Platelet Glycoprotein Ibα Cytoplasmic Tail Exacerbates Thrombosis During Bacterial Sepsis

Abstract

Septic patients, coupling severe disseminated intravascular coagulation (DIC) and thrombocytopenia, have poor prognoses and higher mortality. The platelet glycoprotein Ibα (GPIbα) is involved in thrombosis, hemostasis, and inflammation response. However, it remains unclear whether the GPIbα cytoplasmic tail regulates sepsis-mediated platelet activation and inflammation, especially in Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) infections. Using a mouse model of S. aureus-induced bacteremia, we found that both 10 amino acids of GPIbα C-terminal sequence deficiency and pharmacologic inhibition of protein kinase C (PKC) alleviated pathogenesis by diminishing platelet activation and aggregate formation. Furthermore, the GPIbα cytoplasmic tail promoted the phagocytosis of platelets by Kupffer cells in vivo. The genetically deficient GPIbα cytoplasmic tail also downregulated inflammatory cytokines and reduced liver damage, ultimately improving the survival rate of the septic mice. Our results illustrate that the platelet GPIbα cytoplasmic domain exacerbates excessive platelet activation and inflammation associated with sepsis through a PKC-dependent pathway. Thus, our findings provide insights for the development of effective therapeutic strategies using PKC inhibitor treatment against bacterial infection.

Keywords: GPIbα; PKC; bacterial infection; inflammation; platelet activation.

Scheme 1

Schematic diagram depicting GPIbα-14-3-3ζ regulation of platelet activation during bacterial infection. Dissociation of 14-3-3ζ from GPIbα results in decreased PKC activity and PKC-mediated integrin α_IIbβ₃ activation, and granule secretion. MPαC, a peptide corresponding to the C-terminal 14-3-3ζ binding sequence, compensates for the absence of cytoplasmic domain and restores platelet signaling and function.

Figure 1

Deletion of the GPIbα cytoplasmic tail diminishes bacteria-induced liver damage, inflammatory cytokines, and mortality. (A,B) WT and 10aa^−/− mice were injected with S. aureus (A) or E. coli (B) (1.5 × 10⁸ CFU). The survival rate was observed for up to 8 days (n= 12 mice). (C,D) Blood was collected from WT and 10aa^−/− mice before or at 1, 2, and 4 h after S. aureus injection i.v. The level of TNFalpha (C) and IL-6 (D) in plasma was measured by an enzyme-linked immunosorbent assay (n = 6 independent experiments). (E) 24 h after S. aureus injection, the mice were killed and their livers were collected. (F,G) 24 h after S. aureus treatment, plasma from mice was collected and aspartate aminotransferase (AST) (F), alanine aminotransferase (ALT) (G) concentrations were measured (n = 5 independent experiments). Log-rank (Mantel–Cox) test in (A,B). Two-way ANOVA test in (C,D,F,G). Data are shown as mean ± SEM (C–G). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 2

The GPIbα cytoplasmic tail aggravates S. aureus-induced platelet activation. (A) Platelet counts in the peripheral blood of WT and 10aa^−/− mice after S. aureus injection i.v. at 24 h (n = 5 mice). (B) Representative confocal images and quantification of WT and 10aa^−/− platelet and S. aureus interaction in vivo (n = 10 visual fields from 3 mice of each genotype). Scale bar: 10 μm. (C) Aggregation of platelets from WT and 10aa^−/− treated with fibrinogen in response to S. aureus (n = 6 independent experiments). (D,E) Flow cytometric analysis of P-selectin expression (D) and JON/A binding (E) of WT and 10aa^−/− platelets treated with PAR4-AP (n = 3 mice). (F) The platelets of WT and 10aa^−/− mice were transfused into Mpl^−/− mice or without platelet transfusion (platelets resuspended buffer: MTB as Baseline) following injection with S. aureus (1.5 × 10⁸ CFU). Survival rate was monitored for up to 8 days (n = 8 mice per genotype). Differences between the two groups were assessed using unpaired two-way ANOVA tests (A,D,E) and unpaired two-tailed Student’s t-tests in (B,C). Log-rank (Mantel–Cox) test in (F). Data are shown as the mean ± SEM. * p < 0.05; ** p < 0.01; and *** p < 0.001; ns, no significance.

Figure 3

The GPIbα cytoplasmic tail exacerbates platelet aggregation in S. aureus-infected mice. (A) Representative intravital microscopy (IVM) images of WT and 10aa^−/− platelets (DyLight649-GP1bβ, Green), S. aureus (PH-red, Red) and Kupffer cells (F4/80, Blue) in the liver at 30 min post-injection (n = 3 independent experiments). Scale bars: 25 μm. (B,C) Quantitative analysis of the area of platelets and bacteria in the liver at different time points (n = 3 of mice). (D) Bacterial burden in blood at indicated time points (4 h) (n = 5 mice per group). (E) Representative intravital microscopy (IVM) images and quantitative analysis of platelet aggregates (DyLight649-GP1bβ, Green) in the WT and 10aa^−/− liver after 24 h injection of S. aureus (n = 3 mice per group of different visions). Scale bars: 25 μm. Differences between two groups were assessed using two-way ANOVA test (B,C) and unpaired two-tailed Student’s t-test (D,E). Data are shown as the mean ± SEM from three or five independent experiments. * p < 0.05; *** p < 0.001; ns, no significance.

Figure 4

MPαC recovers platelet aggregation of 10aa^−/− mice in bacterial sepsis. (A) Aggregation and quantification plots of WT and 10aa^−/− murine platelets treated with S. aureus with or without MPαC, (6.25 μM) (n = 3 independent experiments). (B) Representative images and quantification of IVM in the WT and 10aa^−/− livers intravenous injection of MPαCsc/MPαC before S. aureus infection after 24 h (n = 3 mice), Platelets: (DyLight649-GP1bβ, Red), Vessels: (FITC-dextran 2000, green), Scale bars: 25 μm. Differences between two groups were assessed using two-way ANOVA test (A,B). Data are shown as the mean ± SEM from three or five independent experiments. ** p < 0.01; ns, no significance.

Figure 5

The GPIbα cytoplasmic tail regulates S. aureus-induced platelet activation by the PKC-dependent pathway. (A) Representative blots and quantification of the phosphorylation levels of PKC substrate (pleckstrin) in WT and 10aa^−/− platelets treated with S. aureus (n = 5 independent experiments). (B,C) The effect of PKC inhibitor GO6983 on washed murine (B) and human (C) platelets aggregation induced by S. aureus (n = 3 independent experiments). (D) The level of phosphorylation of PKC substrate (pleckstrin) in human platelets pre-treated with GO6983 stimulated by S. aureus (n = 3 independent experiments). Two-way ANOVA test in (A). One-way ANOVA followed by Dunnett’s post hoc test in (B–D). Data are shown as the mean ± SEM. ** p < 0.01; *** p < 0.001; ns, no significance.

Figure 6

Inhibition of PKC protects mice from S. aureus-induced platelet clearance and thrombosis and prolongs survival. (A,B) The effect of PKC inhibitors on the accumulation of calcein-labeled and S. aureus-bound platelets in murine liver observed by intravital confocal microscopy (n = 3 independent experiments), platelets: (DyLight649-GP1bβ, Green), S. aureus: (PH-red, Red), Kupffer cells: (F4/80, Blue), Scale bars: 25 μm. (C) Representative images and quantification of IVM in the Mpl^−/− lung transferred with a mixture of the GO6983-treated platelet and S. aureus (n = 3 independent experiments), platelets: (DyLight649-GP1bβ, Green), S. aureus: (PH-red, Red), Scale bars: 25 μm. (D) The effect of GO6983 on S. aureus-induced platelet aggregates formation after 24 h observed by IVM, Scale bars: 25 μm. (E) The platelets pre-treated with or without GO6983 were transfused into Mpl^−/− mice after being injected with S. aureus (1.5 × 10⁸ CFU). Survival rate was observed for up to 8 days (n = 8 mice per genotype). (F) Representative images and quantification of IVM in the WT and pkcα^−/− livers and lung injected with S. aureus after 24 h (n = 3 mice per group of different visions), platelets: (DyLight649-GP1bβ, Red), Vessels: (FITC-dextran 2000, green), Scale bars: 25 μm. Two-way ANOVA test in (B). Unpaired two-tailed Student’s t-test in (C,D,F). Log-rank (Mantel–Cox) test in (E). Data are shown as the mean ± SEM. * p < 0.05, *** p < 0.001; ns, no significance.