The Mechanism Of Type Oxidized Low Density Lipoprotein Activated Platelets

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 involvedMethods: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-LDL1. Preparation and definition of ox-LDL and platelet and the preparation of plateleta) 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 activationa) 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 plateleta) 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/L2. The impact of ox-LDL on platelet activationa) 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 platelet3.1Ox-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 P2Y12receptora) 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.