Effects of Berbamine on PAF Production in Human Neutrophils and on Platelet Aggregation

The effects of berbamine, an alkaloid of dibenzylisoquinoline, on PAF produc tion in human neutrophils and on platelet aggregation induced by PAF were studied and compared with those of the calcium antagonist verapamil. Preincubation with berbamine (50 mmol / L, 100 mmol / L) or verapamil (10 mmol / L, 100 mmol / L) was shown to significantly inhibit A 23187 stimulated PAF synthesis. Berbamine and verapamil were found to inhibit platelet aggregation induced by PAF 70 pmol / L in a dose dependent manner. These results suggest that the inhibitory effects of berbamine and verapamil on A 23187 stimulated PAF synthesis in human neutrophils and PAF induced platelet aggregation are possibly brought about by inhibiting cellular calcium influx.

Human platelets inhibit liver fibrosis in severe combined immunodeficiency mice

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.

Controlling human platelet activation with calcium-binding nanoparticles

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.

Optimization study on the rehydration process of lyophilized human platelets

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.

Moisture sorption characteristics of freeze-dried human platelets

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.

Physiological Roles of Platelet-activating Factor in Mammalian and Human Reproduction

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.

口服海曲泊帕与皮下注射重组人血小板生成素用于单倍体造血干细胞移植

背景:异基因造血干细胞移植是治疗恶性血液病的重要手段,术后血小板植入延迟是常见并发症,严重影响患者生存质量,然而,目前并无标准方案来提高血小板植入率和预防血小板植入延迟。目的:对比分析口服海曲泊帕与皮下注射重组人血小板生成素促进恶性血液病患者单倍体造血干细胞移植后血小板植入的安全性及有效性。方法:回顾性分析2016年1月至2022年10月进行单倍体造血干细胞移植的163例恶性血液病患者的临床资料。+2 d开始皮下注射重组人血小板生成素的患者共72例,归为重组人血小板生成素组;+2 d开始口服海曲泊帕的患者共27例,归为海曲泊帕组;未应用海曲泊帕及重组人血小板生成素的64例患者归为空白对照组。对3组植入情况、100 d内Ⅱ-Ⅳ度急性移植物抗宿主病发生率、1年生存率、1年复发率及安全性进行分析。结果与结论:(1)中位随访时间52(12-87)个月,空白对照组、重组人血小板生成素组、海曲泊帕组患者中性粒细胞植入时间分别为(12.95±3.88)d,(14.04±3.71)d,(13.89±2.74)d,差异无显著性意义(P=0.352);血小板植入时间分别为(15.16±6.27)d,(17.67±6.52)d,(17.00±4.75)d,差异无显著性意义(P=0.287);(2)空白对照组、重组人血小板生成素组、海曲泊帕组第60天血小板完全植入率分别为64.06%,90.28%,92.59%,差异有显著性意义(P<0.001);亚组分析显示,空白对照组与重组人血小板生成素组比差异有显著性意义(P<0.001),空白对照组与海曲泊帕组比差异有显著性意义(P=0.004),重组人血小板生成素组与海曲泊帕组比差异无显著性意义(P=0.535);(3)空白对照组、重组人血小板生成素组、海曲泊帕组100 dⅡ-Ⅳ度急性移植物抗宿主病发生率分别为25.00%,30.56%,25.93%,差异无显著性意义(P=0.752);(4)巨细胞病毒血症、巨细胞病毒肺炎、肝功能损伤发生率在3组间无显著性差异(P>0.05);(5)随访期内,3组患者均未发生血栓事件;(6)结果表明,重组人血小板生成素、海曲泊帕均可提高恶性血液病患者单倍体造血干细胞移植后血小板的植入率,疗效相当且安全性良好。

Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

Role of platelet plasma membrane Ca^(2+)-ATPase in health and disease

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.

Numerical Evaluation of Residual Water Content after Freezing during the Lyophilization of Platelets

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.

为提升IPTR患者血小板输注后CCI值建立分级规避HLA抗体对应抗原方法及HLAMatchmaker的应用研究

目的:建立分级规避HLA抗体MFI阈值对应抗原方法,联合应用HLAMatchmaker表位计算法,选择供患者表位最小错配评分值,评估两种方法为免疫性血小板输注无效(Immune platelet transfusion refractoriness,IPTR)患者选择HLA相容性血小板供者,在提升血小板输注后校正增加值(CCI)的应用价值。方法:采用SPRCA法完成51例IPTR患者的7807次血小板交叉配型实验,判断其免疫反应阴/阳性结果。采用Luminex单抗原流式微珠法检测患者的HLA-I类抗体,获得不同特异性抗体对应HLA-I类抗原MFI值,并将其分组及分级,强阳性组(MFI>4000,1级)、中阳性组(1000中阳性组>弱阳性组)。强阳性和中阳性组与阴性对照组之间的SPRCA实验免疫反应阳性结果检出数存在统计学差异(P<0.001),弱阳性位组和阴性对照组之间的SPRCA实验免疫反应阳性结果检出数无统计学差异(P>0.05)。设置强阳性组为相应特异性HLA位点对应抗原1级规避阈值,中阳性组为2级规避阈值,弱阳性组为3级规避阈值,在供者血小板紧缺情况下,可以不需要规避弱阳性组。规避1和2级HLA-I类抗体对应供者抗原及选择HLAMatchmaker表位错配评分数≤7血小板供者策略,24 h内CCI值均>4.5×109/L,均可获得临床血小板输注有效。结论:在为IPTR患者选择HLA-I类相容性供者时,分级规避HLA-I类抗体对应供者抗原,综合选择供受者HLAMatchmaker表位错配评分数≤7,经血小板交叉配型实验确认为阴性结果的供者选择策略,对提升IPTR患者血小板计数具有一定实际应用价值。

南京地区HPA、HLA已知基因型血小板供者资料库的建立与库容分析

目的建立南京地区人群HPA、HLA已知基因型血小板供者资料库,从群体资料分析本地人群血小板HPA-1~6w、-15和HLA-A、-B的等位基因多态性,推算患者基因配合的匹配概率与适宜本地的库容水平。方法分别采用PCR-SSP和PCR-SBT方法对HPA-1-6w、-15和HLA-A、-B进行基因分型;统计分析HPA-1~6w、-15和HLA-A、-B的等位基因频率与HLA单倍型频率以及HPA-1~6w、-15的组合表型频率;依次推算找到HPA、HLA匹配供者的概率与适宜的库容大小。结果获得了南京地区HPA-1~6w、-15和HLA-A、-B群体多态性资料;根据统计结果评估,在不考虑ABO同型的情况下,当血小板供者库容为527人时,组合表型频率{>}0.001的患者有95%的概率在库中找到至少1例HPA-1~6w、-15相匹配的供者。建立1个库容为1875人的血小板供者库可满足单倍型{>}0.001的患者有95%的可能找到至少1例HLA-A、-B相合的供者。结论建立了本地区血小板献血者HPA、HLA基因资料库,为后续血小板库的扩建、维护与临床应用提供了重要的数据支持。

贵阳地区血小板捐献者HPA-1~6/10/15/21和HLA-A/B基因多态性研究

目的研究贵阳地区机采血小板捐献者人类血小板抗原HPA-1~6/10/15/21及人类白细胞抗原HLA-A、B基因分布及多态性。方法采用实时荧光定量PCR法(qPCR)对287名贵阳地区机采血小板捐献者进行HPA-1~6/10/15/21和HLA-A、B基因分型,列举aa\ab\bb基因及HLA-A、B等位基因的分布情况、计算aa\ab\bb基因及HLA-A、B等位基因型频率。结果287名血小板捐献者HPA-1~6/10/15/21系统中,HPA-4和HPA-10的基因型均为aa型,不具有多态性;HPA-1,2,5,6和21主要以aa型为主;仅在HPA-3和HPA-15中检出bb型;杂合度最高的是HPA-15,HPA3杂合度居次;HLA-A位点检出14个等位基因,频率最高的3个是A*02(0.36)、A*11(0.33)和A*24(0.162;HLA-B位点检出23个等位基因,频率最高的4个是B*46(0.19)、B*15(0.149、B*40(0.14)和B*13(0.13)。结论贵阳地区机采血小板捐献者HPA-1~6/10/15/21和HLA-A、B基因存在多态性,需建立该地HPA/HLA基因分型血小板供者库服务于临床。

富血小板血浆联合人脐带间充质干细胞治疗3期及以上压力性损伤患者的护理经验总结

总结2例富血小板血浆(PRP)联合人脐带间充质干细胞(huMSCs)治疗3期及以上压力性损伤(PI)患者的护理经验,旨在明确PRP联合huMSCs在压力性损伤修复过程中的重要作用,为干细胞治疗压力性损伤的临床应用提供理论依据。护理要点如下:成立干细胞移植团队,明确职责;huMSCs培养、PRP凝胶制备过程;干细胞移植术治疗全程;移植术后安全性评估;PRP凝胶在伤口愈合过程中的作用机制;PRP联合huMSCs的促进作用;创面换药管理;进行营养筛查,制订营养支持计划;实施个性化体位变换;及时评估心理状态,进行个体化心理疏导。经过精心的治疗及护理,2例患者创面均痊愈。