Fig. 2.

Thrombospondin-1 (TSP-1) isoforms generated in co-cultures of vascular cell populations. Confluent endothelial cell (EC) cultures grown in serum-free medium for 48 hours were either left untreated (ECs) or were supplied with freshly isolated human ( A ) peripheral blood mononuclear cells (PBMCs), ( B ) platelets or ( D ) neutrophils. Where indicated (+) a stimulus was added to activate PBMCs (lipopolysaccharide [LPS] at 1 µg/mL), platelets (thrombin receptor activator peptide 6 [TRAP-6] at 10 µM) or neutrophils (phorbol myristate acetate [PMA] at 100 ng/ml). ( C ) Platelets were also combined with neutrophils or kept separately for control. Supernatant was collected after 0.5, 1, 2 and 4 hours of culture. Reduced protein samples were analysed by immunoblots with Ab11. Each experiment was repeated four times with blood drawn from different donors. Please refer to Supplementary Fig. S2 (available in the online version) for quantitation of immunoblots. M, biotinylated protein marker.