Differential platelet protein release profiles in community-acquired pneumonia and COVID-19

Figures

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  FIGURE 1. Spontaneous mediator release of platelets in patients with community-acquired pneumonia (CAP) or COVID-19, and controls. Boxplots detailing platelet release of proteins: a) proteins released from platelet α-granules; b) proteins released/shed from other locations within or on platelets. For all proteins except von Willebrand factor (VWF): CAP n=31, COVID-19 n=101 and control n=48. For VFW: CAP n=30, COVID-19 n=100 and control n=47. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. PAI-1: plasminogen activator inhibitor 1; WHO std.: World Health Organization standard; CD40L: CD40 ligand; TFPI: tissue factor pathway inhibitor.

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  FIGURE 2. Effect size comparison of platelets’ spontaneous mediator release in patients with community-acquired pneumonia (CAP) or COVID-19. Forest plot depicting the magnitude of spontaneously released platelet-mediator differences (Hedges’ g) between CAP and COVID-19 patients. An effect size between −0.2 and 0.2 was considered negligible, an effect size >0.8 or <−0.8 large, >0.5 or <−0.5 moderate and >0.2 or <−0.2 small. Shades of red depict a positive effect size, with increasing intensity of colour for higher values. PAI-1: plasminogen activator inhibitor 1; VWF: von Willebrand factor; CD40L: CD40 ligand; TFPI: tissue factor pathway inhibitor.

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  FIGURE 3. Platelet surface protein expression. Expression of platelet surface proteins representative of platelet activation, measured using flow cytometry. a) Platelet surface P-selectin expression of community-acquired pneumonia (CAP) and COVID-19 patients normalised to the respective median level of control samples (dotted line) measured concomitantly to samples of interest. Controls n=45, CAP n=17, COVID-19 n=53. b) Platelet surface expression of GPIIbIIIa in active conformation in CAP and COVID-19 patients normalised to the respective median level of control samples (dotted line) measured concomitantly to samples of interest. Control n=40, CAP n=13, COVID-19 n=37. MFI: median fluorescence intensity; Unstim: unstimulated platelets.

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  FIGURE 4. Principal component analysis and hierarchical clustering based on platelet mediator release. a) Variance of the data explained by different principal components (PC), showing that PC1 is able to capture the majority of variance. b) Boxplots comparing PC1 coordinates per group. p-values were derived from a Wilcox-test comparing the PCs between groups. c) Unsupervised hierarchical clustering. Subjects are clustered based on the value of the different mediators. d) Correlation between spontaneous platelet mediator release and modified early warning score (MEWS) in community-acquired pneumonia (CAP) patients; shaded areas indicate 95% confidence interval. *p<0.05; ***p<0.001; ****p<0.0001. PAI-1: plasminogen activator inhibitor 1; VWF: von Willebrand factor; CD40L: CD40 ligand; TFPI: tissue factor pathway inhibitor.

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  FIGURE 5. Release of platelet proteins following GPVI or PAR1 stimulation in patients with community-acquired pneumonia (CAP) or COVID-19, and controls. Boxplots illustrate protein release from platelets following ex vivo stimulation of GPVI (by CRP-XL) or PAR1 (by TRAP-6) treatment for 30 min at 37°C. a) Proteins from platelet α-granules; b) proteins released or shed from other platelet compartments. Sample sizes for each panel, excluding von Willebrand factor (VWF), are as follows: for CRP-XL stimulated samples, CAP n=32, COVID-19 n=97, control n=48; for TRAP-6 stimulated samples, CAP n=28, COVID-19 n=78, control n=41; for VWF, CRP-XL stimulated samples, CAP n=30, COVID-19 n=96, control n=47; and for TRAP-6 stimulated samples, CAP n=28, COVID-19 n=76, control n=41. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. GPVI: glycoprotein VI; PAR1: protease-activated receptor 1; PC: principal component; CRP-XL: synthetic cross-linked collagen-related peptide; TRAP-6: thrombin receptor-activating peptide-6; PAI-1: plasminogen activator inhibitor 1; CD40L: CD40 ligand; TFPI: tissue factor pathway inhibitor; WHO std.: World Health Organization standard.

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  FIGURE 6. Effect size comparison of platelet release and correlations with surface expression following activation in CAP and COVID-19 patients. a) Forest plot depicting the magnitude of platelet–mediator differences (Hedges’ g) between CAP and COVID-19 patients. An effect size between −0.2 and 0.2 was considered negligible, an effect size >0.8 or <−0.8 large, >0.5 or <−0.5 moderate and >0.2 or <−0.2 small. Shades of red depict a positive effect size, with increasing intensity of colour for higher values. b) The upper panel displays the correlation between the release of platelet mediators and P-selectin surface expression following CRP-XL treatment in CAP and COVID-19 patients. The lower panel presents a similar analysis for TRAP-6-treated platelets. c) Correlation between platelet mediator release and GPIIbIIIa expression. For both b and c the vertical coloured bar at the right indicates the Spearman correlation coefficient: darker red signifies a higher rho, while darker blue indicates a lower rho. Correlations significant at p<0.05 are marked with an asterisk.

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  FIGURE 7. Odds ratio for (non)invasive ventilation and/or 30-day mortality. Forest plot illustrating the odds ratio of a composite end-point of (non)invasive ventilation and/or 30-day mortality for a 10% increase in the release of platelet proteins, derived from a logistic regression model. The horizontal lines depict the 95% confidence interval, the colour of the dot represents significance. PAI-1: plasminogen activator inhibitor 1; CD40L: CD40 ligand; GPV1: glycoprotein VI.