AIM:To investigate the role of human platelets in liver fibrosis.METHODS:Severe combined immunodeficiency(SCID)mice were administered CCl4and either phosphate-buffered saline(PBS group)or human platelet transfusions(h...AIM:To investigate the role of human platelets in liver fibrosis.METHODS:Severe combined immunodeficiency(SCID)mice were administered CCl4and either phosphate-buffered saline(PBS group)or human platelet transfusions(hPLT group).Concentrations of hepatocyte growth factor(HGF),matrix metallopeptidases(MMP)-9,and transforming growth factor-β(TGF-β)in the liver tissue were compared between the PBS and the hPLT groups by enzyme-linked immunosorbent assay(ELISA)and Western blotting.The effects of a human platelet transfusion on liver fibrosis included the fibrotic area,hydroxyproline content,and-smooth muscle actin(α-SMA)expression,which were evaluated by picrosirius red staining,ELISA,and immunohistochemical staining using an anti-mouse-SMA antibody,respectively.Phosphorylations of mesenchymal-epithelial transition factor(Met)and SMAD3,downstream signals of HGF and TGF-β,were compared between the two groups by Western blotting and were quantified using densitometry.Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling.Furthermore,the accumulation of human platelets in the liver 2 h after platelet transfusion was compared between normal and fibrotic livers by immunohistochemical staining using an anti-human CD41 antibody.RESULTS:The fibrotic area and hydroxyproline content in the liver were both significantly lower in the hPLT group when compared to the PBS group(fibrotic area,1.7%±0.6%vs 2.5%±0.6%,P=0.03;hydroxyproline content,121±26 ng/g liver vs 156±47 ng/g liver,P=0.04).There was less α-smooth muscle actin staining in the hPLT group than in the PBS group(0.5%±0.1%vs 0.8%±0.3%,P=0.02).Hepatic expression levels of mouse HGF and MMP-9were significantly higher in the hPLT group than in the PBS group(HGF,109±13 ng/g liver vs 88±22 ng/g liver,P=0.03;MMP-9,113%±7%/GAPDH vs 92%±11%/GAPDH,P=0.04).In contrast,the concentration of mouse TGF-β in the liver tissue was significantly lower in the hPLT group than in the PBS group(22±5ng/g liver vs 39±6 ng/g liver,P=0.02).Phosphorylation of Met was more prevalent in the hPLT group than in the PBS group(37%±4%/GAPDH vs 20%±8%/GAPDH,P=0.03).Phosphorylation of SMAD3was weaker in the hPLT group than in the PBS group(60%±12%/GAPDH vs 84%±12%/GAPDH,P=0.1),although this difference was not significant.Furthermore,a lower rate of hepatocyte apoptosis was observed in the hPLT group than in the PBS group(5.9%±1.7%vs 2.9%±2.1%,P=0.02).Significant human platelet accumulation was observed in the fibrotic liver tissues,whereas few platelets accumulated in the normal liver.CONCLUSION:Human platelets inhibit liver fibrosis in SCID mice.Increased concentration of HGF in the liver suppresses hepatic stellate cell activation,induces MMPs,and inhibits hepatocyte apoptosis.展开更多
Human platelets aggregate at sites of blood vessel damage in response to a rise in their cytosolic calcium concentration.Controlling these cytosolic calcium rises would provide a method to inhibit platelet activation ...Human platelets aggregate at sites of blood vessel damage in response to a rise in their cytosolic calcium concentration.Controlling these cytosolic calcium rises would provide a method to inhibit platelet activation and prevent the unwanted blood clots that causes heart attack and strokes.Previously we have predicted that calcium accumulation within the lumen of an infolded portion of the platelet plasma membrane called the open canalicular system(OCS)is essential for maintaining this cytosolic calcium rise.Due to its nanometer dimensions of the OCS,it has been difficult to measure or interfere with the predicted luminal calcium accumulation.Here we utilise iron oxide magnetic nanoparticles coated with the known calcium chelator,citrate,to create calcium-binding nanoparticles.These were used to assess whether an OCS calcium store plays a role in controlling the dynamics of human platelet activation and aggregation.We demonstrate that citrate-coated nanoparticles are rapidly and selectively uptaken into the OCS of activated human platelets,where they act to buffer the accumulation of calcium there.Treatment with these calcium-binding nanoparticles reduced thrombin-evoked cytosolic calcium rises,and slowed platelet aggregation and clot retraction in human platelets.In contrast,nanoparticles that cannot bind calcium have no effect.This study demonstrates that the OCS acts as a key source of calcium for maintaining cytosolic calcium rises and accelerating platelet aggregation,and that calcium-binding nanoparticles targeted to the OCS could provide an anti-platelet therapy to treat patients at risk of suffering heart attacks or strokes.展开更多
Freeze-drying is a promising method for a long-term storage of human platelets.The moisture sorption characteristics of freeze-dried human platelets(FDHPs) were studied in this paper.The moisture sorption isotherms of...Freeze-drying is a promising method for a long-term storage of human platelets.The moisture sorption characteristics of freeze-dried human platelets(FDHPs) were studied in this paper.The moisture sorption isotherms of FDHPs and freeze-dried lyophilization buffer(FDLB) were measured at 4,25,and 37°C.The experimental data were fitted to Brunauer-Emmett-Teller(BET) and Guggenheim-Anderson-de Boer(GAB) equations.There were no sig-nificant statistical differences(P>0.05) between the sorption characteristics of FDHPs and FDLB at 4 and 25°C,while FDHPs absorbed more water at 37°C.The net isosteric heat of sorption was derived.The heat for FDHPs showed an abnormal negative value at low moisture contents when 25 and 37°C data were used.Dynamic sorption experiments were carried out at 25°C with environmental water activity controlled at 0.75,0.85,and 0.90.The moisture diffusion coefficient was fitted to be 8.24×10 -12 m 2 /s when experimental data at initial time were used.These results would be helpful in choosing prehydration and storage condition for FDHPs.展开更多
Long-term preservation of human platelets will greatly reduce the risk of their shortage. Lyophilization has been proved feasible for this purpose. For the recovery of lyophilized platelets,rehydration is an important...Long-term preservation of human platelets will greatly reduce the risk of their shortage. Lyophilization has been proved feasible for this purpose. For the recovery of lyophilized platelets,rehydration is an important process. In this paper,the rehydration proc-esses for 1 mL and 2 mL samples were studied. The effects of prehydration duration(15,30,60,90,120 and 150 min) in 37°C water vapor and the concentration of rehydration solution(25%,50%,75%,100% platelet-poor plasma) on the recovery rate,MPV(mean platelet volume) and PDW(platelet distribution width) were investigated. The mass changes during the prehydration process were weighed. The optimized rehydration conditions are as follows:(1) for 1 mL sample,the prehydration duration was 15 min and for 2 mL sample the prehydration duration was 90 min;(2) the rehydration solution was 75% platelet-poor plasma. Under optimized conditions,the morphology of the rehydrated platelets kept normal and their ultrastructures kept intact,their aggregation capacity to thrombin(1 U/mL) was 82.8% of the fresh ones. These results will be helpful for designing the freeze-drying protocols for human platelets.展开更多
This review described origination, biosynthesis and functions of platelet-activating factor （PAF） in the reproductive system of mammals and human beings. The article mainly focused on biological roles of the phospho...This review described origination, biosynthesis and functions of platelet-activating factor （PAF） in the reproductive system of mammals and human beings. The article mainly focused on biological roles of the phospholipid mediator in sperm fertilization and embryonic implantation. As an autocrine product of sperm and embryos, PAF markedly stimulates sperm motility and fertilization and serves as a capacitation factor in a ligand-receptor manner, After fertilization, embryo-derived PAF improves its own development, especially from fertilized ova to blastocyst stage and is thought to act as an embryo growth factor in the same manner as on sperm. Its mechanism of action was also clarified. At the end, it was presented some advances in its clinical application, followed by discussion of some issues possibly concerning in its current application.展开更多
Pre-freezing is an important stage in freeze-drying processes.For the lyophilization of a cell,freezing not only plays a role for primary dehydration,but it also determines the amount of residual(intracellular or extr...Pre-freezing is an important stage in freeze-drying processes.For the lyophilization of a cell,freezing not only plays a role for primary dehydration,but it also determines the amount of residual(intracellular or extracellular)water,which in turn can influence the solution properties and the choice of operation parameters.The freezing of human platelets in lyoprotectant solution is theoretically investigated here.A two-parameter model and an Arrhenius expression are used to describe cell membrane permeability and its temperature dependency.It is assumed that the intracellular solution is composed of four components:sodium chloride,trehalose,serum protein and water,while the extracellular solution consists of three components.Non-ideal solution behaviors are predicted using measured data.The concentration of maximally freeze-concentrated solution is estimated on the basis of an assumption of solute hydration.The impacts of lyoprotectant composition and extracellular sub-cooling on intracellular supercooling and residual water content in the cell are analyzed.The values of activation energy of hydraulic permeability at low temperatures are tested to study their impact on the critical cooling rate.As the mass fraction extracellular lyoprotectant(trehalose+bovineserum albumin)increases from 5 wt%to 20 wt%,the intracellular water content at the end of freezing does not change,but the intracellular solution undergoes much higher super-cooling degree.Increasing the mass ratio of trehalose to bovine serum albumin does not change the intracellular water content,but can mitigate intracellular super-cooling.While 0.05 mol/kg trehalose is loaded into platelet,the total quantity of residual water at the end of freezing may raise by 4.93%.The inclusion of dimethyl sulfoxide(Me2SO)in protectant may bring negative impacts to the drying stage by increasing the residual water content and lowering the drying temperature.展开更多
Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis.Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders.O...Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis.Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders.On the other hand,hyperactive platelets lead to heart attack and stroke.Calcium is a major second messenger in platelet activation,and elevated intracellular calcium leads to hyperactive platelets.Elevated platelet calcium has been documented in hypertension and diabetes;both conditions increase the likelihood of heart attack and stroke. Thus,proper regulation of calcium metabolism in the platelet is extremely important.Plasma membrane Ca2+-ATPase(PMCA)is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux.In keeping with the important role of calcium in platelet function,PMCA is a highly regulated transporter.In human platelets,PMCA is activated by Ca2+/calmodulin,by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide.It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis.In addition,the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation.Rapid regulation by phosphorylation results in changes in the rate of platelet activation,whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction.In hypertension and diabetes,PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation.Clearly,a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.展开更多
基金Supported by Research grants from University of Tsukubathe Basic Research Support Program for Young Researcher
文摘AIM:To investigate the role of human platelets in liver fibrosis.METHODS:Severe combined immunodeficiency(SCID)mice were administered CCl4and either phosphate-buffered saline(PBS group)or human platelet transfusions(hPLT group).Concentrations of hepatocyte growth factor(HGF),matrix metallopeptidases(MMP)-9,and transforming growth factor-β(TGF-β)in the liver tissue were compared between the PBS and the hPLT groups by enzyme-linked immunosorbent assay(ELISA)and Western blotting.The effects of a human platelet transfusion on liver fibrosis included the fibrotic area,hydroxyproline content,and-smooth muscle actin(α-SMA)expression,which were evaluated by picrosirius red staining,ELISA,and immunohistochemical staining using an anti-mouse-SMA antibody,respectively.Phosphorylations of mesenchymal-epithelial transition factor(Met)and SMAD3,downstream signals of HGF and TGF-β,were compared between the two groups by Western blotting and were quantified using densitometry.Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling.Furthermore,the accumulation of human platelets in the liver 2 h after platelet transfusion was compared between normal and fibrotic livers by immunohistochemical staining using an anti-human CD41 antibody.RESULTS:The fibrotic area and hydroxyproline content in the liver were both significantly lower in the hPLT group when compared to the PBS group(fibrotic area,1.7%±0.6%vs 2.5%±0.6%,P=0.03;hydroxyproline content,121±26 ng/g liver vs 156±47 ng/g liver,P=0.04).There was less α-smooth muscle actin staining in the hPLT group than in the PBS group(0.5%±0.1%vs 0.8%±0.3%,P=0.02).Hepatic expression levels of mouse HGF and MMP-9were significantly higher in the hPLT group than in the PBS group(HGF,109±13 ng/g liver vs 88±22 ng/g liver,P=0.03;MMP-9,113%±7%/GAPDH vs 92%±11%/GAPDH,P=0.04).In contrast,the concentration of mouse TGF-β in the liver tissue was significantly lower in the hPLT group than in the PBS group(22±5ng/g liver vs 39±6 ng/g liver,P=0.02).Phosphorylation of Met was more prevalent in the hPLT group than in the PBS group(37%±4%/GAPDH vs 20%±8%/GAPDH,P=0.03).Phosphorylation of SMAD3was weaker in the hPLT group than in the PBS group(60%±12%/GAPDH vs 84%±12%/GAPDH,P=0.1),although this difference was not significant.Furthermore,a lower rate of hepatocyte apoptosis was observed in the hPLT group than in the PBS group(5.9%±1.7%vs 2.9%±2.1%,P=0.02).Significant human platelet accumulation was observed in the fibrotic liver tissues,whereas few platelets accumulated in the normal liver.CONCLUSION:Human platelets inhibit liver fibrosis in SCID mice.Increased concentration of HGF in the liver suppresses hepatic stellate cell activation,induces MMPs,and inhibits hepatocyte apoptosis.
基金Authors are deeply acknowledged to the Wellcome Trust for funding support(Seed Award in Science,Project grant no 207617/Z/17/Z).
文摘Human platelets aggregate at sites of blood vessel damage in response to a rise in their cytosolic calcium concentration.Controlling these cytosolic calcium rises would provide a method to inhibit platelet activation and prevent the unwanted blood clots that causes heart attack and strokes.Previously we have predicted that calcium accumulation within the lumen of an infolded portion of the platelet plasma membrane called the open canalicular system(OCS)is essential for maintaining this cytosolic calcium rise.Due to its nanometer dimensions of the OCS,it has been difficult to measure or interfere with the predicted luminal calcium accumulation.Here we utilise iron oxide magnetic nanoparticles coated with the known calcium chelator,citrate,to create calcium-binding nanoparticles.These were used to assess whether an OCS calcium store plays a role in controlling the dynamics of human platelet activation and aggregation.We demonstrate that citrate-coated nanoparticles are rapidly and selectively uptaken into the OCS of activated human platelets,where they act to buffer the accumulation of calcium there.Treatment with these calcium-binding nanoparticles reduced thrombin-evoked cytosolic calcium rises,and slowed platelet aggregation and clot retraction in human platelets.In contrast,nanoparticles that cannot bind calcium have no effect.This study demonstrates that the OCS acts as a key source of calcium for maintaining cytosolic calcium rises and accelerating platelet aggregation,and that calcium-binding nanoparticles targeted to the OCS could provide an anti-platelet therapy to treat patients at risk of suffering heart attacks or strokes.
基金Project supported by the Natural Science Foundation of Zhejiang Province,China(No.Y1090409)the Doctoral Fund of Ministry of Education of China(No.20070335145)
文摘Freeze-drying is a promising method for a long-term storage of human platelets.The moisture sorption characteristics of freeze-dried human platelets(FDHPs) were studied in this paper.The moisture sorption isotherms of FDHPs and freeze-dried lyophilization buffer(FDLB) were measured at 4,25,and 37°C.The experimental data were fitted to Brunauer-Emmett-Teller(BET) and Guggenheim-Anderson-de Boer(GAB) equations.There were no sig-nificant statistical differences(P>0.05) between the sorption characteristics of FDHPs and FDLB at 4 and 25°C,while FDHPs absorbed more water at 37°C.The net isosteric heat of sorption was derived.The heat for FDHPs showed an abnormal negative value at low moisture contents when 25 and 37°C data were used.Dynamic sorption experiments were carried out at 25°C with environmental water activity controlled at 0.75,0.85,and 0.90.The moisture diffusion coefficient was fitted to be 8.24×10 -12 m 2 /s when experimental data at initial time were used.These results would be helpful in choosing prehydration and storage condition for FDHPs.
基金supported by the National Natural Science Foundation of China (50606032)Specialized Research Fund for the Doctoral Program of Higher Education (20070335145)Scientific Research Foundation for Advanced Talents,Nanjing University of Aeronautics and Astronautics,China (1001-909382)
文摘Long-term preservation of human platelets will greatly reduce the risk of their shortage. Lyophilization has been proved feasible for this purpose. For the recovery of lyophilized platelets,rehydration is an important process. In this paper,the rehydration proc-esses for 1 mL and 2 mL samples were studied. The effects of prehydration duration(15,30,60,90,120 and 150 min) in 37°C water vapor and the concentration of rehydration solution(25%,50%,75%,100% platelet-poor plasma) on the recovery rate,MPV(mean platelet volume) and PDW(platelet distribution width) were investigated. The mass changes during the prehydration process were weighed. The optimized rehydration conditions are as follows:(1) for 1 mL sample,the prehydration duration was 15 min and for 2 mL sample the prehydration duration was 90 min;(2) the rehydration solution was 75% platelet-poor plasma. Under optimized conditions,the morphology of the rehydrated platelets kept normal and their ultrastructures kept intact,their aggregation capacity to thrombin(1 U/mL) was 82.8% of the fresh ones. These results will be helpful for designing the freeze-drying protocols for human platelets.
文摘This review described origination, biosynthesis and functions of platelet-activating factor （PAF） in the reproductive system of mammals and human beings. The article mainly focused on biological roles of the phospholipid mediator in sperm fertilization and embryonic implantation. As an autocrine product of sperm and embryos, PAF markedly stimulates sperm motility and fertilization and serves as a capacitation factor in a ligand-receptor manner, After fertilization, embryo-derived PAF improves its own development, especially from fertilized ova to blastocyst stage and is thought to act as an embryo growth factor in the same manner as on sperm. Its mechanism of action was also clarified. At the end, it was presented some advances in its clinical application, followed by discussion of some issues possibly concerning in its current application.
基金supported by the National Natural Science Foundation of China[grant number 51876185]archaeological artifact protection technology project of Zhejiang Province grant number 2017008].
文摘Pre-freezing is an important stage in freeze-drying processes.For the lyophilization of a cell,freezing not only plays a role for primary dehydration,but it also determines the amount of residual(intracellular or extracellular)water,which in turn can influence the solution properties and the choice of operation parameters.The freezing of human platelets in lyoprotectant solution is theoretically investigated here.A two-parameter model and an Arrhenius expression are used to describe cell membrane permeability and its temperature dependency.It is assumed that the intracellular solution is composed of four components:sodium chloride,trehalose,serum protein and water,while the extracellular solution consists of three components.Non-ideal solution behaviors are predicted using measured data.The concentration of maximally freeze-concentrated solution is estimated on the basis of an assumption of solute hydration.The impacts of lyoprotectant composition and extracellular sub-cooling on intracellular supercooling and residual water content in the cell are analyzed.The values of activation energy of hydraulic permeability at low temperatures are tested to study their impact on the critical cooling rate.As the mass fraction extracellular lyoprotectant(trehalose+bovineserum albumin)increases from 5 wt%to 20 wt%,the intracellular water content at the end of freezing does not change,but the intracellular solution undergoes much higher super-cooling degree.Increasing the mass ratio of trehalose to bovine serum albumin does not change the intracellular water content,but can mitigate intracellular super-cooling.While 0.05 mol/kg trehalose is loaded into platelet,the total quantity of residual water at the end of freezing may raise by 4.93%.The inclusion of dimethyl sulfoxide(Me2SO)in protectant may bring negative impacts to the drying stage by increasing the residual water content and lowering the drying temperature.
文摘Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis.Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders.On the other hand,hyperactive platelets lead to heart attack and stroke.Calcium is a major second messenger in platelet activation,and elevated intracellular calcium leads to hyperactive platelets.Elevated platelet calcium has been documented in hypertension and diabetes;both conditions increase the likelihood of heart attack and stroke. Thus,proper regulation of calcium metabolism in the platelet is extremely important.Plasma membrane Ca2+-ATPase(PMCA)is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux.In keeping with the important role of calcium in platelet function,PMCA is a highly regulated transporter.In human platelets,PMCA is activated by Ca2+/calmodulin,by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide.It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis.In addition,the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation.Rapid regulation by phosphorylation results in changes in the rate of platelet activation,whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction.In hypertension and diabetes,PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation.Clearly,a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.
