TF supplementation evokes fibrin network formation as well as PS exposure.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055466#pone.0055466-Abbott1" target="_blank">[41]</a> CLSM images show fibrin network (green) formation in the presence of platelets labeled with R-6G (red). TF addition evoked fibrin matrix assembly and concurrent immediate binding of the fibrin/fibrinogen by platelets (white arrows) followed by progressive PS exposure on the platelet surface which was detected by platelet ANX (white) binding. (Bi) An enlarged representative image of single platelet bound to fibrin scaffold before the anionic phospholipids exposure in the outer leaflet of membrane, and (Bii) morphological changes of platelet membrane such as blebbing and shedding of microvesicles from cell surface (arrow heads) which accompany PS exposure. Scale bars show 10 µm.</p

Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through α_IIbβ₃ Evokes Phosphatidylserine Exposure on Their Cell Surface

<div><p>Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an <em>in-vitro</em> experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an α_IIbβ₃ antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.</p> </div

Attenuation of fibrin network formation suppress PS exposure on platelets surface.

<p>(A) Exposure of PS by R-6G labeled platelets (red) in the presence of either (i) FK633 (30 µM), (ii) GPRP (3 mM) or (iii) Cyt-B (100 µg/ml) upon stimulation of diluted PRP containing fbg-488 (green) and ANX (white) with thrombin (1 U/ml). CLSM images were taken 60 minutes after thrombin supplementation. (B) Kinetics of platelet PS exposure expressed as the percentage of ANX fluorescence-positive platelets in thrombin-treated (1 U/ml) samples in CLSM study. Presence of either FK633 (open triangle, n = 7), GPRP (open circle, n = 7) or Cyt-B (closed triangle, n = 7) suppressed platelet anionic phospholipids exposure compared with control (close square, n = 5), suggesting that crosstalk between platelets and the fibrin scaffold is a key feature of the anionic phospholipids exposure. Data are shown as mean ± SD. Scale bar shows 10 µm.</p

[Ca²⁺]_i elevation and PS exposure in platelets stimulated by thrombin and bound to fibrin network.

<p>Representative traces show [Ca²⁺]_i changes illustrated as fluo-4 to Fura Red relative fluorescence intensities ratios (black) in four randomly chosen platelets bound to rigid network of fibrin (n = 37 cells from four independent experiments). There were essentially no changes in the fluorescence intensities of ANX (blue) while spontaneous Ca²⁺ oscillations were observed in platelets. Ultimately, platelet morphological alterations and PS exposures were directly preceded by the sustained [Ca²⁺]_i elevations (arrows).</p

Cyt-B does not affect IMC dependent platelet anionic phospholipids exposure.

<p>Kinetics of platelet PS exposure are expressed as the percentage of ANX fluorescence-positive platelets in CLSM studies. Graph presents exposure of platelet PS either in the absence (closed square, n = 5) or the presence of 100 µg/ml Cyt-B (open square, n = 6) upon supplementation of IMC at a concentration of 10 µM. Data are shown as mean ± SD.</p

Changes in Glu-plg-568 relative fluorescence intensity induced by exogenous tPA administration.

<p>(<b>A</b>) Glu-plg-568 was administered to GFP-mice 10 minutes before each laser injury and 40 minutes after either human tPA (white: square, diamond, circle; three individual experiments) or an equivalent volume of 0.9% NaCl (black: square, diamond, circle; three individual experiments) was infused. Thrombi images were collected every 5 minutes after the laser-evoked endothelial injury, starting 2 minutes before and then every 5 minutes up to 60 minutes after either tPA or saline infusion. Each fluorescence intensity was normalized to the value obtained 2 minutes before either tPA or saline administration. tPA or saline was injected at time 0 (arrow). (B) Shown are the Glu-plg-568 accumulation increase ratios, which were expressed as the average values of the maximum increases in Glu-plg-568 relative fluorescence intensity after administration of either tPA (mean ± SD, n = 3, white column) or saline (mean ± SD, n = 3, black column). This assay was analyzed with a <i>t</i>-test for independent samples (<i>P</i> < 0.05).</p

Time-dependent accumulation of Glu-plg-568 on the platelet surfaces from GFP-mice incorporated into the fibrin network.

<p>Diluted PRP from GFP-mice was treated with thrombin (1 U/ml) in the presence of Glu-plg-568, and accumulation of Glu-plg-568 on the surface of platelets incorporated into the fibrin network was monitored over time by CLSM, either in the absence (A) or in the presence (B) of 100 mM of EACA or 15 U/ml CPB (C). Representative CLSM images were obtained 3 μm from the bottom of the dish at the indicated time points after addition of thrombin (1 U/ml). Fibrin network formation and the incorporation of platelets in the network were clearly demonstrated after addition of thrombin, which was followed by accumulation of Glu-plg-568 on the surface of platelets even prior to the decrease in GFP fluorescence intensity. Binding of Glu-plg-568 was significantly inhibited in the presence of EACA and CPB. The scale bars represent 10 μm. (D) The platelet Glu-plg-568 binding relative ratio was expressed as the integrated fluorescence intensity of Glu-plg-568 bound to the surface of platelet 120 minutes after thrombin supplementation and divided by the area of the platelet (n = 30 activated platelets from three independent experiments in each column). This assay was analyzed with a <i>t</i>-test for independent samples. Results are normalized to the control sample mean value (mean ± SD).</p

TNF-α production in response to LPS.

<p>(A) Comparison of basal levels of <i>TNFA</i> mRNA in granulocytes among healthy controls, Group A and Group B. (B) Comparison of fold increase of <i>TNFA</i> mRNA induced by simulation with 1 μg/mL LPS for indicated times. The numbers in Group A are compatible with patient numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158527#pone.0158527.g003" target="_blank">Fig 3B</a>. (C) TNF-<b>α</b> concentration in culture medium after a 2-hour stimulation with LPS. Granulocytes were cultured in the presence of 1 μg/mL LPS, and TNF-<b>α</b> concentration in medium was measured by ELISA. (D) TNF-<b>α</b> concentration in culture medium after a 3-hour stimulation with LPS.</p