A model of platelet aggregation involving multiple interactions of thrombospondin-1, fibrinogen, and GPIIbIIIa receptor

Thrombospondin-1 (TSP) may, after secretion from platelet alpha granules, participate in platelet aggregation, but its mode of action is poorly understood. We evaluated the capacity of TSP to form inter-platelet cross-bridges through its interaction with fibrinogen (Fg), using either Fg-coated beads or Fg bound to the activated GPIIbIIIa integrin (GPIIbIIIa*) immobilized on beads or on activated fixed platelets (AFP), i.e. in a system free of platelet signaling and secretion mechanisms. Aggregation at physiological shear rates (100-2000 s(-1)) was studied in a microcouette device and monitored by flow cytometry. Soluble TSP bound to and induced aggregation of Fg-coated beads dose-dependently, which could be blocked by the amino-terminal heparin-binding domain of TSP, TSP18. Soluble TSP did not bind to GPIIbIIIa*-coated beads or AFP, unless they were preincubated with Fg. The interaction of soluble TSP with Fg-GPIIbIIIa*-coated beads or Fg-AFP resulted in the formation of aggregates via Fg-TSP-Fg cross-bridges, as demonstrated in a system where direct cross-bridges mediated by GPIIbIIIa*-Fg on one particle and free GPIIbIIIa* on a second particle were blocked by the RGD mimetic Ro 44-9883. Soluble TSP increased the efficiency of Fg-mediated aggregation of AFP by 30-110% over all shear rates and GPIIbIIIa* occupancies evaluated. Surprisingly, TSP binding to Fg already bound to its GPIIbIIIa* receptor appears to block the ability of this occupied Fg to recognize another GPIIbIIIa* receptor, but this TSP can indeed cross-bridge to another Fg molecule on a second platelet. Finally, TSP-coated beads could directly coaggregate at shear rates from 100 to 2000 s(-1). Our studies provide a model for the contribution of secreted TSP in reinforcing inter-platelet interactions in flowing blood, through direct Fg-TSP-Fg and TSP-TSP cross-bridges.     

Platelet-enhanced apolipoprotein E production by human macrophages: a possible role in atherosclerosis

Cholesterol-loaded human monocyte-derived macrophages increase their production of apolipoprotein E (apoE). Although cholesterol loading is often achieved with modified plasma lipoproteins, macrophages can be loaded also by coculture with platelets. Therefore, the relationship between platelet-mediated cholesteryl ester accumulation and apoE secretion was examined. Macrophages were isolated by adherence and cultured for 6 days in serum-free medium. Secreted apoE was measured with a sensitive solid-phase radioimmunoassay. Maximum apoE secretion by the adherent macrophages from 5 x 10(6) peripheral blood mononuclear cells was obtained with 3 x 10(8) platelets and was ten-fold greater than control cells cultured in the absence of platelets. Platelet-mediated apoE secretion was consistently greater than that obtained by culture with either native or acetylated low density lipoproteins. Whereas the 1000 g supernatants of unstimulated platelets were poor inducers of apoE secretion, substances rich in cholesterol that were shed from thrombin-stimulated platelets and recovered in the 1000 g supernatants were almost as active as intact platelets. In all studies, platelet-induced secretion of apoE paralleled the capacity of platelets to induce macrophage cholesterol accumulation, indicating that macrophage apoE secretion was readily influenced by macrophage cholesterol metabolism.     

hESCs/hiPSCs体外诱导产生红细胞的研究进展及其临床应用的展望

人类胚胎干细胞和多功能诱导性干细胞的诞生,标志着干细胞研究已经跨入了全新的应用时代。干细胞研究领域的一个重要方向是特定谱系成熟细胞的定向诱导分化。在诸多的血细胞中,成熟红细胞因为无核而携带着最小量的遗传物质,可能作为最早的干细胞治疗产品而应用于输血替代治疗。最近,干细胞向造血细胞（包括红细胞）的研究正方兴未艾。但由于方法学上的偏差,诱导产生的红细胞的成熟度各有所不同。该文在结合了作者实验室的工作经验的基础上,对目前人类多潜能干细胞向红细胞特定诱导分化的方法做了综合的描述,并提出了该研究领域亟需解决的重大科学问题。     

A mathematical model of haemopoiesis as exemplified by CD34 cell mobilization into the peripheral blood

A mathematical model for the kinetics of haemopoietic cells, including CD34+cells, is proposed. This minimal model reflects the known kinetics of haemopoietic progenitor cells, including peripheral blood CD34+ cells, white blood cells and platelets, in the presence of granulocyte colony-stimulating factor. Reproducing known perturbations within this system, subjected to granulocyte colony-stimulating factor treatment and apheresis of peripheral blood progenitor cells (CD34+ cells) in healthy individuals allows validation of the model. Predictions are made with this model for reducing the length of time with neutropenia after high-dose chemotherapy. Results based on this model indicate that myelosuppressive treatment together with infusion of CD34+ peripheral blood progenitor cells favours a faster recovery of the haemopoietic system than with granulocyte colony-stimulating factor alone. Additionally, it predicts that infusion of white blood cells and platelets can relieve the symptoms of neutropenia and thrombocytopenia, respectively, without drastically hindering the haemopoietic recovery period after high dose chemotherapy.     

Conjugation to hyperbranched polyglycerols improves RGD-mediated inhibition of platelet function in vitro

RGD (arginine-glycine-aspartic acid) is a known peptide sequence that binds platelet integrin GPIIbIIIa and disrupts platelet-fibrinogen binding and platelet cross-linking during thrombosis. RGD peptides are unsuitable for clinical applications due to their high 50% inhibitory concentration (IC50) and low in vivo residence times. We addressed these issues by conjugating RGD peptides to biocompatible macromolecular carriers: hyperbranched polyglycerols (HPG) via divinyl sulfone. The GPIIbIIIa binding activity of RGD was maintained after conjugation and the effectiveness of the HPG-RGD conjugate was dependent upon molecular weight and the number of RGD peptides attached to each HPG molecule. These polyvalent inhibitors of platelet aggregation decreased the IC50 of RGD in an inverse linear manner based on the number of RGD peptides per HPG. Since HPG-RGD conjugates do not cause platelet activation by degranulation and certain substitution ratios do not increase fibrinogen binding to resting platelets, HPG-RGD may serve as a model for a novel class of antithrombotics.     

小鼠腹膜炎模型下血小板的生物学特征及其转录组变化

作为血液中最小的无核血细胞,血小板除具有经典的止凝血功能外,在调节固有免疫和适应性免疫方面也发挥着重要作用。但是,对于血小板参与免疫调节的分子机制,目前研究相对较少。通过给小鼠腹腔注射热灭活的大肠杆菌构建了急性炎症模型,并借助血常规、流式细胞术等实验方法检测了炎症条件下腹腔免疫细胞以及外周血细胞的动态变化。研究发现,在感染早期小鼠外周血中性粒细胞能快速迁移至腹腔参与炎症反应,同时,血小板的数目和大小在感染早期也会发生显著变化。进一步的RNA-Seq分析发现,小鼠血小板的转录组在感染后发生显著改变,其中,免疫反应相关基因显著上调,血小板表面黏附分子的表达也显著增强。研究结果揭示了急性炎症条件下血小板转录组的变化,为了解血小板免疫功能的潜在分子机制提供了新的角度。     

Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, GPIIbIIIa

Platelet membrane GPIIbIIIa is a member of the family receptors named integrins that recognize RGD sequences in their ligands. GPIIbIIIa interacts with at least three different adhesive ligands: fibrinogen, fibronectin, and von Willebrand factor. These interactions are inhibited by RGD-containing peptides and by peptides corresponding to a sequence unique to fibrinogen in the COOH-terminal domain of its gamma chain (HLGGAKQAGDV). Two RGD sequences are present in fibrinogen A alpha chain: an RGDS sequence at A alpha 572-575, and an RGDF sequence at A alpha 95-98. Polyclonal antibodies raised against the RGDF sequence and the gamma COOH-terminal domain both reacted specifically with fibrinogen in solid phase enzyme-linked immunosorbent assays and immunoprecipitated the protein in solution. The Fab fragments prepared from these antibodies inhibited fibrinogen-platelet interaction and aggregation. These results demonstrate that these two sequences are both accessible within the fibrinogen molecule and are both implicated in ligand binding and cell-cell interaction. In addition, by further examining the interaction of the gamma chain peptide with platelets, it was found that RGDF and the gamma peptide produced a similar dose-dependent inhibition of the binding of the labeled gamma peptide to ADP-stimulated platelets. These results provide evidence that the RGDF sequence present at the A alpha 95-98 constitutes with the gamma 401-411 sequence two recognition sites interacting with the same site or with mutually exclusive sites on GPIIbIIIa.     

Human induced pluripotent stem cell–derived lung progenitor and alveolar epithelial cells attenuate hyperoxia-induced lung injury

Background aims

Bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by disrupted lung growth, is the most common complication in extreme premature infants. BPD leads to persistent pulmonary disease later in life. Alveolar epithelial type 2 cells (AEC2s), a subset of which represent distal lung progenitor cells (LPCs), promote normal lung growth and repair. AEC2 depletion may contribute to persistent lung injury in BPD. We hypothesized that induced pluripotent stem cell (iPSC)-derived AECs prevent lung damage in experimental oxygen-induced BPD.

Characterization of Quinolinic Acid Phosphoribosyltransferase in Human Blood and Observations in Huntington''s Disease

Quinolinic acid (QUIN), an excitotoxic compound present in the mammalian CNS and periphery, has been hypothetically linked to human neurodegenerative disorders such as Huntington's disease and epilepsy. Quinolinic acid phosphoribosyltransferase (QPRT), the catabolic enzyme of QUIN, is found in the CNS and peripheral organs where it may be a major influence on the tissue levels of QUIN. We have measured QPRT activity in human blood as a means of assessing one aspect of QUIN metabolism in humans. The enzyme was present in blood cells, platelets having a sixfold greater activity than erythrocytes, but was essentially absent from the plasma. In a blood cell fraction, enzyme activity was potently inhibited by phthalic acid (IC50 = 6.1 microM). Kinetic analyses conducted over a range of QUIN concentrations yielded Km values of 1.89-3.75 microM and Vmax values of 33.4-72.5 fmol nicotinic acid mononucleotide/h/mg protein. Enzyme activity varied 2.2-fold between normal individuals, was reasonably constant over a series of sampling intervals, and showed some diminution when blood was stored for 1 month at -20 degrees C. No differences of enzyme activity in erythrocytes or platelets were apparent between three Huntington's disease patients and their unaffected spouses. These data indicate that measurements of QPRT activities in blood are a convenient means to monitor QUIN metabolism in human subjects and that a deficiency of the enzyme is not apparent in Huntington's disease.     

Comparison of three kinetic models of HIV-1 infection: implications for optimization of treatment

Clinical markers in the peripheral blood guide the treatment of human immunodeficiency virus type 1 (HIV-1). Likewise, many of the theoretical models developed to simulate infection only incorporate variables in the blood. To test the suitability of blood-only models, three distinct models of HIV infection kinetics are compared: "full model" including latently and actively infected cells and virus in the peripheral blood and lymphoid tissue (LT); "reduced model", including peripheral blood and LT without latent cells; and "blood model" including only actively infected cells and virus in the peripheral blood. Using the same parameter values for all three, qualitative differences are demonstrated between the blood model and its more inclusive counterparts. Additionally, optimization studies show that the reduced and blood models generate progressively lower optimal treatment levels relative to the full model when constant-level treatment is considered. These findings indicate that including the lymphoid tissue and latently infected cells into kinetic models may lead to differing conclusions with regard to optimal treatment and could be useful in guiding therapy even when plasma viral levels are below detectable limits.     

WASP levels in platelets and lymphocytes of wiskott-aldrich syndrome patients correlate with cell dysfunction

Wiskott-Aldrich syndrome, an inherited blood cell disorder due to mutations of the X-chromosome gene WASP (Wiskott-Aldrich syndrome protein), was characterized originally by thrombocytopenia, immunodeficiency, and eczema. Whereas platelet dysfunction is severe and consistent, immune defects are clinically variable, ranging from negligible to life threatening. To understand this heterogeneity, we quantified WASP in PBMC and platelets, and also in neutrophils, of patients with diverse mutations. A surprisingly complex pattern of WASP expression found for lymphoid cells formed the basis for dividing the patient mutations into four groups. Group A have low WASP levels in PBMC and higher levels in EBV cell lines, as well as near normal WASP RNA levels (7 patients, most with mild disease), suggesting that group A WASP molecules are hypersusceptible to proteolysis. Group B have low WASP levels in PBMC and EBV cells and similar low RNA levels (2 patients, moderate disease). Group C have discordant expression: WASP-positive peripheral T cells and WASP-negative peripheral B cells and EBV cell lines (9 patients, variable disease severity). Noteworthy among group C kindred are several instances of B cell lymphomas. In group D, PBMC and EBV cell lines are WASP negative (7 patients, severe disease). In contrast to the complex lymphoid cell expression patterns, all patient platelets examined were WASP negative (18 diverse patients). WASP absence in platelets provides an apparent molecular explanation for the universally severe platelet dysfunction in this disease, and the cumulative lymphoid cell findings suggest that WASP levels play a substantial role in determining immune outcome.     

Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics

Blood cells and biofluid proteomics are emerging as a valuable tool to assess effects of interventions on health and disease. This study is aimed to assess the amount and variability of proteins from platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva from ten healthy volunteers for proteomics analysis, and whether protein yield is affected by prolonged fasting. Volunteers provided blood, saliva and morning urine samples once a week for 4 weeks after an overnight fast. Volunteers were fasted for a further 24 h after the fourth sampling before providing their final samples. Each 10 mL whole blood provided 400–1,500 μg protein from platelets, and 100–600 μg from PBMC. 30 μL plasma depleted of albumin and IgG provided 350–650 μg protein. A sample of morning urine provided 0.9–8.6 mg protein/dL, and a sample of saliva provided 70–950 μg protein/mL. None of these yields were influenced by the degree of fasting (overnight or 36 h). In conclusion, in contrast to the yields from plasma, platelets and PBMC, the protein yields of urine and saliva samples were highly variable within and between subjects. Certain disease conditions may cause higher or lower PBMC counts and thus protein yields, or increased urinary protein levels.     

Distribution of glycoconjugates on the plasma membrane and on membranes of the open-canalicular system in human platelets

Summary Distribution of carbohydrate moieties in the membrane system of the human blood platelet was studied by electron microscopy employing lectins as a probe. Glutaraldehyde-fixed platelets were treated with biotinylatedlectins (ConA, RCA, WGA, PNA, SBA, DBA and UEA-1) and labeled with horseradish peroxidase-conjugated avidin. Among the lectins used, ConA bound uniformly to the plasma membrane as well as to the membrane of the opencanalicular system (OCS). Other lectins showed more or less reduced binding on the OCS membrane compared with that on the plasma membrane, indicating that there exist regional differences in the distribution pattern of glycoconjugates in the membrane system of the platelet. The relationship of the distribution pattern of the glycoconjugates with the distribution of the major platelet glycoproteins GPIb and GPIIbIIIa is discussed.     

Distribution of glycoconjugates on the plasma membrane and on membranes of the open-canalicular system in human platelets. A cytochemical study

Distribution of carbohydrate moieties in the membrane system of the human blood platelet was studied by electron microscopy employing lectins as a probe. Glutaraldehyde-fixed platelets were treated with biotinylated-lectins (ConA, RCA, WGA, PNA, SBA, DBA and UEA-1) and labeled with horseradish peroxidase-conjugated avidin. Among the lectins used, ConA bound uniformly to the plasma membrane as well as to the membrane of the open-canalicular system (OCS). Other lectins showed more or less reduced binding on the OCS membrane compared with that on the plasma membrane, indicating that there exist regional differences in the distribution pattern of glycoconjugates in the membrane system of the platelet. The relationship of the distribution pattern of the glycoconjugates with the distribution of the major platelet glycoproteins GPIb and GPIIbIIIa is discussed.     

Characteristics of hematopoietic stem cells of umbilical cord blood

Umbilical cord blood collected from the postpartum placenta and cord is a rich source of hematopoietic stem cells (HSCs) and is an alternative to bone marrow transplantation. In this review we wanted to describe the differences (in phenotype, cytokine production, quantity and quality of cells) between stem cells from umbilical cord blood, bone marrow and peripheral blood. HSCs present in cord blood are more primitive than their counterparts in bone marrow or peripheral blood, and have several advantages including high proliferation. With using proper cytokine combination, HSCs can be effectively developed into different cell lines. This process is used in medicine, especially in hematology.     

High-resolution CryoFESEM of individual cell adhesion molecules (CAMs) in the glycocalyx of human platelets: detection of P-selectin (CD62P), GPI-IX complex (CD42A/CD42B alpha,B beta), and integrin GPIIbIIIa (CD41/CD61) by immunogold labeling and stereo imaging.

The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPIba in the GPI-IX complex (CD42a/CD42b alpha,b beta), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with 10-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes. (J Histochem Cytochem 49:809-819, 2001)     

Thrombocytes Correlate with Lymphangiogenesis in Human Esophageal Cancer and Mediate Growth of Lymphatic Endothelial Cells In Vitro

Recent data provide evidence for an important role of thrombocytes in lymphangiogenesis within human malignant disease. The aim of this study was to investigate the role of thrombocytes in lymphangiogenesis in human esophageal cancer. Perioperative peripheral blood platelet counts (PBPC) were evaluated retrospectively in 320 patients with esophageal cancer, comprising 184 adenocarcinomas (AC), and 136 squamous cell carcinomas (SCC). Data on lymphangiogenesis evaluated by anti-podoplanin immunostaining were available from previous studies, platelets within the tumor tissue were assessed by CD61 immunostaining. For in vitro studies, human lymphatic endothelial cells (LECs) were isolated and co-cultured with peripheral blood platelets. Stromal thrombocytic clusters (STC) were evident in 82 samples (25.6%), and vascular thrombocytic clusters (VTC) in 56 (17.5%). STC and VTC were associated with a significantly higher PBPC at investigation of all cases. The presence of STC was associated with higher lymphatic microvessel density (p<0.001), PBPC and STC were associated with lymphovascular invasion of tumor cells in a regression model. The presence of STCs was associated with shorter DFS of all patients (p = 0.036, Breslow test), and VTC with shorter DFS in in SCC (p = 0.025, Breslow test). In cell culture, LEC proliferation was enhanced by co-culture with human platelets in a dose- and time-dependent manner mediated by the release of PDGF-BB and VEGF-C. Platelets play an important role in lymphangiogenesis and lymphovascular invasion in esophageal cancer, influencing prognosis. So the disruption of signaling pathways between platelets, tumor cells and lymphatic endothelium might be of benefit for patients.     

The clearance mechanism of chilled blood platelets

Platelet transfusion is a very common lifesaving medical procedure. Not widely known is the fact that platelets, unlike other blood cells, rapidly leave the circulation if refrigerated prior to transfusion. This peculiarity requires blood services to store platelets at room temperature, limiting platelet supplies for clinical needs. Here, we describe the mechanism of this clearance system, a longstanding mystery. Chilling platelets clusters their von Willebrand (vWf) receptors, eliciting recognition of mouse and human platelets by hepatic macrophage complement type 3 (CR3) receptors. CR3-expressing but not CR3-deficient mice exposed to cold rapidly decrease platelet counts. Cooling primes platelets for activation. We propose that platelets are thermosensors, primed at peripheral sites where most injuries occurred throughout evolution. Clearance prevents pathologic thrombosis by primed platelets. Chilled platelets bind vWf and function normally in vitro and ex vivo after transfusion into CR3-deficient mice. Therefore, GPIb modification might permit cold platelet storage.