Fig 1.
Weight loss and bacterial dissemination increases over time during invasive infection.
Mice infected with S. pyogenes AP1 were weighed before infection and at 5, 12, and 18 hours post infection and the decrease in body weight was calculated (A). At 0, 5, 12, and 18 hours post infection the mice were sacrificed the bacterial load in blood (B), spleen (C), and liver (D) was determined by serial dilution of homogenates and viable count determination. Horizontal line (A) represents the mean, p-value calculated using a t-test. Horizontal line (B-D) represents median, p-value calculated using a Mann-Whitney U test.
Fig 2.
Circulating neutrophil counts increase rapidly during invasive infection, while platelet counts decrease progressively over time.
At 0, 5, 12, and 18 hours post infection with S. pyogenes the mice were sacrificed and a blood sample was taken by cardiac puncture. Flow cytometry of citrated blood was used to determine neutrophil (A) and platelet counts (B) in each animal. Horizontal line represents median, p-value calculated using a Mann-Whitney U test.
Fig 3.
Activated platelets are not found in ex-vivo blood samples during invasive infection however plasma levels of released CD62P are significantly increased over time.
At 0, 5, 12, and 18 hours post infection with S. pyogenes the mice were sacrificed and a blood sample was taken by cardiac puncture. Flow cytometry of citrated blood was used to determine the activation status of the circulating platelet population activated platelets as median of the fluorescence (MFI) for CD62P (A) and JONA (B). Plasma was prepared and soluble CD62P was determined by ELISA (C). Horizontal line represents median, p-value calculated using a Mann-Whitney U test.
Fig 4.
Platelet-Neutrophil complex formation is increased during the early stages of invasive infection and decreased during late stage infection.
At 0, 5, 12, and 18 hours post infection with S. pyogenes the mice were sacrificed and a blood sample was taken by cardiac puncture. Flow cytometry of citrated blood was used to determine formation of platelet-neutrophil complexes in vivo (A) or post addition of thrombin ex vivo (B). Upregulation of CD11b on neutrophils was also determined (C). The median of the fluorescence for CD11b was measured for each sample with and without prior stimulation with fMLF ex-vivo. Stimulation with fMLF for each sample was set to 100% and the values displayed are for unstimulated samples. Horizontal line represents mean, p-value calculated using a t-test with Welch’s correction for unequal variances.
Fig 5.
Platelets are progressively accumulated in the liver sinusoids at during invasive infection.
At 0, 12, and 18 hours post S. pyogenes infection the mice were sacrificed and the liver was harvested, fixed and embedded in paraffin blocks, sectioned and immunohistochemistry was performed with anti CD41 to detect platelets. Representative images are shown and quantification of the number of platelet aggregates per slide is illustrated as a column graph, where each dot is an individual mouse.
Fig 6.
Organ damage increases over time during invasive infection and correlates with platelet activation and thrombocytopenia.
At 0, 5, 12 and 18 hours post S. pyogenes infection the mice were sacrificed and a blood sample was taken by cardiac puncture. Plasma was prepared and AST activity was determined using a commercial kit (A). Horizontal line represents median, p-value calculated using a Mann-Whitney U test. Correlation between plasma AST activity and platelet count (B) or plasma AST activity and plasma CD62 levels (C) was performed using a Spearman’s rank correlation.