Tunable activation of therapeutic platelet-rich plasma by pulse electric field: Differential effects on clot formation, growth factor release, and platelet morphology

PLoS One. 2018 Sep 26;13(9):e0203557. doi: 10.1371/journal.pone.0203557. eCollection 2018.

Abstract

Background: Activation of platelet-rich plasma (PRP) by pulse electric field (PEF) releases growth factors which promote wound healing (e.g., PDGF, VEGF for granulation, EGF for epithelialization).

Aims: To determine after PEF activation of therapeutic PRP: 1) platelet gel strength; 2) profile of released growth factors; 3) alpha- and T-granule release; 4) platelet morphology.

Methods: Concentrated normal donor PRP was activated by 5 μsec (long) monopolar pulse, ~4000 V/cm (PEF A) or 150 nsec (short) bipolar pulse, ~3000 V/cm (PEF B) in the presence of 2.5 mM (low) or 20 mM (high) added CaCl2. Clot formation was evaluated by thromboelastography (TEG). Surface exposure of alpha granule (P-selectin) and T-granule (TLR9 and protein disulfide isomerase [PDI]) markers were assessed by flow cytometry. Factors in supernatants of activated PRP were measured by ELISA. Platelet morphology was evaluated by transmission electron microscopy (TEM).

Results: Time to initial clot formation was shorter with thrombin (<1 min) than with PEF A and B (4.4-8.7 min) but clot strength (elastic modulus, derived from TEG maximum amplitude) was greater with PEF B than with either thrombin or PEF A (p<0.05). Supernatants of PRP activated with PEF A had higher EGF levels than supernatants from all other conditions. In contrast, levels of PF4, PDGF, and VEGF in supernatants were not significantly different after PEF A, PEF B, and thrombin activation. T-granule markers (TLR9 and PDI) were higher after thrombin than after PEF A or B with low or high CaCl2. By TEM, platelets in PEF-treated samples retained a subset of granules suggesting regulated granule release.

Conclusion: Pulse length and polarity can be modulated to produce therapeutic platelet gels as strong or stronger than those produced by thrombin, and this is tunable to produce growth factor profiles enhanced in specific factors important for different stages of wound healing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Electrochemical Techniques
  • Enzyme-Linked Immunosorbent Assay
  • Epidermal Growth Factor / metabolism
  • Healthy Volunteers
  • Humans
  • Intercellular Signaling Peptides and Proteins / metabolism*
  • Microscopy, Electron, Transmission
  • P-Selectin / metabolism
  • Platelet Activation / physiology*
  • Platelet-Derived Growth Factor / metabolism
  • Platelet-Rich Plasma / metabolism*
  • Thrombelastography
  • Toll-Like Receptor 9 / metabolism
  • Vascular Endothelial Growth Factor A / metabolism

Substances

  • Intercellular Signaling Peptides and Proteins
  • P-Selectin
  • Platelet-Derived Growth Factor
  • Toll-Like Receptor 9
  • Vascular Endothelial Growth Factor A
  • Epidermal Growth Factor

Grants and funding

This work was funded by a research grant from GE Healthcare to Boston Children’s Hospital, Principal Investigator, A.L. Frelinger. The funders participated in study design, data collection, and preparation of the manuscript, but had no role in data analysis or the decision to publish. Authors V.B. Neculaes, A.S. Torres, and A. Caiafa are employed by a commercial company: GE Global Research Center. V.B. Neculaes and A.S. Torres contributed to design of the studies and provided critical revisions to the manuscript. A. Caiafa contributed to design of the studies, performed experiments and provided critical revisions to the manuscript. Neither these authors nor the funders had a role in data analysis or the decision to publish.