Figure 2

Cold preconditioning promotes the release of growth factors and cytokines from platelets. (A) PRP was incubated at 37 °C, 23 °C, or 4 °C for 30 min, and the levels of VEGF, EGF, bFGF, IL-17, IL-8, and PDGF in releasates of PRP before clotting (CaCl₂−) or after clotting (CaCl₂+) were determined by ELISA. Total intra-platelet levels were measured in platelet lysates. (B) PRP was incubated at 37 °C, 23 °C, or 4 °C for 30 min, clotted with CaCl₂ (22 mM) for 40 min, and releasates were used to induce HMEC-1 (15 × 10³) proliferation. Saline plus FBS 2% was used as control. (C) HMEC-1 (15 × 10³) were incubated, individually or in combinations, with anti-human VEGFR2/KDR/Flk-1 (10 µg/ml), anti-human-EGFR (40 µg/ml) or irrelevant IgG, for 30 min. Next, endothelial proliferation was induced by addition of recombinant VEGF (20 ng/ml), EGF (20 ng/ml) or with PRPr preincubated at 37 °C or 4 °C and then clotting was induced with CaCl₂ (22 mM). Saline plus FBS 2% was used as control (n = 4–5, *P < 0.05, **P < 0.01, ***P < 0.001 vs. unstimulated; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 vs. 37 °C; ^‡P < 0.05, ^‡‡P < 0.01, ^‡‡‡P < 0.001 vs. lysis; ^#P < 0.05 vs. without neutralizing antibodies (Abs)). VEGF: vascular endothelial growth factor; EGF: epidermal growth factor; bFGF: basic fibroblast growth factor; PDGF: platelet derived growth factor; IL: interleukin.