The septic milieu triggers expression of spliced tissue factor mRNA in human platelets

Abstract

Background: Activated platelets have previously-unrecognized mechanisms of post-transcriptional gene expression that may influence hemostasis and inflammation. A novel pathway involves splicing of pre-mRNAs in resting platelets to mature, translatable mRNAs in response to cellular activation.

Objectives: We asked if bacterial products and host agonists present in the septic milieu induce tissue factor pre-mRNA splicing in platelets from healthy subjects. In parallel, we asked if spliced tissue factor (TF) mRNA is present in platelets from septic patients in a proof-of-principle analysis.

Patients/methods: TF pre-mRNA and mRNA expression patterns were characterized in platelets from septic patients and in platelets isolated from healthy subjects activated with bacteria, toxins and inflammatory agonists. Procoagulant activity was also measured.

Results and conclusions: Live bacteria, staphylococcal α-toxin and lipopolysaccharide (LPS) induced TF pre-mRNA splicing in platelets isolated from healthy subjects. Toxin-stimulated platelets accelerated plasma clotting, a response that was blocked by a previously-characterized splicing inhibitor and by an anti-tissue factor antibody. Platelets from septic patients expressed spliced TF mRNA, whereas it was absent from unselected and age-matched control subjects. Tissue factor-dependent procoagulant activity was elevated in platelets from a subset of septic patients. Thus, bacterial and host factors induce splicing of TF pre-mRNA, expression of TF mRNA and tissue factor-dependent clotting activity in human platelets. TF mRNA is present in platelets from some septic patients, indicating that it may be a marker of altered platelet phenotype and function in sepsis and that splicing pathways are induced in this syndrome.

© 2011 International Society on Thrombosis and Haemostasis.

Figures

Figure 1. LPS induces splicing of TF pre-mRNA by human platelets

(A) The intron-exon structure of the unspliced TF pre-mRNA and the mature, spliced TF mRNA are shown in diagrammatic form. Primers for exons 4 and 5 that span the intervening intron (bracket and asterisk) were constructed and used to examine platelets for unspliced and spliced TF mRNA transcripts as previously described [18]. (B) Platelets isolated from healthy controls were incubated in medium alone or with LPS for 30–240 minutes. The box-stick diagrams to the right identify human pre-mRNA for TF (pHTF) and mature mRNA for TF (mHTF) based on PCR analysis of the exon 4–5 region outlined in (A) and Methods. Size markers (M) are shown on the left and a lane without PCR primers (Neg. PCR) is shown on the right. The transcript expression patterns shown in this figure are representative of three independent experiments. (C) Platelets from healthy controls were assayed at time zero (second lane) or were activated with 100 ng/ml of LPS for 120 minutes in the presence or absence of the splicing inhibitor Tg003. Left Panel: TF mRNA expression patterns were examined as in (B). Right panel: Membranes from platelets preincubated with Tg003 or control buffer and then activated with LPS were added to human plasma and clotting times were measured in the presence or absence of a neutralizing antibody against TF. The bars represent the mean±SEM for four independent experiments. The asterisk (*) indicates a significant difference (p<0.05) between activated platelets and quiescent or inhibitor-treated platelets. (D) Platelets isolated from healthy subjects were analyzed at baseline (lane 2) or after stimulation with LPS (100ng/ml, 120 min) following preincubation (15 min) in buffer alone (lane 3), with CLI-095 dissolved in DMSO (lanes 4, 5), or with the same final concentrations of DMSO (lanes 6, 7). TF pre-mRNA and mRNA were analyzed as in B and C. CLI-095, an inhibitor of signaling via TLR4 [27], partially blocked TF splicing at the low concentration (2 μm; lane 4) and almost completely blocked it at a higher concentration (3 μm; lane 5). This figure is representative of three experiments. (E) Isolated platelets (2.1+0.2 ×109) or monocytes (4.0+0.03 ×106) were incubated with LPS (100 ng/ml) or with thrombin (0.1 U/ml) for 120 minutes. The number of platelets and monocytes for each study were based on the number of each cell type present in 10 ml of the volunteer donor’s blood, which was determined by differential platelet and monocyte counts assessed on the day of the experiment. TF-dependent procoagulant activity associated with isolated cell membranes was measured in 3 independent experiments. The bars indicate mean±SEM of the experiments and the data are displayed as fold increase over baseline TF activity in unstimulated membranes from platelets and monocytes.

Figure 2. α-toxin induces splicing of TF pre-mRNA and expression of TF-dependent procoagulant activity by human platelets

(A) TF mRNA expression patterns were assessed in platelets from healthy controls that were assayed at time zero (lane 2) or were activated with staphylococcal α-toxin for 120 minutes (lane 3) as described in Figure 1. This gel is representative of 3 independent experiments. (B) Platelets from healthy volunteers were incubated with control buffer or activated with α-toxin for 120 minutes in the presence or absence of Tg003. Plasma clotting was measured as described in Figure 1C. The bars represent the mean ± SEM for four independent experiments and the asterisk (*) indicates a significant difference (p<0.05) between activated platelets and quiescent or inhibitor treated platelets.

Figure 3. E. coli and S. aureus induce splicing of TF pre-mRNA in human platelets

(A) Platelets isolated from healthy controls were incubated in medium alone (control) or in the presence of S. aureus or E. coli bacteria for 30 minutes. The platelets and bacteria were then fixed in suspension and subsequently stained with Alexa 488 phalloidin (for F-actin; green staining in platelets) and TOPRO-3 (for DNA; magenta in bacteria). This figure is representative of multiple independent studies. (B) TF pre-mRNA and mRNA expression patterns were assessed in platelets from healthy controls that were incubated with E. coli or S. aureus for 30 minutes. This gel is representative of 3 independent experiments.

Figure 4. Platelets isolated from patients with sepsis express spliced TF mRNA

(A) Patterns of TF pre-mRNA and mature TF mRNA expression were examined in platelets from healthy control donors and septic patients as outlined in Figure 1. No exogenous activating agonist was added. A representative experiment examining platelets isolated from a healthy control and a septic patient, studied in parallel, is shown. The pie chart below the gel illustrates the number of platelet preparations from septic patients (46 total), isolated within 72 hours of ICU admission, that expressed unspliced or spliced TF transcripts. Unspliced and spliced transcripts were present together in platelets from some septic subjects as shown, consistent with patterns detected in vitro (Figures 1–3). (B) A representative example of serial examination of platelets isolated from a septic patient at day 1, 3, 5, and 10 post-admission to the ICU. Platelets from control donors were assayed in parallel with each sample from the patient; one representative blot is shown in lane 2. The pie chart below the gel illustrates the total number of septic patients (13 of 16) whose platelets expressed spliced TF mRNA on at least one day during the septic episode and serial analysis.

Figure 5. Platelets from patients with sepsis have increased TF-dependent procoagulant activity compared to activity associated with platelets from control subjects

TF-dependent procoagulant activity was measured using membranes and microparticles isolated from platelets in samples from healthy control subjects (n=5) and patients with sepsis (n=5). No exogenous activating agonist was added. Samples from control subjects and septic patients were assayed in side-by-side fashion on the same day. The paired samples are indicated by matching symbols in each panel. The single asterisk (*) indicates a statistical significance of p=0.03. The double asterisks (**) denotes a statistical significance of p