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Tissue-factor–bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation

Ian del Conde, Corie N. Shrimpton, Perumal Thiagarajan and José A. López

Article Figures & Data

Figures

  • Figure 1.

    Content of PSGL-1, TF, and CD45 in monocytes and their microvesicles (MVs). THP-1 cells or blood monocytes were stimulated with LPS; microvesiculation was induced with calcium ionophore. Cells were separated from their shed MVs by sedimenting the cells at 5000g for 5 minutes. The entire cell pellet and MV suspension were resuspended and diluted to the same extent, and lysed in SDS sample buffer. Equal volumes of the lysates were subjected to SDS-PAGE and analyzed by Western blot. (A) PSGL-1 and TF content in THP-1 cells and their shed MVs. (B) TF and CD45 content in monocytes, THP-1 cells, their respective MVs, and in MVs isolated from fresh plasma. (C) PSGL-1 and CD45 content in monocytes and their shed MVs. (D) Densitometric analysis of the MV/cell ratio of PSGL-1, TF, and CD45. These data are represented as the mean value for the MV/cell ratio in 6 different experiments plus or minus SD. *P < .003 CD45 versus PSGL-1 and versus TF.

  • Figure 2.

    Isolation of lipid rafts from monocytes. Triton X-100 lysates from monocytes were fractionated by centrifugation over a discontinuous sucrose gradient. Eleven equal fractions were obtained and assessed by Western blotting for the presence of (A) PSGL-1, (B) TF, and (C) CD45. Lipid rafts were identified by the presence of the raft-marker, flotillin-1. (D) Effect of membrane cholesterol depletion with MβCD on the localization of PSGL-1 and TF to lipid rafts. (E) As assessed by flow cytometry, cholesterol depletion did not affect the surface expression of PSGL-1 (top panels) or TF (bottom panels) on LPS-treated THP-1 cells. Background fluorescence was set with a fluorescent mouse IgG control (empty histograms). (F) Effect of membrane cholesterol depletion with 5 mM MβCD on the generation of microvesicles induced by calcium ionophore in THP-1 cells. The results shown are representative of 3 separate experiments. Values are shown as mean plus or minus SD. *P = .04 (n = 4).

  • Figure 3.

    Monocyte microvesicles transfer TF and PSGL-1 to activated platelets via PSGL-1. (A) Collagen-activated or unstimulated washed platelets were incubated with either monocyte microvesicles or control buffer. Platelets were sedimented and unbound microvesicles were removed by washing 5 times. The platelets were pelleted, lysed in SDS sample buffer, and analyzed by SDS-PAGE and Western blotting for PSGL-1 and TF. (B) Flow cytometric analysis of platelets incubated with either microvesicles or buffer, handled as described in (A). Samples using resting platelets plus microvesicles, activated platelets alone, and samples probed with control fluorescent IgG served as the negative controls in these experiments. (C) Treatment with anti–PSGL-1 (KPL-1) or an anti–P-selectin antibody, but not a nonspecific mouse IgG, blocked the transfer of TF and PSGL-1.

  • Figure 4.

    Microvesicles fuse with activated platelets. (A) Fluorescence microscopy (NBD channel) of THP-1 cells labeled with NBD-PE and Rh-PE (left panel, top) and of their shed microvesicles (left panel, bottom). Original magnifications, × 40 and × 400 on a Nikon Eclipse EP00 upright microscope equipped with 4 ×/0.13 NA and 40 ×/0.75 NA objective lenses (Nikon, Melville, NY), respectively. Images were captured using a Photometrics CoolSnap CS digital camera (Photometrics, Tucson, AZ). Solubilization of the labeled microvesicles with 1% SDS diluted the probes, resulted in an increase in NBD fluorescence, and a decrease in Rh fluorescence (right panel). (B) Labeled microvesicles were incubated with either: (x) unstimulated platelets; (▪) activated platelets; (♦) activated platelets + annexin V (100 μg/mL); (▴) activated platelets + anti–PSGL-1 antibody, KPL-1 (5 μg/mL). (C) Transfer of PSGL-1 and TF to activated platelets by monocyte microvesicles, as assessed by flow cytometry. Annexin V (100 μg/mL) did not affect the number of platelets that acquire these proteins. Background fluorescence was determined using fluorescent nonspecific mouse IgG. n = 3. (D) Membrane fusion between activated platelets and THP-1 microvesicles at 37°C (▪), and at 4°C (▪). (E) Fluorescence microscopy in rhodamine channel (×40) showing the transfer of the fluorescent lipid R18 from labeled microvesicles to platelets adhered on immobilized fibrinogen. Transfer of the lipid resulted in the spread platelets becoming fluorescent, an effect that was blocked by annexin V. The small fluorescent spots in the annexin V–treated sample correspond to the labeled microvesicles. These images are representative of 4 experiments. Act indicates activated; Plts, platelets; Unstim, unstimulated; An V, annexin V. Error bars indicate ± standard deviation (SD) in panels B-D.

  • Figure 5.

    Effect of membrane fusion on TF-VIIa activity. (A) TF-bearing microvesicles were incubated with 100 μg/mL of either bovine serum albumin (BSA) or unlabeled annexin V, and then washed. Microvesicles (MVs) treated with annexin V exhibited no binding of FITC-conjugated annexin V above an EDTA-treated sample, indicating blockade of phosphatidylserine on their surfaces. (B) TF-VIIa activity was measured using a chromogenic assay based on Xa generation. These values are adjusted for the direct effect of annexin V on the chromogenic assay. (C) Annexin V–coated microvesicles alone had approximately 35% less TF-VIIa activity than an equal number of BSA-treated microvesicles. n = 4; *P < .05. Error bars indicate ± SD.