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To compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4), IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta-1 (TGF-β1) from two platelet-rich fibrin (PRF) preparations from equine blood obtained at either 240 g/8 min or 416 g/10 min.Whole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1 h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6 h, 24 h and then every 48 h to 21 days. Cytokines and GFs were measured by ELISA at 1 h (serum supernatants from PRF clots) and all time points of culture medium change. A negative control (plasma) and a positive control (blood lysate) were also included.There were no relevant differences between the two protocols for the temporal release of proteins. However, a significant (p = 0.01) effect of time was noted. All cytokines were detected after 6 h of PRF clot culture until day 21. GF were detected at 1 h until day 21. The concentrations for these proteins diminished gradually over time. A highly significant (p = 0.01) correlation was noticed between all the proteins evaluated.Leukocytes enmeshed in PRF clots were able to produce cytokines, TGF-β1 and PDGF-BB. These findings demonstrate a paramount role of leukocytes in wound healing induced or modified by PRF clots in mammals.PRF is a technology for the treatment of wounds in man and horses.Equine PRF retained and released cytokines and anabolic GFs over three weeks.Leukocytes are of paramount importance to produce cytokines in PRF.GFs are initially released od platelets from PRF and then released from leukocytes.TNF-α acts more as a regulatory protein than a proinflammatory cytokine in PRF.