两者相比有显著统计学差异(p<0.05),提示ox-LDL的脂质被有效氧化。脂蛋白电泳也证实,ox-LDL琼脂糖电泳相对迁移率升高,提示LDL已被有效氧化,可用于后续实验。经BCA法测定蛋白浓度为2.56g/L。
2. ox-LDL对血小板活化指标的影响
2.1ox-LDL促进ADP诱导的血小板聚集
2.1.1在未使用阿司匹林阻断TXA2生成的情况下：单纯ox-LDL不能引起血小板聚集；当ox-LDL与ADP同时加入血小板悬液时,ox-LDL未能促进ADP诱导的血小板最大聚集率；当用60μg/ml的ox-LDL与血小板预先孵育5mmin时,可明显促进ADP诱导的血小板最大聚集率(14%±1%vs34%±2%,p<0.05)。
2.1.2使用阿司匹林阻断TXA2生成的情况下,ADP诱导的血小板最大聚集率较未加阿司匹林时下降,且聚集的血小板很快发生解聚；但用60μg／ml的ox-LDL预先孵育血小板5mmin后,发现ox-LDL仍可促进ADP诱导的聚集(12%±2%vs18%±1%,p<0.05),且形成的聚集稳定,不发生解聚反应。
2.2ox-LDL促进血小板在纤维蛋白原表面的铺展实验组与对照组血小板均发生铺展,在40倍物镜下观察发现实验组未充分铺展血小板(荧光较强且面积较小)较对照组减少；放大5倍后,实验组和对照组各选取4个视野,每个视野选取铺展面积较大的3个血小板,以photoshopCS4计算铺展面积(以像素值表示),比较两组血小板铺展面积,对照组vs实验组为21646.83±2488.06vs32197.33±9134.24,p<0.05,表明ox-LDL可以促进血小板在纤维蛋白原表面的铺展。
2.3ox-LDL促进全血状态下血小板GpⅡb/Ⅲa受体的活化以80μg／ml的ox-LDL预先与全血孵育后,可明显增加血小板表面活化的GpⅡb/Ⅲa复合物数目(38%±2%vs46.1%±l%,p<0.05),并且在不同时间点(30min、1h、2h),随着孵育时间的延长,血小板表面活化的GpⅡb/Ⅲa复合物数目呈增长趋势。
3. ox-LDL活化血小板的具体机制探讨
3.1. ox-LDL未促进血小板致密颗粒内ADP的释放在纤维蛋白原(Fib)存在的情况下,用ADP刺激血小板时,虫荧光素-荧光素酶标记的ATP曲线明显上升,表明血小板致密颗粒内容物释放,从而引起血小板的二相聚集；不加Fib的情况下,用ox-LDL预先孵育血小板5min,ADP刺激的ATP曲线未上升,且没有明显的二相聚集发生,表明ox-LDL不能促进致密颗粒内ADP的释放。
3.2ox-LDL对P2Y12受体下游信号的影响
3.2.1ox-LDL对血小板内cAMP水平无影响以公式{[3H]cAMP/([3H]ATP+[3H]cAMP) x103}计算自ATP转换的cAMP,发现ox-LDL并未使ADP诱导的cAMP水平发生明显变化(3.88±0.01vs4.02±0.78,p>0.05),且预先使用P2Y12受体特异性拮抗剂ARC69931MX阻断ADP作用后,cAMP水平明显上升,但是ox-LDL对cAMP水平无明显影响(9.90±2.11vs1162±0.20,p>0.05)。
3.2.2ox-LDL对磷酸化的VASP无影响在未使用阿司匹林阻断TXA2生成的情况下,ox-LDL预先与血小板孵育5分钟未能影响磷酸化的VASP水平(以photoshop计算实验组与对照组的电泳条带灰度值,24.77±0.28vs21.45±1.73,p>0.05),说明ox-LDL对VASP的水平无影响。
3.2.3ox-LDL可增加磷酸化Akt的水平经ox-LDL预先孵育5min后,实验组血小板内磷酸化的Akt条带较对照组明显增宽(以photoshop计算条带面积2139.00±320.58vs55333±165.13,p<0.05)；使用阿司匹林阻断TXA2生成后的结果类似；在使用ARC69931MX阻断P2Y12受体的情况下,用ox-LDL预先孵育血小板5min,并使用TXA2激活血小板,Akt的磷酸化未被阻断(3588.50±239.71vs3456.50±736.10,p>0.05),表明ox-LDL对Akt磷酸化的增强作用不受ARC69931MX的影响且与P2Y12受体无关。
结论：
1. ox-LDL可明显增强血小板的活性,表现在可促进ADP诱导的血小板最大聚集率的升高、促进血小板在纤维蛋白原表面的铺展、活化血小板表面的GpⅡb/Ⅲa受体；
2. ox-LDL促进血小板活化的作用与P2Y12受体无关；也与致密颗粒内ADP的释放无关；
3. ox-LDL活化血小板的过程中,Akt的磷酸化发挥重要作用,但具体作用途径有待进一步研究。
Background:
With the social development and the improvement of living conditions, the cholesterol intake increases and the incidence of hyperlipidemia and atherosclerosis disease is increasing year by year. As the major disease of atherosclerosis, coronary heart disease(CHD) has become the main cause of death in China. It is clear that, the thrombosis caused by the formation and instability of atherosclerotic plaque is the important pathological mechanism of CHD and acute coronary disease(ACS). Studies have showed that, oxidized low-density lipoprotein(ox-LDL), which is the main active ingredient of low-density lipoprotein(LDL), and platelet play an centrol role during the formation of thrombosis, while ox-LDL also has some impact on the platelet activation, but the exact mechanism is still a lack of in-depth study. Therefore, to study the impact of ox-LDL on platelet activation is of great significance on elucidating the pathogenesis of CHD and preventing the clinical events of ACS.
As a core part of arterial thrombosis, although there are many specific aspects to be clarified, the main steps of platelet activation has been researched adequately. When platelet is activated, thromboxane A2(TXA2) is synthesized from arachidonic acid (AA) through the cyclooxygenase-1(COX-1) pathway. Once formed, TXA2can diffuse across the membrane and activate other platelets. In platelets, there are two variants of the TXA2receptor:TPa and TPβ,which couple to the proteins Gq and G12or G13.Thrombin is rapidly generated at sites of vascular injury from circulating prothrombin and, besides mediating fibrin generation, represents the most potent platelet activator. Platelet responses to thrombin are largely mediated through G-protein-linked protease-activated receptors (PARs). Human platelets express PAR1and PAR4PAR1couples to members of the G12/13, Gq, and Gi protein families, which can promote the release of density granule. Platelets express at least two ADP receptors, P2Y1and P2Y12, which couple to Gq and Gi, respectively. The actibating Gq protein can activate phospholipase C (PLC), which can degrades the membrane phosphoinositides, releasing the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG). DAG activates intracellular protein kinase C (PKC), which causes protein phosphorylation. The release of IP3increases cytosolic levels of Ca2+The Gi can suppress the adenylate cyclase(AC) while activate the phosphatidylinositol3-kinase (PI3K). The suppression of AC can degrade the level of cyclic adenosine monophosphate(cAMP), thus dephosphorylate the vasodilator-stimulated phosphoprotein(VASP). The PI3K can promote the phosphorylation of Akt and together with VASP, activate the GpIIb/IIIa. The α-subunits of G12and G13bind Rho guanine-nucleotide exchange factors (RhoGEFs), providing for Rho-mediated cytoskeletal responses that are probably involved in the change in platelet shape.
During the activation of platelet, P2Y12receptor occupies a Key position. Because its activation can not only activate platelets, also zoom in platelet activation induced by TXA2and thrombin, and maintain stability of blood clots. Given that ox-LDL is the most important factor of hyperlipidemia and P2Y12receptor occupies a key position during platelet activation, to study the impact of ox-LDL on platelet acrivation and whether ox-LDL can activate plaelet via P2Y12receptor is a key entry point, and of great significance to clarify the mechanism of arterial thrombosis.
Objective:
1. To further clarify the effection of ox-LDL on different platelet acrivation markers;
2. To investigate the specific mechanism of ox-LDL activating platelet, and focus on whether P2Y12receptor is involved
Methods:
Ox-LDL was prepared by recipetation and oxidation of copper sulfate. Refer to literature, platelet was incubated with ox-LDL in different concentration, and then to study the aggregation, spreading induced by ADP and fibrinogn respectively, while the activated GpⅡb/Ⅲa was detectived by FUCS. Then the release of ADP, the level of cAMP, VASP and Akt were invesgated to study the specific mechanism of platelet activation indued by ox-LDL
1. Preparation and definition of ox-LDL and platelet and the preparation of platelet
a) Preparation and definition of ox-LDL:Low density lipoprotein was prepared by recipetation, and then was oxidized by copper sulfate for24-48hours to generate ox-LDL. And the degree of oxidation is tested by thiobarbituric acid(TBA), while the consentration is tested by BCA method.
b) Preparation of the platelet:Whole blood was drawn by venipunctune from healthy volunteers, and was anticoagulated by1/7volume of acid citrate dextrose (ACD). The blood was centrifuged at300g for10minutes at22℃, and platelet-rich plasma(PRP) was collected. The PRP was recentrifuged at900g for10minutes. Pelleted platelets were finally resuspended at a concentration of2.5*108/mL in Tyrode's buffer.
2. The impact of ox-LDL on platelet activation
a) The platelet was incubated with ox-LDL in different concentration, then ADP(lOuM) was added and aggregater was used to measure the impact of ox-LDL on the maximum of platelet aggregation.
b) Th chamber was incubated with fibrinogen(20μg/ml) over night, then platelet incubated with ox-LDL was added to spread for45min, then phaloidin and fluorescence microscope were used to observe the impact of ox-LDL on platelet spreading on the surface of fibrinogen.
c) To invesgate the express of fibrinogen receptor GpⅡb/Ⅲa(PAC-1) via folw cytometry.270μl tyrode's buffer was added to30μl whole blood,30μl of which e was added to the flow tubes A,B, and60ub/ml ox-LDL was added to incubated with the platelet.10μl fluid was aspirate and incubated with PAC-1and CD42b for15minutes, and then1%PFA was added to sotp the reaction. The sample was tested via FUCS in less than1hour, and SystemⅡ, EXPO TM32Multicomp software were used to analyse the date for the percent of PAC-1-FITC positive platelet.
3. The investigation of specific mechanism for ox-LDL activating platelet
a) Platelet aggregater was used to measure the impact of ox-LDL on the ATP release. When platelet activating, ATP and ADP releases from platelet density granule. The platelet was incubated with ox-LDL for5minutes, and ADP was added to activate the platelet. Platelet aggregater was used to paint the curve of ATP releasing.
b) The test of cAMP downstream of P2Y12receptor Platelet rich plasma was incubated with2μ Ci/mL [74kBq/mL][3H]-adenine and1mM asprin, then washed platelet got by centrifuge was suspensioned in tyrode's buffer. After incubation with ox-LDL for5minutes, ADP was added to active the platelet, and then cAMP and ATP were separated and detected by Liquid scintillation counter. cAMP conversed from ATP could be calculated via {[3H]cAMP/([3H]ATP+[3H]cAMP) x103}.
c) The test of phosphorylated VASP dowmstream of P2Y12receptor Platelet was incubated with ox-LDL for5minutes, and ADP was added to activate platelet. Sample were boiled with lysis buffer and loading buffer for15minutes.10%resolving gel and5%stacking gel were added to separate phosphorylated-VASP via SDS-PAGE assay. Then sample was transfered to nitrocellulosemembrance, and stained with Pierce West Pico. And finally, the membrance was exposed to film for30seconds.
d) The test of phosphorylated Akt downstream of P2Y12receptor Platelet was incubated with ox-LDL for5minutes, and ADP was added to activate platelet. Sample were boiled with lysis buffer and loading buffer for15minutes.10%resolving gel and5%stacking gel were added to separate phosphorylated-Akt via SDS-PAGE assay. Then sample was transfered to nitrocellulosemembrance, and stained with Pierce West Pico. And finally, the membrance was exposed to film for30seconds.
4Statistical analysis: The normal distribution of measurement data was expressed with X±S and the difference among the groups was tested by t test, via Microsoft Excel. A two-tailed P value<0.05was considered as significant.
Results:
1. Preparation and definition of ox-LDL LDL was prepared by recipetation, was definited by single band in the5%gel electrophoresis.The TBARS value of native LDL is3.69±0.47,and ox-LDL is30.72±2.24. Compare with native LDL group p<0.05.And the REM of ox-LDL in the5%gel electrophoresis is increased. And the consentration is2.56g/L
2. The impact of ox-LDL on platelet activation
a) ox-LDL promotes the aggregation induced by ADP Incubation with platelet for5minutes, ox-LDL(60ug/ml) can promote the aggregation induced by ADP(14%±1%vs34%±2%, p<0.05), in the absence of asprin; when the ox-LDL and ADP were added to the platelet on the same time without incubation, there is no difference in the aggregation induced by ADP; the ox-LDL can not induce platelet aggregate without ADP; there is not consentration-dependent relationship between the ox-LDL and the platelet aggregation; ox-LDL can still promote the aggregation of platelet induced by ADP in the presence of asprin(12%±2%vs18%±1%, p<0.05).
b) Ox-LDL promote the platelet spreading on fibrinogen In the presence of ox-LDL, platelets spread on the suiface of chamber more obviously in the40-fold objective. Five times magnification, three platelets covering the most surface in each four fold were selected and the covered surface was detected by software of photoshop CS4and the surface of platelet incubated with ox-LDL was vaster(21646.83±2488.06vs32197.33±9134.24, p<0.05).
c) Ox-LDL promote the activation of the GpⅡb/Ⅲa Incubated with the Ox-LDL(80μg/ml) for5minutes, the activated GpIIb/IIIa receptors of platelet were monitered by the FACS(38%±2%vs46.1%±1%, p<0.05). As time goes by, the number of activated GpⅡb/Ⅲa receptors of platelet showed a growed trend.
3. The investigation of mechanism for ox-LDL activating platelet
3.1Ox-LDL can not promote the release of ATP from density granule In the platelet, ADP and ATP are both stored in the density granule. During the platelet activating, ADP and ATP were released from the granule and transfered to the outside. So the release of ATP can reflect the release of ADP. Ox-LDL can not promote the release of ATP from density granule without fibrinogen, because the curve didn't change when the platelets were incubered with ox-LDL.
3.2The impact of ox-LDL on the down stream singnaling pathway of P2Y12receptor
a) Ox-LDL had no impact on the level of cAMP The cAMP conversed from ATP can be calculated by{[3H]cAMP/([3H]ATP+[3H]cAMP) x103}.Ox-LDL didn't change the level of cAMP no matter whether the ARC69931MX is present or not(3.88±0.01vs4.02±0.78, p>0.05;9.90±2.11vs11.62±0.20, p>0.05).
b) Ox-LDL has no effect on the phosphorylation of VASP The surface of the phosphorylation of VASP was calculated by photoshop, and there is no difference between the experimental groupe and the control group (24.77±0.28vs21.45±1.73, p>0.05). It indicats that ox-LDL has no effect on the phosphorylation of VASP.
c) Ox-LDL promote the phosphorylation of Akt Ox-LDL promote the the phosphorylation of Akt induced by ADP (the surface of Akt was mesured by photoshop553.33±165.13vs2139.00±320.58,p<0.05), and the same when the asprine is added; ARC69931MX has no effect on the phosphorylation of Akt (3588.50±239.71vs3456.50±736.10, p>0.05); indicate that the promotion of ox-LDL on the phosphorylation on Akt is not via P2Y12receptor.
Conclusion:
1. Ox-LDL can promote the activation of platelet reflected by the enhanced aggregation of platelet induced by ADP, the more spreading platelets and bigger spreading suiface on fibrinogen and the activation of the GpⅡb/Ⅲa;
2. There is no relationship between the ox-LDL activating platelet and the P2Y12receptor, so is the release of ADP from density granule;
3. The phospharylation of Akt plays an important part in the platelet activation by ox-LDL, and the further reaearch of definite mechanism is needed.
随着社会的发展和人们生活水平的提高、胆固醇摄入的增加,高脂血症及动脉粥样硬化疾病的发病率逐年上升。作为动脉粥样硬化的主要疾病、目前冠心病已成为我国人群死亡的主要病因。已明确动脉粥样硬化斑块的形成及斑块不稳定导致的局部血栓是冠心病、急性冠脉综合征(ACS)发生的重要病理环节,研究已经肯定低密度脂蛋白及血小板在其中扮演主要角色,两者是冠心病研究的重要课题。氧化型低密度脂蛋白(oxidized low-density lipoprotein, ox-LDL)是低密度脂蛋白致动脉粥样硬化的主要活性成份,近期文献报道ox-LDL除致斑块形成外、对血小板活化也有直接的促进作用,在动脉血栓的形成过程中可能发挥重要作用,但具体机制尚缺乏深入研究。因此,研究ox-LDL对血小板活化的影响对阐明冠心病的发病机理、防治ACS临床事件的发生意义重大。
作为动脉血栓形成的核心环节,尽管还有许多有待阐明的具体环节,血小板活化的主要步骤已得到充分的研究。研究显示：血小板活化时,其胞膜上的花生四烯酸(AA)在环氧化酶1(COX-1)的催化下合成血栓烷A2(TXA2),后者可扩散到胞膜外进而活化其他血小板。血小板上有两种TXA2受体：TPα和TPβ,两者均可与蛋白Gq、G12/13偶联。在血管损伤部位,循环中的凝血酶原迅速生成凝血酶,后者可与血小板表面的蛋白酶激活受体(PAR1和PAR4)反应,激活与之偶联的Gq、G12/13和Gi蛋白,启动活化过程,并促进血小板胞浆颗粒内容物的释放。其中血小板致密颗粒内的ADP,可扩散到胞膜外激活血小板表面的P2Y1和P2Y12受体,并使偶联的Gq和Gi蛋白发生构型改变。构型改变了的Gq蛋白可激活磷脂酶C (PLC),后者可催化降解磷酸肌醇生成三磷酸肌醇(IP3)和二酰基甘油(DAG). DAG能够激活血小板内蛋白激酶C (PKC)进而引起蛋白磷酸化,IP3则促进内质网内Ca2+的释放,使胞浆中Ca2+的水平升高。Gi蛋白可抑制腺苷酸环化酶(AC)的活性并促进PI3K的活性。AC活性的下降可降低胞浆环一磷酸腺苷(cAMP)的水平,使磷酸化的血管舒张剂刺激磷蛋白(VASP)去磷酸化。PI3K活性升高可使Akt磷酸化增强,进而通过mTORs并与VASP-起活化GpⅡb/Ⅲa受体。G12/13蛋白可与Rho鸟苷酸交换因子(RhoGEFs)结合,诱发Rho因子介导的细胞骨架反应,从而导致血小板形状改变。最后促进血小板的粘附、聚集,导致血小板血栓形成。
在上述血小板活化过程中,P2Y12受体占据中心地位,因为其活化不但可以激活血小板,还可以放大和维持TXA2和凝血酶引起的血小板的活化,稳定形成的血栓。在研究高脂血症对血小板功能影响的过程中,考虑到高脂血症中最重要的致病因素是ox-LDL,而P2Y12受体在血小板活化中又占有中心地位,因而选择ox-LDL对血小板活性的影响以及ox-LDL是否可通过P2Y12受体促进血小板活化是关键的切入点,对阐明动脉血栓机制具有重要的意义。
目的：
1.进一步明确ox-LDL对不同血小板活化指标的影响；
2.初步探讨ox-LDL活化血小板的具体机制,重点明确ox-LDL是否可通过P2Y12受体活化血小板。
方法：
通过改良沉淀和硫酸铜氧化法制备ox-LDL。按文献报道,将不同浓度的ox-LDL分别与成年健康者的血小板孵育5分钟后,分别检测ADP诱导的血小板最大聚集率、纤维蛋白原诱导的铺展面积和血小板表面活化的GpⅡb/Ⅲa受体数目,从而观察ox-LDL对上述血小板活化指标的影响。然后通过检测致密颗粒内ADP的释放、P2Y12受体下游信号cAMP、VASP和Akt的水平来初步探讨ox-LDL活化血小板的机制。
1. ox-LDL制备和鉴定及血小板的准备：
1.1ox-LDL的制备和鉴定：采用改良沉淀法提取人血浆低密度脂蛋白(low density lipoprotein,LDL),然后以0.01mmol/L的硫酸铜氧化24-48小时制备ox-LDL,以硫代巴比妥酸(thiobarbituric acid, TBA)检测ox-LDL的氧化修饰程度,并以BCA法测定蛋白浓度；
1.2血小板的制备：自志愿者肘部静脉取血36m1,以1：6的右旋枸橼酸ACD抗凝,常温300g离心10分钟,分离富血小板血浆,然后900g离心10分钟,分离血小板,重悬在含0.02U/mL apyrase的苔氏液(Tyrode buffer)中,并将血小板终浓度调整至2.5×108个/毫升。
2. ox-LDL对血小板活化指标的影响
2.1以10uM的ADP刺激不同浓度ox-LDL孵育后的血小板,使用双通道血小板聚集仪以1cm/mi n的走纸速度、测定苔氏液参比下的最大聚集率,观察ox-LDL对血小板聚集率的影响；
2.2血小板铺展及面积测量。实验组血小板预先与ox-LDL共孵育5min,对照组未做处理。用20μg/mL的纤维蛋白原包被chamber后,将标本接种于chamber内,以玫瑰红-鬼笔环肽荧光染料染色,37℃孵育45分钟,4%多聚甲醛固定,染色,荧光显微镜下观察。在低倍视野下比较未铺展血小板数目,在高倍视野下观察血小板铺展面积；
2.3全血法流式细胞术检测干预前后血小板膜表面纤维蛋白原受体(PAC-1) GpⅡb/Ⅲa复合物的表达。取出30μl全血,加入缓冲液稀释至300μl；每一标本取流式管A、B,均加入上述液体30μl；加入60μg/ml的ox-LDL共孵育,在30分钟、60分钟、120分钟分别取出10μll。A管PAC-1(FITC标记)、CD42b (APC标记),B管：PAC-1同型、CD42b (APC标记)；室温避光孵育15min；最后加入1%多聚甲醛,置于4℃避光保存,1小时内流式细胞仪上检测。血小板PAC-1及CD62p含量以其阳性血小板百分率表示。使用SystemⅡ软件采集样本,参照阴性空白对照,用EXPO TM32Multicomp软件分析数据,得出PAC-1-FITC抗体荧光的百分率；
3. ox-LDL活化血小板的具体机制探讨
3.1虫荧光素-荧光素酶标记的ATP曲线测定。血小板活化时,其致密颗粒内容物(如ADP和ATP)释放,可以通过血小板聚集仪和虫荧光素-荧光素酶观察血小板胞浆内内ATP含量的变化来间接反映ADP的释放。加用ox-LDL预先孵育血小板5min,再用ADP刺激血小板时,测量虫荧光素-荧光素酶标记的ATP曲线,并与不加ox-LDL的标本对照,观察ox-LDL对血小板致密颗粒内ADP释放的影响；
3.2血小板P2Y12受体下游cAMP的测定：获得富血小板血浆,加入2μCi/mL [74kBq/mL][3H]腺嘌呤和1mM的阿司匹林,37℃孵育1小时,通过[3H]腺嘌呤标记血小板内的ATP,离心分离血小板,重悬在含apyrase0.02U/mL的苔氏液中。加入ox-LDL预先孵育5分钟,恒定搅拌速度900rpm,将血小板与ox-LDL共孵育后,加入ADP刺激,然后利用柱层析法分离ATP和cAMP,并使用液闪仪测定含[3H]标记的cAMP和ATP,最后通过公式：{[3H]cAMP/([3H]ATP+[3H]cAMP) x103},计算血小板内自ATP转化生成的cAMP浓度；
3.3血小板P2Y12受体下游磷酸化VASP的测量采用ox-LDL处理血小板,加入SDS细胞裂解液和SDS蛋白上样缓冲液液制备样品,以10%的分离胶和5%的浓缩胶分别加入80μ1预染蛋白和10μl的血小板样品,60V-110V电压聚丙烯酰胺凝胶电泳1小时分离VASP；结束后进行蛋白质全湿法电转移至硝酸纤维素膜上1.5小时,加入终止液1.5小时；然后加入1：1000的p-VASP兔抗人单克隆抗体,4℃过夜孵育；以洗涤液洗涤3次(摇床上连续摇动),再加入1：2000标有辣根过氧化物酶的山羊抗兔二抗,常温孵育2小时；洗涤3次后加1：1的发光试剂发光约5分钟；暗室曝光30秒,显像,定影。通过western-blot检测分析磷酸化的VASP水平。
3.4ox-LDL影响血小板内Akt浓度的测量：采用上述方法用ox-LDL处理血小板,血小板裂解后液制备样品,以10%的分离胶和5%的浓缩胶分别加入80μl预染蛋白和10μl的血小板样品,60-110V电压聚丙烯酰胺凝胶电泳1小时分离Akt；结束后进行蛋白质全湿法电转移至硝酸纤维素膜上1.5小时,加入终止液1.5小时；然后加入1：1000加入p-Akt兔抗人单克隆抗体,4℃过夜孵育；洗涤液洗涤3次,再加入1：2000标有辣根过氧化物酶的山羊抗兔二抗,常温孵育2小时；洗涤3次后加1：1的发光试剂发光约5分钟；暗室曝光30秒,显像,定影。通过western-blot检测分析Akt的水平；
4.统计分析。每个实验重复3～6次,文中采用的图片均为有代表性的图片。实验数据中,正态分布的计量资料录入Microsoft excel工作表,计算组内均数±标准差(mean±SE)表示,组间比较采用t检验,以p<0.05为具有统计学显著性差异标准。
结果：
1. ox-LDL制备和鉴定：人血浆分离LDL,经5%琼脂糖电泳证实为单一条带。LDL氧化程度由TBARS值(单位：nM MDA/ml)反映。实验结果显示：新鲜LDL的值为3.69±0.47,而ox-LDL的值为30.72±2.24, ox-LDL的MDA含量明显增高,且ox-LDL的TBARS值大于LDL的TBARS值5倍以上,两者相比有显著统计学差异(p<0.05),提示ox-LDL的脂质被有效氧化。脂蛋白电泳也证实,ox-LDL琼脂糖电泳相对迁移率升高,提示LDL已被有效氧化,可用于后续实验。经BCA法测定蛋白浓度为2.56g/L。
2. ox-LDL对血小板活化指标的影响
2.1ox-LDL促进ADP诱导的血小板聚集
2.1.1在未使用阿司匹林阻断TXA2生成的情况下：单纯ox-LDL不能引起血小板聚集；当ox-LDL与ADP同时加入血小板悬液时,ox-LDL未能促进ADP诱导的血小板最大聚集率；当用60μg/ml的ox-LDL与血小板预先孵育5mmin时,可明显促进ADP诱导的血小板最大聚集率(14%±1%vs34%±2%,p<0.05)。
2.1.2使用阿司匹林阻断TXA2生成的情况下,ADP诱导的血小板最大聚集率较未加阿司匹林时下降,且聚集的血小板很快发生解聚；但用60μg／ml的ox-LDL预先孵育血小板5mmin后,发现ox-LDL仍可促进ADP诱导的聚集(12%±2%vs18%±1%,p<0.05),且形成的聚集稳定,不发生解聚反应。
2.2ox-LDL促进血小板在纤维蛋白原表面的铺展实验组与对照组血小板均发生铺展,在40倍物镜下观察发现实验组未充分铺展血小板(荧光较强且面积较小)较对照组减少；放大5倍后,实验组和对照组各选取4个视野,每个视野选取铺展面积较大的3个血小板,以photoshopCS4计算铺展面积(以像素值表示),比较两组血小板铺展面积,对照组vs实验组为21646.83±2488.06vs32197.33±9134.24,p<0.05,表明ox-LDL可以促进血小板在纤维蛋白原表面的铺展。
2.3ox-LDL促进全血状态下血小板GpⅡb/Ⅲa受体的活化以80μg／ml的ox-LDL预先与全血孵育后,可明显增加血小板表面活化的GpⅡb/Ⅲa复合物数目(38%±2%vs46.1%±l%,p<0.05),并且在不同时间点(30min、1h、2h),随着孵育时间的延长,血小板表面活化的GpⅡb/Ⅲa复合物数目呈增长趋势。
3. ox-LDL活化血小板的具体机制探讨
3.1. ox-LDL未促进血小板致密颗粒内ADP的释放在纤维蛋白原(Fib)存在的情况下,用ADP刺激血小板时,虫荧光素-荧光素酶标记的ATP曲线明显上升,表明血小板致密颗粒内容物释放,从而引起血小板的二相聚集；不加Fib的情况下,用ox-LDL预先孵育血小板5min,ADP刺激的ATP曲线未上升,且没有明显的二相聚集发生,表明ox-LDL不能促进致密颗粒内ADP的释放。
3.2ox-LDL对P2Y12受体下游信号的影响
3.2.1ox-LDL对血小板内cAMP水平无影响以公式{[3H]cAMP/([3H]ATP+[3H]cAMP) x103}计算自ATP转换的cAMP,发现ox-LDL并未使ADP诱导的cAMP水平发生明显变化(3.88±0.01vs4.02±0.78,p>0.05),且预先使用P2Y12受体特异性拮抗剂ARC69931MX阻断ADP作用后,cAMP水平明显上升,但是ox-LDL对cAMP水平无明显影响(9.90±2.11vs1162±0.20,p>0.05)。
3.2.2ox-LDL对磷酸化的VASP无影响在未使用阿司匹林阻断TXA2生成的情况下,ox-LDL预先与血小板孵育5分钟未能影响磷酸化的VASP水平(以photoshop计算实验组与对照组的电泳条带灰度值,24.77±0.28vs21.45±1.73,p>0.05),说明ox-LDL对VASP的水平无影响。
3.2.3ox-LDL可增加磷酸化Akt的水平经ox-LDL预先孵育5min后,实验组血小板内磷酸化的Akt条带较对照组明显增宽(以photoshop计算条带面积2139.00±320.58vs55333±165.13,p<0.05)；使用阿司匹林阻断TXA2生成后的结果类似；在使用ARC69931MX阻断P2Y12受体的情况下,用ox-LDL预先孵育血小板5min,并使用TXA2激活血小板,Akt的磷酸化未被阻断(3588.50±239.71vs3456.50±736.10,p>0.05),表明ox-LDL对Akt磷酸化的增强作用不受ARC69931MX的影响且与P2Y12受体无关。
结论：
1. ox-LDL可明显增强血小板的活性,表现在可促进ADP诱导的血小板最大聚集率的升高、促进血小板在纤维蛋白原表面的铺展、活化血小板表面的GpⅡb/Ⅲa受体；
2. ox-LDL促进血小板活化的作用与P2Y12受体无关；也与致密颗粒内ADP的释放无关；
3. ox-LDL活化血小板的过程中,Akt的磷酸化发挥重要作用,但具体作用途径有待进一步研究。
Background:
With the social development and the improvement of living conditions, the cholesterol intake increases and the incidence of hyperlipidemia and atherosclerosis disease is increasing year by year. As the major disease of atherosclerosis, coronary heart disease(CHD) has become the main cause of death in China. It is clear that, the thrombosis caused by the formation and instability of atherosclerotic plaque is the important pathological mechanism of CHD and acute coronary disease(ACS). Studies have showed that, oxidized low-density lipoprotein(ox-LDL), which is the main active ingredient of low-density lipoprotein(LDL), and platelet play an centrol role during the formation of thrombosis, while ox-LDL also has some impact on the platelet activation, but the exact mechanism is still a lack of in-depth study. Therefore, to study the impact of ox-LDL on platelet activation is of great significance on elucidating the pathogenesis of CHD and preventing the clinical events of ACS.
As a core part of arterial thrombosis, although there are many specific aspects to be clarified, the main steps of platelet activation has been researched adequately. When platelet is activated, thromboxane A2(TXA2) is synthesized from arachidonic acid (AA) through the cyclooxygenase-1(COX-1) pathway. Once formed, TXA2can diffuse across the membrane and activate other platelets. In platelets, there are two variants of the TXA2receptor:TPa and TPβ,which couple to the proteins Gq and G12or G13.Thrombin is rapidly generated at sites of vascular injury from circulating prothrombin and, besides mediating fibrin generation, represents the most potent platelet activator. Platelet responses to thrombin are largely mediated through G-protein-linked protease-activated receptors (PARs). Human platelets express PAR1and PAR4PAR1couples to members of the G12/13, Gq, and Gi protein families, which can promote the release of density granule. Platelets express at least two ADP receptors, P2Y1and P2Y12, which couple to Gq and Gi, respectively. The actibating Gq protein can activate phospholipase C (PLC), which can degrades the membrane phosphoinositides, releasing the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG). DAG activates intracellular protein kinase C (PKC), which causes protein phosphorylation. The release of IP3increases cytosolic levels of Ca2+The Gi can suppress the adenylate cyclase(AC) while activate the phosphatidylinositol3-kinase (PI3K). The suppression of AC can degrade the level of cyclic adenosine monophosphate(cAMP), thus dephosphorylate the vasodilator-stimulated phosphoprotein(VASP). The PI3K can promote the phosphorylation of Akt and together with VASP, activate the GpIIb/IIIa. The α-subunits of G12and G13bind Rho guanine-nucleotide exchange factors (RhoGEFs), providing for Rho-mediated cytoskeletal responses that are probably involved in the change in platelet shape.
During the activation of platelet, P2Y12receptor occupies a Key position. Because its activation can not only activate platelets, also zoom in platelet activation induced by TXA2and thrombin, and maintain stability of blood clots. Given that ox-LDL is the most important factor of hyperlipidemia and P2Y12receptor occupies a key position during platelet activation, to study the impact of ox-LDL on platelet acrivation and whether ox-LDL can activate plaelet via P2Y12receptor is a key entry point, and of great significance to clarify the mechanism of arterial thrombosis.
Objective:
1. To further clarify the effection of ox-LDL on different platelet acrivation markers;
2. To investigate the specific mechanism of ox-LDL activating platelet, and focus on whether P2Y12receptor is involved
Methods:
Ox-LDL was prepared by recipetation and oxidation of copper sulfate. Refer to literature, platelet was incubated with ox-LDL in different concentration, and then to study the aggregation, spreading induced by ADP and fibrinogn respectively, while the activated GpⅡb/Ⅲa was detectived by FUCS. Then the release of ADP, the level of cAMP, VASP and Akt were invesgated to study the specific mechanism of platelet activation indued by ox-LDL
1. Preparation and definition of ox-LDL and platelet and the preparation of platelet
a) Preparation and definition of ox-LDL:Low density lipoprotein was prepared by recipetation, and then was oxidized by copper sulfate for24-48hours to generate ox-LDL. And the degree of oxidation is tested by thiobarbituric acid(TBA), while the consentration is tested by BCA method.
b) Preparation of the platelet:Whole blood was drawn by venipunctune from healthy volunteers, and was anticoagulated by1/7volume of acid citrate dextrose (ACD). The blood was centrifuged at300g for10minutes at22℃, and platelet-rich plasma(PRP) was collected. The PRP was recentrifuged at900g for10minutes. Pelleted platelets were finally resuspended at a concentration of2.5*108/mL in Tyrode's buffer.
2. The impact of ox-LDL on platelet activation
a) The platelet was incubated with ox-LDL in different concentration, then ADP(lOuM) was added and aggregater was used to measure the impact of ox-LDL on the maximum of platelet aggregation.
b) Th chamber was incubated with fibrinogen(20μg/ml) over night, then platelet incubated with ox-LDL was added to spread for45min, then phaloidin and fluorescence microscope were used to observe the impact of ox-LDL on platelet spreading on the surface of fibrinogen.
c) To invesgate the express of fibrinogen receptor GpⅡb/Ⅲa(PAC-1) via folw cytometry.270μl tyrode's buffer was added to30μl whole blood,30μl of which e was added to the flow tubes A,B, and60ub/ml ox-LDL was added to incubated with the platelet.10μl fluid was aspirate and incubated with PAC-1and CD42b for15minutes, and then1%PFA was added to sotp the reaction. The sample was tested via FUCS in less than1hour, and SystemⅡ, EXPO TM32Multicomp software were used to analyse the date for the percent of PAC-1-FITC positive platelet.
3. The investigation of specific mechanism for ox-LDL activating platelet
a) Platelet aggregater was used to measure the impact of ox-LDL on the ATP release. When platelet activating, ATP and ADP releases from platelet density granule. The platelet was incubated with ox-LDL for5minutes, and ADP was added to activate the platelet. Platelet aggregater was used to paint the curve of ATP releasing.
b) The test of cAMP downstream of P2Y12receptor Platelet rich plasma was incubated with2μ Ci/mL [74kBq/mL][3H]-adenine and1mM asprin, then washed platelet got by centrifuge was suspensioned in tyrode's buffer. After incubation with ox-LDL for5minutes, ADP was added to active the platelet, and then cAMP and ATP were separated and detected by Liquid scintillation counter. cAMP conversed from ATP could be calculated via {[3H]cAMP/([3H]ATP+[3H]cAMP) x103}.
c) The test of phosphorylated VASP dowmstream of P2Y12receptor Platelet was incubated with ox-LDL for5minutes, and ADP was added to activate platelet. Sample were boiled with lysis buffer and loading buffer for15minutes.10%resolving gel and5%stacking gel were added to separate phosphorylated-VASP via SDS-PAGE assay. Then sample was transfered to nitrocellulosemembrance, and stained with Pierce West Pico. And finally, the membrance was exposed to film for30seconds.
d) The test of phosphorylated Akt downstream of P2Y12receptor Platelet was incubated with ox-LDL for5minutes, and ADP was added to activate platelet. Sample were boiled with lysis buffer and loading buffer for15minutes.10%resolving gel and5%stacking gel were added to separate phosphorylated-Akt via SDS-PAGE assay. Then sample was transfered to nitrocellulosemembrance, and stained with Pierce West Pico. And finally, the membrance was exposed to film for30seconds.
4Statistical analysis: The normal distribution of measurement data was expressed with X±S and the difference among the groups was tested by t test, via Microsoft Excel. A two-tailed P value<0.05was considered as significant.
Results:
1. Preparation and definition of ox-LDL LDL was prepared by recipetation, was definited by single band in the5%gel electrophoresis.The TBARS value of native LDL is3.69±0.47,and ox-LDL is30.72±2.24. Compare with native LDL group p<0.05.And the REM of ox-LDL in the5%gel electrophoresis is increased. And the consentration is2.56g/L
2. The impact of ox-LDL on platelet activation
a) ox-LDL promotes the aggregation induced by ADP Incubation with platelet for5minutes, ox-LDL(60ug/ml) can promote the aggregation induced by ADP(14%±1%vs34%±2%, p<0.05), in the absence of asprin; when the ox-LDL and ADP were added to the platelet on the same time without incubation, there is no difference in the aggregation induced by ADP; the ox-LDL can not induce platelet aggregate without ADP; there is not consentration-dependent relationship between the ox-LDL and the platelet aggregation; ox-LDL can still promote the aggregation of platelet induced by ADP in the presence of asprin(12%±2%vs18%±1%, p<0.05).
b) Ox-LDL promote the platelet spreading on fibrinogen In the presence of ox-LDL, platelets spread on the suiface of chamber more obviously in the40-fold objective. Five times magnification, three platelets covering the most surface in each four fold were selected and the covered surface was detected by software of photoshop CS4and the surface of platelet incubated with ox-LDL was vaster(21646.83±2488.06vs32197.33±9134.24, p<0.05).
c) Ox-LDL promote the activation of the GpⅡb/Ⅲa Incubated with the Ox-LDL(80μg/ml) for5minutes, the activated GpIIb/IIIa receptors of platelet were monitered by the FACS(38%±2%vs46.1%±1%, p<0.05). As time goes by, the number of activated GpⅡb/Ⅲa receptors of platelet showed a growed trend.
3. The investigation of mechanism for ox-LDL activating platelet
3.1Ox-LDL can not promote the release of ATP from density granule In the platelet, ADP and ATP are both stored in the density granule. During the platelet activating, ADP and ATP were released from the granule and transfered to the outside. So the release of ATP can reflect the release of ADP. Ox-LDL can not promote the release of ATP from density granule without fibrinogen, because the curve didn't change when the platelets were incubered with ox-LDL.
3.2The impact of ox-LDL on the down stream singnaling pathway of P2Y12receptor
a) Ox-LDL had no impact on the level of cAMP The cAMP conversed from ATP can be calculated by{[3H]cAMP/([3H]ATP+[3H]cAMP) x103}.Ox-LDL didn't change the level of cAMP no matter whether the ARC69931MX is present or not(3.88±0.01vs4.02±0.78, p>0.05;9.90±2.11vs11.62±0.20, p>0.05).
b) Ox-LDL has no effect on the phosphorylation of VASP The surface of the phosphorylation of VASP was calculated by photoshop, and there is no difference between the experimental groupe and the control group (24.77±0.28vs21.45±1.73, p>0.05). It indicats that ox-LDL has no effect on the phosphorylation of VASP.
c) Ox-LDL promote the phosphorylation of Akt Ox-LDL promote the the phosphorylation of Akt induced by ADP (the surface of Akt was mesured by photoshop553.33±165.13vs2139.00±320.58,p<0.05), and the same when the asprine is added; ARC69931MX has no effect on the phosphorylation of Akt (3588.50±239.71vs3456.50±736.10, p>0.05); indicate that the promotion of ox-LDL on the phosphorylation on Akt is not via P2Y12receptor.
Conclusion:
1. Ox-LDL can promote the activation of platelet reflected by the enhanced aggregation of platelet induced by ADP, the more spreading platelets and bigger spreading suiface on fibrinogen and the activation of the GpⅡb/Ⅲa;
2. There is no relationship between the ox-LDL activating platelet and the P2Y12receptor, so is the release of ADP from density granule;
3. The phospharylation of Akt plays an important part in the platelet activation by ox-LDL, and the further reaearch of definite mechanism is needed.
引文
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