Ex-vivo Platelet Activation using Electric Pulse Stimulation

Nicole LaPlante, V. Bogdan Neculaes, Brian D. Lee, Andrew S. Torres, Kenneth Conway,

Steve Klopman and Antonio Caiafa

GE Global Research Center, Niskayuna, NY, U.S.A.

Keywords: Activated Platelets, Platelet Gel, Wound Healing, Pulsed Electric Field, Growth Factor.

Abstract: Activated platelet rich plasma (PRP), also known as platelet gel, is an encouraging autologous cell therapy

with numerous applications in areas including: wound healing, haemostasis and wound infection control.

Activation of PRP using electric pulse stimulation is a promising alternative to activation via biologics such

as bovine thrombin. By removing the need for biologics, it is possible to deliver a cost-effective, fast, truly

autologous platelet gel option. In this position paper, we describe parameters for effective ex-vivo release of

several growth factors from human platelets in PRP using electric field pulses with the duration of hundreds

of nanoseconds. Growth factor release levels with nanosecond pulse electric fields seem at the same level or

higher compared to bovine thrombin, the standard platelet activator used in clinical practice. These findings

suggest that electric pulse stimulation has the potential to become not only a viable alternative to

biochemical platelet activators, but to actually enhance the desired in vivo biological effects, such as wound

healing.

1 INTRODUCTION

Whole blood contains several components, including

red blood cells, white blood cells, plasma and

platelets. Platelets have a typical lifespan of about

seven to ten days and will concentrate and aggregate

at the site of injury as part of the body’s response to

promote haemostasis, tissue regeneration and

revascularization (Tate and Crane 1999). Platelets

are formed in the bone marrow and contain

populations of granules – such as alpha granules and

dense granules. Normal platelet count in whole

blood is about 200,000 platelets/ul (Tate and Crane,

1999).

Platelet cell therapy is an approach to harvest the

natural ability of the body to stop the bleeding and

promote wound healing. By collecting one’s

platelets, activating them ex-vivo, and placing them

back on the wound, a novel therapeutic approach has

been developed, that dramatically enhances what the

body has been programmed to accomplish naturally.

Platelet gel is a substance containing a

concentrated amount of platelets which are activated

to release proteins found within the alpha granules.

These proteins, which include numerous growth

factors, are released upon platelet activation and

include platelet-derived growth factor (PDGF),

transforming growth factor-beta (TGF- β), vascular

endothelial growth factor (VEGF) and epidermal

growth factor (EGF). Activated platelets, platelet

gel, have been shown to enhance wound healing

(Driver, 2006,, Lacci, 2010), induce hemostasis

(Gunyadin, 2008), and provide antibacterial

protection for the wound as it heals. The application

landscape is quite broad for platelet gel. Among the

many potential clinical applications, effective

therapy has been shown for diabetic foot ulcers,

dentistry, cardiac surgery, cosmetic surgery,

orthopaedic surgery, sports medicine.

Typical workflow for generating platelet gel

performed at the bed side includes a blood draw

from the patient, platelet separation/concentration

(centrifugation is the state of the art method), and,

finally, ex-vivo platelet activation using a

combination of thrombin and calcium chloride. After

activation, platelet concentrates have a gel-like

consistency. The final step is the application of this

gel on the pre-determined wound. Currently, platelet

activation is performed using bovine thrombin (state

of the art in US) or other types of thrombin in

Canada and Europe (recombinant thrombin,

autologous thrombin or human thrombin isolated

from donor plasma). Various types of thrombin

currently used are rather expensive and can have

202

LaPlante N., Bogdan Neculaes V., D. Lee B., S. Torres A., Conway K., Klopman S. and Caiafa A..

Ex-vivo Platelet Activation using Electric Pulse Stimulation.

DOI: 10.5220/0004328602020208

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2013), pages 202-208

ISBN: 978-989-8565-34-1

 2013 SCITEPRESS (Science and Technology Publications, Lda.)

significant side effects. For example, bovine

thrombin–associated immune-mediated

coagulopathy will incur a cost per patient from $

16,584 to $ 163,072 (Alexander, 2009). The

workflow of generating autologous thrombin can be

complex and lengthy, about 40 minutes

(http://www.biomet.com/biologics/international/prin

t/BBI0004.1_081508.pdf). Human thrombin from

donor plasma carries a risk for transmitting of

infection diseases (Cada, 2008). A truly autologous,

fast and significantly less expensive platelet

activation method would eliminate potential side

effects and will lower the cost for the platelet gel

treatment, opening opportunities for increased

access of this therapeutic approach.

This autologous, non-animal derived, non-

biochemical activation method would allow a fast

and completely autologous platelet gel solution in

the clinic, for difficult to heal wounds. Autologous

platelet gel could become the standard of care for

difficult to heal wounds, such as diabetic foot ulcers.

2 PLATELET ACTIVATION

USING ELECTRIC PULSE

STIMULATION

Short duration pulse electric fields, in the

nanosecond range, have been shown to have

significant effects on the intracellular structures

(Beebe, 2005). Typically, long pulse electric fields,

larger than 0.1 ms, have been efficiently utilized for

cell membrane permeabilization termed

“electroporation”, with a variety of applications such

as exogenous molecule delivery, transfection/gene

delivery, and tumor cell death using irreversible

electroporation. The use of modelling tools in the

context of an electrical model for biological cells,

predicts that pulses with duration shorter than the

charging time of the outer cellular membrane, can

affect the intracellular organelles (Beebe, 2005).

Experiments have indeed confirmed this

theoretical prediction; numerous experimental

demonstrations have pointed out biological effects

of electric pulse stimulation in the nanosecond

range: modulation of caspase activity (caspases

modulate a variety of cell functions – proliferation,

differentiation, cell cycle), apopotosis – programmed

cell death and calcium mobilization (Beebe, 2005).

These short, nanosecond electric field pulses are

thought to create small pores – nanopores- in the

organelle membranes.

Nanosecond pulse electric field effects on

calcium transport have been recently introduced as a

novel method for ex-vivo platelet activation (Zhang,

2008). It has been hypothesized that nanosecond

pulse electric fields, nsPEF, cause calcium to leak

out from the intracellular stores as of result of

nanopores being created in organelles membrane, as

well as an influx of extracellular calcium through

plasma membrane nanopores (Zhang, 2008). This

calcium transport with nsPEF has been correlated

with platelet activation; initial experiments using

newly outdated platelets from the American Red

Cross showed platelet aggregation with nsPEF, with

evaluation of one growth factor following activation,

PDGF (Zhang, 2008). Measurements of PDGF

release from washed platelets with nsPEF were

compared to PDGF release using bovine thrombin;

PDGF levels with nsPEF were generally close to the

release measured using bovine thrombin (Zhang,

2008).

While these experiments have been very

promising for the proof of concept platelet activation

using nsPEF, there has been a need for a more

complete characterization and benchmarking of this

novel activation method. This paper presents a study

of growth factor release from human platelets by

looking at several growth factors, and reveals

significant differences between thrombin-mediated

activation and nsPEF-mediated activation of

platelets.

2.1 Platelet Rich Plasma (PRP)

Preparation

For each experiment, one unit of human whole blood

from single donor was purchased from a commercial

vendor (Bioreclamation) and shipped following lab

testing for standard pathogens; the vendor used ACD

as an anticoagulant. Blood was therefore 3 days old

at time of PRP preparation. Haematological

measurements are performed on the day of PRP

preparation, including density of red blood cells

(RBCs), platelets (PLTs) and haemoglobin.

Standard preparation of PRP was performed

using a commercial kit and centrifuge (SmartPReP2

APC+, Harvest Technologies) per manufacturer’s

protocol. Briefly, 60mL of whole blood is placed in

separation device and up to 7mL of PRP is

recovered following centrifugation steps, which

usually take about 15 minutes. Typical enrichment

of PLTs is 3 times the amount of the starting density

in whole blood. PRP is aliquoted (1mL per aliquot)

into 4mm cuvettes (Molecular BioProducts catalog

#212373) or 1.5mL Eppendorf tubes for activation

Ex-vivo Platelet Activation using Electric Pulse Stimulation

203

studies and allowed to sit at room temperature until

used for experiment.

2.2 Activation of Platelets

2.2.1 Thrombin-mediated Activation

Reagents were prepared and stored on ice on the day

of experiment. Bovine thrombin (BioPharm

Laboratories catalog #91-010) was prepared in

saline solution (0.9% NaCl) at a stock concentration

to allow for 1:10 (vol/vol) standard dilution in all

experiments.

Bovine thrombin preparation details are below:

124 mg/bottle = 10000 U/bottle in 1 mL 0.9% NaCl

for injection = 10 U/uL. Do 1:10 dilution so that

concentration = 1U/uL. For 50 U experiments, add

50 uL (we used 1, 5, 50, 500, 1000 U for our

experiments of platelet activation with bovine

thrombin). Unless stated – data presented in this

paper will focus on results obtained with 1 U. CaCl2

(Sigma Aldrich) was prepared at stock concentration

to allow for 1:100 (vol/vol) standard dilution in all

experiments but is maintained in these studies at

10mM CaCl2.

We add bovine thrombin to 1 mL of PRP in 4mm

cuvette (Molecular BioProducts catalog #212373)

and allow sample to sit at room temperature;

typically clotting with bovine thrombin occurs

within roughly 30 seconds. The PRP is then

centrifuged at 10,000 rpm for 10 minutes in an

Eppendorf tube. The supernatant is pipetted from

tube and either used in assay immediately or stored

at ≤ -20 C.

2.2.2 Activation Studies: nsPEF-mediated

Activation

For each experiment, we applied electric field pulses

to 1 mL of freshly prepared PRP in 4mm cuvette

(Molecular BioProducts catalog #212373) and allow

sample to sit at room temperature for up to 30

minutes (clotting takes place within roughly 5

minutes). The PRP is then centrifuged at 10,000 rpm

for 10 minutes in a Eppendorf tube. The supernatant

is pipetted from tube and either used in assay

immediately or at ≤ -20 C.

2.2.3 Growth Factor Measurements

All measurements are performed using commercial

enzyme-linked immunosorbent assays (ELISAs)

using manufacturer’s protocols: PDGF (R&D

Systems, #DAA00B), IGF-1 (R&D Systems,

#DG100), EGF (R&D Systems, DEG00), VEGF

(R&D Systems, DVE00).

3 PULSE GENERATOR FOR

PLATELET ACTIVATION AND

EXPERIMENTAL RESULTS

Electric pulse generation relies on a few approaches,

the most common being capacitive energy discharge

and pulse forming networks. Capacitive energy

discharge methods are the simplest, but they provide

pulses that can be very difficult to regulate in

amplitude and duration. Pulse forming networks

organized as lines or an ensemble of passive

elements such as inductors and capacitors are by far

the most common topology to generate short square

pulse with specified pulse width.

The pulse generator for ex-vivo activation of

platelets has been designed and built at Old

Dominion University (ODU) and delivers 300 ns

pulses to the load; the load is a 4-mm cuvette

containing platelet rich plasma. The output voltage

of the instrument is 12 kV and creates an electric

field of 30 kV/cm in the 4 mm cuvettes. The

instrument was designed so that the impedance of

the 4 mm cuvette with platelet rich plasma matches

its output impedance. Typically, the 4 mm cuvette

with platelet rich plasma will behave like a resistive

load, with an impedance of roughly 15 ohm.

The nanosecond pulse generator is powered by

standard 110 V AC and can deliver 1 – 9

nanosecond electric field pulses in a single

sequence. The device also provides a cuvette holder

that is designed for standard electroporation cuvettes

that are commercially available for in vitro

workflows.

The nanosecond pulse generator uses a pulse

forming network to generate the 300 ns pulses. The

pulse forming network consists of a combination of

capacitors and inductors arranged in a Blumlein-line

configuration, as shown in Figure 1. The generator

uses an AC-DC rectifier and a DC-DC converter to

step up the voltage from 110 V AC to about 12 kV

DC. A spark gap switch is used to determine the

output voltage of the pulse forming network.

The cuvette holder is easily accessed through an

opening that is placed on the top of the device.

A picture of the internal components of the

nanosecond pulse electric field generator is shown in

Figure 1.

Generally, experiments for platelet activation

using the instrument described here used electric

BIODEVICES 2013 - International Conference on Biomedical Electronics and Devices

204

Figure 1: Picture of internal architecture of the nanosecond

pulse electric field generator; the pulse forming network is

highlighted in blue.

fields on the order or 30 kV/cm. Five pulses at 5 Hz,

were used for platelet activation here. Previous

experiments at Old Dominion University have

identified the optimal number of pulses for

activation as five.

Figure 2 shows an example of activated platelets

using nanosecond pulse electric fields. The gel like

consistency is easily observed – as a result of

platelet aggregation during activation. There are red

blood cells in the platelet rich plasma typically

separated by the Harvest Technologies instrument –

therefore the platelet gel is red. Other platelet

separation technologies can leave out the red blood

cells – the platelet gel will be yellowish in color.

Figure 2: Example of platelet gel created at GE Global

Research using nanosecond pulse electric fields.

Finally, Figure 3 gives a simple visual

representation of PRP activation in cuvettes: nsPEF

versus negative control (no pulsing). Platelet

aggregation and clot formation prevent the PRP to

flow to the bottom of the cuvette after nanosecond

pulsing; in the control cuvette PRP flows to the

bottom, as no activation or clotting occurs.

Several growth factors were evaluated in terms

of release, using nanosecond pulse electric fields

(nsPEF) and bovine thrombin: TGF – β1, PDGF –

Figure 3: Effects of nsPEF on platelet rich plasma: left

hand side cuvette was used as control (no nsPEF), while

the right hand side cuvette was pulsed with nsPEF. Pulsed

PRP cuvette shows clotting (platelet activation) – not PRP

flow. In the control cuvette the PRP flows to the bottom,

as no clotting occurs. Here cuvettes are turned upside

down.

aa, IGF, VEGF and EGF. The platelet enrichment

obtained was around three times higher compared to

whole blood. Generally platelet rich plasma has a

platelet concentration about three to five times

higher compared to whole blood (Whitlow, 2008).

Platelet-derived growth factor (PDGF) is responsible

with cell replication, stimulates angiogenesis, and

regulates collagen synthesis (Tate and Crane, 1999).

Transforming growth factor-beta (TGF- β)

stimulates undifferentiated mesenchymal cell

proliferation, stimulates angiogenesis and regulates

mitogenic effects of other growth factors (Tate and

Crane, 1999). Vascular endothelial growth factor

(VEGF) stimulates angiogenesis and acts as

mitogenetic factor for endothelial cells (Tate and

Crane, 1999). Epidermal growth factor (EGF)

stimulates angiogenesis and promotes growth and

differentiation of chondrocytes and osteoblasts (Tate

and Crane, 1999). Insulin-like growth factor 1 (IGF-

1) has effects on differentiation, peripheral growth,

and survival in various cells and tissues.

Blood from several human donors was used for

these experiments. As pointed in literature, there are

donor-to-donor variations with respect to amount of

growth factor released upon platelet activation. As a

general trend for the work presented here, we

observed that differences between nsPEF and bovine

thrombin for VEGF and EGF levels can be

considerable.

As expected, nsPEF and bovine thrombin do not

increase IGF-1 levels upon platelet activation

(Everts, 2006). IGF-1 levels for nsPEF and bovine

thrombin are roughly equivalent (data not shown).

The fact that IGF-1 levels in activated platelets are at

the same levels as in non-activated platelets is

Ex-vivo Platelet Activation using Electric Pulse Stimulation

205

Figure 4: Example of data for PDGF-aa release for nsPEF

compared to bovine thrombin (BT) for one donor; 1 and 2

designate the number of cuvettes tested; from each

cuvette, three samples of supernatant were tested for

growth factor release, and data averaged for each bar

graph.

explained by considering that the plasma pool of

IGF-1 is greater than the platelet pool; IGF-1 is

mainly excreted by the liver in the plasma.

Additionally, PDGF-aa and TGF – β1 levels have

been observed as largely equivalent between nsPEF

and bovine thrombin (TGF – β1 data not shown

here).

Representative data from our studies are shown

here. “nsPEF” indicates the samples activated with

nanosecond pulse electric fields, while “BT”

indicates samples activated with bovine thrombin.

First, PDGF-aa levels nsPEF versus bovine thrombin

are displayed here for a single donor (Figure 4).

We observed that growth factor levels are

significantly increased compared to whole blood and

platelet rich plasma (PRP) that was not activated.

Unless specified, the bovine thrombin activation was

performed with 1 U/ul. Each bar graph was obtained

from a different cuvette – using PRP from the same

donor.

Figure 5 shows that the use of nsPEF increases

the levels of VEGF and EGF compared to negative

controls, whole blood and non-activated platelet rich

plasma. As pointed here, significantly higher levels

of VEGF and EGF seem to be released with nsPEF

compared to bovine thrombin. This was an

unexpected result. Various measurements of growth

factor release with several bovine thrombin

concentrations – from 1 to 1000 units – did not

trigger similar VEGF and EGF levels as obtained

with nsPEF (data not shown here). Therefore the

results in Figures 5 do not seem to be caused by an

insufficient amount of added bovine thrombin.

Finally, if one looks at donor to donor variability

for EGF and VEGF release – our data seem to

indicate much higher variability for bovine thrombin

Figure 5: Example of data for VEGF and EGF release for

nsPEF compared to bovine thrombin (BT) for one donor. 1

and 2 designate the number of cuvettes tested; from each

cuvette, three samples of supernatant were tested for

growth factor release, and data averaged for each bar

graph.

Figure 6: Example of data for EGF release for nsPEF

compared to bovine thrombin (BT), for three donors – D1,

D2 and D3.

compared to nsPEF. As an example, Figure 6

displays EGF release data for three donors, bovine

thrombin (1 U) versus nsPEF.

What is striking about the data in Figure 6 is not

necessarily the high donor to donor variability for

EGF release with bovine thrombin – this has been

noted previously in literature. The much lower

variability of EGF release with nsPEF from donor to

donor is intriguing and unexpected.

BIODEVICES 2013 - International Conference on Biomedical Electronics and Devices

206

4 DISCUSSION OF RESULTS

Experimental data for growth factor release seem to

indicate that using electric pulse stimulation in the

nanosecond range for platelet activation would result

in a different growth factor profile compared to

bovine thrombin. For example, existing data in

literature show that typical physiological platelet

activators – thrombin, ADP, collagen – tend to have

relatively similar growth release profiles for VEGF

(Maloney, 1998). While the use of nsPEF can offer

the advantage of platelet activation without the use

animal based activators already on the market –

bovine thrombin, bovine derived collagen – one

would have to establish the in vivo wound healing

effects of this different growth factor mix.

There are additional questions to be answered

with in vitro experiments before considering a

clinical path, such as the effects of nsPEF on other

components in the platelet rich plasma (white blood

cells - WBC, red blood cells - RBC). Additionally,

one would need to understand how nsPEF act on

platelet rich plasma compositions produced by the

numerous platelet separation machines

commercially available.

Different devices produce various versions of

platelet rich plasmas – RBC count, WBC count,

platelet enrichment, viability of PRP components

can vary. These versions of PRP may not only be

different from a biology point of view, but also they

could exhibit different electrical behaviours, which

may need to be accounted when one would design a

commercial instrument. Finally, experiments

described in this work use typical electroporation

cuvettes that may need additional qualification for

any human in vivo work.

5 CONCLUSIONS

The use of nanosecond electric field pulses for ex-

vivo platelet activation is an exciting novel

technology, which opens promising opportunities for

a truly autologous solution in the platelet gel space,

by accomplishing platelet aggregation and growth

factor release without using animal derived

activators. It should be noted that previous

researchers demonstrated various means for platelet

activation that do not include the use of thrombin or

other bio-chemical vectors – based on the use of

physical means such as ultrasound (Poliachik, 2001),

light (Verhaar, 2008) and high speed centrifugation

(Mazzucco, 2009). However the use of nsPEF for

platelet activation has significant advantages over

previous attempts to bypass the use of biochemical

activators: speed (~ 1 s exposure to electric field

pulses), process control, low cost, simplified

workflow. The wide potential applicability of

platelet gel therapy – healing of non-healing wounds

such as diabetic foot ulcers, haemostasis, and

reduction of wound infection – may be further

fostered by the introduction of this rapid, low cost,

easy access, truly autologous, non-animal derived

platelet activation method.

ACKNOWLEDGEMENTS

The authors of this paper would like to thank

Barbara Hargrave, Richard Heller (Old Dominion

University) and Reginald Smith (GE Global

Research) for valuable discussions and suggestions

throughout this research. The authors thank Yeong-

Jer Chen (Old Dominion University) for building

and providing technical assistance with the

nanosecond pulse generator used for experiments

presented here.

REFERENCES

Tate, K. S. and Crane, D. M., 2010. Platelet rich plasma

grafts in musculoskeletal medicine. In Journal of

Prolotherapy, Volume 2, Issue 2.

Driver, V. R., Hanft, J., Fylling, C. P., Beriou, J. M., 2006.

A prospective, randomized, controlled trial of

autologous platelet-rich plasma gel for the treatment of

diabetic foot ulcers. In Ostomy/Wound Management,

52(6).

Lacci, K. M. and Dardik, A., 2010. Platelet-rich plasma :

support for its use in wound healing. In Yale Journal

of biology and medicine, 83.

Gunaydin, S., McCusker, K., Sari, T., Onur, M., Gurpinar,

A., Sevim, H., Atasoy, P., Yorgancioglu, C., Zorlutuna

Y., 2008. Clinical impact and biomaterial evaluation

of autologous platelet gel in cardiac surgery. In

Perfusion, 23, 179-186.

Alexander, W., 2009. Meeting highlights of the 30th

American College of Clinical Pharmacy annual

meeting. In Pharmacy & Therapeutics, vol. 34, no. 12.

Cada, D. J., Levien, T. Baker, D. E., 2008. Thrombin,

Topical (Recombinant). In Hospital Pharmacy

Volume 43, Number 7, Wolters Kluwer Health, Inc.

Beebe, S. J. and Schoenbach, K. H., 2005. Nanosecond-

pulsed electric fields: a new stimulus to activate

intracellular signalling. In Journal of Biomedicine and

Biotechnology. 4:297-300.

Zhang, J., Blackmore, P. F., Hargrave, B. Y., Xiao, S.,

Beebe, S.J, Schoenbach, K.H., 2008. Nanosecond

Ex-vivo Platelet Activation using Electric Pulse Stimulation

207

electric field pulse (nanopulse): a novel non-ligand

agonist for platelet activation. In Archives of

biochemistry and biophysics, 417.

Whitlow, J., Shackelford, A. G., Sievert, A. N., Sistino, J.

J., 2008. Barriers to the acceptance and use of

autologous platelet gel. In Perfusion, 23: 283–289

Everts, P. A. M., Mahoney, C.B., Hoffmann, J.J.M.L.,

Schonberger, J. P. A. M., Box, H. A. M., van Zundert,

A., Knape, J. T. A., 2006. Platelet rich plasma

preparation using three devices: Implications on

platelet activation and platelet growth factor release. In

Growth Factors. 24:165-171.

Maloney, J. P., Silliman, C.C., Ambruso, D. R., Wang, J.,

Tuder, R. M., Voelkel, N.F., 1998. In vitro release of

vascular endothelial growth factor during platelet

aggregation. In American Journal of Physiology-

Heart and Circulatory Physiology 275:H1054-H1061.

Poliachik, S. L., Chandler, W. L., Mourad, P. D., Ollos, R.

J., and Crum, L. A., 2001. Activation, aggregation and

adhesion of platelets exposed to high-intensity focused

ultrasound. In Ultrasound in Medicine and Biology,

Vol. 27, No. 11, pp. 1567–1576.

Verhaar, R., Dekkers, D. W. C., De Cuyper, I. M.,

Ginsberg, M. H., Korte, D. Verhoeven, A. J., 2008.

UV-C irradiation disrupts platelet surface disulfide

bonds and activates the platelet integrin _IIb_3. In

Blood, Volume 112, Number 13

Mazzucco, L., Balbo, V., Cattana, E., Guaschino, R., and

Borzini, P., 2009. Not every PRP-gel is born equal -

Evaluation of growth factor availability for tissues

through four PRP-gel preparations: Fibrinet®,

RegenPRP-Kit®, Plateltex® and one manual

procedure. In Vox Sanguinis 97, 110–118

BIODEVICES 2013 - International Conference on Biomedical Electronics and Devices

208