## 2 Materials and Methods
### Pulse Electric Fields Application  
The pulse electric fields were applied in batch mode using a cuvette connected to a pulse generator. A total of 1 mL of PC was added into the cuvette chamber and pulses from a pulse generator, with a frequency of 1 Hz, were applied through the cuvette electrodes, as seen in Figure 1a,b. The pulse voltage and current was measured using a digital oscilloscope (LeCroy WaveAce 224), with a sampling rate of 1 GS/s, connected to a current transformer (Pearson 110) and a high voltage probe (North Start High Voltage PVM-1). As an operation example, a 2 \(\upmu\)s pulse with 4.5 kV and 150 A is presented in Figure 1c, giving about 11 kV/cm.  
The commercial pulse generator EPULSUS-PM1B-10 (from EnergyPulse Systems, Lisbon, Portugal), was used. This generator is able to deliver positive pulses up to 10 kV and 400 A. The pulses can last 1 \(\upmu\)s to 200 \(\upmu\)s, with repetition rates of 1 Hz to 200 Hz, at a maximum average output power of 3 kW. The pulse generator is based on a solid-state Marx generator, where various n capacitors are first charged in parallel from a relatively low voltage \(U_{dc}\), and then connected in series with the load, applying about \(nU_{dc}\)[20]. As this generator is based on a direct capacitive discharge, it can deliver almost rectangular pulses, independent of the load impedance, as long as the generator's stored energy is much higher than the pulse energy, which is the case in this application.  
A standard electroporation cuvette was used, with a 4 mm distance between electrodes and an 800 \(\upmu\)L volume.  
For PEF applications the most important parameters are the electric field amplitude, \(E\) (kV/cm), and the specific energy, \(W_{s}\) (kJ/kg), given by  
\[E=\frac{U}{d} \tag{1}\]  
where \(U\) refers to the pulse voltage amplitude applied in kV, and \(d\) the distance between the electrodes of the treatment chamber in cm.  
\[W_{s}=\frac{W_{t}}{m} \tag{2}\]  
where \(W_{t}\) is the total applied energy, and \(m\) is the mass of the sample inside the treatment chamber in kg. The total applied energy is given, in \(J\), by  
\[W_{t}=UII_{on}N \tag{3}\]  
where \(I\) is the pulse current amplitude in \(A\), \(t_{on}\) the pulse width in \(\upmu\)s, and \(N\) the number of pulses applied.  
The main objective of using PEF is to avoid the thermal impact, so the increase in temperature, \(\Delta T\) in \({}^{\circ}C\), from the application of PEF, is important, given by  
\[\Delta T=\frac{W_{s}}{C_{p}} \tag{4}\]  
where \(C_{p}\) is the specific heat capacity (kJ/(kg\(\cdot\)K)) of the treated material, which was assumed to be similar to water: 4.18 kJ/(kg\(\cdot\)K).  
Figure 1: **(a)** Schematic design of PEF application to platelets. (**b**) Details of the cuvette treatment chamber, with electrical connections. (**c**) Voltage and current pulse example of the experiments, (green) voltage 1 kV/div, (yellow) current 50 A/div, 1 \(\upmu\)s/div.  
Prior to PEF application, PCs were initially centrifuged at 1000\(\times\)\(g\) for 10 min. The resulting platelet pellet was resuspended in plasma additive solution (PAS; InterSol) to a final concentration of 1 \(\times\)\(10^{9}\) platelets/mL. Platelets in PAS (1 mL) were then added to the cuvettes and exposed to 9 PEF conditions, comprising a range of 10-15 kV/cm and 1-15 pulses of 2 \(\upmu\)s duration.