http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, Early Online: 1–4 ! 2014 Informa UK Ltd. DOI: 10.3109/09537104.2013.863859

ORIGINAL ARTICLE

Human platelet gel supernatant inactivates opportunistic wound pathogens on skin Chelsea M. Edelblute1, Amy L. Donate1, Barbara Y. Hargrave1,2, & Loree C. Heller1,2 Frank Reidy Research Center for Bioelectrics, Old Dominion University, Suite 300, Norfolk, VA, USA and 2School of Medical Diagnostic & Translational Sciences, College of Health Sciences, Old Dominion University, Norfolk, VA, USA

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Abstract

Keywords

Activation of human platelets produces a gel-like substance referred to as platelet rich plasma or platelet gel. Platelet gel is used clinically to promote wound healing; it also exhibits antimicrobial properties that may aid in the healing of infected wounds. The purpose of this study was to quantify the efficacy of human platelet gel against the opportunistic bacterial wound pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus on skin. These opportunistic pathogens may exhibit extensive antibiotic resistance, necessitating the development of alternative treatment options. The antimicrobial efficacy of platelet gel supernatants was quantified using an in vitro broth dilution assay, an ex vivo inoculated skin assay, and in an in vivo skin decontamination assay. Human platelet gel supernatants were highly bactericidal against A. baumannii and moderately but significantly bactericidal against S. aureus in vitro and in the ex vivo skin model. P. aeruginosa was not inactivated in vitro; a low but significant inactivation level was observed ex vivo. These supernatants were quite effective at inactivating a model organism on skin in vivo. These results suggest application of platelet gel has potential clinical applicability, not only in the acceleration of wound healing, but also against relevant bacteria causing wound infections.

Acinetobacter baumannii, platelet gel, platelet rich plasma, Pseudomonas aeruginosa, Staphylococcus aureus

Introduction Platelet rich plasma or platelet gel (PG) is produced after differential centrifugation of uncoagulated whole blood. Platelets are activated by the addition of bovine thrombin or other molecules [1]. Alternatively, platelets can be activated by the application of nanosecond pulsed electric fields (PEFs) [2]. The activated platelet releases the contents of its a-granules [3, 4], including many growth factors. The clinical delivery of this high concentration growth factor mixture may aid in tissue healing [5–7]. For this reason, PG is gaining popularity in clinical applications including dental applications [8], sports injuries [9, 10], wound healing [11, 12], for example in plastic surgery [13] and bone healing [14] applications, and improving heart function [15]. Although PG application is gaining clinical popularity, additional controlled studies are essential for confirmation of its efficacy [9, 10, 14, 16–19]. Proteins or peptides with antimicrobial properties are well described [20–22], and platelets secrete platelet microbicidal proteins (PMPs) [23]. The antimicrobial activity associated with PG may aid in the healing of infected wounds. Supernatants from activated platelets were assayed for several reasons. PG is difficult to pipette accurately. Proteins are secreted and may be found in the supernatant. Finally, PG supernatants were assayed for antimicrobial activity to avoid platelet activation by direct platelet-bacterial contact [24]. Correspondence: Loree C. Heller, Frank Reidy Research Center for Bioelectrics, Old Dominion University, 4211 Monarch Way, Suite 300, Norfolk, VA 23508, USA. Tel: +1 757 683 2416. Fax: +1 757 451 1010. E-mail: [email protected]

History Received 6 September 2013 Revised 22 October 2013 Accepted 30 October 2013 Published online 16 January 2014

The current study quantifies the antimicrobial activity of human PG supernatant against three clinically relevant opportunistic wound pathogens, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa. A. baumannii is an emerging opportunist associated with skin and other serious infections [25–27]. S. aureus asymptomatically colonizes the skin and nasal passages; many types of infections, including wound infections, originate from this niche [28]. P. aeruginosa is ubiquitous in the environment and has a long history as an opportunistic wound pathogen [27, 29, 30]. Due to the capacity of these microorganisms to develop multi-drug resistance, the development of new antibiotics and alternative antibacterial treatments is imperative [31]. In these experiments, antimicrobial activity was quantified in vitro, in an ex vivo model, and in an in vivo model.

Methods Bacterial strains S. aureus (ATCC 25923), A. baumannii (ATCC 19606), and P. aeruginosa (ATCC 27853) were purchased from the American Type Culture Collection (Manassas, VA). E. coli DH5a was purchased from Invitrogen (Life Technologies, Grand Island, NY). Platelet gel One-day expired platelets were purchased from the American Red Cross (ARC, Norfolk, VA). Each 250 ml single donor unit contained 3  1011 platelets suspended in donor plasma containing approximately 35 ml ACD-A. After centrifugation for 10 min at 550xg, the platelet pellet was resuspended in Tyrode’s

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buffer pH 7.2. The control group was the minimally manipulated, quiescent platelet pellet. The experimental groups included the platelet pellet enriched with 2 mM CaCl2 or additionally activated with 100 US units/ml bovine thrombin (BioPharm, Bluffdale, UT) or PEFs (five 30 kV/cm 300 ns pulses at 2 Hz in 4 mm cuvettes). After clotting, samples were centrifuged one minute at 12 800  g at room temperature and the supernatant was assayed. Broth dilution assays Broth dilution assays were performed per a revision of clinical laboratory guidelines [32]. Briefly, 103 bacterial cells in Mueller Hinton broth were incubated 18 hours with platelet supernatant (final dilution 1:5) or Tyrode’s buffer in 96-well polypropylene microtiter plates. Growth was quantified visually and at OD620; bactericidal effect was determined by plating wells with no visible growth.

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Ex vivo assays One inch square pieces of swine skin were acquired post-mortem from an unrelated protocol as approved by the Old Dominion University Institutional Animal Care and Use Committee (IACUC). Skin pieces were inoculated with 104 bacterial cells, allowed to dry, and 300ml of PG supernatant was applied. After incubation at 37  C for 30 min, tissue pieces were homogenized in 2 ml sterile phosphate-buffered saline (PBS) (Stomacher 80 lab blender, Tekmar Co., Cincinnati, OH). The homogenates were serially diluted and plated to MacConkey or mannitol salt agar as appropriate. After incubation overnight at 37  C, colony forming units (CFUs) were quantified (Flash & Go Automated Colony Counter; IUL Instruments, Barcelona, Spain) and CFU reduction was calculated as percent or log10 by comparison to an untreated control group. In vivo assays All procedures were approved by the Old Dominion University IACUC and by the United States Army Medical Research and Materiel Command Animal Care and Use Review Office. Male Sprague Dawley rats (4300 g, Charles River, Wilmington, MA) were anesthetized by isoflurane inhalation. The shaved right flank was inoculated with 10 ml PBS or PBS containing 1  104 Escherichia coli cells and allowed to dry five minutes. An untreated positive control served to confirm the expected bacterial load. Three hundred microliters of PG supernatant was applied and allowed to act for 30 min. After treatment, the animals were euthanized by CO2 exposure and skin samples assayed as described previously for ex vivo assays. Statistical analysis Group comparisons were performed by analysis of variance (ANOVA) followed by the Tukey–Kramer post-test (GraphPad, La Jolla, CA).

Results and discussion In the broth dilution assays (Table I), the A. baumannii and S. aureus strains were sensitive to both control and experimental PG supernatants (p50.001). A. baumannii inactivation approached 100% while S. aureus inactivation approached 40%. P. aeruginosa growth was not significantly inhibited by any PG supernatant group in vitro. Bacterial inactivation was independent of platelet activation state, since inactivation by the supernatants from quiescent platelets, calcium enriched platelets, and the activated platelet groups did not vary significantly. This could indicate the presence of a constitutively expressed bactericidal

Platelets, Early Online: 1–4

Table I. Inactivation of bacterial cells by platelet gel supernatants by broth dilution assay. % Reduction Supernatant

A. baumannii

P. aeruginosa

S. aureus

Quiescent Plus 2 mM CaCl2 Plus thrombin Plus PEF

98.8  2.3*** 99.2  1.7*** 99.3  1.3*** 98.4  1.9***

1.6  3.6 0.4  0.9 0.0  0.0 1.5  2.9

33.7  4.1*** 38.1  5.1*** 34.5  8.0*** 27.4  8.7***

Mean  SD, n ¼ 4–6 independent experiments. ***p50.001 with respect to control. Table II. Inactivation of bacterial cells by platelet gel supernatants on swine skin pieces. Log10 reduction Supernatant

A. baumannii

P. aeruginosa

S. aureus

Quiescent Plus 2 mM CaCl2 Plus thrombin Plus PEF

3.3  0.4*** 1.9  0.7*** 3.1  0.7*** 3.1  0.5***

0.6  0.5 0.4  0.4 0.5  0.3 0.7  0.3*

0.4  0.3 0.3  0.2 0.5  0.2* 0.5  0.5*

Mean  SD, n ¼ 5–7 independent experiments. ***p50.001; *p50.05 with respect to control. Table III. Inactivation of bacterial cells by platelet gel supernatants on rat skin in vivo. Supernatant Quiescent Plus thrombin Plus PEF

Log10 reduction 3.2  0.0** 2.8  0.8** 2.2  1.2*

Mean  SD, n ¼ 3–4 independent experiments. **p50.01; *p50.05 with respect to control.

component, such as complement or antigen-specific antibody. Alternatively, these expired platelets may have been pre-activated during collection, storage, and preparation, producing PMP secretion [33]. Swine skin was used as a model for human skin colonization [34]. Skin samples inoculated with the three opportunistic pathogens were treated with PG supernatant (Table II). A. baumannii was significantly (p50.001) inactivated by 43 log10 by both control and activated PG supernatants. In this model, S. aureus was significantly (p50.05) inactivated by 0.5 log10 only by the thrombin- and PEF-activated supernatants; no significant inactivation was observed in the quiescent or CaCl2 enriched groups. This level of inactivation was lower than that observed in vitro even though in this experiment PG supernatant was undiluted, while the in vitro assay by necessity was broth diluted. PG supernatant from PEF-activated platelets also achieved a statistically detectable (p50.05) inactivation of P. aeruginosa, which differed from the in vitro results. This ex vivo swine skin model may be a more accurate indicator of PMP activity after platelet activation than the classic broth dilution assay. Finally, microbial inactivation was confirmed in vivo on rat skin (Table III). Viable skin differs in several ways from ex vivo skin samples, including that fact it is thermoregulated and acidic [35]. E. coli DH5a was used as a model organism in this experiment to avoid the necessity for animal BSL2 precautions. As expected, 43 log10 E. coli was recovered from the positive control group. All PG supernatants inactivated E. coli by approximately 3 log10 (p 5 0.01). No significant difference in

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DOI: 10.3109/09537104.2013.863859

inactivation level was detected between the treatment groups. This observation supports the idea that expired platelets may have been pre-activated during collection or storage. Platelet-based therapies are gaining clinical acceptance worldwide [5–14, 16–18]. Overall, with respect to antibacterial function, this study demonstrated a limited difference between the tested platelet activation methods. All platelet supernatants inactivated A. baumannii. However, platelet activation was necessary for significantly detectable activity against S. aureus and P. aeruginosa on swine skin samples. The marginal difference in antimicrobial activity observed between activated platelets and quiescent control samples suggests several potential mechanisms. It is well known that platelets are subjected to mechanical stress during collection, storage, and PG preparation [32]. This stress can induce preactivation of platelets absent of an agonist. Samples used in this study were acquired eight days after collection, as one-day expired human platelets are permitted for research use. Time between donation and experimental testing could facilitate platelet degradation. Additionally, the non-specific activity of the complement system and antigen-specific antibodies present in donor plasma could play an operative role [36, 37]. Complement acts against non-self invaders by detecting pathogen-associated molecular patterns present on the surface of most bacteria [36]. Once a pathogen has been identified, the processes of opsonization, chemotaxis [36], and formation of the membrane attack complex [38] enhance phagocytosis of foreign microorganisms. In the classical pathway, convertases bind the Fc portion of antibodies complexed with foreign antigens, thereby acting in a specific manner [36]. In the current study, pelleted platelets were resuspended in Tyrode’s buffer, replacing plasma as the diluent and presumably eliminating residual plasma proteins including complement and circulating antibody. We therefore suggest the antibacterial activity observed in these experiments could be attributed to PMPs secreted from a-granules of activated platelets [2, 3, 19–21], and subsequently collected in PG supernatant [22]. Most importantly, PG supernatants exhibited a significant antibacterial effect against these opportunistic pathogens. Although different assays were used, these results support the antimicrobial effects previously noted against A. baumannii [39] and S. aureus [40, 41]. Published studies differ concerning efficacy of platelet products against P. aeruginosa [40–43]; however, some efficacy was observed here. Future studies should assess the efficacy of PG supernatants against multi-drug resistant strains of these organisms, particularly A. baumannii, as the propensity for resistance development is a principal clinical issue. Ultimately, in addition to its potential for accelerating wound healing, PG application may aid in the therapy of wound infections with these opportunistic pathogens.

Declaration of interest BYH is an inventor on an unlicensed patent, number 13/129,076 ‘‘Activation and Aggregation of Human Platelets and Formation of Platelet Gels by Nanosecond Pulsed Electric Fields’’Inventors: Stephen Beebe, Peter Blackmore, Karl Schoenbach and Barbara Hargrave. This research and development project is being conducted by Old Dominion University and is made possible by a research grant that was awarded and administered by the U.S. Army Medical Research & Materiel Command and the Telemedicine & Advanced Technology Research Center, at Fort Detrick, MD under Contract Number W81XWH-11-1-0740. The views, opinions and/or findings contained in this research are those of the author(s) and do not necessarily reflect the views of the Department of Defense and should not be construed as an official DoD/Army position, policy or decision unless so designated by other documentation. No official endorsement should be made.

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Human platelet gel supernatant inactivates opportunistic wound pathogens on skin.

Activation of human platelets produces a gel-like substance referred to as platelet rich plasma or platelet gel. Platelet gel is used clinically to pr...
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