BLOOD COMPONENTS Leukoreduced whole blood–derived platelets treated with antimicrobial peptides maintain in vitro properties during storage Marta Bosch-Marcé,1 Shalini Seetharaman,2 James Kurtz,2 Ketha V.K. Mohan,1 Stephen J. Wagner,2 and Chintamani D. Atreya1
BACKGROUND: Bacterial sepsis is still a complication in patients transfused with stored platelets (PLTs). We have recently demonstrated that selected antimicrobial peptides (AMPs) have bactericidal activity in bacteriaspiked PLTs. In a subsequent preclinical study, we have also shown that these AMPs do not elicit antibody response in rabbits and treatment of PLTs before transfusion does not affect their in vivo recovery and survival in severe combined immunodeficient mice. Here we have selected two such AMPs, Arg-Trp (RW) repeats of tri- and tetra-peptides (RW3 and RW4) in combination (i.e., cocktail), and evaluated their effect on the in vitro properties of PLTs. STUDY DESIGN AND METHODS: Leukoreduced ABOand D-identical whole blood–derived PLT concentrates were pooled and divided into two 60-mL aliquots in CLX storage bags. On Day 0, one bag received a peptide cocktail of RW3 plus RW4 at 0.01 mmol/L final concentration (test) and the other bag received only phosphate-buffered saline (PBS), the AMP solvent (control). The treated PLTs were stored for 7 days at 20 to 24°C. Samples were collected on Days 1, 5, and 7 to evaluate the in vitro properties of PLTs with standard assays. RESULTS: In vitro properties of the RW3 plus RW4 cocktail–treated PLTs were similar to those incubated with PBS only. There were no significant differences between the control and test PLTs during the 7-day storage. CONCLUSION: Leukoreduced whole blood–derived PLTs treated with a mixture of RW3 and RW4 peptides maintain their in vitro properties during 7 days of storage.
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residual rate of bacterial sepsis from transfusion components, especially platelets (PLTs), continues to occur in the United States despite successful maneuvers to reduce risk, which include improvements in skin disinfection, automated blood culture, and sample diversion of a small volume of initially collected blood.1 It is estimated that 1 in 2000 to 1 in 3000 units of PLT concentrates (PCs) are contaminated.1,2 The prevalence of transfusion-associated sepsis is estimated at approximately 1 in 25,000 PLT transfusions;1 roughly 100 to 150 individuals transfused per year suffer severe adverse reactions, some resulting in fatalities.3 Bacterial sepsis is the second major cause of death from transfusion in the United States.4 We have recently demonstrated that two different types of synthetic antimicrobial peptides (AMPs), the thrombininduced human PLT microbicidal protein sequence– derived AMPs and the arginine-tryptophan (RW) repeat peptides, have antibacterial effect against both Gram-negative and Gram-positive bacteria in PCs.5 We
ABBREVIATIONS: AMP(s) = antimicrobial peptide(s); ESC = extent of shape change; HSR = hypotonic shock response; PC(s) = platelet concentrate(s); PRP(s) = platelet-rich plasma(s); RT = room temperature; RW = Arg-Trp. From the 1Section of Cell Biology, Laboratory of Cellular Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland; and the 2 Blood Components Department, American Red Cross, Rockville, Maryland. Address reprint requests to: Chintamani D. Atreya, PhD, Building 29A, Room 2C-11, NIH Campus, 8800 Rockville Pike, Bethesda, MD 20892; e-mail: [email protected]. Received for publication July 27, 2013; revision received September 25, 2013, and accepted October 9, 2013. doi: 10.1111/trf.12534 Published 2014. This article is a U.S. Government work and is in the public domain in the USA. TRANSFUSION 2014;54:1604-1609.
AMPs DO NOT AFFECT PLT IN VITRO PROPERTIES
have also demonstrated that some of these peptides also show antiviral activity against vaccinia virus.6 AMPs are a unique group of small peptides considered to be part of the host defense system that are produced by all types of living organisms from single-cell microorganisms to mammals.7 AMPs have broadspectrum antimicrobial activity against bacteria, viruses, fungi, and protozoa8 and may be effective against antibiotic-resistant bacteria.9 AMPs constitute a very heterogeneous group of molecules that differ in size (6 to 100 amino acids), sequence, and structure, although they share common features such as often being positively charged and an amphipathic structure.10,11 Although most AMPs target microbes through membrane disruption and pore formation, other mechanisms of their antimicrobial activity include intracellular targeting of cytoplasmic components and inhibition of intracellular functions. With respect to antiviral effects, these peptides prevent infection and viral spread by inhibiting the viral fusion and egress.11 The bactericidal and red blood cell hemolytic activity of the RW peptides have been reported to increase with the number of repeats. RW with three repeats (RW3) has been described as the optimal length for the greatest antimicrobial activity with the least hemolytic activity.12,13 We have recently demonstrated that selected AMPs have bactericidal activity in bacteria-spiked PLTs.5 In a subsequent preclinical study, we have also shown that these AMPs do not elicit antibody response in rabbits and treatment of PLTs before transfusion does not affect their in vivo recovery and survival in severe combined immunodeficient mice.14 In this study, we have selected two such AMPs, Arg-Trp (RW) repeats of tri- and tetra-peptides (RW3 and RW4) in combination (i.e., cocktail) that we have reported as highly bactericidal15 and evaluated their effect on the in vitro properties of stored PLTs.
MATERIALS AND METHODS Blood collection and PC preparation Blood was collected by Holland Laboratory Research Blood Program staff at their facility from healthy donors under informed consent. The exclusion criteria for preparing PCs were that 1) PLT count on Day 1 should be more than 5.5 × 1010 and 2) avoid whole blood that has visible lipemia. Based on these criteria, of 13 whole blood donations, only 10 were used for the experiments (n = 10). For each experiment, 2 units of ABO- and D-identical whole blood (500 mL) were collected in triple collection sets (ATS-LPL, Leukotrap PL system, Pall Corp., East Hills, NY). Following collection, whole blood units were held at room temperature (RT) for a minimum of 1 hour and the PCs were prepared within 8 hours of collection, as previously described (Day 0).16 PCs were prepared by the PLT-rich plasma (PRP) method, consisting of a first cen-
trifugation of the whole blood at 2620 × g at 20 to 24°C and an accumulated centrifugal effect equal to 1.70 × 107 in a centrifuge with a rotor (Sorvall RC3BP and H6000A/ HLR-6, respectively; Thermo Electron Corp., Asheville, NC). PRPs were then expressed through ATS-LPL filters into two satellite CLX containers, and the two leukoreduced PRP units were pooled at RT in a 1-L transfer pack (Fenwal, Inc., Lake Zurich, IL) using a sterile connection device. The PRP pool was thoroughly but gently mixed and then equally divided into two identical PRPs into the original CLX containers. The two PRPs were centrifuged at 4203 × g (Sorvall RC3BP, Thermo Electron Corp.) at 20 to 24°C with an accumulated centrifugal effect equal to 5.50 × 107, and plasma was expressed to produce two identical 60-mL PCs. One-hundred milliliters of PLTpoor plasma was prepared by whole blood centrifugation at 3313 × g for 30 minutes using a centrifuge (Sorvall RT7 Plus, Thermo Electron Corp.), to be used for assay dilutions. PLT-poor plasma was then stored in the refrigerator.
Peptide synthesis, reconstitution, and addition to the PCs Two AMPs (RW3 and RW4) were synthesized at our Core Facility at Center for Biologics Evaluation and Research (CBER). They are cationic peptides containing three and four repeats of the Arg-Trp motif, respectively (RWRWRW and RWRWRWRW).5,6,12 The peptides were purified using reverse-phase high-performance liquid chromatography as previously described.17 Peptide reconstitution was done as previously reported.6 The two peptide stock solutions were prepared at 10 mmol/L in phosphate-buffered saline (PBS) and stored at 4°C. After the PCs rested for 1 hour at RT, 60 μL of the peptide stock solutions (test) or PBS (control) alone was added by syringe through a sterile injection site coupler (Fenwal, Inc.) to a 60-mL PLT pool to yield final peptide concentrations of 0.01 mmol/L. After AMP inoculation, the PLTs were subjected to reciprocal agitation on a flatbed agitator (Helmer, Inc., Noblesville, IN) and stored for 7 days at 20 to 24°C with continuous agitation.
In vitro PLT testing All PCs were sampled on Day 1 and on the afternoons of Day 5 (for 5-day storage) and Day 7 (for 7-day storage). The samples were obtained aseptically through a sterile injection site coupler and the following assays were performed: 1.
PLT concentration and mean PLT volume were measured in ethylenediaminetetraacetate tubes using two hematology analyzers (Cell-Dyn 3700, Abbott, Abbott Park, IL; and XE-2100D, Sysmex America, Inc.,
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2.
3.
4.
5.
6.
Mundelein, IL). PLT content was calculated as the product of PLT concentration and volume. Volume was determined by weight using a specific gravity of 1.03. The pH was measured at RT (20 to 24°C) with a pH meter (Orion, Thermo Scientific, Beverly, MA) with an electrode (Accu-pHast, Fisher Scientific, Pittsburgh, PA). pO2, pCO2, glucose, lactate, and pH (37°C) were determined with a blood gas analyzer (Cobas b221, Roche Diagnostics, Indianapolis, IN). Bicarbonate levels were calculated from CO2 and pH measurements (37°C). The extent of shape change (ESC) and the hypotonic shock response (HSR) were measured turbidimetrically (SPA 2000, Chronolog, Havertown, PA).18 PLT activation was evaluated through measuring CD61, CD62, and CD63 by using their corresponding isotype controls19 in a flow cytometer (FACScan, BD Biosciences, San Jose, CA). The expression of GP1bα was measured by flow cytometry with a CD42b antibody and its isotype control.20 PLT aggregation was measured at 37°C using a lumiaggregometer with 10 μmol/L adenosine 5′-diphosphate and 10 μg/mL collagen as agonists which were added near simultaneously to initiate aggregation. Aggregation was expressed as either the slope of a plot of turbidity versus time or the amplitude relative to an internal aggregation standard. PLT morphology was determined by phase contrast microscopy and calculated as the percentage of PLTs that presented discoid morphology. Swirling, a noninvasive qualitative measure of discoid PLTs, was estimated by rotating a section of the PC bag in front of a light. A scale of 0 to 2 was used; a score of 2 indicated good swirling, 1 was the score for a reduced amount of swirling, and 0 was the score when swirling could not be visually observed.21-23
Statistical analysis Calculation of means and standard deviations (SDs) of experimental values and two-tailed paired t test were performed using standard statistical software (Instat, GraphPad software, GraphPad, San Diego, CA). A p value of 0.001 was considered significant for the t tests, in light of the multiple comparisons involving three sampling days and 18 assays.
RESULTS A total of 10 sets of whole blood–derived PCs were prepared as described under Materials and Methods and evaluated. No differences were found on Day 1 in any of the variables evaluated between control (PBS) and test (RW3 and RW4) PCs (Fig. 1 and Table 1, p > 0.001). 1606
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Fig. 1. Evaluation of pH during storage of pooled leukoreduced PCs and control. Ten leukoreduced whole blood–derived PCs were pooled and divided into two identical pools. One pool was incubated with PBS, control (○), and the other pool was incubated with AMPs RW3 and RW4, test (■). Values represent the mean ± SD (n = 10).
Figure 1 shows the mean pH values for PCs treated with the cocktail of AMPs RW3 and RW4 and PCs incubated with PBS during 7-day storage. pH values decreased over time in both groups of PCs but there were no significant differences between test and control PCs as can be seen in Fig. 1 (p > 0.001), where the values of pH for both groups of PCs decreased in parallel. The effect of the RW3 and RW4 cocktail treatment on additional in vitro PLT variables is depicted in Table 1. After 5- and 7-day storage, there were no significant differences between test and control PCs in any of the variables evaluated (p > 0.001). Some of the variables measured changed over the time of storage but they decreased (glucose, bicarbonate, ESC, HSR, morphology, aggregation, CD42b) or increased (lactate) at a similar rate for both arms.
DISCUSSION PCs are stored at RT with constant agitation to preserve their viability. However, these conditions provide an ideal environment for bacterial growth. In this study, we evaluated the effects of RW3 and RW4 cocktail on an array of in vitro PLT properties during storage. Our results demonstrate that this peptide cocktail did not affect the in vitro properties of whole blood–derived leukoreduced PLTs during 7 days of storage. The AMPs evaluated in this study belong to the synthetic (RW)n tandem repeat (n = 1-5) peptide family. Others have evaluated these AMPs for both their bactericidal and their hemolytic activities; the RW3 peptide has been reported to have an optimal antimicrobial activity with no detectable hemolytic activity (in sheep blood).12
Test 6.0 ± 1.5 52.51 ± 1.33 1147.15 ± 285.89 14.0 ± 5.5 54.7 ± 9 7.68 ± 0.57 57.9 ± 5.4 2±0 151.2 ± 7.9 22.3 ± 3.1 6.5 ± 1.3 518.9 ± 35.4 19.8 ± 2.6 62.4 ± 16.6 71.9 ± 20.4 30.9 ± 10.3 7.4 ± 4.6 544 ± 95 Day 7 Control 6.0 ± 1.5 52.69 ± 1.45 1132.65 ± 272.62 13.6 ± 5.4 55.8 ± 8.7 7.75 ± 0.55 57.6 ± 5.0 2±0 152.3 ± 6.1 21.1 ± 2.4 6.4 ± 1.2 528.8 ± 40.2 20.1 ± 3.0 61.3 ± 16.3 67.6 ± 17.7 31.0 ± 10.5 6.9 ± 4.7 541 ± 90 Test 6.7 ± 1.6 57.13 ± 1.28 1173.10 ± 287.40 17.4 ± 6.3 59.1 ± 7.6 7.57 ± 0.50 65.4 ± 5.7 2±0 143.5 ± 10.0 25.3 ± 3.4 10 ± 1.1 563.8 ± 27.8 14.7 ± 1.8 71.0 ± 16.3 85.6 ± 19.6 28.8 ± 13.8 8.9 ± 10.6 609 ± 88 Day 5 Control 6.6 ± 1.6 57.46 ± 1.71 1153.75 ± 268.30 16.7 ± 6 61.0 ± 7.8 7.65 ± 0.50 65.7 ± 6.2 2±0 144.1 ± 8.3 23.9 ± 2.8 9.7 ± 1.1 572.5 ± 28.2 14.8 ± 1.5 67.8 ± 17.3 78.5 ± 16.1 29.0 ± 13.5 8.7 ± 9.5 601 ± 84 Test 7.2 ± 1.7 61.77 ± 1.34 1166.65 ± 273.48 23.0 ± 5.7 65.5 ± 7.2 7.45 ± 0.46 72.6 ± 5.3 2±0 128.8 ± 10.2 36.8 ± 4.3 17.7 ± 2.3 641.2 ± 20.9 5.7 ± 0.5 77.7 ± 12.6 94.7 ± 20.5 28.4 ± 8.0 6.2 ± 5.3 692 ± 111 Day 1
* Values represent mean ± 1 SD. MPV = mean PLT volume.
Control 7.2 ± 1.7 62.06 ± 1.69 1161.85 ± 274.56 22.8 ± 5.7 66.5 ± 8.8 7.54 ± 0.43 72.2 ± 6.0 2±0 129.9 ± 9.9 35.5 ± 4.6 17.4 ± 1.8 641.3 ± 42.4 5.7 ± 0.6 75.8 ± 13.0 97.3 ± 16.7 26.9 ± 9.2 5.5 ± 5.1 696 ± 98 Assay Content (×1010) Volume (mL) Concentration (×109/L) ESC (%) HSR (%) MPV (fL) Morphology (% disc) Swirling pO2 (mmHg) pCO2 (mmHg) Bicarbonate (mmol/L) Glucose (mmol/L) Lactate (mmol/L) Aggregation amplitude (%) Aggregation (% slope) CD62P (%) CD63 (%) CD42b (mean fluorescence)
TABLE 1. In vitro PLT storage variables for pooled whole blood–derived PLTs incubated with RW3 and RW4 (test, n = 10) or PBS (control, n = 10)*
AMPs DO NOT AFFECT PLT IN VITRO PROPERTIES
In our study, we have used both RW3 and RW4 at 0.01 mmol/L concentrations. This concentration of the AMPs was selected for the current study since we had previously demonstrated significant antibacterial and antiviral activities of RW3 and RW4 at this concentration.5,6 In these studies, the peptide activities on selected bacteria and viruses were evaluated in the presence of human plasma and PLTs under similar conditions as reported in this study.5,6 New technologies continue to be developed to improve the collection and extend the storage time of PCs. These technologies require in vitro and in vivo evaluation for their effect on PLTs quality. At the moment, the gold standard is to evaluate in vivo PLT quality by measuring the recovery and survival of radiolabeled human autologous PLTs in normal human volunteers.24,25 Because of the small risk from exposure to radiation from PLT radiolabel studies as well as the cost of in vivo testing in humans, two surrogate methods have been in use as a first step toward assessing new PLT collection and storage technologies before embarking on a clinical trial with normal human volunteers. The surrogate methods evaluate the changes of in vitro properties of PLTs during storage26 and the survival and recovery of human PLTs in mice.27,28 Unfortunately no in vitro test can completely predict the survival and recovery of PLTs infused to healthy volunteers,29,30 and investigators and regulators believe that a battery of in vitro tests provide data that characterize PLT quality.31 Our group has recently demonstrated that selected individual AMPs have bactericidal activity in PCs5 and that all of these AMPs, including RW3 and RW4, do not affect the survival and recovery of human PLTs in severe combined immunodeficient mice. In addition, most of these peptides, including RW3 and RW4, do not produce an immunogenic response in rabbits.14 In this study, we evaluated the in vitro properties of whole blood–derived leukoreduced PLTs when incubated with a cocktail of two AMPs, RW3 and RW4, at bactericidal concentrations as reported by us.5 It is interesting to note that the RW3 and RW4 are short peptides each consisting of six and eight amino acids, respectively, and tempting to speculate that upon degradation by proteases may or may not result in any harmful by-products that could induce toxicologic effects in the recipient. However, we are cognizant of the fact that toxicologic tests for establishing the safety profile of these selected AMPs in a suitable animal model must be conducted before considering this concept toward reducing the microbial burden in stored PLTs. Using a battery of PLT assays, we demonstrated that the in vitro storage properties of whole blood–derived leukoreduced PLTs treated with RW3 and RW4 were maintained during 7 days of storage similar to those observed in control PLTs. These results indicate that mixtures of RW3 and RW4 peptides at concentrations that exhibit potent bactericidal activity, are not harmful to PLTs. Of Volume 54, June 2014
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interest, PLTs treated with RW3 and RW4 had levels of CD42b similar to those of control PLTs. The decline in CD42b levels during storage without decline of PLT concentration in our study is similar to previously reported results.32 The loss of CD42b during storage has been implicated in enhanced clearance of transfused PLTs in murine and rabbit animal models.20,32 Based on the results of our study, together with other published studies,5,14,15 evidence is provided to support continued development of AMPs for possible clinical evaluation. ACKNOWLEDGMENTS MBM is the recipient of a postdoctoral fellowship at the Center for Biologics Evaluation and Research (CBER) administered by the
9. Bals R. Epithelial antimicrobial peptides in host defense against infection. Respir Res 2000;1:141-50. 10. Nakatsuji T, Gallo RL. Antimicrobial peptides: old molecules with new ideas. J Invest Dermatol 2012;132: 887-95. 11. Yeaman MR, Yount NY. Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 2003;55:2755. 12. Liu Z, Brady A, Young A, et al. Length effects in antimicrobial peptides of the (RW)n series. Antimicrob Agents Chemother 2007;51:597-603. 13. Chan DI, Prenner EJ, Vogel HJ. Tryptophan- and argininerich antimicrobial peptides: structures and mechanisms of action. Biochim Biophys Acta 2006;1758:1184-202. 14. Bosch-Marcé M, Mohan KV, Gelderman MP, et al. Preclini-
Oak Ridge Institute for Science and Education (ORISE) through
cal safety evaluation of human platelets treated with anti-
an intraagency agreement between the US Department of Energy and the US Food and Drug Administration. The authors thank Ms Sandhya Kulkarni for her help with timely procurement of mate-
microbial peptides in severe combined immunodeficient mice. Transfusion 2014;54:569-76. 15. Mohan KV, Sainath Rao S, Gao Y, et al. Enhanced antimi-
rials used in this work.
crobial activity of peptide-cocktails against common bacterial contaminants of ex vivo stored platelets. Clin Microbiol Infect 2014;20:39-46.
CONFLICT OF INTEREST The authors report no conflicts of interest or funding sources.
REFERENCES
16. Wagner SJ, Skripchenko A, Myrup A, et al. Evaluation of in vitro storage properties of prestorage pooled whole bloodderived platelets suspended in 100 percent plasma and treated with amotosalen and long-wavelength ultraviolet
1. Blajchman MA, Beckers EA, Dickmeiss E, et al. Bacterial detection of platelets: current problems and possible reso-
light. Transfusion 2009;49:704-10. 17. Shaw C. Peptide purification by reverse-phase HPLC. Methods Mol Biol 1994;32:275-87.
lutions. Transfus Med Rev 2005;19:259-72. 2. Blajchman MA. Incidence and significance of the bacterial
18. Holme S, Moroff G, Murphy S. A multi-laboratory evaluation of in vitro platelet assays: the tests for extent of shape
contamination of blood components. Dev Biol (Basel) 2002;108:59-67. 3. Yomtovian R, Lazarus HM, Goodnough LT, et al. A prospective microbiologic surveillance program to detect and prevent the transfusion of bacterially contaminated plate-
change and response to hypotonic shock. Biomedical Excellence for Safer Transfusion Working Party of the International Society of Blood Transfusion. Transfusion 1998;38: 31-40. 19. Skripchenko A, Myrup A, Thompson-Montgomery D, et al.
lets. Transfusion 1993;33:902-9. 4. US Food and Drug Administration. Fatalities reported to FDA following blood collection and transfusion. Annual summary for fiscal year 2011. 2012. [cited 2013 Dec 30]. Available from: http://www.fda.gov/downloads/ BiologicsBloodVaccines/SafetyAvailability/ ReportaProblem/TransfusionDonationFatalities/ UCM300764.pdf 5. Mohan KV, Rao SS, Atreya CD. Evaluation of antimicrobial peptides as novel bactericidal agents for room temperature-stored platelets. Transfusion 2010;50: 166-73. 6. Mohan KV, Rao SS, Atreya CD. Antiviral activity of selected antimicrobial peptides against vaccinia virus. Antiviral Res 2010;86:306-11. 7. Jenssen H, Hamill P, Hancock RE. Peptide antimicrobial agents. Clin Microbiol Rev 2006;19:491-511. 8. Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:389-95.
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Periods without agitation diminish platelet mitochondrial function during storage. Transfusion 2010;50:390-9. Bergmeier W, Piffath CL, Cheng G, et al. Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates GPIbalpha shedding from platelets in vitro and in vivo. Circ Res 2004;95:677-83. Wagner SJ, Vassallo R, Skripchenko A, et al. The influence of simulated shipping conditions (24- or 30-hr interruption of agitation) on the in vitro properties of apheresis platelets during 7-day storage. Transfusion 2008;48:1072-80. Wagner SJ, Vassallo R, Skripchenko A, et al. Comparison of the in vitro properties of apheresis platelets during 7-day storage after interrupting agitation for one or three periods. Transfusion 2008;48:2492-500. Moroff G, Kurtz J, Seetharaman S, et al. Storing apheresis platelets without agitation with simulated shipping conditions during two separate periods: immediately after collection and subsequently between Day 2 and Day 3. Transfusion 2011;51:636-42.
AMPs DO NOT AFFECT PLT IN VITRO PROPERTIES
24. Murphy S. Radiolabeling of PLTs to assess viability: a proposal for a standard. Transfusion 2004;44:131-3. 25. AuBuchon JP. Radioisotopic reflections. Transfusion 2005; 45:28S-32S. 26. Wagner SJ, Seetharaman S, Kurtz J. Comparison of the in vitro storage properties of Amicus apheresis platelets collected using single- and double-needle procedures from the same donors. Transfusion 2012;52:524-8. 27. Piper JT, Gelderman MP, Vostal JG. In vivo recovery of human platelets in severe combined immunodeficient mice as a measure of platelet damage. Transfusion 2007;47: 1540-9. 28. Newman PJ, Aster R, Boylan B. Human platelets circulating in mice: applications for interrogating platelet function
29. Rinder HM, Smith BR. In vitro evaluation of stored platelets: is there hope for predicting posttransfusion platelet survival and function? Transfusion 2003; 43:2-6. 30. Dijkstra-Tiekstra MJ, Pietersz RN, Huijgens PC. Correlation between the extent of platelet activation in platelet concentrates and in vitro and in vivo parameters. Vox Sang 2004;87:257-63. 31. Moroff G, Kurtz J, Seetharaman S, et al. Comparative in vitro evaluation of apheresis platelets stored with 100% plasma or 65% platelet additive solution III/35% plasma and including periods without agitation under simulated shipping conditions. Transfusion 2012;52:834-43. 32. Leytin V, Gwozdz AA, Garvey B, et al. Role of platelet
and survival, the efficacy of antiplatelet therapeutics, and
surface glycoprotein 1bα and P-selectin in the clearance
the molecular basis of platelet immunological disorders.
of transfused platelet concentrates. Transfusion
J Thromb Haemost 2007;5 Suppl 1:305-9.
2004;44:1487-95.
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BACKGROUND: Bacterial sepsis is still a complication in patients transfused with stored platelets (PLTs). We have recently demonstrated that selected antimicrobial peptides (AMPs) have bactericidal activity in bacteriaspiked PLTs. In a subsequent preclinical study, we have also shown that these AMPs do not elicit antibody response in rabbits and treatment of PLTs before transfusion does not affect their in vivo recovery and survival in severe combined immunodeficient mice. Here we have selected two such AMPs, Arg-Trp (RW) repeats of tri- and tetra-peptides (RW3 and RW4) in combination (i.e., cocktail), and evaluated their effect on the in vitro properties of PLTs. STUDY DESIGN AND METHODS: Leukoreduced ABOand D-identical whole blood–derived PLT concentrates were pooled and divided into two 60-mL aliquots in CLX storage bags. On Day 0, one bag received a peptide cocktail of RW3 plus RW4 at 0.01 mmol/L final concentration (test) and the other bag received only phosphate-buffered saline (PBS), the AMP solvent (control). The treated PLTs were stored for 7 days at 20 to 24°C. Samples were collected on Days 1, 5, and 7 to evaluate the in vitro properties of PLTs with standard assays. RESULTS: In vitro properties of the RW3 plus RW4 cocktail–treated PLTs were similar to those incubated with PBS only. There were no significant differences between the control and test PLTs during the 7-day storage. CONCLUSION: Leukoreduced whole blood–derived PLTs treated with a mixture of RW3 and RW4 peptides maintain their in vitro properties during 7 days of storage.
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residual rate of bacterial sepsis from transfusion components, especially platelets (PLTs), continues to occur in the United States despite successful maneuvers to reduce risk, which include improvements in skin disinfection, automated blood culture, and sample diversion of a small volume of initially collected blood.1 It is estimated that 1 in 2000 to 1 in 3000 units of PLT concentrates (PCs) are contaminated.1,2 The prevalence of transfusion-associated sepsis is estimated at approximately 1 in 25,000 PLT transfusions;1 roughly 100 to 150 individuals transfused per year suffer severe adverse reactions, some resulting in fatalities.3 Bacterial sepsis is the second major cause of death from transfusion in the United States.4 We have recently demonstrated that two different types of synthetic antimicrobial peptides (AMPs), the thrombininduced human PLT microbicidal protein sequence– derived AMPs and the arginine-tryptophan (RW) repeat peptides, have antibacterial effect against both Gram-negative and Gram-positive bacteria in PCs.5 We
ABBREVIATIONS: AMP(s) = antimicrobial peptide(s); ESC = extent of shape change; HSR = hypotonic shock response; PC(s) = platelet concentrate(s); PRP(s) = platelet-rich plasma(s); RT = room temperature; RW = Arg-Trp. From the 1Section of Cell Biology, Laboratory of Cellular Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland; and the 2 Blood Components Department, American Red Cross, Rockville, Maryland. Address reprint requests to: Chintamani D. Atreya, PhD, Building 29A, Room 2C-11, NIH Campus, 8800 Rockville Pike, Bethesda, MD 20892; e-mail: [email protected]. Received for publication July 27, 2013; revision received September 25, 2013, and accepted October 9, 2013. doi: 10.1111/trf.12534 Published 2014. This article is a U.S. Government work and is in the public domain in the USA. TRANSFUSION 2014;54:1604-1609.
AMPs DO NOT AFFECT PLT IN VITRO PROPERTIES
have also demonstrated that some of these peptides also show antiviral activity against vaccinia virus.6 AMPs are a unique group of small peptides considered to be part of the host defense system that are produced by all types of living organisms from single-cell microorganisms to mammals.7 AMPs have broadspectrum antimicrobial activity against bacteria, viruses, fungi, and protozoa8 and may be effective against antibiotic-resistant bacteria.9 AMPs constitute a very heterogeneous group of molecules that differ in size (6 to 100 amino acids), sequence, and structure, although they share common features such as often being positively charged and an amphipathic structure.10,11 Although most AMPs target microbes through membrane disruption and pore formation, other mechanisms of their antimicrobial activity include intracellular targeting of cytoplasmic components and inhibition of intracellular functions. With respect to antiviral effects, these peptides prevent infection and viral spread by inhibiting the viral fusion and egress.11 The bactericidal and red blood cell hemolytic activity of the RW peptides have been reported to increase with the number of repeats. RW with three repeats (RW3) has been described as the optimal length for the greatest antimicrobial activity with the least hemolytic activity.12,13 We have recently demonstrated that selected AMPs have bactericidal activity in bacteria-spiked PLTs.5 In a subsequent preclinical study, we have also shown that these AMPs do not elicit antibody response in rabbits and treatment of PLTs before transfusion does not affect their in vivo recovery and survival in severe combined immunodeficient mice.14 In this study, we have selected two such AMPs, Arg-Trp (RW) repeats of tri- and tetra-peptides (RW3 and RW4) in combination (i.e., cocktail) that we have reported as highly bactericidal15 and evaluated their effect on the in vitro properties of stored PLTs.
MATERIALS AND METHODS Blood collection and PC preparation Blood was collected by Holland Laboratory Research Blood Program staff at their facility from healthy donors under informed consent. The exclusion criteria for preparing PCs were that 1) PLT count on Day 1 should be more than 5.5 × 1010 and 2) avoid whole blood that has visible lipemia. Based on these criteria, of 13 whole blood donations, only 10 were used for the experiments (n = 10). For each experiment, 2 units of ABO- and D-identical whole blood (500 mL) were collected in triple collection sets (ATS-LPL, Leukotrap PL system, Pall Corp., East Hills, NY). Following collection, whole blood units were held at room temperature (RT) for a minimum of 1 hour and the PCs were prepared within 8 hours of collection, as previously described (Day 0).16 PCs were prepared by the PLT-rich plasma (PRP) method, consisting of a first cen-
trifugation of the whole blood at 2620 × g at 20 to 24°C and an accumulated centrifugal effect equal to 1.70 × 107 in a centrifuge with a rotor (Sorvall RC3BP and H6000A/ HLR-6, respectively; Thermo Electron Corp., Asheville, NC). PRPs were then expressed through ATS-LPL filters into two satellite CLX containers, and the two leukoreduced PRP units were pooled at RT in a 1-L transfer pack (Fenwal, Inc., Lake Zurich, IL) using a sterile connection device. The PRP pool was thoroughly but gently mixed and then equally divided into two identical PRPs into the original CLX containers. The two PRPs were centrifuged at 4203 × g (Sorvall RC3BP, Thermo Electron Corp.) at 20 to 24°C with an accumulated centrifugal effect equal to 5.50 × 107, and plasma was expressed to produce two identical 60-mL PCs. One-hundred milliliters of PLTpoor plasma was prepared by whole blood centrifugation at 3313 × g for 30 minutes using a centrifuge (Sorvall RT7 Plus, Thermo Electron Corp.), to be used for assay dilutions. PLT-poor plasma was then stored in the refrigerator.
Peptide synthesis, reconstitution, and addition to the PCs Two AMPs (RW3 and RW4) were synthesized at our Core Facility at Center for Biologics Evaluation and Research (CBER). They are cationic peptides containing three and four repeats of the Arg-Trp motif, respectively (RWRWRW and RWRWRWRW).5,6,12 The peptides were purified using reverse-phase high-performance liquid chromatography as previously described.17 Peptide reconstitution was done as previously reported.6 The two peptide stock solutions were prepared at 10 mmol/L in phosphate-buffered saline (PBS) and stored at 4°C. After the PCs rested for 1 hour at RT, 60 μL of the peptide stock solutions (test) or PBS (control) alone was added by syringe through a sterile injection site coupler (Fenwal, Inc.) to a 60-mL PLT pool to yield final peptide concentrations of 0.01 mmol/L. After AMP inoculation, the PLTs were subjected to reciprocal agitation on a flatbed agitator (Helmer, Inc., Noblesville, IN) and stored for 7 days at 20 to 24°C with continuous agitation.
In vitro PLT testing All PCs were sampled on Day 1 and on the afternoons of Day 5 (for 5-day storage) and Day 7 (for 7-day storage). The samples were obtained aseptically through a sterile injection site coupler and the following assays were performed: 1.
PLT concentration and mean PLT volume were measured in ethylenediaminetetraacetate tubes using two hematology analyzers (Cell-Dyn 3700, Abbott, Abbott Park, IL; and XE-2100D, Sysmex America, Inc.,
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2.
3.
4.
5.
6.
Mundelein, IL). PLT content was calculated as the product of PLT concentration and volume. Volume was determined by weight using a specific gravity of 1.03. The pH was measured at RT (20 to 24°C) with a pH meter (Orion, Thermo Scientific, Beverly, MA) with an electrode (Accu-pHast, Fisher Scientific, Pittsburgh, PA). pO2, pCO2, glucose, lactate, and pH (37°C) were determined with a blood gas analyzer (Cobas b221, Roche Diagnostics, Indianapolis, IN). Bicarbonate levels were calculated from CO2 and pH measurements (37°C). The extent of shape change (ESC) and the hypotonic shock response (HSR) were measured turbidimetrically (SPA 2000, Chronolog, Havertown, PA).18 PLT activation was evaluated through measuring CD61, CD62, and CD63 by using their corresponding isotype controls19 in a flow cytometer (FACScan, BD Biosciences, San Jose, CA). The expression of GP1bα was measured by flow cytometry with a CD42b antibody and its isotype control.20 PLT aggregation was measured at 37°C using a lumiaggregometer with 10 μmol/L adenosine 5′-diphosphate and 10 μg/mL collagen as agonists which were added near simultaneously to initiate aggregation. Aggregation was expressed as either the slope of a plot of turbidity versus time or the amplitude relative to an internal aggregation standard. PLT morphology was determined by phase contrast microscopy and calculated as the percentage of PLTs that presented discoid morphology. Swirling, a noninvasive qualitative measure of discoid PLTs, was estimated by rotating a section of the PC bag in front of a light. A scale of 0 to 2 was used; a score of 2 indicated good swirling, 1 was the score for a reduced amount of swirling, and 0 was the score when swirling could not be visually observed.21-23
Statistical analysis Calculation of means and standard deviations (SDs) of experimental values and two-tailed paired t test were performed using standard statistical software (Instat, GraphPad software, GraphPad, San Diego, CA). A p value of 0.001 was considered significant for the t tests, in light of the multiple comparisons involving three sampling days and 18 assays.
RESULTS A total of 10 sets of whole blood–derived PCs were prepared as described under Materials and Methods and evaluated. No differences were found on Day 1 in any of the variables evaluated between control (PBS) and test (RW3 and RW4) PCs (Fig. 1 and Table 1, p > 0.001). 1606
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Fig. 1. Evaluation of pH during storage of pooled leukoreduced PCs and control. Ten leukoreduced whole blood–derived PCs were pooled and divided into two identical pools. One pool was incubated with PBS, control (○), and the other pool was incubated with AMPs RW3 and RW4, test (■). Values represent the mean ± SD (n = 10).
Figure 1 shows the mean pH values for PCs treated with the cocktail of AMPs RW3 and RW4 and PCs incubated with PBS during 7-day storage. pH values decreased over time in both groups of PCs but there were no significant differences between test and control PCs as can be seen in Fig. 1 (p > 0.001), where the values of pH for both groups of PCs decreased in parallel. The effect of the RW3 and RW4 cocktail treatment on additional in vitro PLT variables is depicted in Table 1. After 5- and 7-day storage, there were no significant differences between test and control PCs in any of the variables evaluated (p > 0.001). Some of the variables measured changed over the time of storage but they decreased (glucose, bicarbonate, ESC, HSR, morphology, aggregation, CD42b) or increased (lactate) at a similar rate for both arms.
DISCUSSION PCs are stored at RT with constant agitation to preserve their viability. However, these conditions provide an ideal environment for bacterial growth. In this study, we evaluated the effects of RW3 and RW4 cocktail on an array of in vitro PLT properties during storage. Our results demonstrate that this peptide cocktail did not affect the in vitro properties of whole blood–derived leukoreduced PLTs during 7 days of storage. The AMPs evaluated in this study belong to the synthetic (RW)n tandem repeat (n = 1-5) peptide family. Others have evaluated these AMPs for both their bactericidal and their hemolytic activities; the RW3 peptide has been reported to have an optimal antimicrobial activity with no detectable hemolytic activity (in sheep blood).12
Test 6.0 ± 1.5 52.51 ± 1.33 1147.15 ± 285.89 14.0 ± 5.5 54.7 ± 9 7.68 ± 0.57 57.9 ± 5.4 2±0 151.2 ± 7.9 22.3 ± 3.1 6.5 ± 1.3 518.9 ± 35.4 19.8 ± 2.6 62.4 ± 16.6 71.9 ± 20.4 30.9 ± 10.3 7.4 ± 4.6 544 ± 95 Day 7 Control 6.0 ± 1.5 52.69 ± 1.45 1132.65 ± 272.62 13.6 ± 5.4 55.8 ± 8.7 7.75 ± 0.55 57.6 ± 5.0 2±0 152.3 ± 6.1 21.1 ± 2.4 6.4 ± 1.2 528.8 ± 40.2 20.1 ± 3.0 61.3 ± 16.3 67.6 ± 17.7 31.0 ± 10.5 6.9 ± 4.7 541 ± 90 Test 6.7 ± 1.6 57.13 ± 1.28 1173.10 ± 287.40 17.4 ± 6.3 59.1 ± 7.6 7.57 ± 0.50 65.4 ± 5.7 2±0 143.5 ± 10.0 25.3 ± 3.4 10 ± 1.1 563.8 ± 27.8 14.7 ± 1.8 71.0 ± 16.3 85.6 ± 19.6 28.8 ± 13.8 8.9 ± 10.6 609 ± 88 Day 5 Control 6.6 ± 1.6 57.46 ± 1.71 1153.75 ± 268.30 16.7 ± 6 61.0 ± 7.8 7.65 ± 0.50 65.7 ± 6.2 2±0 144.1 ± 8.3 23.9 ± 2.8 9.7 ± 1.1 572.5 ± 28.2 14.8 ± 1.5 67.8 ± 17.3 78.5 ± 16.1 29.0 ± 13.5 8.7 ± 9.5 601 ± 84 Test 7.2 ± 1.7 61.77 ± 1.34 1166.65 ± 273.48 23.0 ± 5.7 65.5 ± 7.2 7.45 ± 0.46 72.6 ± 5.3 2±0 128.8 ± 10.2 36.8 ± 4.3 17.7 ± 2.3 641.2 ± 20.9 5.7 ± 0.5 77.7 ± 12.6 94.7 ± 20.5 28.4 ± 8.0 6.2 ± 5.3 692 ± 111 Day 1
* Values represent mean ± 1 SD. MPV = mean PLT volume.
Control 7.2 ± 1.7 62.06 ± 1.69 1161.85 ± 274.56 22.8 ± 5.7 66.5 ± 8.8 7.54 ± 0.43 72.2 ± 6.0 2±0 129.9 ± 9.9 35.5 ± 4.6 17.4 ± 1.8 641.3 ± 42.4 5.7 ± 0.6 75.8 ± 13.0 97.3 ± 16.7 26.9 ± 9.2 5.5 ± 5.1 696 ± 98 Assay Content (×1010) Volume (mL) Concentration (×109/L) ESC (%) HSR (%) MPV (fL) Morphology (% disc) Swirling pO2 (mmHg) pCO2 (mmHg) Bicarbonate (mmol/L) Glucose (mmol/L) Lactate (mmol/L) Aggregation amplitude (%) Aggregation (% slope) CD62P (%) CD63 (%) CD42b (mean fluorescence)
TABLE 1. In vitro PLT storage variables for pooled whole blood–derived PLTs incubated with RW3 and RW4 (test, n = 10) or PBS (control, n = 10)*
AMPs DO NOT AFFECT PLT IN VITRO PROPERTIES
In our study, we have used both RW3 and RW4 at 0.01 mmol/L concentrations. This concentration of the AMPs was selected for the current study since we had previously demonstrated significant antibacterial and antiviral activities of RW3 and RW4 at this concentration.5,6 In these studies, the peptide activities on selected bacteria and viruses were evaluated in the presence of human plasma and PLTs under similar conditions as reported in this study.5,6 New technologies continue to be developed to improve the collection and extend the storage time of PCs. These technologies require in vitro and in vivo evaluation for their effect on PLTs quality. At the moment, the gold standard is to evaluate in vivo PLT quality by measuring the recovery and survival of radiolabeled human autologous PLTs in normal human volunteers.24,25 Because of the small risk from exposure to radiation from PLT radiolabel studies as well as the cost of in vivo testing in humans, two surrogate methods have been in use as a first step toward assessing new PLT collection and storage technologies before embarking on a clinical trial with normal human volunteers. The surrogate methods evaluate the changes of in vitro properties of PLTs during storage26 and the survival and recovery of human PLTs in mice.27,28 Unfortunately no in vitro test can completely predict the survival and recovery of PLTs infused to healthy volunteers,29,30 and investigators and regulators believe that a battery of in vitro tests provide data that characterize PLT quality.31 Our group has recently demonstrated that selected individual AMPs have bactericidal activity in PCs5 and that all of these AMPs, including RW3 and RW4, do not affect the survival and recovery of human PLTs in severe combined immunodeficient mice. In addition, most of these peptides, including RW3 and RW4, do not produce an immunogenic response in rabbits.14 In this study, we evaluated the in vitro properties of whole blood–derived leukoreduced PLTs when incubated with a cocktail of two AMPs, RW3 and RW4, at bactericidal concentrations as reported by us.5 It is interesting to note that the RW3 and RW4 are short peptides each consisting of six and eight amino acids, respectively, and tempting to speculate that upon degradation by proteases may or may not result in any harmful by-products that could induce toxicologic effects in the recipient. However, we are cognizant of the fact that toxicologic tests for establishing the safety profile of these selected AMPs in a suitable animal model must be conducted before considering this concept toward reducing the microbial burden in stored PLTs. Using a battery of PLT assays, we demonstrated that the in vitro storage properties of whole blood–derived leukoreduced PLTs treated with RW3 and RW4 were maintained during 7 days of storage similar to those observed in control PLTs. These results indicate that mixtures of RW3 and RW4 peptides at concentrations that exhibit potent bactericidal activity, are not harmful to PLTs. Of Volume 54, June 2014
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interest, PLTs treated with RW3 and RW4 had levels of CD42b similar to those of control PLTs. The decline in CD42b levels during storage without decline of PLT concentration in our study is similar to previously reported results.32 The loss of CD42b during storage has been implicated in enhanced clearance of transfused PLTs in murine and rabbit animal models.20,32 Based on the results of our study, together with other published studies,5,14,15 evidence is provided to support continued development of AMPs for possible clinical evaluation. ACKNOWLEDGMENTS MBM is the recipient of a postdoctoral fellowship at the Center for Biologics Evaluation and Research (CBER) administered by the
9. Bals R. Epithelial antimicrobial peptides in host defense against infection. Respir Res 2000;1:141-50. 10. Nakatsuji T, Gallo RL. Antimicrobial peptides: old molecules with new ideas. J Invest Dermatol 2012;132: 887-95. 11. Yeaman MR, Yount NY. Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 2003;55:2755. 12. Liu Z, Brady A, Young A, et al. Length effects in antimicrobial peptides of the (RW)n series. Antimicrob Agents Chemother 2007;51:597-603. 13. Chan DI, Prenner EJ, Vogel HJ. Tryptophan- and argininerich antimicrobial peptides: structures and mechanisms of action. Biochim Biophys Acta 2006;1758:1184-202. 14. Bosch-Marcé M, Mohan KV, Gelderman MP, et al. Preclini-
Oak Ridge Institute for Science and Education (ORISE) through
cal safety evaluation of human platelets treated with anti-
an intraagency agreement between the US Department of Energy and the US Food and Drug Administration. The authors thank Ms Sandhya Kulkarni for her help with timely procurement of mate-
microbial peptides in severe combined immunodeficient mice. Transfusion 2014;54:569-76. 15. Mohan KV, Sainath Rao S, Gao Y, et al. Enhanced antimi-
rials used in this work.
crobial activity of peptide-cocktails against common bacterial contaminants of ex vivo stored platelets. Clin Microbiol Infect 2014;20:39-46.
CONFLICT OF INTEREST The authors report no conflicts of interest or funding sources.
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16. Wagner SJ, Skripchenko A, Myrup A, et al. Evaluation of in vitro storage properties of prestorage pooled whole bloodderived platelets suspended in 100 percent plasma and treated with amotosalen and long-wavelength ultraviolet
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AMPs DO NOT AFFECT PLT IN VITRO PROPERTIES
24. Murphy S. Radiolabeling of PLTs to assess viability: a proposal for a standard. Transfusion 2004;44:131-3. 25. AuBuchon JP. Radioisotopic reflections. Transfusion 2005; 45:28S-32S. 26. Wagner SJ, Seetharaman S, Kurtz J. Comparison of the in vitro storage properties of Amicus apheresis platelets collected using single- and double-needle procedures from the same donors. Transfusion 2012;52:524-8. 27. Piper JT, Gelderman MP, Vostal JG. In vivo recovery of human platelets in severe combined immunodeficient mice as a measure of platelet damage. Transfusion 2007;47: 1540-9. 28. Newman PJ, Aster R, Boylan B. Human platelets circulating in mice: applications for interrogating platelet function
29. Rinder HM, Smith BR. In vitro evaluation of stored platelets: is there hope for predicting posttransfusion platelet survival and function? Transfusion 2003; 43:2-6. 30. Dijkstra-Tiekstra MJ, Pietersz RN, Huijgens PC. Correlation between the extent of platelet activation in platelet concentrates and in vitro and in vivo parameters. Vox Sang 2004;87:257-63. 31. Moroff G, Kurtz J, Seetharaman S, et al. Comparative in vitro evaluation of apheresis platelets stored with 100% plasma or 65% platelet additive solution III/35% plasma and including periods without agitation under simulated shipping conditions. Transfusion 2012;52:834-43. 32. Leytin V, Gwozdz AA, Garvey B, et al. Role of platelet
and survival, the efficacy of antiplatelet therapeutics, and
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