Leukocyte- and Platelet-Derived Microvesicle Interactions following In Vitro and In Vivo Activation of Toll-Like Receptor 4 by Lipopolysaccharide

BACKGROUND: Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS). METHODOLOGY/PRINCIPAL FINDINGS: Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens. CONCLUSIONS/SIGNIFICANCE: Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood

Syncytiotrophoblast Microvesicles Released from Pre-Eclampsia Placentae Exhibit Increased Tissue Factor Activity

Background: Pre-eclampsia is a complication of pregnancy associated with activation of coagulation. It is caused by the placenta, which sheds increased amounts of syncytiotrophoblast microvesicles (STBM) into the maternal circulation. We hypothesized that STBM could contribute to the haemostatic activation observed in pre-eclampsia. Methodology/Principal Findings: STBM were collected by perfusion of the maternal side of placentae from healthy pregnant women and women with pre-eclampsia at caesarean section. Calibrated automated thrombography was used to assess thrombin generation triggered by STBM-borne tissue factor in platelet poor plasma (PPP). No thrombin was detected in PPP alone but the addition of STBM initiated thrombin generation in 14/16 cases. Pre-eclampsia STBM significantly shortened the lag time (LagT, P = 0.01) and time to peak thrombin generation (TTP, P = 0.005) when compared to normal STBM. Blockade of tissue factor eliminated thrombin generation, while inhibition of tissue factor pathway inhibitor significantly shortened LagT (p = 0.01) and TTP (P,0.0001), with a concomitant increase in endogenous thrombin potential. Conclusions/Significance: STBM triggered thrombin generation in normal plasma in a tissue factor dependent manner, indicating that TF activity is expressed by STBM. This is more pronounced in STBM shed from pre-eclampsia placentae. As more STBM are shed in pre-eclampsia these observations give insight into the disordered haemostasis observed in thi

Loss of estrogen receptor β decreases mitochondrial energetic potential and increases thrombogenicity of platelets in aged female mice

Platelets derived from aged (reproductively senescent) female mice with genetic deletion of estrogen receptor beta (βER) are more thrombogenic than those from age-matched wild-type (WT) mice. Intracellular processes contributing to this increased thrombogenicity are not known. Experiments were designed to identify subcellular localization of estrogen receptors and evaluate both glycolytic and mitochondrial energetic processes which might affect platelet activation. Platelets and blood from aged (22–24 months) WT and estrogen receptor β knockout (βERKO) female mice were used in this study. Body, spleen weight, and serum concentrations of follicle-stimulating hormone and 17β-estradiol were comparable between WT and βERKO mice. Number of spontaneous deaths was greater in the βERKO colony (50% compared to 30% in WT) over the course of 24 months. In resting (nonactivated) platelets, estrogen receptors did not appear to colocalize with mitochondria by immunostaining. Lactate production and mitochondrial membrane potential of intact platelets were similar in both groups of mice. However, activities of NADH dehydrogenase, cytochrome bc1 complex, and cytochrome c oxidase of the electron transport chain were reduced in mitochondria isolated from platelets from βERKO compared to WT mice. There were a significantly higher number of phosphatidylserine-expressing platelet-derived microvesicles in the plasma and a greater thrombin-generating capacity in βERKO compared to WT mice. These results suggest that deficiencies in βER affect energy metabolism of platelets resulting in greater production of circulating thrombogenic microvesicles and could potentially explain increased predisposition to thromboembolism in some elderly females

Venous gas embolism as a predictive tool for improving CNS decompression safety

A key process in the pathophysiological steps leading to decompression sickness (DCS) is the formation of inert gas bubbles. The adverse effects of decompression are still not fully understood, but it seems reasonable to suggest that the formation of venous gas emboli (VGE) and their effects on the endothelium may be the central mechanism leading to central nervous system (CNS) damage. Hence, VGE might also have impact on the long-term health effects of diving. In the present review, we highlight the findings from our laboratory related to the hypothesis that VGE formation is the main mechanism behind serious decompression injuries. In recent studies, we have determined the impact of VGE on endothelial function in both laboratory animals and in humans. We observed that the damage to the endothelium due to VGE was dose dependent, and that the amount of VGE can be affected both by aerobic exercise and exogenous nitric oxide (NO) intervention prior to a dive. We observed that NO reduced VGE during decompression, and pharmacological blocking of NO production increased VGE formation following a dive. The importance of micro-nuclei for the formation of VGE and how it can be possible to manipulate the formation of VGE are discussed together with the effects of VGE on the organism. In the last part of the review we introduce our thoughts for the future, and how the enigma of DCS should be approached

Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles

Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy

Circulating microparticles: square the circle

Background: The present review summarizes current knowledge about microparticles (MPs) and provides a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance. Results: MPs are a heterogeneous population of cell-derived vesicles, with sizes ranging between 50 and 1000 nm. MPs are capable of transferring peptides, proteins, lipid components, microRNA, mRNA, and DNA from one cell to another without direct cell-to-cell contact. Growing evidence suggests that MPs present in peripheral blood and body fluids contribute to the development and progression of cancer, and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases. MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform. Conclusions: Improvements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics, but also for the evaluation of treatment regimens and predicting disease outcomes

Fetal corticotrophin-releasing hormone mRNA, but not phosphatidylserine-exposing microparticles, in maternal plasma are associated with factor VII activity in pre-eclampsia

Background: Pre-eclampsia is associated with increased placental debris circulating in maternal plasma. Objectives: This study related placental debris to maternal markers of coagulation and endothelial activation in pre-eclampsia. Patients/methods: Circulating fetal corticotrophin-releasing hormone (CRH) mRNA and phosphatidylserine (PS)-exposing microparticles were assayed in third trimester plasma from women with pre-eclampsia (n = 32) and controls (n = 32) matched for age, body mass index, parity, and gestational age at sampling. Markers of maternal hemostasis and endothelial function were assessed. Results: Fetal CRH mRNA levels were higher in pre-eclampsia [mean 0.75 (SD 2.77) CRH/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio] than in control pregnancies [0.20 (0.74), P = 0.014]. PS-exposing microparticle levels were not different between the groups. Women with pre-eclampsia had higher levels of tissue factor pathway inhibitor (TFPI), prothrombin F1+2 fragment (F1+2), factor XIIa, soluble vascular cell adhesion molecule 1, von Willebrand factor and plasminogen activator inhibitor 1 than controls. Fetal CRH mRNA correlated with TFPI in pre-eclampsia and control groups (r = 0.38, P = 0.031, and r = 0.37, P = 0.039, respectively). Fetal CRH mRNA correlated with FVII activity (r = 0.43, P = 0.017) and PS-exposing microparticles correlated inversely with F1+2 (r = −0.64, P < 0.001) in pre-eclampsia. Conclusions: Placental debris, assessed by fetal CRH mRNA levels in maternal blood, is related to coagulation potential, i.e. FVII activity, but not to markers of coagulation or endothelial activation in pre-eclampsia