Platelet-derived factors selectively upregulate tumor cell IL-8. To identify factors in platelet releasate that could be driving IL-8 production in tumor cells, TRAP-, MCF-7-, and MDA-231-activated platelet releasates were analyzed using the angiogenesis array described in Figure 1. (A) Proteins identified in APR at the highest abundance were used to direct future experiments. (B) To support the array results, platelets (plt) were activated with either breast tumor cell lines (3 × 10⁶/mL) or TRAP (5 μM), and release of CCL5 was measured by ELISA. (C) To determine whether CCL5 alone is sufficient to drive tumor cell IL-8, tumor cells were treated with 100 to 1000 ng/mL rhCCL5, and supernatant IL-8 was measured 24 hours later by ELISA. (D) IL-8 secretion in response to platelets was measured in the presence or absence of the CCL5 receptor antagonist, maraviroc (100 μM), to determine whether CCL5 is the specific factor in platelet releasate that drives tumor cell IL-8. CCR5 expression was determined by immunofluorescence in MDA-MB-231, MCF-7, BT-20, and SKBR-3 cell lines (E) and human platelets (F). (G-H) To determine whether platelet-derived CCL5 can drive metastasis, maraviroc was used to block CCR5 in invasion assays. Scale bars represent 10 μm. P values for panels B and D were determined by separate unpaired Student t tests. P values for panels C and G were determined by 1-way ANOVA, with post hoc Dunnett’s or Tukey’s multiple comparisons testing, respectively. n = 1 single experiment in panel A and 3 to 4 independent replicates per treatment group in panels B-D and G-H.