Angiogenesis regulators loaded into platelets are not released with agonists of platelet activation. (A) A total of 1 mL PRP was incubated with 600 ng/mL of either VEGF or bFGF for 30 minutes. This resulted in “loading” of these proteins into the α-granules of platelets similarly to Figure 1. After the incubation, the platelets were aggregated using a mild aggregation agonist (ADP), a more potent agonist (thrombin), or spun down without any stimulation (control). The resulting supernatants/sera were then analyzed for VEGF and bFGF using a commercially available ELISA. Neither of the platelet agonists was capable of releasing bFGF from platelets. In the case of VEGF, ADP-induced aggregation failed to release the VEGF and even the more potent aggregation with thrombin resulted in only a modest release of the loaded VEGF. (B) PRP was incubated with 100 ng bFGF/mL on gentle rocker at room temperature for 45 minutes, spun at 150g, and the plasma containing excess of the protein was removed. The platelets were then resuspended in 1 mL saline to which 20 mM ADP or 1 unit thrombin was added. The sample was then spun again at 900g to pellet the platelets, and the supernatant and platelet analyzed using bFGF ELISA. As evident, platelets retained the majority of the loaded protein, and protein was released with either agonist. Both experiments were repeated on 2 separate occasions, and the graph represents the mean of 5 samples per dose level plus or minus SEM. Loading concentration in panel A corresponds to 600 ng VEGF or bFGF per mL.