Tumor necrosis factor alpha (TNFα ) does not increase platelet aggregation in platelet-rich plasma (PRP) in vitro. (a) ADP-induced (5 μmol/L) platelet aggregation was not increased in human PRP stimulated with TNFα (5 ng/mL, 15 minutes to 4 hours) compared with non-stimulated PRP (n = 10). (b) Addition of supernatant from TNFα (5 ng/mL, 4 hours)-stimulated human microvascular endothelial cells (HMECs) after small interfering RNA (siRNA) knockdown of TNFR1 or TNFR2 to PRP for 90 minutes did not change platelet aggregation (n = 6). (c) No difference in platelet aggregation was observed after incubation with TNFα (5 ng/mL, 30 minutes) in PRP from TNFR1−/− mice. The aggregation traces are representative of five independent experiments. Ctr., control; T1-KD, tumor necrosis factor alpha receptor subtype 1 knockdown; T2-KD, tumor necrosis factor alpha receptor subtype 2 knockdown; TNFR1−/−, tumor necrosis factor alpha receptor subtype 1 knockout; WT, wild-type.