Influence Of High Hydrostatic Pressure On Platelet And The Role Of PPARγ In The Protection Of Platelet Under High Hydrostatic Pressures

Background and designFindings have already revealed that both platelet clumping and cohesion are sthenic (overactive) in patients with hypertension. Pertinent researches on the relationship of hypertension and platelet mainly focus on clinical observation. However, other factors besides blood pressure can also be involved in platelet activation as hypertension patients often suffer from many other abnormal conditions. Thus, the effect of hydrostatic pressure on platelet was tested in vitro in order to determine platelet activation and its extent influenced by the single factor of blood pressure.Recent researches found that platelets can not only synthesize proteins but also translate mRNA into proteins via splicing pre-mature mRNA pre-stored in polyribosome into mature mRNA. When Platelet activated, signal transduction reaction starts up and results in subsequent specific RNA translating and protein synthesis. As platelet originates from megakaryocyte, megakaryocyte mRNA has a direct influence on the functions of activated platelet. The changes of platelet in hypertension patients could be a subsequent event of megakaryocytes alternation. However, it has not been studied yet, whether high pressure can influence peripheral vascular megakaryocytes and further affect platelet states and functions.Peroxisome proliferator activated receptors (PPARs), which have a wide range of diverse functions, expresses highly in Platelet. Studies showed that platelet PPARγis active and can prohibit the activation of platelet. Platelet PPARγchanges in hypertension patients and the signal transduction ways via which PPARγcan exert its influence deserve further study.Based on the unresolved problems mentioned above, the present research firstly cultured platelet and megakaryocyte in vitro under different hydrostatic pressure(a self-designed high-hydrostatic-pressure device is made to treat platelet under different hydrostatic pressure) and then studied in the following respects: high hydrostatic pressure's influence on the shape, structure and function of both platelet and megakaryocyte; the protection of PPARγafter platelet has been intrigued by high hydrostatic pressure. This study aims to enhance the understanding of activation of platelet under high blood pressure so as to evaluate therapeutic effect of antihypertensive in new perspective. The experimental data could provide valuable basis for more effectively clinical prevention and treatment of damage in target hypertensive organSection one: influence of high hydrostatic pressure on platelet shape, structure and functionObjective:By utilizing the model of culturing platelet under high hydrostatic pressure in vitro and treating platelet under 180mmHg hydrostatic pressure for 4 different period of time(0h,1h,6h and 18h) or treating platelet for 18h but under 4 different hydrostatic pressure(0mmHg,120mmHg,180mmHg and 240mmHg), changes of platelet shape, structure and function have been studied.Method:Firstly the model of platelet culturing under high hydrostatic pressure has been established. Then platelet has been treated under 180mmHg hydrostatic pressure for 4 different period of time(0h, 1h, 6h and 18h) or treated for 18h but under 4 different hydrostatic pressure (0mmHg, 120mmHg, 180mmHg and 240mmHg), and the following indices has been tested: 1) platelet ultrastructure changes were observed by electron microscope, platelet shape parameters were determined by full automatic blood cell analyzer, LDH in platelet culture medium detected by full automatic biochemical analyzer; 2) platelet aggregation rate, 3) PAC-1 and CD40L(CD154) (surface marker of platelet activation) determined by flow cytometry; 4) the expression of CD40L(CD154), P-selectin, PPARγand Caspase3 in platelet determined by immunoblot; 5) PPARγactivity.Results:1) Platelet ultrastructure: after 18h culturing under normal atmosphere pressure, platelet cells are of the similar shape and size and in a static state with a small number of dendritic extensions and many dense granule while after high hydrostatic pressure treatment, platelet cells become different in shape and size with an increasing number of dendritic extensions. With the decrease of dense granule platelet becomes active and some specific platelet cells take on a picture of breakdown.2) Platelet parameters: have no significant difference among various time-treatment groups (all under the same atmosphere pressure). When treated for 6h or 18h under180mmHg hydrostatic pressure the mean platelet volume(MPV) declined dramatically , the platelet component distribution width(PDW) rose remarkably in the 18h time-treatment group; among the different hydrostatic pressure treatment groups, PLT dropped essentially in 240mmHg treatment group; compared with the control group, MPV and PDW decreased dramatically when treated in 180 mmHg and 240mmHg.3) LDH changes: when platelet cultured under normal atmosphere pressure LDH in upper clear solution among different time-treatment groups have no statistic difference. However, when cultured under180mmHg hydrostatic pressure, LDH concentration increases gradually and its value in 18h time-treatment group is remarkably higher than other groups. Among different hydrostatic pressure treatment groups, LDH concentration in both 240mmHg and 180mmHg treatment groups is notably higher than control group.4) Platelet aggregation rate: after 1 hour treatment of 180mmHg hydrostatic pressure the largest aggregation rate enhances greatly and after 6 hour or 18 hour treatment the 2min, 4min and largest aggregation rate elevate significantly. Among different hydrostatic pressure groups (180mmHg and 240mmHg), the 2min, 4min and largest aggregation rate increased significantly and all three aggregation rates of 180mmHg group are significant higher than those of 120mmHg group. However, the maximal aggregation rate of 240mmHg group is smaller than that of 180mmHg group but with no statistical significance.5) Platelet PAC-1: PAC-1concentration in all three hydrostatic pressure groups elevate greatly; after 1 hour treatment of 180mmHg hydrostatic pressure, the activation of platelets increase but not significant while after 6 hour or 18 hour treatment remarkable platelet activation can be detected when compared with 0 hour or 1 hour treatment. 6) As the elevation of hydrostatic pressure, the volume of both P-selectin protein increases gradually: P-selectin expression level in 180mmHg and 240mmHg group increases more dramatically than that of 120mmHg group.7) CD154 expression: The quantity of CD40L (CD154) in platelet lyses in 180mmHg and 240mmHg group declines notably; Tests by flow cytometry show that CD40L (CD154) expression in platelet surface increases remarkably with hydrostatic pressure elevating.8) PPARγexpression in platelet: PPARγexpression levels among different pressure-treat groups and different time-treat groups show no statistical difference. Yet, PPARγactivity augmented remarkably when pressure rose from 120mmHg to 180 mmHg. However, PPARγactivity under 240mmHg is greatly lower than that of 180mmHg group.9) Expression level of caspase 3 in platelet: with pressure elevation 17KD fragment of caspase 3 gradually emerges and accumulates while 32KD fragment drops notably. 17KD fragment quantity enhances remarkably in 180mmHg and 240mmHg group when compared with control and 120mmHg group. Conclusion:1) As pressure elevating, MPV↓and PCDW↑; when cultured under 180mmHg hydrostatic pressure, as time progressing similar alterations shows up.2) Higher hydrostatic pressures can boost the activated complex of GPIIb/GPIIIa and platelet aggregation.3) High hydrostatic pressures can elevate P-slectin expression level; give rise to CD40L (CD154) splitted.4) Under high hydrostatic pressure PPARγquantity has no remarkable changes but its activity increases to some extent as pressure elevates and time progress.5) Hydrostatic pressures can activate Caspase 3 and cause platelet apoptosis. Section two: The effects of high hydrostatic pressure on megakaryocyteObject:To investigate the effects of high hydrostatic pressure on the morphrage, structure of megakaryocyte 01(Meg-01) and the expressions of platelet functional related gene, and approach the influence of high hydrostatic pressure on megakaryocyte function. Method:After the model of megakaryocyte culturing under high pressure has been constructed, Meg-01 cells were cultured under 4 different high pressures (0, 120, 180, 240mmHg), then cells proliferation and apoptosis percentage were measured by MTT and TUNEL respectively, the gene expressions of GPIIb, GPIIIa, P-selectin and PPARγwere measured by RT-PCR.Result:1) Proliferation rate: compared with control, cells had significant proliferations in 120mmHg group, but in higher pressure groups, 180mmHg and 240mmHg, the proliferation of cells were remarkable fewer than that of control and 120mmHg groups and in 240mmHg group cell proliferation rate was notably smaller than that of 180 mmHg group.2) Apoptosis index: there was no significant difference in apoptosis index between 120mmHg and the control, but the apoptosis percentage of cells increased significantly in 180mmHg and 240mmHg groups.3) mRNA volumes of GPIIb/ GPIIIa: mRNA levels of GPIIb and GPIIIa in higher hydrostatic pressure groups (180mmHg and 240mmHg) increased obviously when compared with the control and 120mmHg groups. And GPIIIa mRNA level in 240mmHg group was significant higher than that of 180mmHg group.4) Volumes of P-selectin mRNA: P-selectin mRNA level in 180 mmHg group elevated significantly while in 240mmHg group it increased but with no significance.5) Volumes of PPARγmRNA: compared with control, PPARγmRNA expression was stimulated obviously when treated with 120mmHg hydrostatic pressure, but there was no significant difference among 180mmHg, 240mmHg and the control groups.Conclusion:1) Moderate hydrostatic pressure (120mmHg) can stimulate Meg-01 proliferation, but higher hydrostatic pressures (180mmHg and 240mmHg) play the opposite role, inhibit proliferation and facilitate apoptosis.2) Higher hydrostatic pressures (180mmHg and 240mmHg) can stimulate and promote the expressions of GPIIb/ GPIIIa and P-slectin in Meg-01.3) 120mmHg hydrostatic pressure facilitates PPARγexpression but under higher hydrostatic pressures PPARγexpression mainly remains the same as usual. Section three: The role of PPARγin the protection of platelet under high hydrostatic pressuresObject:To study the role of PPARγin the protection of platelet cultured in high hydrostatic pressure.Method:Platelets were divided into six groups randomly, C (normal atmosphere ), C+wy( interfered with PPARγagonist under normal atmosphere), C+gw( interfered with PPARγinhibitor under normal atmosphere), H ( high hydrostatic pressure, 180mmHg), H+wy(interfered with PPARγagonist under high hydrostatic pressure), H+gw(interfered with PPARγinhibitor under high hydrostatic pressure). Platelet aggregation rate, the concentration of calcium and NO, the expressions of iNOS,CD40L(CD154),NFκB and P-selectin protein in platelet, NO levels in culture medium were measured after being cultured for 18h.Result:1) Platelet aggregation rates: Aggregation rates among C, C+wy and C+gw groups have no statistical difference; 2min, 4min and Max aggregation rates in group H are notable larger than those of C group; All three aggregation rates of group H+wy are significant smaller than group H; those of group H+gw is relatively higher (with no significance) than those of group H.2) PAC-1: PAC-1 expressions had no statistical difference among C, C+wy and C+gw groups; PAC-1 expression in group H is remarkable higher than that of group C; PAC-1 expression in group C+wy is obvious fewer than that of group H; PAC-1 volume of group H+gw is larger than that of group H but with no statistical significance.3)CD40L(CD154) expression: CD40L(CD154) expressions in both platelet membrane and cytoplasm have no statistical difference among C, C+wy and C+gw groups; CD40L(CD154) expression in platelet membrane of group H is notably higher than that of group C while when it comes to CD40L(CD154) expression in cytoplasm the opposite is true; CD40L(CD154) expression in platelet membrane of group H+wy is obviously fewer than that of group H while when it comes to CD40L(CD154) expression in cytoplasm the opposite is also true; CD40L(CD154) expression in platelet membrane of group H+gw is slightly more than that of group H while when it comes to CD40L(CD154) expression in cytoplasm the opposite is also true ,however, both difference is insignificant;4) Calcium concentration in platelet: calcium concentrations have no statistical difference among C, C+wy and C+gw groups but calcium concentration in group H is remarkably higher than that of control group; calcium concentration in group H+wy, which is obviously smaller than that of group H, is still notably higher than that of control group; calcium concentration in group H+gw is higher than that of control and H+wy group, but the latter comparison had no significant difference. 5) NO levels in platelet and platelet upper clear solution: NO levels in platelet and platelet upper clear solution have no statistical difference among C, C+wy and C+gw groups; NO levels in platelet and platelet upper clear solution of group H are significantly lower than that of control; NO levels in platelet and platelet upper clear solution of group H+wy are remarkably higher than that of high hydrostatic pressure groups but when compared with control that still have remarkable decline; NO levels of group H+gw are obviously lower than group control and H+wy but have no significant difference when compared with group H.6) expressions of eNOS and iNOS in platelet: eNOS and iNOS expressions in platelet have no statistical difference among C, C+wy and C+gw groups; when group H is compared with control, iNOS expression increases notably and eNOS expression decreases remarkably while when group H+wy compared with group H the opposite is true; eNOS expression in group H+gw is much fewer(but with no significance)than that of group H while iNOS expression in group H+gw is significantly higher than that of group H.7) Expression of P-selectin: P-selectin expression has no statistical difference among C, C+wy and C+GW groups and H, H+wy and H+gw groups. P-selectin expression in group H is remarkably higher than that of control.8) Expression of NF-κB: NF-κB expression has no statistical difference among C, C+wy and C+gw groups and H, H+wy and H+gw groups. NF-κB expression in group H is notably greater than that of control.Conclusion:1) After cultured for 18hrs under 180mmHg hydrostatic pressure, PPARγagonist Wy14643 can greatly reduce not only platelet aggregation and activation but also the expressions of PAC-1 and CD40L (CD154) in platelet membrane. This can be achieved by either way of reducing calcium concentration and increasing eNOS expression in platelet or decreasing iNOS expression and enhancing NO volume in platelet and culture medium.2) After cultured for 18hrs under 180mmHg hydrostatic pressure, PPARγinhibitor Gw9662 can not further activate platelet which maybe because PPARγhas already been in the highly prohibitive state.