Key Points• NET formation is stimulated by platelet or soluble P-selectin.Neutrophil extracellular traps (NETs) can be released in the vasculature. In addition to trapping microbes, they promote inflammatory and thrombotic diseases. Considering that P-selectin induces prothrombotic and proinflammatory signaling, we studied the role of this selectin in NET formation. NET formation (NETosis) was induced by thrombin-activated platelets rosetting with neutrophils and was inhibited by anti-P-selectin aptamer or anti-P-selectin glycoprotein ligand-1 (PSGL-1) inhibitory antibody but was not induced by platelets from P-selectin 2/2 mice. Moreover, NETosis was also promoted by P-selectin-immunoglobulin fusion protein but not by control immunoglobulin. We isolated neutrophils from mice engineered to overproduce soluble P-selectin (P-selectin DCT/DCT mice). Although the levels of circulating DNA and nucleosomes (indicative of spontaneous NETosis)were normal in these mice, basal neutrophil histone citrullination and presence of P-selectin on circulating neutrophils were elevated. NET formation after stimulation with platelet activating factor, ionomycin, or phorbol 12-myristate 13-acetate was significantly enhanced, indicating that the P-selectin DCT/DCT neutrophils were primed for NETosis. In summary, P-selectin, cellular or soluble, through binding to PSGL-1, promotes NETosis, suggesting that this pathway is a potential therapeutic target for NET-related diseases. (Blood. 2015;126(2):242-246)
Although platelets are widely recognized as having a critical role in primary hemostasis and thrombosis, increasing experimental and clinical evidence identifies these enucleated cells as relevant modulators of other physiopathological processes including inflammation and tissue regeneration. These phenomena are mediated through the release of growth factors, cytokines, and extracellular matrix modulators that sequentially promote (i) revascularization of damaged tissue through the induction of migration, proliferation, differentiation, and stabilization of endothelial cells in new blood vessels; (ii) restoration of damaged connective tissue through migration, proliferation, and activation of fibroblasts; and (iii) proliferation and differentiation of mesenchymal stem cells into tissue-specific cell types. For these reasons, platelet-rich plasma (PRP) derivatives are used in regenerative medicine for the treatment of several clinical conditions including ulcers, burns, muscle repair, bone diseases, and tissue recovery following surgery. The benefits of PRP administration are associated with an economical advantage, taking into consideration that PRP administration does not require complex equipment or training for its execution. Moreover, due to their primary autologous origin, concerns of disease transmission or immunogenic reactions can be disregarded. Thus, platelet-enriched materials have become highly relevant in the last decade and constitute a growing focus of experimental and clinical study in the context of wound healing and tissue regeneration. However, despite the diverse applications, the efficacy of regenerative treatments using PRP is being called into question due to the lack of large controlled clinical trials and the lack of consensus regarding the PRP preparation techniques. This review describes the biological mechanisms underlying PRP's regenerative effects, the different methods of preparation and application of these biomaterials, and the controversies and future prospects related to the use of PRP in regenerative medicine.
The formation of neutrophil extracellular traps (NETs) is a newly described phenomenon that increases the bacteria-killing ability and the inflammatory response of neutrophils. Because NET generation occurs in an inflammatory microenvironment, we examined its regulation by anti-inflammatory drugs. Treatment of neutrophils with dexamethasone had no effect, but acetylsalicylic acid (ASA) treatment prevented NET formation. NETosis was also abrogated by the presence of BAY 11-7082 [(E)-3-[4-methylphenylsulfonyl]-2-propenenitrile] and Ro 106-9920 [6-(phenylsulfinyl)tetrazolo [1,5-b]pyridazine], two structurally unrelated nuclear factor-kB (NF-kB) inhibitors. The decrease in NET formation mediated by ASA, BAY-11-7082, and Ro 106-9920 was correlated with a significant reduction in the phosphorylation of NF-kB p65 subunit, indicating that the activation of this transcription factor is a relevant signaling pathway involved in the generation of DNA traps. The inhibitory effect of these drugs was also observed when NET generation was induced under acidic or hyperthermic conditions, two stress signals of the inflammatory microenvironment. In a mouse peritonitis model, while pretreatment of animals with ASA or BAY 11-7082 resulted in a marked suppression of NET formation along with increased bacteremia, dexamethasone had no effect. Our results show that NETs have an important role in the local control of infection and that ASA and NF-kB blockade could be useful therapies to avoid undesired effect of persistent neutrophil activation.
Although platelet-rich plasma (PRP) is used as a source of growth factors in regenerative medicine, its effectiveness remains controversial, partially due to the absence of PRP preparation protocols based on the regenerative role of platelets. Here, we aimed to optimise the protocol by analysing PRP angiogenic and regenerative properties. Three optimising strategies were evaluated: dilution, 4 °C pre-incubation, and plasma cryoprecipitate supplementation. Following coagulation, PRP releasates (PRPr) were used to induce angiogenesis in vitro (HMEC-1 proliferation, migration, and tubule formation) and in vivo (chorioallantoic membrane), as well as regeneration of excisional wounds on mouse skin. Washed platelet releasates induced greater angiogenesis than PRPr due to the anti-angiogenic effect of plasma, which was decreased by diluting PRPr with saline. Angiogenesis was also improved by both PRP pre-incubation at 4 °C and cryoprecipitate supplementation. A combination of optimising variables exerted an additive effect, thereby increasing the angiogenic activity of PRPr from healthy donors and diabetic patients. Optimised PRPr induced faster and more efficient mouse skin wound repair compared to that induced by non-optimised PRPr. Acetylsalicylic acid inhibited angiogenesis and tissue regeneration mediated by PRPr; this inhibition was reversed following optimisation. Our findings indicate that PRP pre-incubation at 4 °C, PRPr dilution, and cryoprecipitate supplementation improve the angiogenic and regenerative properties of PRP compared to the obtained by current methods.
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