Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma

Abstract

Background: Activated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma.

Aims: To determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity.

Methods: PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin.

Results: PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived microparticles, platelets, and platelet aggregates whereas SMHEF pulses primarily resulted in platelet-derived microparticles. Microparticles and platelets in PRP activated with SMLEF bipolar pulses had significantly lower annexin V-positivity than those following SMHEF activation. In contrast, the % P-selectin positivity and surface P-selectin expression (MFI) for platelets and microparticles in SMLEF bipolar pulse activated PRP was significantly higher than that in SMHEF-activated PRP, but not significantly different from that produced by thrombin activation. Higher levels of EGF were observed following either SMLEF bipolar pulses or SMHEF pulses of PRP than after bovine thrombin activation while VEGF, PDGF, and PF4 levels were similar with all three activating conditions. Cell proliferation was significantly increased by releasates of both SMLEF bipolar pulse and SMHEF pulse activated PRP compared to plasma alone.

Conclusions: PEF activation of PRP at bipolar low vs. monopolar high field strength results in differential platelet-derived microparticle production and activation of platelet surface procoagulant markers while inducing similar release of growth factors and similar capacity to induce cell proliferation. Stimulation of PRP with SMLEF bipolar pulses is gentler than SMHEF pulses, resulting in less platelet microparticle generation but with overall activation levels similar to that obtained with thrombin. These results suggest that PEF provides the means to alter, in a controlled fashion, PRP properties thereby enabling evaluation of their effects on wound healing and clinical outcomes.

Fig 1

Representative electrical tracings for A) SMLEF bipolar pulse and B) SMHEF monopolar pulse. A) SMLEF bipolar pulse was ~150 ns pulse width, ~650 ns interval between pulses of opposite polarity,~4kV/cm electric field. B) SMHEF monopolar pulse was pulse ~650 ns, 20 kV/cm electric field. Samples received a total of 80 pairs of bipolar SMLEF pulses at 1 second intervals (the spacing between the opposite polarity pulses within a pair of bipolar pulses was about 650 ns, as shown in Fig 1A; a pair of two bipolar pulses, as shown in Fig 1A, was applied every 1 second–a total of 80 pairs) or 5 monopolar pulses at 1 second intervals. Black tracing: voltage; red tracing: current.

Fig 2. Flow cytometric analysis of platelets and platelet-derived microparticles (PDMP) in PRP following activation with SMLEF bipolar pulses, SMHEF monopolar pulses and thrombin.

A) Representative forward- and side-light scatter profiles of (CD41/CD42b double positive) particles in activated and unactivated PRP samples. The oval indicates the location of the normal forward and side-light scatter distribution for intact platelets; CD41+/CD42b+ particles with lower forward and side light scatter are considered PDMP. B) PDMP as % of all CD41/CD42b double positive particles. Platelet count prior to stimulation was 1095.2 ± 192.9 x 10⁹/L (mean ± SD). C) Percentage of PDMP positive for surface phosphatidylserine as detected by annexin V binding; D) Percentage of platelets positive for surface phosphatidylserine as detected by annexin V binding; E) Percentage of all CD41/CD42b double positive particles positive for surface P-selectin. F) P-selectin mean fluorescence intensity (MFI) per particle. Upper and lower boundaries of boxes represent 25^th and 75^th %tile, whiskers represent 10^th and 90^th %tiles, line indicates median, n = 5. *p<0.05, **p<0.01, ***p<0.001.

Fig 3. Growth factor release and stimulation of cell proliferation.

PRP, treated as described in the Methods, was centrifuged, the supernatant recovered and assayed for pro- and anti-angiogenic factors by ELISA and for stimulation of cell proliferation using serum-starved epithelial cells (MCF10A). Cell proliferation in response to the plasma supernatants of unactivated or activated PRP (panel E) is normalized to that obtained with supernatants of unactivated PRP. Purified recombinant human EGF 100 ng/mL added to serum-free media increased cell proliferation 1.75-fold relative to media alone (data not shown). Results shown are means ± SEM, n = 5. *p<0.05, **p<0.01, ***p<0.001.