Platelet-activation factor (PAF), one of the potent proinflammatory mediators, is produced from a large range of cells, including polymorphonuclear neutrophils, monocytes, and natural killer cells. To study the role of PAF in the pathogenesis of silicosis, we determined the PAF in silicotic patients and in healthy persons. The results showed that the concentration of PAF in the plasma of silicotic patients was significantly higher than that of healthy persons. Our in vitro experimental results showed that the total numbers of fibroblasts were markedly raised with added PAF from 0 to 1 μ g/ml. Adding 1 μ g/ml PAF significantly increased the total numbers of fibroblasts after culture for 48, 72, 96 hrs. Therefore, we suggest that PAF be possibly involved in the pathogenesis of silicosis. However, the mechanism remains to be further elucidated.
Platelet Activating Factor ; g <3> ; Pathogenesis ; /mL ; Effective

BACKGROUND: Although apoptosis occurs in nucleated cells, studies show that this event also occurs in some anucleated cells such as platelets. During storage of platelets, the viability of platelets decreased, storage lesions were observed, and cells underwent apoptosis. We investigated the effects of caspase-3 inhibitor on the survival and function of platelets after different periods of storage. METHODS: Platelet concentrates were obtained from the Iranian Blood Transfusion Organization in plastic blood bags. Caspase-3 inhibitor (Z-DEVD-FMK) was added to the bags. These bags along with control bags to which no inhibitor was added were stored in a shaking incubator at 22degrees C for 7 days. The effects of Z-DEVD-FMK on the functionality of platelets were analyzed by assessing their ability to bind to von Willebrand factor (vWF) and to aggregate in the presence of arachidonic acid and ristocetin. Cell survival was surveyed by MTT assay. RESULTS: At day 4 of storage, ristocetin-induced platelet aggregation was significantly higher in the inhibitor-treated (test) than in control samples; the difference was not significant at day 7. There was no significant difference in arachidonic acid-induced platelet aggregation between test and control samples. However, at day 7 of storage, the binding of platelets to vWF was significantly higher in test than in control samples. The MTT assay revealed significantly higher viability in test than in control samples at both days of study. CONCLUSION: Treatment of platelets with caspase-3 inhibitor could increase their functionality and survival.
Apoptosis ; Arachidonic Acid ; Blood Platelets* ; Blood Transfusion ; Caspase 3* ; Cell Survival ; Incubators ; Plastics ; Platelet Aggregation ; Ristocetin ; von Willebrand Factor

Animals ; Liver ; pathology ; Liver Cirrhosis, Experimental ; metabolism ; pathology ; physiopathology ; Male ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Platelet-Derived Growth Factor beta ; metabolism

This study was aimed to investigate the effect of platelet-derived microparticles (PMP) on stimulating the proliferation of granulocyte-macrophage progenitors (CFU-GM) from umbilical cord blood. Different concentrations of thrombin were adopted to activate the platelets for releasing PMP. Flow cytometry was adopted to evaluate the efficiencies of different concentrations of thrombin to produce PMP. Umbilical cord blood mononuclear cells (MNC) were obtained from healthy donors and the MNC were isolated by Ficoll density gradient centrifugation. MNC were cultured in 2.7% methylcellulose containing different concentration of PMP and colonies were counted under an inverted microscope after 7 days. The result showed that the release rate of PMP activated by 2.0, 1.5, 1.0 and 0.5 U/ml thrombin were 28.7%, 47.7%, 50.1% and 43.9% respectively. The PMP enhanced colony formation in dose-dependent manner. The number of colonies per 2 x 10(5) MNCs in groups of PMP at different concentrations (10, 50 and 100 microg/ml) were 119.8 +/- 32.2,142.8 +/- 45.2 and 180.8 +/- 85.1 respectively. The number of colonies in the groups of PMP at 100 microg/ml and 50 microg/ml were statistically significant when compared with control group (103.0 +/- 24.8) (P < 0.05). The number of colonies per 2 x 10(5) MNC in the group of PMP (10 microg/ml) was 119.8 +/- 32.2 which was higher than that in control group, but there was no statistical significance between two groups. It is concluded that platelet activated with 1.0 U/ml thrombin can get the best release efficiency of PMP and PMP can enhance the proliferation of granulocyte-macrophage progenitor cells of umbilical cord blood.
Blood Platelets ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Fetal Blood ; cytology ; Granulocyte Precursor Cells ; drug effects ; Humans ; Macrophages ; drug effects ; Phosphatidylserines ; metabolism ; Platelet Activation ; Platelet Factor 3 ; analysis

OBJECTIVE: To elucidate the underlying mechanism of Panax notoginseng saponin (PNS) on gastric epithelial cell injury and barrier dysfunction induced by dual antiplatelet (DA). METHODS: Human gastric mucosal epithelial cell (GES-1) was cultured and divided into 4 groups: a control, a DA, a PNS+DA and a LY294002+PNS+DA group. GES-1 apoptosis was detected by flow cytometry, cell permeability were detected using Transwell, level of prostaglandins E2 (PGE2), 6-keto-prostaglandin F1α (6-keto-PGF1α) and vascular endothelial growth factor (VEGF) in supernatant were measured by enzyme linked immunosorbent assay (ELISA), expression of phosphatidylinositide 3-kinase (PI3K), phosphorylated-PI3K (p-PI3K), Akt, phosphorylated-Akt (p-Akt), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), glycogen synthase kinase-3β (GSK-3β) and Ras homolog gene family member A (RhoA) were measured by Western-blot. RESULTS: DA induced apoptosis and hyper-permeability in GES-1, reduced supernatant level of PGE2, 6-keto-PGF1α and VEGF (P<0.05). Addition of PNS reduced the apoptosis of GES-1 caused by DA, restored the concentration of PGE2, 6-keto-PGF1α and VEGF (P<0.05). In addition, PNS attenuated the alteration of COX-1 and COX-2 expression induced by DA, up-regulated p-PI3K/p-Akt, down-regulated RhoA and GSK-3β. LY294002 mitigated the effects of PNS on cell apoptosis, cell permeability, VEGF concentration, and expression of RhoA and GSK-3β significantly. CONCLUSIONS PNS attenuates the suppression on COX/PG pathway from DA, alleviates DA-induced GES-1 apoptosis and barrier dysfunction through PI3K/Akt/ VEGF-GSK-3β-RhoA network pathway.
Cyclooxygenase 1 ; Epithelial Cells/metabolism* ; Glycogen Synthase Kinase 3 beta ; Humans ; Panax notoginseng ; Phosphatidylinositol 3-Kinases/metabolism* ; Platelet Aggregation Inhibitors ; Proto-Oncogene Proteins c-akt/metabolism* ; Saponins/pharmacology* ; Vascular Endothelial Growth Factor A ; rhoA GTP-Binding Protein

PURPOSE: We investigated whether serum levels of insulin growth factor-1 (IGF-1) and insulin growth factor binding protein-3 (IGFBP-3) are valuable in predicting clinical outcomes or are correlated with other laboratory findings in children with Henoch-Schönlein purpura (HSP). METHODS: We examined 27 children who were consecutively admitted to our hospital with HSP between January 2011 and February 2012. Blood tests (C-reactive protein, white blood cell count, platelet count, erythrocyte sedimentation rate, albumin, immunoglobulin A, complement C3, antineutrophil cytoplasmic antibody, IGF-1, IGFBP-3) and urine tests were performed upon admission. IGF-1 and IGFBP-3 were resampled in the recovery phase. Controls included 473 children whose IGF-1 and IGFBP-3 were sampled for evaluating their growth, at the outpatient department of pediatric endocrinology in our hospital. IGF-1 and IGFBP-3 were compared between the HSP children and controls, and between the acute and recovery phases in HSP children. The ability of these values to predict clinical outcomes including renal involvement was analyzed using bivariate logistic regression analysis (BLRA). RESULTS: IGF-1 and IGFBP-3 were not different between the HSP children and controls (148.7±117.6 vs. 69.2±96.9, P=0.290: 3465.9±1290.9 vs. 3597.2±1,127.6, P=0.560, respectively). There was no significant difference in IGF-1 or IGFBP-3 between acute and recovery phases. Based on the BLRA, no variable, including IGF-1 and IGFBP-3, could predict clinical outcomes including the presence of nephritis. CONCLUSION: We concluded that IGF-1 and IGFBP-3 do not predict clinical outcomes of HSP, including renal involvement, in this study.
Antibodies, Antineutrophil Cytoplasmic ; Blood Sedimentation ; Child* ; Complement C3 ; Endocrinology ; Hematologic Tests ; Humans ; Immunoglobulin A ; Insulin* ; Insulin-Like Growth Factor Binding Protein 3 ; Insulin-Like Growth Factor I ; Leukocyte Count ; Logistic Models ; Nephritis ; Outpatients ; Platelet Count ; Purpura*

PURPOSE: von Willebrand disease is a common inherited bleeding disorder characterized by high degree of variable clinical presentation due to either quantitative or qualitative defects in von Willebrand factor. Its incidence in Korea is not well studied while that in western countries is extensively studied. METHODS: We classified 16 cases of vWD from 14 unrelated families based on vWF antigen, ristocetin cofactor activity, factor VIII activity and vWF multimeric patterns analysed by agarose gel electrophoresis, according to a revised classification by ISTH. RESULTS: There were 12 cases (75%) of type 1 vWD or 2M/2N with normal multimeric pattern, 3 cases (18.75%) of type 2 vWD lacking high molecular weight multimers and only 1 case of type 3 vWD with no multimers. CONCLUSION: The proportion of each vWD subtype in Korea is similar to that in western countries, however, accurate diagnosis based on ristocetin induced platelet aggregation test, factor VIII binding assay and molecular genetic diagnosis seems to be necessary for a more complete classification of vWD.
Classification ; Diagnosis ; Electrophoresis, Agar Gel ; Factor VIII ; Hemorrhage ; Humans ; Incidence ; Korea* ; Molecular Biology ; Molecular Weight ; Platelet Aggregation ; Ristocetin ; von Willebrand Disease, Type 3 ; von Willebrand Diseases* ; von Willebrand Factor

Platelet aggregation is not only an essential part of hemostasis, but also initiates acute coronary syndrome or ischemic stroke. The precise understanding of the activation mechanism of platelet aggregation is fundamental for the development of more effective agents against platelet aggregation. Adenosine diphosphate, thrombin, and thromboxane A2 activate platelet integrin alphaIIbbeta3 through G protein-coupled receptors. G protein-mediated signaling pathways, which are initiated by Gq, G12/G13 or Gi, include phospholipase C with calcium signaling, Rho signaling, protein kinase C and phosphatidylinositol 3-kinase. Rap1b, Ca2+ and diacylglycerol-regulated guanine nucleotide exchange factor I, Rap1-GTP-interacting adaptor molecule, and Akt are important proteins involved in G protein-mediated activation of integrin alphaIIbbeta3. Binding of talin-1 and kindlin-3 to cytoplasmic domains of beta3-integrin triggers a conformational change in the extracellular domains that increases its affinity for ligands, such as fibrinogen or von Willebrand factor. Fibrinogens act as bridges between adjacent platelets to generate a platelet aggregate.
Acute Coronary Syndrome ; Adenosine Diphosphate ; Blood Platelets ; Calcium Signaling ; Cytoplasm ; Fibrinogen ; Guanine Nucleotide Exchange Factors ; Hemostasis ; Ligands ; Phosphatidylinositol 3-Kinase ; Platelet Activation ; Platelet Aggregation ; Platelet Glycoprotein GPIIb-IIIa Complex ; Protein Kinase C ; Proteins ; Receptors, G-Protein-Coupled ; Stroke ; Thrombin ; Thromboxane A2 ; Type C Phospholipases ; von Willebrand Factor

Prolonged thrombocytopenia is a puzzling problem following umbilical cord blood (CB) transplantation. It might be the result of inadequate megakaryocyte progenitors and the arrest in the megakaryocyte maturation. It is an important method to solve the problem by transfusing ex-vivo expanded CB megakaryocyte progenitor cells into the patients to shorten the duration of platelet recovery. However, the most optimal condition of expansion has not been established so far. In the study, cord blood mononuclear cells (MNC) were cultured in serum-free medium with TPO, IL-3, SCF and IL-6. The numbers of MNC, CFU-MK and CD41(+) cells were measured at 0, 6, 10 and 14 days, in order to find the best cytokine combination and optimal harvest time point for clinical use. The results showed that the megakaryocyte progenitors most efficiently expanded with the cytokine combination including TPO, IL-3, SCF and IL-6. The time point of maximal CFU-MK growth is day 10. At 10 days, the numbers of CFU-MK and CD41(+) cells expanded by 6.8- and 8.8-fold respectively. In conclusion, in vitro, the cytokine cocktail including TPO, IL-3, SCF and IL-6 was the most optimal cytokine combination which stimulates the ex vivo expansion of megakaryocyte progenitors in CB MNC and serum-free medium. The maximal CFU-MK colonies were harvested at 10 days, that may be an optimal harvest time for clinical transfusion.
Antigens, CD34 ; analysis ; Cell Division ; Culture Media, Serum-Free ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Interleukin-3 ; pharmacology ; Interleukin-6 ; pharmacology ; Megakaryocytes ; cytology ; Platelet Membrane Glycoprotein IIb ; analysis ; Stem Cell Factor ; pharmacology ; Thrombopoietin ; pharmacology

OBJECTIVETo study the effects on adherence of hematopoietic stem/progenitor cells, PF4 was assessed alone or in combination with IL-3 for effects on the total adherence and various kinds of adhesion molecules of KG1a cells as well as actin polymerization in KG1a cells.

METHODSThe total adherence was assayed by crystal violet dye staining. The adhesion molecule expression was determined by FACS analysis. These adhesion molecule monoclonal antibodies individually blocked total adherence by MTT. F-actin content was monitored by fluorospectrophotometry.

RESULTS100 ng/ml PF4 could increase the total adherence of KG1a cells by 80%. 20 ng/ml IL-3 could increase the total adherence of KG1a cells by 96%. When PF4 and IL-3 were combined, the total adherence could be promoted by 97%. Exposure of 1 x 10(6) cells/ml of KG1a cells to 100 ng/ml PF4 the increased total adherence of KG1a cells was mediated by PECAM-1 (CD31), CD44, LFA-1 (CD11a) and Mac-1 (CD11b) but not by P-selectin (CD62P) and E-selectin (CD62E). These adhesion molecule monoclonal antibodies could individually block total adherence for 34%-43%. Similar phenomenon was observed when IL-3 was added onto KG1a cells. Further study found that PF4 induced actin polymerization of KG1a cells.

CONCLUSIONSOur study indicated that PF4 promoted total adherence, as well as several adhesion molecule expression and actin polymerization of KG1a cells. The results suggest that PF4 may have therapeutic utility along with other cytokines by enhancing the total adhesion of hematopoietic stem/progenitor cells to promote the homing.

Actins ; metabolism ; Cell Adhesion ; Cell Adhesion Molecules ; metabolism ; Chemokines, CXC ; metabolism ; Endothelium, Vascular ; metabolism ; Hematopoietic Stem Cells ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-3 ; pharmacology ; Platelet Factor 4 ; pharmacology ; Polymers