The use of formulations based on thrombocyte concentrate lysates (platelet-released growth factors, PRGF) revealed promising effects in the treatment of chronic wounds. The underlying mechanisms are not well understood. We could show that PRGF induced the expression of antimicrobial peptides (AMPs) such as the human beta-defensin-2 and -3 (hBD-2, hBD-3) in keratinocytes which may contribute to the observed beneficial effects of PRGF-based formulations in wound treatment. Given that it is unclear which components of platelets induce AMPs in keratinocytes we aimed to biochemically identify PRGF-derived factors that induce hBD-2 in human keratinocytes. As PRGF contain a wide variety of different molecules we first investigated if larger (>10 kDa), medium (10 to 3kDa) or smaller (<3 kDa) molecules induced hBD-2. Using molecular weight filtration we found that the fraction >10 kDa was most active although induction levels were lower compared to native PRGF. Notably, reassembly of all three fractions (>10 kDa, 10 to 3 kDa and <3 kDa) restored the effect of the native PRGF indicating a synergistic action of different PRGF-components to induce hBD-2 in keratinocytes. Gel filtration experiments combined by mass spectrometric analyses revealed the presence of several proteins between 30-40 kDa in the hBD-2 inducing fraction. Further purification of the hBD-2 inducing protein(s) by conventional reversed-phase (RP)-HPLC failed due to acid labile properties of the hBD-2-inducing molecules. Optimized RP-HPLC separation strategies using low acidic conditions revealed a distinct hydrophobic fraction containing hBD-2- but no hBD-3-inducing activity. Interestingly, a less hydrophobic fraction contained hBD-3-, but no hBD-2-inducing activity suggesting that PRGF contains distinct proteins that selectively induce hBD-2 and hBD-3 in keratinocytes. These experiments are the basis for the final identification of PRGF-derived mediators that support healing of chronic wounds by their capacity to induce the expression of AMPs in keratinocytes.

Characterization of platelets-derived compounds that induce antimicrobial peptides in human keratinocytes