OBJECTIVETo study the pathological and clinical characteristics of a patient with spontaneous platelet aggregation of his giant and morphologically abnormal platelets.

METHODSPlatelet size and structure were observed under light microscope and electron microscope. Platelet aggregation was measured turbidometrically. Platelet glycoproteins (GP) were analyzed using flow cytometry. PCR and DNA sequencing were performed to identify the gene abnormality.

RESULTSThe patient had spontaneous platelet aggregation of giant platelets with thickened plasma membrane and increased number of granules in various shapes. Aspirin and ticlopidine did not affect the spontaneous aggregation. The expression of GP I b, GP II b, GP III a and P-selectin in the platelet membrane were in normal range. Results of gene analyses for GP I balpha, GP I bbeta and GPIX were also normal.

CONCLUSIONBoth morphological and functional abnormalities of the platelets from the patient were clearly distinguishable from that of other hereditary giant platelet disorders. It would probably represent a novel platelet disorder which had not been reported to date.

Aspirin ; pharmacology ; Bernard-Soulier Syndrome ; metabolism ; pathology ; Blood Platelet Disorders ; metabolism ; pathology ; Cell Size ; physiology ; Child ; Cytoplasmic Granules ; pathology ; ultrastructure ; Female ; Humans ; Platelet Aggregation ; drug effects ; physiology ; Platelet Aggregation Inhibitors ; pharmacology ; Platelet Membrane Glycoproteins ; genetics ; metabolism ; Ticlopidine ; pharmacology

Lysosomal dysfunction is a common pathological feature of neurodegenerative diseases. GTP-binding protein type A1 (GBA1) encodes beta-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase. Homozygous mutations in GBA1 cause Gaucher disease, the most common lysosomal storage disease, while heterozygous mutations are strong risk factors for Parkinson's disease. However, whether loss of GCase 1 activity is sufficient for lysosomal dysfunction has not been clearly determined. Here, we generated human neuroblastoma cell lines with nonsense mutations in the GBA1 gene using zinc-finger nucleases. Depending on the site of mutation, GCase 1 activity was lost or maintained. The cell line with GCase 1 deficiency showed indications of lysosomal dysfunction, such as accumulation of lysosomal substrates, reduced dextran degradation and accumulation of enlarged vacuolar structures. In contrast, the cell line with C-terminal truncation of GCase 1 but with intact GCase 1 activity showed normal lysosomal function. When alpha-synuclein was overexpressed, accumulation and secretion of insoluble aggregates increased in cells with GCase 1 deficiency but did not change in mutant cells with normal GCase 1 activity. These results demonstrate that loss of GCase 1 activity is sufficient to cause lysosomal dysfunction and accumulation of alpha-synuclein aggregates.
Cell Line ; Enzyme Activation/genetics ; Gene Knockout Techniques ; Gene Order ; Genetic Loci ; Glucosylceramidase/genetics/*metabolism ; Humans ; Lysosomes/*metabolism ; Mutation ; *Protein Aggregation, Pathological/genetics ; Protein Binding ; Zinc Fingers ; alpha-Synuclein/chemistry/*metabolism

OBJECTIVETo investigate the effects and underlying molecular mechanisms of icariin (ICA) on self-renewal and differentiation of neural stem cells (NSCs).

METHODSNSCs were derived from forebrains of mice embryos by mechanical dissociation into single cell suspension. The self-renewal of NSCs was measured by neurosphere formation assay. The proliferation of NSCs was detected by water-soluble tetrazolium (WST) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Protein expression of neuron-specific marker tubulin-βIII(TuJ1) and astrocyte-specific marker glial fibrillary acidic protein (GFAP) were measured by immunofluorescence and Western blotting. Using microarray, the differentially expressed genes (DEGs) were screened between NSCs with or without ICA treatment. The signaling pathways enriched by these DEGs and their role in mediating effects of ICA were analyzed.

RESULTSICA significantly promoted neurosphere formation of NSCs cultured in growth protocol in a dose-dependent manner and achieved the maximum effects at 100 nmol/L. ICA also increased optical absorbance value and EdU incorporation into nuclei of NSCs. ICA had no significant effects on the percentage of TuJ1 or GFAP-positive cells, and TuJ1 or GFAP protein expression in NSCs cultured in differentiation protocol. A total of 478 genes were found to be differentially regulated. Among signaling pathways significantly enriched by DEGs, mitogen activated protein kinase (MAPK) pathway was of interest. Blockade of extracellular signal-regulated kinase (ERK)/MAPK, other than p38/MAPK subfamily pathway partially abolished effects of ICA on neurosphere formation and EdU incorporation of NSCs.

CONCLUSIONICA can promote the selfrenewal of NSCs at least partially through ERK/MAPK signaling pathway.

Animals ; Cell Aggregation ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; genetics ; Deoxyuridine ; analogs & derivatives ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flavonoids ; pharmacology ; Gene Expression Regulation ; drug effects ; MAP Kinase Signaling System ; drug effects ; genetics ; Mice ; Neural Stem Cells ; cytology ; drug effects ; enzymology

OBJECTIVETo assess the platelet and plasma concentrations of fibronectin (Fn) and fibrinogen (Fg) in congenital fibrinogenopenic (FgP) patients and explore their role in inducing platelet adhesion and aggregation.

METHODSA FgP family was selected as study group and the platelets isolated and purified to assess concentrations of Fn and Fg in platelets, alpha-granules and plasma with Western blotting, immuofluoresence staining and flow cytometry (FACS), respectively, the expression of platelets GP II b/III a by FACS.

RESULTSThe concentration of platelets Fn in FgP patients is higher than that in controls, and is higher in homozygote than in heterozygote. In contrast, plasma Fn levels were identical in all samples. The amount of platelet Fg from FgP patients is lower than that from the controls and positively correlated with the concentration of their plasma Fg. No difference in the expression of platelet GP II b/III a had been found.

CONCLUSIONIt suggested that increased platelet Fn could partially compensate the lack of Fg and lead the platelet adhesion and aggregation.

Afibrinogenemia ; congenital ; metabolism ; pathology ; Blood Platelets ; metabolism ; pathology ; Cell Adhesion ; physiology ; Female ; Fibrinogen ; genetics ; metabolism ; Fibronectins ; blood ; genetics ; metabolism ; Heterozygote ; Homozygote ; Humans ; Male ; Pedigree ; Platelet Aggregation ; physiology ; Platelet Membrane Glycoproteins ; metabolism

OBJECTIVETo test the function of endothelial cells transfected eNOS gene.

METHODSTo test the function of eNOS-overexpressing endothelial cells inhibiting smooth muscle cell proliferation and platelet aggregation in vitro by colorimetry, MTT and (3)H-TdR permeate, respectively, in four different endothelial cells groups.

RESULTSThe platelet aggregation is significantly decreasing between the groups of EC and eNOS at the different time (P < 0.05), is 42.2 and 32.6 separately at the time of 120 h. The SMC proliferation is significantly decreasing between the groups of EC and eNOS at the different time (P < 0.05). It is 0.28 and 0.22 by MTT test, 4691 and 3995 by (3)H-TdR permeate separately at the time of 120 h.

CONCLUSIONSeNOS-overexpressing endothelial cells inhibited significantly smooth muscle cells proliferation and platelet aggregation in vitro; which shows powerful effect 48 hours post transfection and lasts up to 120 hours at the least; and were held back by L-NAME.

Animals ; Cell Communication ; Cell Proliferation ; Coculture Techniques ; Dogs ; Endothelial Cells ; cytology ; Endothelium, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; Nitric Oxide ; metabolism ; physiology ; Nitric Oxide Synthase Type III ; genetics ; Platelet Aggregation ; physiology ; Transfection

In response to viral infection, RIG-I-like RNA helicases detect viral RNA and signal through the mitochondrial adapter protein VISA. VISA activation leads to rapid activation of transcription factors IRF3 and NF-κB, which collaborate to induce transcription of type I interferon (IFN) genes and cellular antiviral response. It has been demonstrated that VISA is activated by forming prion-like aggregates. However, how this process is regulated remains unknown. Here we show that overexpression of HSC71 resulted in potent inhibition of virus-triggered transcription of IFNB1 gene and cellular antiviral response. Consistently, knockdown of HSC71 had opposite effects. HSC71 interacted with VISA, and negatively regulated virus-triggered VISA aggregation. These findings suggest that HSC71 functions as a check against VISA-mediated antiviral response.
Adaptor Proteins, Signal Transducing ; biosynthesis ; chemistry ; genetics ; metabolism ; Cell Aggregation ; genetics ; GPI-Linked Proteins ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; HSC70 Heat-Shock Proteins ; genetics ; metabolism ; Heat-Shock Response ; genetics ; Humans ; Interferon Regulatory Factor-3 ; genetics ; metabolism ; Interferon-beta ; genetics ; NF-kappa B ; genetics ; Prions ; metabolism ; Receptors, Retinoic Acid ; metabolism ; Viruses ; drug effects ; metabolism ; pathogenicity

To investigate the functional role of KAI1/CD82, a metastasis suppressor for human prostate cancer, in the regulation of homotypic cell adhesion, we transfected KAI1 cDNA into DU 145 human prostate cancer cells and established stable transfectant clones with high KAI1/CD82 expression. The KAI1 transfectant cells exhibited significantly increased homotypic cell aggregation in comparison with the control transfectant cells. This aggregation of the KAI1 transfectants was further enhanced upon exposure to anti-CD82 antibody, suggesting that KAI1/CD82 may be involved in the intracellular signaling for the cell adhesion. Among several signal pathway inhibitors tested, PP1, an inhibitor of Src family kinases, significantly suppressed homotypic aggregation of the KAI1 transfectant cells. Ligation of KAI1/CD82 with anti-CD82 antibody increased endogenous Src kinase activity of the KAI1 transfectant cells. When different types of src expression constructs were retransfected into the KAI1-transfected DU 145 cells, kinase-negative mutant src transfectant cells exhibited much lower homotypic aggregation than the mock cells transfected with an empty vector. Moreover, homotypic aggregation of the mutant src transfectant cells was not enhanced by KAI1/CD82 ligation with anti- CD82 antibody. These results suggest that Src mediates the intracellular signaling pathway of KAI1/CD82 for the induction of homotypic adhesion of human prostate cancer cells.
Adenocarcinoma/*metabolism/pathology/*secondary ; Antigens, Surface ; Cell Adhesion/genetics ; Cell Aggregation/genetics ; Gene Expression Regulation ; Genes, Tumor Suppressor ; Genes, src ; Human ; Male ; Membrane Glycoproteins/genetics/*metabolism ; Prostatic Neoplasms/*metabolism/pathology/*secondary ; Signal Transduction/genetics ; Tumor Cells, Cultured ; src-Family Kinases/genetics/metabolism

The opening of mitochondrial permeability transition pore (MPTP) plays a critical role in platelet activation. However, the potential trigger of the MPTP opening in platelet activation remains unknown. Inflammation is the crucial trigger of platelet activation. In this study, we aimed to explore whether and how the important inflammatory cytokine IL-17 is associated with MPTP opening in platelets activation by using MPTP inhibitor cyclosporine-A (CsA). The mitochondrial membrane potential (ΔΨm) was detected to reflect MPTP opening levels. And the platelet aggregation, activation, and the primary signaling pathway were also tested. The results showed that the MPTP opening levels were increased and Δψm reduced in platelets administrated with IL-17. Moreover, the levels of aggregation, CD62P, PAC-1, P53 and the phosphorylation of ERK2 were enhanced along with the MPTP opening in platelets pre-stimulated with IL-17. However, CsA attenuated these effects triggered by IL-17. It was suggested that IL-17 could induce MPTP opening through ERK2 and P53 signaling pathway in platelet activation and aggregation.
Blood Platelets ; cytology ; drug effects ; metabolism ; Cell Separation ; Cyclosporine ; pharmacology ; Dual Specificity Phosphatase 2 ; genetics ; metabolism ; Gene Expression Regulation ; Humans ; Interleukin-17 ; metabolism ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; agonists ; antagonists & inhibitors ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; P-Selectin ; genetics ; metabolism ; Phosphorylation ; drug effects ; Platelet Activation ; drug effects ; Platelet Aggregation ; drug effects ; Primary Cell Culture ; Signal Transduction ; Tumor Suppressor Protein p53 ; genetics ; metabolism

BACKGROUND: High on-clopidogrel platelet reactivity could be partially explained by loss-of-function alleles of CYP2C19, the enzyme that converts clopidogrel into its active form. Shexiang Tongxin Dropping Pill (STDP) is a traditional Chinese medicine to treat angina pectoris. STDP has been shown to improve blood flow in patients with slow coronary flow and attenuate atherosclerosis in apolipoprotein E-deficient mice. However, whether STDP can affect platelet function remains unknown. OBJECTIVE: The purpose of this study is to examine the potential effects of STDP on platelet function in patients undergoing percutaneous coronary intervention (PCI) for unstable angina. The interaction between the effects of STDP with polymorphisms of CYP2C19 was also investigated. DESIGN, PARTICIPANTS AND INTERVENTION: This was a single-center, randomized controlled trial in patients undergoing elective PCI for unstable angina. Eligible subjects were randomized to receive STDP (210 mg per day) plus dual antiplatelet therapy (DAPT) with clopidogrel and aspirin or DAPT alone. MAIN OUTCOME MEASURES: The primary outcome was platelet function, reflected by adenosine diphosphate (ADP)-induced platelet aggregation and platelet microparticles (PMPs). The secondary outcomes were major adverse cardiovascular events (MACEs) including recurrent ischemia or myocardial infarction, repeat PCI and cardiac death; blood biomarkers for myocardial injury including creatine kinase-MB isoenzyme (CK-MB) and high-sensitive troponin I (hsTnI); and biomarkers for inflammation including intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1) and galectin-3. RESULTS: A total of 118 subjects (mean age: [66.8 ± 8.9] years; male: 59.8%) were included into analysis: 58 in the control group and 60 in the STDP group. CYP2C19 genotype distribution was comparable between the 2 groups. In comparison to the control group, the STDP group had significantly lower CK-MB (P < 0.05) but similar hsTnI (P > 0.05) at 24 h after PCI, lower ICAM-1, VCAM-1, MCP-1 and galectin-3 at 3 months (all P < 0.05) but not at 7 days after PCI (P > 0.05). At 3 months, the STDP group had lower PMP number ([42.9 ± 37.3] vs. [67.8 ± 53.1] counts/μL in the control group, P = 0.05). Subgroup analysis showed that STDP increased percentage inhibition of ADP-induced platelet aggregation only in slow metabolizers (66.0% ± 20.8% in STDP group vs. 36.0% ± 28.1% in the control group, P < 0.05), but not in intermediate or fast metabolizers. The rate of MACEs during the 3-month follow-up did not differ between the two groups. CONCLUSION: STDP produced antiplatelet, anti-inflammatory and cardioprotective effects. Subgroup analysis indicated that STDP inhibited residual platelet reactivity in slow metabolizers only. TRIAL REGISTRATION This study was registered on www.chictr.org.cn: ChiCTR-IPR-16009785.
Adenosine Diphosphate ; Angina, Unstable/chemically induced* ; Animals ; Biomarkers ; Clopidogrel ; Cytochrome P-450 CYP2C19/genetics* ; Drugs, Chinese Herbal ; Galectin 3 ; Humans ; Intercellular Adhesion Molecule-1 ; Male ; Mice ; Percutaneous Coronary Intervention/adverse effects* ; Platelet Aggregation Inhibitors/adverse effects* ; Vascular Cell Adhesion Molecule-1/genetics*

BACKGROUNDTissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds.

METHODSDecellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 x 10(6) cells/cm(2) and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA).

RESULTSEPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer. Platelets adhesion experiments suggested that the neo-endothelium was non-thrombogenic. The expression levels of eNOS and t-PA genes in the neo-endothelium were quite similar to those in human umbilical vein endothelial cells.

CONCLUSIONSEPCs isolated from the human umbilical cord blood can differentiate into endothelial cells in vitro and form a functional endothelium atop decellularized heart valve scaffolds. Thus, EPCs may be a promising cell source for constructing tissue-engineered heart valves.

Animals ; Cell Proliferation ; Endothelial Cells ; cytology ; metabolism ; Heart Valve Prosthesis ; Heart Valves ; cytology ; metabolism ; ultrastructure ; Humans ; Immunohistochemistry ; Microscopy, Electron, Scanning ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Platelet Aggregation ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; Swine ; Tissue Engineering ; methods ; Tissue Plasminogen Activator ; genetics ; metabolism ; Umbilical Cord ; cytology