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Blood, Vol. 109, Issue 12, 5242-5250, June 15, 2007
Human platelets synthesize and express functional tissue factor
Blood Panes et al.
109: 5242
Supplemental materials for: Panes et al, Vol 109, Issue 12, 5242-5250
Files in this Data Supplement:
- Figure S1. Activation-increase of platelet procoagulant activity is not affected by puromycin (PDF, 14.1 KB) -
Isolated platelets as described in Methods were pre-incubated for 1 hour with or without 1mM puromycin. At the indicated times after 5µM TRAP activation, platelets were incubated with FVIIa (1U/mL) for 5 min at 37°C. The reaction with FX, CaCl2 and substrate of FXa yielded a chromogenic product measurable at 405nm. The PCA was read in a calibration curve obtained with serial dilutions of hrTF (Innovin). Time 0 represents the nonstimulated platelet PCA. The time–course of PCA curves with and without puromycin were not statistically different. Basal platelet PCA, without activation, was significantly lower than PCA at 60 minutes (p=0.035) and 120 minutes (p=0.0009) after activation. Each time point represents the mean ± SD of up to 19 different experiments. (General linear model, with Bonferroni pairwise comparisons). When the experiments were performed in platelets isolated at 4°C, nonstimulated platelets had undetectable PCA, which after TRAP-activation attained similar levels than those of platelets processed at room temperature.
- Figure S2. Platelet procoagulant activity is mainly dependent on TF-FVIIa (PDF, 50.4 KB) -
Platelets suspensions were obtained by room temperature processing. Black bars show the PCA values standardized at 100% of nonstimulated (T-0) and TRAP-activated platelets (T-15 minutes, T-60 minutes, T-120 minutes), without inhibitors. The effect of each inhibitor was expressed as percentage of the control value at each specified time. The effects of activated protein C (aPC) and  -FVIIIC, intrinsic pathway inhibitors, were not statistically different from their respective controls at any time. Corn trypsin inhibitor (CTI) induced a slight, nonsignificant inhibition at T-15 minutes (60% of residual activity, p = 0.08). However, -TF polyclonal antibody and -FVIIa inhibited the PCA at all times, in resting and stimulated platelets. Of the 4 measurements, -TF Ab inhibited the PCA by a mean of 48% (p=0.02 at T-60 minutes and p<0.001 at T-15 minutes and T-120 minutes). -FVIIa induced a mean inhibition of 76%, with a p value <0.001 for all time-points. (Wilcoxon test for paired samples). The omission of FVIIa in the assay had no effect on platelet PCA. The bars represent the mean ± SE.
- Figure S3. Western blot of TF in platelet membranes (PDF, 33.4 KB) -
Freshly isolated platelets were either pre-incubated or not with puromycin, in both nonstimulated and TRAP-activated conditions (as described in Methods). Solubilized platelet membranes were subjected to Western blot analysis using polyclonal -TF Ab. Platelet activation induced some increase in the main, approximately 47kDa TF band, as well as the appearance of others, most of which were about 35kDa. The fact that pre-incubation with puromycin had no inhibitory effect, above all in nonstimulated platelets, highlights the notion that platelets contain preformed TF. Results were similar using ADP or collagen as agonist. This figure is consistent with the lack of inhibitory effect of puromycin on platelet PCA (Figure S1).
- Figure S4. De novo synthesis of TF in PBMC suspensions (PDF, 65.7 KB) -
The flow cytometry of this PBMC preparation showed that 13% and 5% of all events were CD14+ and CD61+, respectively. The cells were pre-incubated with actinomycin D, or RNA polymerase II inhibitor DRB or puromycin before incubation with 35S-methionine and stimulation with LPS (2 hours at 37°C). After cell lysis and centrifugation, (18|000g for 15 minutes), the membrane fraction and the supernatant were immunoprecipitated, subjected to electrophoresis and protein transfer. 35TF was detected by autoradiography. This figure shows the 35TF in the supernatant fraction. A weak band of approximately 35kDa is barely visible after LPS stimulation (lane 1). Paradoxically, this band is amplified after incubation with actinomycin D and DRB, (lanes 2 and 3). Puromycin abolished all neosynthesis (lane 4). The same analysis in the membrane fraction showed that the band of approximately 35kDa was very weakly expressed or undetectable (not shown). Actinomycin D and puromycin had the same effect on TF synthesis by pure platelet suspensions. These results strongly suggest that TF neosynthesized early (2 hours) after LPS activation is originated in platelets. An experiment with extended activation (5 hours) of PBMC´s is shown in the Manuscript (Figure 4C).
- Figure S5. Confocal immunofluorescence of TF and GPIbα in nonstimulated PBMC preparations (PDF, 140 KB) -
Upper panel shows platelets (indicated by arrows) bound to PBMC, expressing TF and GPIb. Leukocytes have no TF signal. Lower panel is another example of a PBMC, presumably a monocyte, which does not express TF, whereas the surrounding, bound platelets are identified by their strong, co-localized, GPIb and TF labels.
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