Platelets sequester circulating histone H2A in plasma from dengue-infected patients.

<p>(<b>A-B</b>) Western blot analysis for histone H2A and β-actin in (<b>A</b>) freshly isolated platelets from three healthy volunteers (Control) and three patients with dengue; and in (<b>B</b>) platelets from three healthy volunteers that were kept unstimulated (Unst) or stimulated with thrombin (Thr), DENV or Mock for 6h. Human peripheral blood mononuclear cells (PBMC) were used as positive control for histone H2A expression. (<b>C</b>) Histone H2A concentration in plasma from control subjects or patients with mild dengue or dengue with warning signs and severe dengue (WS+Sev). Boxes indicate the median and interquartile ranges and whiskers indicate minimal and maximal values in each group. (<b>D-E</b>) Platelets were isolated from a healthy volunteer and incubated with 20% plasma from five dengue-infected patients (dengue plasma) or five healthy volunteers (control plasma) for 4 hours in the presence or absence of cyclohexamide (CHX), cytochalasin B (CTB) or DMSO (vehicle). (<b>F</b>) Histone H2A concentration in plasma from control subjects or patients with dengue, zika or chikungunya fever. Each dot represents the level of histone H2A in plasma from one patient or control. Lines represent median and interquartile range. (<b>G</b>) Western blot analysis for histone H2A and β-actin in platelets incubated with 20% plasma from three control subjects or three patients with dengue, zika or chikungunya. * means p<0.05 compared to control, zika or chikungunya; # indicates p<0.05 between patients with mild and WS+Sev dengue syndromes. Western blots (<b>D, E</b> and <b>G</b>) are representative of three independent experiments from individual platelet donors.</p

Characteristics of dengue-infected patients classified as mild dengue or dengue with warning signs and severe dengue.

<p>Characteristics of dengue-infected patients classified as mild dengue or dengue with warning signs and severe dengue.</p

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue

<div><p>Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. <i>In silico</i> analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.</p></div

Characteristics of dengue-infected patients and healthy volunteers in proteome and validation cohorts.

<p>Characteristics of dengue-infected patients and healthy volunteers in proteome and validation cohorts.</p

Platelets sequester circulating histone H2A in plasma from dengue-infected patients.

<p>(<b>A-B</b>) Western blot analysis for histone H2A and β-actin in (<b>A</b>) freshly isolated platelets from three healthy volunteers (Control) and three patients with dengue; and in (<b>B</b>) platelets from three healthy volunteers that were kept unstimulated (Unst) or stimulated with thrombin (Thr), DENV or Mock for 6h. Human peripheral blood mononuclear cells (PBMC) were used as positive control for histone H2A expression. (<b>C</b>) Histone H2A concentration in plasma from control subjects or patients with mild dengue or dengue with warning signs and severe dengue (WS+Sev). Boxes indicate the median and interquartile ranges and whiskers indicate minimal and maximal values in each group. (<b>D-E</b>) Platelets were isolated from a healthy volunteer and incubated with 20% plasma from five dengue-infected patients (dengue plasma) or five healthy volunteers (control plasma) for 4 hours in the presence or absence of cyclohexamide (CHX), cytochalasin B (CTB) or DMSO (vehicle). (<b>F</b>) Histone H2A concentration in plasma from control subjects or patients with dengue, zika or chikungunya fever. Each dot represents the level of histone H2A in plasma from one patient or control. Lines represent median and interquartile range. (<b>G</b>) Western blot analysis for histone H2A and β-actin in platelets incubated with 20% plasma from three control subjects or three patients with dengue, zika or chikungunya. * means p<0.05 compared to control, zika or chikungunya; # indicates p<0.05 between patients with mild and WS+Sev dengue syndromes. Western blots (<b>D, E</b> and <b>G</b>) are representative of three independent experiments from individual platelet donors.</p

Interaction network of proteins differentially expressed between dengue and control platelets.

<p>(<b>A</b>) List of all 905 interactions between 252 differentially abundant proteins detected by the proteomic analysis. (<b>B</b>) A GO analysis validated the following biological processes: “platelet activation”, “antigen processing and presentation”, “proteasome activity”, “inflammatory response” and “histones”. Proteins labeled with two colors (fill and boundary colors) are involved in two biological processes. Larger circles represent proteins upregulated in dengue. Small circles indicate proteins downregulated in dengue. Squares represent proteins detected only dengue (large) or control (small) conditions.</p

Increased HLA class I on DENV-infected platelets depends on protein translation and proteasome activity.

<p><b>(A)</b> Western blot analysis for HLA class I and β-actin in freshly isolated platelets from two healthy control volunteers and two dengue-infected patients. (<b>B-H</b>) Platelets isolated from healthy volunteers were kept unstimulated (Unst) or stimulated with thrombin (Thr), DENV or Mock for the indicated times. Panel <b>B</b> shows the overall HLA class I expression in platelets from two independent donors at 6 hours post stimulation; and panel <b>C</b> shows the percent of platelets with high surface expression of HLA class I (HLA class I^High) in each condition. (<b>D-H</b>) Platelets were exposed to DENV or Mock in the presence of DMSO (vehicle), bortezomib (1 μM) or cyclohexamide (10 μM). Panel <b>D</b> show the HLA class I expression at 6 hours post infection, panels <b>E</b>-<b>F</b> show the percent of HLA class I ^High expression and panels <b>G-H</b> depicts the P-selectin (CD62P) surface expression in platelets incubated in each condition. Bars represent mean ± standard error of the mean of 3 to 7 independent experiments from individual platelet donors. * indicates p<0.05 compared to unstimulated platelets or Mock; # means p<0.05 between platelets treated with Vehicle and Bortezomib or Cyclohexamide. Representative histograms are shown.</p

Schematic representation of proteome changes in platelets from dengue-infected patients.

<p>(<b>A</b>) Platelets from dengue-infected patients exhibit changes in their proteome, phenotype and function. Alterations in the platelet proteome in dengue appear to influence five main biological processes based on our analysis: (i) platelet activation; (ii) antigen processing and presentation; (iii) processes influenced by proteasome dependent protein catabolism; (iv) inflammatory response and; (v) histone expression and signaling. (<b>B</b>) DENV infection activates platelets triggering granule release of stored chemokines (i.e. PF4/CXCL4 and RANTES/CCL5) and P-selectin surface expression. (<b>C</b>) DENV also increases HLA class I protein expression and its surface presentation through mechanisms requiring proteasome protein processing. The nature of peptides processed by proteasome activity and presented in HLA class I remains unknown. (<b>D</b>) Platelets sequester circulating cell-free histone H2A from plasma, which contributes to platelet activation during dengue infection.</p