Figure 4.

Platelet-derived CXCL4 drives profibrotic Spp1⁺ macrophage activation

(A) Flow cytometric analysis of Platelet-CMFDA⁺CD11b⁺ platelet-monocyte aggregates after co-culture of WT PBMC with either CMFDA-positive WT or Cxcl4^−/− platelets and stimulation with Vehicle or LPS and Thrombin (n = 4).

(B) RT-qPCR analysis for Arg1, Fn1, and Spp1 in sorted CD11b⁺ monocytes after WT or Cxcl4^−/− platelet-induced activation of WT PBMC. Plt, platelets.

(C) Volcano plot showing differentially expressed genes in CD11b⁺ monocytes activated with either WT or Cxcl4^−/− platelets (n = 4). p-val., p value; plt, platelets; stim., stimulated.

(D) DoRothEA transcription factor analysis of differentially expressed genes in CD11b⁺ monocytes co-cultured with either WT or Cxcl4^−/− platelets.

(E) Expression of a platelet-Cxcl4 activation signature (top upregulated genes defined by an adjusted p value <0.01 and log2FC > 0.5 in WT versus Cxcl4^−/− co-cultured CD11b⁺ monocytes) in cardiac immune cells plotted on the UMAP embedding shown in Figure 1A.

(F) Platelet-Cxcl4 activation signature in cardiac immune cells stratified by immune cell type.

(G) Experimental design of Gli1⁺-fibroblast co-culture with WT or Cxcl4^−/− platelet-stimulated Raw264.7 macrophages. Mac, macrophages; Fibro, fibroblasts; stim, stimulated.

(H) RT-qPCR analysis of Col1a1 and Fn1 expression in Gli1⁺ cardiac fibroblasts after co-culture with WT or Cxcl4^−/− platelet pretreated Raw264.7 macrophages as shown in (C) (n = 6).

For (A) and (B), a two-way ANOVA was computed using Tukey corrections. For (F) and (H), a two-tailed unpaired t test was performed. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. See also Figure S5.