Appropriation of GPIb from platelet-derived extracellular vesicles supports monocyte recruitment in systemic inflammation

Interactions between platelets, leukocytes and the vessel wall provide alternative pathological routes of thrombo-inflammatory leukocyte recruitment. We found that when platelets were activated by a range of agonists in whole blood, they shed platelet-derived extracellular vesicles which rapidly and preferentially bound to blood monocytes compared to other leukocytes. Platelet-derived extracellular vesicle binding to monocytes was initiated by P-selectin-dependent adhesion and was stabilised by binding of phosphatidylserine. These interactions resulted in the progressive transfer of the platelet adhesion receptor GPIbα to monocytes. GPIbα+-monocytes tethered and rolled on immobilised von Willebrand Factor or were recruited and activated on endothelial cells treated with TGF-β1 to induce the expression of von Willebrand Factor. In both models monocyte adhesion was ablated by a function-blocking antibody against GPIbα. Monocytes could also bind platelet-derived extracellular vesicle in mouse blood in vitro and in vivo. Intratracheal instillations of diesel nanoparticles, to model chronic pulmonary inflammation, induced accumulation of GPIbα on circulating monocytes. In intravital experiments, GPIbα+-monocytes adhered to the microcirculation of the TGF-β1-stimulated cremaster muscle, while in the ApoE−/− model of atherosclerosis, GPIbα+-monocytes adhered to the carotid arteries. In trauma patients, monocytes bore platelet markers within 1 hour of injury, the levels of which correlated with severity of trauma and resulted in monocyte clearance from the circulation. Thus, we have defined a novel thrombo-inflammatory pathway in which platelet-derived extracellular vesicles transfer a platelet adhesion receptor to monocytes, allowing their recruitment in large and small blood vessels, and which is likely to be pathogenic

Delineating zinc influx mechanisms during platelet activation

Zinc (Zn2+) is released by platelets during the haemostatic response to injury. Extracellular zinc ([Zn2+]o) initiates platelet activation following influx into the platelet cytosol. However, the mechanisms which permit Zn2+ influx are unknown. Fluctuations in intracellular zinc ([Zn2+]i) were measured in fluozin-3-loaded platelets using fluorometry and flow cytometry. Platelet ac-tivation was assessed using light transmission aggregometry. Detection of phosphoproteins was performed by Western blotting. [Zn2+]o influx and subsequent platelet activation was abrogated by blocking the sodium-calcium exchanger, TRP channels and ZIP7. Cation store depletion regulated Zn2+ influx. [Zn2+]o stimulation resulted in phosphorylation of PKC substates, MLC, and the β3 integrin. Platelet activation via GPVI or Zn2+ resulted in ZIP7 phosphorylation in a casein kinase 2 dependent manner, and initiated elevations of [Zn2+]i that were sensitive to inhibition of Orai1, ZIP7, or IP3R-mediated pathways. These data indicate that platelets detect and respond to changes in [Zn2+]o via influx into the cytosol through TRP channels and NCX exchanger. Platelet activation results in externalisation of ZIP7 which further regulates Zn2+ influx. Increases in [Zn2+]i contribute to the activation of cation-dependent enzymes. Sensitivity of Zn2+ influx to thapsigar-gin indicates a store-operated pathway that we term store-operated Zn2+ entry (SOZE). These mechanisms may affect platelet behaviour during thrombosis and haemostasis

Changes in the pattern of plasma extracellular vesicles after severe trauma

Background Extracellular vesicles (EV) released into the circulation after traumatic injury may influence complications. We thus evaluated the numbers of EV in plasma over 28 days after trauma and evaluated their pro-coagulant and inflammatory effects. Methods and findings 37 patients suffering trauma with an injury severity score >15 were studied along with 24 healthy controls. Plasma samples were isolated by double centrifugation (2000g 20min; 13000g 2min) from blood collected from within an hour up to 28 days after injury. Plasma EV were counted and sized using nanoparticle tracking analysis (NTA); counts and cellular origins were also determined by flow cytometry (FC) using cell-specific markers. Functional effects were tested in a procoagulant phospholipid assay and in flow-based, leukocyte adhesion assay after endothelial cells (EC) were treated with EV. We found that EV concentrations measured by NTA were significantly increased in trauma patients compared to healthy controls, and remained elevated over days. In addition, or FC showed that patients with trauma had higher numbers of EV derived from platelets (CD41+), leukocytes (CD45+) and endothelial EC (CD144+). The increases were evident throughout the 28-day follow-up. However, the FC count represented 400nm. The procoagulant phospholipid activity assay showed that patient plasma accelerated coagulation on day 1 and day 3 after trauma, with coagulation times correlated with EV counts. Furthermore, treatment of EC for 24 hours with plasma containing EV tended to increase the recruitment of peripheral flowing blood mononuclear cells. Conclusions EV counted by FC represent a small sub-population of the total load detected by NTA. Both methods however indicate a significant increase in plasma EV after severe traumatic injury that have pro-coagulant and pro-inflammatory effects that may influence outcomes

Delineating Zinc Influx Mechanisms during Platelet Activation

Zinc (Zn2+) is released by platelets during a hemostatic response to injury. Extracellular zinc ([Zn2+]o) initiates platelet activation following influx into the platelet cytosol. However, the mechanisms that permit Zn2+ influx are unknown. Fluctuations in intracellular zinc ([Zn2+]i) were measured in fluozin-3-loaded platelets using fluorometry and flow cytometry. Platelet activation was assessed using light transmission aggregometry. The detection of phosphoproteins was performed by Western blotting. [Zn2+]o influx and subsequent platelet activation were abrogated by blocking the sodium/calcium exchanged, TRP channels, and ZIP7. Cation store depletion regulated Zn2+ influx. [Zn2+]o stimulation resulted in the phosphorylation of PKC substates, MLC, and β3 integrin. Platelet activation via GPVI or Zn2+ resulted in ZIP7 phosphorylation in a casein kinase 2-dependent manner and initiated elevations of [Zn2+]i that were sensitive to the inhibition of Orai1, ZIP7, or IP3R-mediated pathways. These data indicate that platelets detect and respond to changes in [Zn2+]o via influx into the cytosol through TRP channels and the NCX exchanger. Platelet activation results in the externalization of ZIP7, which further regulates Zn2+ influx. Increases in [Zn2+]i contribute to the activation of cation-dependent enzymes. Sensitivity of Zn2+ influx to thapsigargin indicates a store-operated pathway that we term store-operated Zn2+ entry (SOZE). These mechanisms may affect platelet behavior during thrombosis and hemostasis

Effects of plasma containing EV on leukocyte adhesion to endothelial cells from flow.

<p>HUVEC was pre-treated for 24h with plasma EV from healthy controls or trauma patients, with or without treatment with TNF (5U/ml) for the last 4h. Peripheral blood mononuclear cells (PBMC) or neutrophils (PMN) were perfused at a wall shear stress of 0.05 Pa for 4min followed by 5min washout, and adherent cells counted. Results for patient plasma were expressed relative to healthy control plasma tested on the same day with the same HUVEC and leukocytes. A, B. PBMC adhesion after the absence or presence TNF. C, D. Neutrophil adhesion after the absence or presence TNF. All data are mean ± SEM (number of patients tested at each time in brackets). In A, the ratio of adhesion for patient:control plasma was significantly >1 for all samples tested (p<0.01), but the increase was not statistically significant at any particular day.</p

Percentage of extracellular vesicles with diameter >400nm detected by NTA.

<p>Percentage of extracellular vesicles with diameter >400nm detected by NTA.</p

Concentrations of EV from different cellular origins in plasma of healthy controls and patients analysed by flow cytometry.

<p>EV in plasma from patients or controls were labelled with antibodies recognising markers for A. platelets (CD41⁺), B. leukocytes (CD45⁺) or C. endothelial cells (CD144⁺) and analysed by flow cytometry. In each case, Kruskal-Wallis test showed significant variation between samples but not with time post injury for samples from patients alone; * = p<0.05, ** = p<0.01 compared to control by post hoc Dunn test. D. Plot of numbers of CD41⁺ vs. CD42⁺ EV counts for all samples. Linear regression showed a significant correlation (R² = 0.1909, p<0.01) with equation: CD41⁺ = 2.1x10⁵ + 0.25 CD42b⁺.</p

Characteristics of patients.

<p>Characteristics of patients.</p