Aggregation of human platelets by gingipain-R from Porphyromonas gingivalis cells and membrane vesicles

The hypothesis that there is an association between periodontitis and cardiovascular disease suggests new lines of research on the mechanism whereby oral bacteria might exert systemic effects. This study was conducted to ascertain and quantitate the effect of Porphyromonas gingivalis on human platelets in vitro . A second related objective was to purify and identify the aggregating vector. Aggregation was measured by platelet turbidometry and gingipain-R was purified from P. gingivalis membrane vesicles by Sepharose 2B and hydroxyapatite chromatography. The in vitro aggregation of platelets requires that at least 1.0 2 10 4 cells be stirred with 1.35 2 10 8 platelets. The specific activity is substantially increased in the membrane vesicles that are shed by this bacterium. Aggregability was due to gingipain-R activity, a potent cysteine protease that was found to be highly concentrated in the membrane vesicle fraction. The enzyme was purified 18-fold in high yield from the membrane vesicles, and consists of two noncovalently linked proteins that migrate at 49 and 44 kDa on SDS-PAGE. Aggregation of platelets by gingipain-R was shown to be dose-dependent, and inhibited by leupeptin and arginine, but not by anti-thrombin III. This is the first report enumerating the specific number of cells and lowest concentration of membrane vesicles necessary to evoke a full human platelet response, and the first report to assign this activity to gingipain-R.     

Aggregation of human platelets by gingipain-R from Porphyromonas gingivalis cells and membrane vesicles

The hypothesis that there is an association between periodontitis and cardiovascular disease suggests new lines of research on the mechanism whereby oral bacteria might exert systemic effects. This study was conducted to ascertain and quantitate the effect of Porphyromonas gingivalis on human platelets in vitro. A second related objective was to purify and identify the aggregating vector. Aggregation was measured by platelet turbidometry and gingipain-R was purified from P. gingivalis membrane vesicles by Sepharose 2B and hydroxyapatite chromatography. The in vitro aggregation of platelets requires that at least 1.0 x 10(4) cells be stirred with 1.35 x 10(8) platelets. The specific activity is substantially increased in the membrane vesicles that are shed by this bacterium. Aggregability was due to gingipain-R activity, a potent cysteine protease that was found to be highly concentrated in the membrane vesicle fraction. The enzyme was purified 18-fold in high yield from the membrane vesicles, and consists of two noncovalently linked proteins that migrate at 49 and 44 kDa on SDS-PAGE. Aggregation of platelets by gingipain-R was shown to be dose-dependent, and inhibited by leupeptin and arginine, but not by anti-thrombin III. This is the first report enumerating the specific number of cells and lowest concentration of membrane vesicles necessary to evoke a full human platelet response, and the first report to assign this activity to gingipain-R.     

Comparison of the release of microRNAs and extracellular vesicles from platelets in response to different agonists

On activation platelets release microRNAs and extracellular vesicles (EV) into circulation. The release of EV from platelets has been shown to be dependent on the agonist; in this study, we investigated whether the microRNA profile or EV released from platelets was also agonist specific.

Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (Glycoprotein VI (GPVI)), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block cyclooxygenase-1 and apyrase to remove ADP. The released microRNAs were profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterized by size (Nanoparticle Tracking Analysis, NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70.

Platelet activation triggered the release of 57–79 different microRNAs, dependent upon agonist, with a core of 46 microRNAs observed with all agonists. There was a high level of correlation between agonists (r² > 0.98; p < 0.0001 for all), and with the microRNA content of the parent platelets (r² > 0.98; p < 0.0001). The 46 microRNAs seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control, and differentiation. MiR-223-3p was the most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have anti-inflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect.

These data suggest that all tested agonists trigger the release of a similar microRNA profile while the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNAs and pdEV.     

Formation and release of arrestin domain-containing protein 1-mediated microvesicles (ARMMs) at plasma membrane by recruitment of TSG101 protein

Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses.     

Cardiopulmonary dysfunctions caused by intravenous collagen-induced release of ADP and ATP from platelets.

Bovine tendon collagen suspension (4.5 mg/kg) was injected rapidly into the femoral vein of 14 normal (untreated) and 8 busulfan treated rats. Trasient effects included decreased platelet counts and arterial PO2, increased central venous pressure, apnea, bradycardia and variable A-V block. These findings were most prominent within 1 minute after injection and subsided or disappeared by 10 minutes. During this period, ADP and ATP in platelet-free plasma from carotid arterial blood were measured in a liquid scientillation counter using the firefly luciferase assay. In normal (untreated) rats, collagen injection was followed by increases in plasma ADP and ATP, a rise in plasma hemoglobin and minimal changes of fibrinogen and hematcrit. Pathological observations indicated the platelet emboli in pulmonary vessels. In contrast, rats made thrombocytopenic by intraperitoneally infected busulfan prior to collagen injection had minimal or no change in platelet count, plasma ADP, ATP, hemoglobin, fibrinogen, or cardiopulmonary functions following collagen injection. These findings suggest that collagen injection causes release of ADP and ATP from platelets; released ADP induces platelets to form aggregates which lodge in the coronary and pulmonary microcirculations and elsewhere, resulting in thrombocytopenia and the cardiopulmonary dysfunction, in the presence of shear, of red cells with vessel surfaces altered by platelet aggregates.     

The roles of the quorum-sensing system in the release of extracellular DNA, lipopolysaccharide, and membrane vesicles from Pseudomonas aeruginosa

Biofilms play an important role in the establishment of chronic infection caused by Pseudomonas aeruginosa. It has been suggested that membrane vesicles (MVs) are released into the surrounding medium during normal growth and might supply the bacterial extracellular DNA that is required for early biofilm formation, as MVs released from the bacterial outer membrane are suspected to be the source of extracellular DNA. MVs possess lipopolysaccharide (LPS), extracellular DNA, and several hydrolytic enzymes. It is well known that the quorum-sensing (QS) system is important in controlling virulence factors in P. aeruginosa and biofilm formation. In the current study, we investigated extracellular LPS and DNA in the supernatants of culture solutions from PAO1, the wild-type P. aeruginosa, and those of QS mutants. As compared to that of las QS mutants, the amount of LPS and DNA released was significantly higher in PAO1 and in las QS mutants complemented with N-(3-oxododecanoyl) homoserine lactone. Our study indicated that the QS is among the regulators involved in the release of extracellular DNA and LPS. It is possible that these extracellular components are supplied from MVs. Investigation of the mechanism of biofilm formation is of particular interest, as it may be useful for designing treatments for severe P. aeruginosa infection.     

Purification of two types of gonadotropin receptors from carp ovarian follicles: overlapping recognition by two different ligands

Teleostean gonadotropin receptors were solubilized from the plasma membrane preparation of carp ovarian follicles by lithium diiodosalicylate and Triton X-100. Solubilization resulted fourfold increase in GTH binding activity as compared to the crude plasma membrane preparation. An addition of 25% glycerol and protease inhibitors to the solubilized receptor retained more than 90% original activity at -20 degrees C for 30 days. Gel filtration through Sephadex G 100 significantly increased the specific binding capacity from 70fmol/mg protein (soluble receptor) to 250fmol/mg protein. Peak I of gel filtration showing the receptor activity was further purified by affinity chromatography on purified salmon GTH II - Sepharose with a remarkable increase in specific binding capacity from 250fmol/mg protein (gel filtration peak) to 2300pmol/mg protein for salmon GTH I ligand and 2800pmol/mg protein for GTH II ligand. In SDS-PAGE affinity eluate the active peak showed two distinct bands corresponding to 66 and 62kDa molecular masses. These two proteins were clearly separated in FPLC Mono S chromatography, 62kDa as GTHR I and 66kDa as GTHR II. The former preferably binds to GTH I ligand, while the latter to GTH II, although both demonstrated overlapping recognition to both the ligands. GTH receptor protein I (GTHR I) and II (GTHR II) were purified 42,000- and 54,000-fold, respectively. Competitive binding inhibition studies indicate GTH I and ovine FSH a better ligand for GTHR I, while GTH II and ovine LH were preferable ligands for GTHR II. Biological relevance of these two receptor proteins was ascertained by monitoring the specific binding capacity of GTHR I and II at different stages of the annual reproductive cycle. GTHR I-GTH I was a prevailing complex during preparatory and pre-spawning stages, while GTHR II-GTH II was the dominant one at the maturational and final maturational stages. It may be concluded there are two GTH receptor proteins, each having a preferred ligand. The overlapping ligand-binding activity and profile of each receptor ligand complex suggest their link to seasonal development and maturation of carp ovarian follicle.     

Calcium translocation by Golgi and lateral-basal membrane vesicles from rat intestine: decrease in vitamin D-deficient rats.

Intestinal Ca2+ transport was studied in membrane vesicles isolated from microvillus, Golgi, and lateral-basal membrane preparations. Ca2+ uptake by these vesicles was measured by determination of 45Ca2+ associated with these membranes after collection by micropore filtration. Golgi membranes showed the highest initial rate and equilibration level of Ca2+ uptake. Approximately 90% of this Ca2+ uptake was into an osmotically responsive space, suggesting that what was measured was predominantly Ca2+ translocation. Vitamin D-deficient rats showed a markedly diminished rate of uptake and level of equilibration. These data indicate that a Ca2+-translocating process was associated with Golgi membranes to a greater extent than with surface membranes and that this process was markedly decreased in vitamin D-deficient rats. The results suggest that the Golgi apparatus participates in intestinal Ca2+ absorption.     

Transport of D-glucose by membrane vesicles from normal and avian sarcoma virus-transformed chicken embryo fibroblasts.

Transport of D-glucose was examined in membrane vesicles from normal and avian sarcoma virus (ASV)-transformed chicken embryo fibroblasts. The initial rates of D-glucose transport were found to be 3- to 5-fold higher for vesicles from glucose-starved normal cells and ASV-transformed cells when compared with transport rates for vesicles from normal cells and serum-starved normal cells. Cytochalasin B, phloretin, and diethylstilbestrol inhibited the initial rate of transport in all types of vesicles, and 2-deoxyglucose, 3-O-methylglucose, and galactose were competitive inhibitors. At D-glucose concentrations between 0.25 and 5 mM, vesicles from normal and ASV-transformed cells displayed saturation kinetics with a Km value of 5 mM for both types of vesicles, with transformed cell vesicles showing a 3-fold increase in Vmax compared with normal cell vesicles. At D-glucose concentrations between 5 and 25 mM the initial rate of D-glucose transport was proportional to D-glucose concentration. The vesicles also showed an inhibitor-sensitive efflux at rates similar to those observed for influx.     

Restoration of active transport of solutes and oxidative phosphorylation by naphthoquinones in irradiated membrane vesicles from Mycobacterium phlei

Irradiation of the inverted membrane vesicles of Mycobacterium phlei with light at 360 nm inactivated the natural menaquinone [MK₉(II-H)] and resulted in a loss of substrate oxidation, pH gradient, membrane potential, active transport of proline or calcium ions, and oxidative phosphorylation. Restoration of the protonmotive force and active transport occurred on addition of naphthoquinones such as vitamin K₁, menadione, or lapachol to the irradiated membrane vesicles. However, coupled phosphorylation was restored only by vitamin K₁. Menadione and lapachol did not act as uncoupling agents. The magnitude of the pH gradient and membrane potential in the quinone-restored system was a reflection of the rate of oxidation and was correlated with the rate of uptake of proline or Ca²⁺. These results are consistent with the chemosmotic hypothesis proposed for the energy transducing mechanism for active transport and further demonstrate that the complete respiratory chain is not required to drive active transport. In contrast, the data suggest that in addition to the driving force (protonmotive force) necessary to establish oxidative phosphorylation, a specific spatial orientation of the respiratory components, such as the naphthaquinones, is essential for the utilization of the proton gradient or membrane potential or both. Bypass of electrons from the respiratory chain with menadione may explain the inability of this quinone to restore oxidative phosphorylation; however, lapachol restores oxidation by the same electron transport pathway as the natural menaquinone but fails to restore phosphorylation. Because all three quinones restore the protonmotive force, other factors that are discussed must be considered in understanding the mechanism of oxidative phosphorylation.     

弓形虫主要表面抗原P30两个不同片段的重组表达、纯化及应用

目的将刚地弓形虫（简称弓形虫）主要表面抗原P30基因两个不同片段重组表达，纯化所获重组蛋白用于弓形虫病免疫学诊断。方法根据弓形虫P30基因的全长序列，用PCR法从弓形虫RH株基因组DNA中扩增出P30基因的两个不同片段，并构建相应克隆进行诱导表达，表达蛋白以western blot鉴定。用Amylase Resin以亲和层析法纯化所表达的蛋白。以弓形虫RH株感染家兔，用粗抗原和纯化重组抗原以ELISA法比较检测各种寄生虫病患者、感染家兔血清及疟原虫感染小鼠血清。结果1．成功构建相应克隆，并成功诱导表达，表达产物经纯化后获得高纯度日的蛋白。2．重组抗原与粗抗原相比。具有相同的敏感性和更高的特异性。结论成功获得弓形虫主要表面抗原P30的2个表达产物。以此重组抗原与粗抗原对比检测弓形虫相应抗体，结果证明重组抗原特异性高于粗抗原。     

Direct stimulation of naive T cells by membrane vesicles from antigen-presenting cells: distinct roles for CD54 and B7 molecules

T cell stimulation usually requires direct contact with viable antigen-presenting cells (APCs). However, we show here that small exosome-like membrane vesicles shed from APCs can be recognized by na?ve CD8+ T cells in the absence of viable APCs. T cell antigen receptor-dependent binding of vesicles by CD8+ cells is MHC class I/peptide-specific and requires that the vesicles coexpress intercellular adhesion molecule 1 (ICAM-1, CD54), although not B7 (B7-1). In the absence of B7, T cell binding of vesicles is nonimmunogenic. By contrast, vesicles expressing both ICAM-1 and B7 are strongly immunogenic and cause purified APC-depleted CD8+ cells to mount peptide-specific proliferative responses and differentiate into effector cells.     

Original article: relaxin reduces the number of circulating platelets and depresses platelet release from megakaryocytes: studies in rats

In previous studies we have shown that relaxin (RLX), a peptide hormone produced predominantly during pregnancy, is a powerful inhibitor of platelet aggregation. The current study shows that RLX, given systemically to rats, also affects the number of circulating platelets and their release by spleen megakaryocytes. Male albino rats were treated acutely or chronically with RLX, and the effects of the peptide evaluated by platelet count and morphological analysis of spleen megakaryocytes. The results obtained show that RLX causes a decrease of circulating platelets, which is already appreciable 1 h after a single administration and becomes significant upon a May treatment with the peptide (790000 ± 18000/ml in the RLX-treated rats versus 865 000 ± 23 000/ml in the controls; P<0.01). Coincidentally, spleen megakaryocytes show clearcut changes consistent with a depression of platelet release, namely the appearance of a thick peripheral cytoplasmic halo, filled with actin microfilaments and without platelet territories, and a virtual absence of images of thrombocytopoiesis. Based on the properties of RLX in inhibiting hemostasis, recognized in previous investigations and further proved by the present study, a role of the peptide is proposed in counteracting the hypercoagulable state which currently takes place during normal pregnancy.     

Arrest and metastasis of blood-borne tumor cells are modified by fusion of plasma membrane vesicles from highly metastatic cells.

B16 mouse melanoma sublines in culture spontaneously shed intact plasma membrane vesicles. These vesicles can be fused with the plasma membrane of cells from homologous and heterologous B16 sublines by using polyethylene glycol and phytohemagglutinin-P. Fusion of vesicles from a highly metastatic subline (F10) that localizes exclusively in the lung with cells from a poorly metastatic subline (F1) significantly increased the ability of F1 cells to become arrested in the lung and form metastases in this organ. In contrast, fusion of F1 vesicles with F10 cells did not alter the ability of vesicle-modified cells to localize in the lung or form lung metastases. F10 vesicle-modified F1 cells reverted to their original arrest behavior and metastatic capacity after removal of F10 vesicle components from the plasma membrane. The changes in the arrest and metastatic behavior of F10 vesicle-modified F1 cells were highly highly specific. Vesicles from other B16 sublines that are poorly metastatic and show limited localization in the lung (F1, FLLr, and F10Lr) did not modify the arrest behavior and metastatic capacity of FU cells. These results suggest that the differences in the abilities of the F1 and F10 sublines to localize in the lung are determined by differences in cell surface properties.     

The calcium paradoxon of renin release: calcium suppresses renin exocytosis by inhibition of calcium-dependent adenylate cyclases AC5 and AC6

An increase in the free intracellular calcium concentration promotes exocytosis in most secretory cells. In contrast, renin release from juxtaglomerular (JG) cells is suppressed by calcium. The further downstream signaling cascades of this so called "calcium paradoxon" of renin secretion have been incompletely defined. Because cAMP is the main intracellular stimulator of renin release, we hypothesized that calcium might exert its suppressive effects on renin secretion via the inhibition of the calcium-regulated adenylate cyclases AC5 and AC6. In primary cultures of JG cells, calcium-dependent inhibitors of renin release (angiotensin II, endothelin-1, thapsigargin) suppressed renin secretion, which was paralleled by decreases in intracellular cAMP levels [cAMP]. When [cAMP] was clamped by membrane permeable cAMP derivates, renin release was not suppressed by any of the calcium liberators. Additionally, both endothelin and thapsigargin suppressed cAMP levels and renin release in isoproterenol or forskolin-pretreated As4.1 cells, a renin-producing cell line that expresses AC5 and AC6. The calcium-dependent inhibition of intracellular cAMP levels and renin release was prevented by small interfering RNA-mediated knockdown of AC5 and/or AC6 expression, underlining the functional significance of these AC isoforms in renin-producing cells. Finally, in isolated perfused mouse kidneys, angiotensin II completely inhibited the stimulation of renin secretion induced by adenylate cyclase activation (isoproterenol) but not by membrane permeable cAMP analogs, supporting the conclusion that the suppressive effect of calcium liberators on renin release is mediated by inhibition of adenylate cyclase activity.     

Functional lac carrier protein in cytoplasmic membrane vesicles isolated from Escherichia coli: Temperature and pH dependence of dansyl-galactoside binding

6'-(N-Dansyl)aminohexyl-1-thio-beta-D-galactopyranoside binds specifically to the lac carrier protein in cytoplasmic membrane vesicles isolated from Escherichia coli. Binding can be induced by substrate oxidation (generation of an electrochemical gradient of protons), by potassium efflux in the presence of valinomycin (generation of a potassium diffusion potential), and by passive, carrier-mediated lactose efflux. We show that in all three cases the number of binding sites is temperature dependent. Binding is maximal and constant above 20 degrees ; it decreases between 20 degrees and 10 degrees . Oxidation of substrate (D-lactate) leads to the development of an electrochemical gradient of protons across the membrane (interior negative and alkaline), which is composed of interconvertible electrical and chemical gradients. We show that both the electrical potential across the membrane and the chemical difference in proton concentrations across the membrane are independent of temperature between 5 degrees and 25 degrees . We show that the number of binding sites induced by D-lactate oxidation depends on pH. At both 25 degrees and 5 degrees , the number of binding sites increases from pH 5 to pH 6.5, remains constant between pH 6.5 and 7, and decreases from pH 7 to pH 8. In contrast, the number of binding sites induced by passive, carrier-mediated lactose efflux is independent of pH between pH 5.5 and pH 8.From these findings, we conclude that the pH- and temperature-dependent effects on the number of 6'-(N-dansyl)aminohexyl-1-beta-thio-D-galactopyranoside binding sites have different origins. The pH dependence of binding is energy linked and reflects in part the pH dependence of the electrochemical gradient of protons across the membrane generated by substrate oxidation. The temperature dependence is not an energy-linked phenomenon. The decrease of the number of binding sites at low temperature probably reflects the aggregation of the lac carrier protein with other membrane proteins. This aggregation takes place as a consequence of the conformational disorder-to-order transition of the membrane lipids and the concomitant preferential segregation of the lac carrier protein in the membrane domains containing the disordered lipids.