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

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.

Fig 1. Representative electrical tracings for PEF A (A) and PEF B (B).

Fig 2. Characteristics of clot formation following exposure of PRP to PEF or thrombin at low (2.5 mM) and high (20 mM) concentrations of CaCl₂.

A) Representative TEG plots, B) clotting time (R in min), C) clot strength (maximum amplitude [MA] in mm), D) prothrombin fragment F.1.2 (nmol/L). Individual results are plotted as mean ± SEM (n = 5). Asterisks indicate p<0.05 vs. thrombin, high CaCl₂ (filled blue squares) by Dunnett’s multiple comparison test (following ANOVA).

Fig 3. Platelet surface markers of degranulation for alpha granules (P-selectin) and T granules (TLR9 and PDI), and soluble PDI, following exposure of PRP to PEF, thrombin, or control conditions.

Individual results are plotted as mean ± SEM. Asterisks indicate p<0.05 vs. thrombin, high CaCl₂ (filled blue squares) by Dunnett’s multiple comparison test (following ANOVA). MFI, mean fluorescence intensity.

Fig 4. Growth factors present in the supernatant following PEF, thrombin, or control treatment of PRP at low and high CaCl₂.

Results below the detection limit of the assay are plotted at the lower limit of detection. Individual results are plotted as mean ± SEM. Asterisks indicate p<0.05 vs. thrombin, high CaCl₂ (filled blue squares) by Dunnett’s multiple comparison test (following ANOVA).

Fig 5. Correlation of granule markers with growth factor release.

Fig 6. Platelet morphology by transmission electron microscopy following activation of PRP.

Concentrated PRP supplemented with the indicated concentrations of CaCl₂ were activated by A) PEF A, B) PEF B, C) bovine thrombin, or D) vehicle, then processed as described in Methods. Direct magnification is indicated. Representative images are shown for one representative donor.

Fig 7. Platelet alpha granule content following activation of PRP and correlation with supernatant growth factor levels.

A) Quantitation of alpha granules in TEM images of platelets following activation of PRP. Asterisks indicate p<0.05 vs. thrombin, high CaCl₂ (filled blue squares) by Dunnett’s multiple comparison test (following ANOVA). B—E) Correlation of residual platelet alpha granule numbers with growth factor concentrations in supernatants following PEF, thrombin, or control treatment of PRP at low and high CaCl₂.