| Background:Several studies indicated platelet plays an important role in the pathogenesis of atherosclerosis and cardiovascular disease. Platelet activation may trigger the formation of atherosclerosis, rupture of unstable plaque, and initiate thrombosis, which leads to the occurrences of fatal events.There are several signaling pathways initiate platelet activation. ADP receptor, P2Y12 plays a critical role in mediating the formation of arterial thrombosis. When ADP binds to P2Y12 receptor, stimulation of P2Y12 subsequently activate phosphatidylinositol 3-kinase (PI3K) and inhibit Adenylyl Cyclase (AC). The inhibition of AC further decrease platelet cAMP concentration, results in dephosphorylation of Vasodilator Stimulated phosphoprotein (VASP), VASP is associated with the changing of platelet morphology and the reorganization of actin cyskeleton, which mediating the spreading of platelet and platelet aggreagation. On the other hand, activated PI3K leads to the phosphorylation of Akt. Finally, it causes the conformation change of GPⅡb/Ⅲa and triggers the formation of platelet aggregates.Several studies indicated hypercholesterolemia induces platelet hypersenstivity to agonist. It has reported that low-density lipoprotein cholesterol (LDL-C) increase platelet function. However, the mechanism of LDL-C effect on platelet function remains controversy. Furthermore, the contribution of P2Y12 receptor-mediated pathway among high level LDL-C is still poorly understood.Objective:1. To investigate the effect of high levels LDL-C to platelet function among hypercholesterolemia via clinical research and evaluate the contribution of high levels LDL-C to P2Y12 receptor-mediated pathway.2. To clarify the impact of high levels LDL-C combined with low high-density lipoprotein cholesterol (HDL-C) to platelet function in the patients with hypercholesterolemia.3. To observe the difference of platelet function after the treatment of statins within high levels LDL-C combined with low HDL-C patients.Methods:1. Blood collection and preparation of platelet The blood was collected in the morning from the resting and fasting patients by a 21G needle without stasis. The blood was then stored in acid/citrate/dextrose (ACD,1:9) for platelet studies; in serum vacutainers for lipid profile. Pelleted platelets were finally resuspended at a concentration of 3.0* 105/ul.:2. Lipid assay Lipid profile, included Total Cholesterol(TC), Triglycerides(TG), HDL-C and LDL-C were determined enzymatically on a Hitachi 7600 Automatic Biochemical Analyzer.3. Platelet Aggregation Measurement The examination was performed by optical aggregometry in Platelet-Rich-Plasma (PRP) using a platelet aggregometer. By adopting PPP as reference, the PRP was incubated at 37℃ in the aggregometer followed by stimulated with Adenosine Diphosphate 10μmol/L in a constant stirring rate of 1000 rpm. Platelet aggregation was recorded for 5 min and the analyzed parameter was Maximal Platelet Aggregation (MPAG%).4. Whole Blood Flow Cytometry We obtained PAC-1 FITC, anti-CD62p PE, and anti-CD42b APC to evaluate the function of platelet by Becton Dickinson FACSCanto Flow Cytometer. Fluorescence of 10000 platelets was recorded using BD FACSDiva Software 6.1.3.5.. The test of phosphorylated Akt and VASP downstream of P2Y12 receptor Platelet pellets was resuspended in Tyrodes buffer containing apyrase and ADP was added to activate platelet. Sample were boiled with SDS lysis buffer and loading buffer for 10 minutes. Proteins were separated on 10% resolving gel and 5% stacking gel and then transferred onto NC membranes. Finally, the membrane was exposed to film. Densitometric analyses were performed by Photoshop CS4.ResultsPart 1:The effect of high levels LDL-C to platelet function among hypercholesterolemia.We included 33 high levels LDL-C patients and the control group consisted 35 normocholesterolemic volunteers. The maximal platelet aggregation was higher aurong high levels LDL-C patients than controls though no statistical difference between them (33.03±15.87% vs 29.05±17.75%, P>0.05). The surface expression of platelet CD62p and PAC-1 were increase in high levels LDL-C group as compared with control with statistical difference (1.72±0.96%vs 0.90±0.77%, P<0.001 and 4.33±2.42% vs 2.63±2.03%, P<0.01, respectively). The maximal platelet aggregation was decrease after the treatment of statins (33.03±15.87% vs 26.73±±9.91%, P=0.073). By administration statins, the expression of platelet activation marker, CD62P and PAC-1 were reduced significantly among high levels LDL-C patients (1.72±0.96% vs 0.85±0.72%, P<0.001 and 4.33±2.42% vs 1.50±1.07%, P<0.001, respectively).Part 2:The preliminary study of high levels LDL-C effect on P2Y12 receptor--mediated pathwayADP-induced platelet aggregation was increased in high levels LDL-C patients than control (33.03±15.87% vs 29.05±17.75%, P>0.05). ADP-induced PAC-1 expression was increased among high levels LDL-C patients as compared with control (69.26±12.23% vs 62.60±11.72%, P<0.05). In response to ADP, a significant increase in Akt phosphorylation was detected in high levels LDL-C patients compared with controls (the surface of p-Akt was mesured by photoshop 3679.00±438.59 vs 6116.33±1488.62, P<0.05). ADP-induced VASP dephosphorylation was increased in high levels LDL-C patients than controls (the surface of p-VASP was mesured by photoshop 7855.66±793.69 vs 6349.00±890.20, P=0.094).Part 3:The effect of high levels LDL-C combined with low HDL-C to platelet function in the patients with hypercholesterolemia along with observe the difference of platelet function after the treatment of statins.We included 62 high levels LDL-C patients,14 patients were excluded in case of loss to follow up. Therefore, a consecutive cohort of 48 patients was analysed.23 of these patients were high levels LDL-C combined with low HDL-C patients.25 of these patients were high levels LDL-C combined with normal HDL-C. The control group consisted 35 normocholesterolemic volunteers. Lipid profile, including TC, TG, HDL-C and LDL-C was decrease after the treatment of statins.The maximal platelet aggregation was higher among high levels LDL-C combined with low HDL-C than high levels LDL-C combined with normal HDL-C patients and controls though no statistical difference between them (29.05±17.75%, 31.54±15.12%,37.15±18.85%, P=0.222). Our data showed the increase platelet surface expression of CD62p and PAC-1 in high levels LDL-C combined with normal HDL-C as compare with controls (1.62±1.01% vs 0.90±0.77%, P=0.004 and 4.21 ±2.50% vs 2.63±2.03%, P=0.014, respectively). Moreover, the expression of platelet CD62p and PAC-1 were greater among high levels LDL-C combined with low HDL-C patients than high levels LDL-C combined with normal HDL-C patients with statistical differences (2.60±1.19% vs 1.62±1.01%, P=0.004 and 5.90±2.58% vs 4.21 ±2.50%, P=0.026, respectively).There were significant negative linear relationship between HDL-C and platelet CD62p, HDL-C and platelet PAC-1 (r=0.195, P=0.002 and r=0.431, P=0.034, respectively) in high levels LDL-C patients. Furthermore, the data showed a positive correlation between LDL-C/HDL-C with platelet CD62p and PAC-1 within high levels LDL-C patients (r=0.345, P=0.001and r=0.395, P=0.024, respectively).By administration statins, the maximal platelet aggregation was decrease in both and high levels LDL-C combined with normal HDL-C patients and high levels LDL-C combined with low HDL-C patients (high levels LDL-C combined with low HDL-C group:37.15±18.85% vs 26.18±12.59%, P=0.023, high levels LDL-C combined with low HDL-C group:31.54±15.12% vs 26.85±10.62%, P=0.231).The platelet activation marker, CD62p and PAC-1 were decreased significantly after the treatment of statins (high levels LDL-C combined with low HDL-C group:platelet CD62p:2.60±1.19% vs 1.51±1.67%, P<0.01, platelet PAC-1:5.90±2.58% vs 2.33±1.57%, P<0.001. High levels LDL-C combined with low HDL-C group:platelet CD62p:1.62±1.01%妇vs 0.88±0.74%, P<0.01, platelet PAC-1:4.21±2.50% vs 1.36±1.02%,P<0.001).The expression of platelet CD62p and PAC-1 remains higher in high levels LDL-C combined with low HDL-C than high levels LDL-C combined with normal HDL-C group after 2 months of statins therapy (platelet PAC-12.33±1.57% vs 1.36±1.02%, P=0.0201, and platelet CD62p,1.51±1.67% vs 0.88±0.74%, P=0.116, respectively).Conclusion1. High levels LDL-C enhance platelet activation and platelet aggregation. P2Y12 receptor pathway was involved in platelet hyperactivity among high levels LDL-C patients.2. High levels LDL-C combined with low HDL-C further increase platelet reactivity in the patients with hypercholesterolemia.3. The platelet reactivity remains higher among high levels LDL-C combined with low HDL-C patients as compare with high levels LDL-C combined with normal HDL-C patients regardless the treatment of statins. |
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