(A) TNF prestimulation of SMC enhances the response to platelets. SMC were incubated in 24-well plates until they reached confluency, washed, and medium (None) or recombinant TNF (50 ng/mL) was added. After 4 hours SMC were washed and 1,000 platelets per SMC were added. Plates were centrifuged (500g, 10 minutes), and medium lacking thrombin (Medium) or medium containing thrombin (1 U/mL; Thrombin) was added for a further 24 hours. IL-6 activity in supernatants was measured in 7TD1-assay. Four similar experiments were performed. (B) Prestimulation of SMC with LPS, TNF, or IL-1. In a separate experiment SMC were cultured and prestimulated for 4 hours with LPS (1 μg/mL), TNF-α (TNF, 50 ng/mL), or IL-1α (IL-1, 10 ng/mL) as described in (panel A) and thrombin-activated platelets were added. The IL-6 production of SMC not prestimulated but incubated with thrombin-activated platelets (None) was determined 0% and the maximal IL-6 production after IL-1 prestimulation determined 100%. Two similar experiments were performed.