Platelet P-selectin triggers rapid surface exposure of tissue factor in monocytes

Figures

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  Figure 1. Platelet activators rapidly trigger monocyte TF exposure in whole blood. (a,b) PPACK-anticoagulated human whole blood was stimulated with PAR1-AP (1–100 µM; 5–25 mins) prior to staining with conjugated primary antibodies. Median fluorescence intensity (MFI) is shown for TF on CD14 + monocytes (a) and P-selectin on CD41a + platelets (b) (mean + S.E.M.; n = 5; n.s. not significant p > 0.05; *p < 0.05; **p < 0.01 compared to unstimulated at the same timepoint; Friedman’s non-parametric test with Dunn’s multiple comparison post-test). (c) Whole blood was stimulated with a range of concentrations of the platelet activators, CRP-XL, PAR4-AP, and ADP, for 10 minutes. Monocyte TF and platelet P-selectin are shown on the same axes for comparison of concentration-dependency of their effects.

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  Figure 2. P2Y12 inhibition reduces monocyte TF and platelet P-selectin exposure. Whole blood was treated with aspirin (100 µM), the P2Y12 antagonist, AR-C69906 (10 µM), or their solvents as control, for 10 min prior to stimulation with PAR1-AP (10 µM). Data are mean + S.E.M. (n = 5; n.s. not significant; *p < 0.05; **p < 0.01 for indicated comparison).

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  Figure 3. Platelets are necessary and sufficient for rapid monocyte TF exposure. (a) Isolated monocytes were treated with PAR1-AP (10 µM, 5–10 min) in the absence or presence of washed platelets. (n = 5; ***P < 0.001 for indicated comparison) (b) Washed platelets were stimulated with PAR1-AP, fixed with paraformaldehyde (PFA) then collected by centrifugation to separate the releasate (supernatant) and washed activated-fixed (W A-F) platelets (pellet). As a control, some platelets left unstimulated prior to fixation (washed unactivated-fixed, W U-F) (c,d) Isolated monocytes were incubated with washed unactivated-fixed (W U-F), washed activated-fixed platelets (W A-F) and/or releasate fractions for 5 min. Monocyte TF exposure (c) and monocyte-bound P-selectin (d) are shown as mean + S.E.M. (n = 5; *p < 0.05; **p < 0.01 compared to monocytes alone; Friedman’s non-parametric test with Dunn’s multiple comparison post-test).

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  Figure 4. P-selectin is necessary and sufficient for rapid monocyte TF exposure. PPACK-anticoagulated whole blood was treated with blocking antibodies against ICAM1, ICAM2, CD40, CD11b, CD18, CD42b, P-selectin and PSGL1 (a-b) or the αIIbβ3 antagonist, eptifibatide (10 µM) before stimulation with PAR1-AP (10 μM, 10 min; c-d). Monocyte TF (a, c) and platelet P-selectin (b, d) were detected by flow cytometry (mean + S.E.M; n = 3–6; *p < 0.05; **p < 0.01; ***p < 0.001 compared to PAR1-AP-stimulated samples in absence of inhibitor or blocking antibody; one-way ANOVA with Dunnett’s multiple comparisons post-test). (e,f) Isolated monocytes were stimulated with platelets and PAR1-AP (as positive control) or with soluble recombinant P-selectin at the concentrations indicated for 30 min (e,f) or with control beads or P-selectin-coated beads (g,h). Monocyte TF exposure and monocyte-bound P-selectin are shown in (e,g) and (f,h), respectively. (***p < 0.001 for indicated comparison; one-way ANOVA with Dunnett’s multiple comparison post-test; in e, n.s. indicate not significantly different from monocytes alone).

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  Figure 5. Rapid monocyte TF exposure does not require protein synthesis. PPACK-anticoagulated blood (a,b) or isolated monocytes (c,d) were treated with cycloheximide (100 µM) or vehicle control. Whole blood was stimulated with PAR1-AP for the indicated times and isolated monocytes were stimulated with P-selectin-coated beads for 30 min. (n.s. indicates no significant difference for indicated comparison; one-way ANOVA with Tukey’s multiple comparison post-test.) (e) Summary of proposed rapid platelet-dependent exposure of monocyte TF. Platelet activators trigger α-granule secretion leading to P-selectin exposure on the platelet surface. P-selectin interacts with monocyte PGSL-1, rapidly triggering TF exposure without new protein expression.