Platelet lysate stimulates wound repair of HaCaT keratinocytes.
BACKGROUND
Platelets play a pivotal role in wound healing. Their beneficial effect is attributed to the release of bioactive substances, although the involved mechanisms are mostly unknown.


OBJECTIVE
To investigate mechanisms underlying platelet-induced wound healing using HaCaT keratinocytes, representing an in vitro model of proliferating and migrating keratinocytes.


METHODS
Cells were exposed to platelet lysate (PL) purified from whole blood samples. Cell metabolism and proliferation were assessed using MTS and crystal violet assays, respectively, wound healing was assessed by scratch wound assay and cell migration by transwell assay. Extracellular signal-regulated kinase (ERK) 1/2 and p38 activations were studied using Western immunoblotting and intracellular Ca(2+) dynamics by confocal imaging.


RESULTS
Wound closure rates showed a significant increase at 6 and 24 h in cells exposed to nontoxic 20% PL. The cell migration assay showed a strong chemotactic effect toward PL. The intracellular Ca(2+) chelator BAPTA-AM induced 100% inhibition of the PL effect on wound closure rate, while among the kinase inhibitors, SB203580 exerted about 50% inhibition, and PD98059, wortmannin and LY294002 about 30% inhibition. SB203580 and BAPTA-AM induced 100% inhibition of the PL effect on cell migration, PD98059 about 50% inhibition, and wortmannin and LY294002 no significant inhibition. Confocal imaging allowed detection of a sustained Ca(2+) transient in PL-treated cells, while Western blot showed a more rapid activation of p38 than of ERK1/2.


CONCLUSIONS
Data indicate that PL increases wound healing rate by stimulating keratinocyte migration through a calcium- and p38-dependent mechanism. ERK1/2 and phosphoinositide-3 kinase seem to play minor roles.
Ranzato
E
E
Department of Environmental and Life Sciences, University of Piemonte Orientale, via Bellini 25/G, 15100 Alessandria, Italy. ranzato@mfn.unipmn.it
Patrone
M
M
Mazzucco
L
L
Burlando
B
B
D000704:Analysis of Variance; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000032:analysis; D020013:Calcium Signaling; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D018920:Coculture Techniques; D004789:Enzyme Activation; D006801:Humans; D015603:Keratinocytes / Q000502:physiology*; D020935:MAP Kinase Signaling System; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010976:Platelet Count; D014945:Wound Healing / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D019869:Phosphatidylinositol 3-Kinases; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D002118:Calcium