Microbial reduction of prebagged human plasma using 405 nm light and its effects on coagulation factors.

Bacterial contamination is the most prevalent infectious complication of blood transfusion in the developed world. To mitigate this, several ultraviolet light-based pathogen reduction technologies (PRTs), some of which require photo-chemicals, have been developed to minimize infection transmission. Relative to UV light, visible 405-nm light is safer and has shown potential to be developed as a PRT for the in situ treatment of ex vivo human plasma and platelet concentrates, without the need for photo-chemicals. This study investigates the effect of 405-nm light on human plasma, with focus on the compatibility of antimicrobial light doses with essential plasma clotting factors. To determine an effective antimicrobial dose that is compatible with plasma, prebagged human plasma (up to 300 mL) was seeded with common microbial contaminants and treated with increasing doses of 405-nm light (16 mW cm-2; ≤ 403 J cm-2). Post-exposure plasma protein integrity was investigated using an AOPP assay, in vitro coagulation tests, and ELISA-based measurement of fibrinogen and Protein S. Microbial contamination in 300 mL prebagged human plasma was significantly reduced (P ≤ 0.05) after exposure to ≤ 288 J cm-2, with microbial loads reduced by > 96.2%. This dose did not significantly affect the plasma protein quality parameters tested (P > 0.05). Increased doses (≥ 345 J cm-2) resulted in a 4.3% increase in clot times with no statistically significant change in protein activity or levels. Overall, this study has demonstrated that the effective microbicidal 405 light dose shows little to no negative effect on plasma quality.

Human platelet concentrates treated with microbicidal 405 nm light retain hemostasis activity.

Chemical and UV light-based pathogen reduction technologies are currently in use for human platelet concentrates (PCs) to enhance safety from transfusion-transmitted infections. Relative to UV light, 405 nm violet-blue light in the visible spectrum is known to be less harmful. Hence, in this report for the first time, we have assessed the global hemostasis activity of PCs stored in plasma and the activities of six plasma coagulation factors (CFs) as a measure of in vitro hemostatic activity following exposure to the microbicidal 405 nm light. Apheresis PC samples collected from each screened human donor (n = 22) were used for testing of PCs and platelet poor plasma (PPP). Both PCs and PPPs were treated for 5 h with 405 nm light to achieve a previously established microbicidal light dose of 270 J/cm2. Activated partial thromboplastin time and prothrombin time-based potency assays using a coagulation analyzer and hemostatic capacity via Thromboelastography were analyzed. Thromboelastography analysis of the light-treated PCs and plasma present in the PCs showed little difference between the treated and untreated samples. Further, plasma present in the PCs during the light treatment demonstrated a better stability in potency assays for several coagulation factors compared to the plasma alone prepared from PCs first and subjected to the light treatment separately. Overall, PCs stored in plasma treated with 405 nm violet-blue light retain activity for hemostasis.

The Preclinical Validation of 405 nm Light Parasiticidal Efficacy on Leishmania donovani in Ex Vivo Platelets in a Rag2-/- Mouse Model.

Violet-blue light of 405 nm in the visible spectrum at a dose of 270 J/cm2 alone has been shown to be an effective microbicidal tool for inactivating several bacteria, HIV-1, and Trypanosoma cruzi in ex vivo plasma and platelets. Unlike chemical- and ultraviolet (UV)-based pathogen inactivation methods for plasma and platelet safety, 405 nm light is shown to be less toxic to host cells at light doses that are microbicidal. In this report, we evaluated the parasiticidal activity of a 405 nm light treatment on platelets spiked with the Leishmania donovani parasite. Following the light treatment, parasite viability was observed to be near zero in both low- and high-titer-spiked platelets relative to controls. Furthermore, to test the residual infectivity after inactivation in vivo, the light-treated low-titer L. donovani-spiked platelets were evaluated in an immunodeficient Rag2-/- mouse model and monitored for 9 weeks. The parasiticidal efficacy of 405 nm light was evident from the lack of a presence of parasites in the mice spleens. Parasiticidal activity was confirmed to be mediated through 405 nm light-induced reactive oxygen species (ROS), as quantitatively measured by a 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA)-based assay. Overall, these results confirm the complete inactivation of L. donovani spiked in ex vivo platelets by 405 nm light treatment and exemplify the utility of the Rag2-/- mouse infection model for the preclinical validation of the parasiticidal efficacy of 405 nm light and this light-based technology as a potential PRT for ex vivo platelets.

Metabolomics evaluation of the photochemical impact of violet-blue light (405 nm) on ex vivo platelet concentrates.

INTRODUCTION: Microbicidal violet-blue light in the visible spectrum (405 nm) has been under evaluation for pathogen inactivation in ex vivo human plasma and platelets (PLTs) stored in plasma. Results to date have demonstrated that several blood-borne infectious disease-causing pathogens can be successfully reduced to significantly low levels in the light-treated plasma and PLTs. METHOD: In order to evaluate whether the microbicidal 405 nm light is safe for the treatment of PLT concentrates for pathogen inactivation, LC/MS-based metabolomics analyses were performed to evaluate the overall impact of 405 nm violet-blue light treatment on ex vivo PLT concentrates suspended in plasma and on plasma itself, and to identify metabolome changes in intra-platelet and extra-cellular medium (i.e., plasma). RESULTS: The metabolomics data identified that platelet activating factors (PAFs), agonists and prostaglandins, which can influence PLT basic functions such as integrity, activation, and aggregation potential were unaltered, suggesting that 405 nm light illumination is safe regarding PLT basic functions. Distinct increases in hydroxyl fatty acids and aldehydes, as well as decreases in antioxidant metabolites indicated that reactive oxygen species (ROS) were generated at high levels after only one hour of exposure to 405 nm light. Distinctly changed endogenous photosensitizer metabolites after 1 h of light exposure provided good evidence that 405 nm light was an effective microbicide acting through ROS mechanism and no external additive photosensitizers were required.

Antimicrobial 405 nm violet-blue light treatment of ex vivo human platelets leads to mitochondrial metabolic reprogramming and potential alteration of Phospho-proteome.

Continued efforts to reduce the risk of transfusion-transmitted infections (TTIs) through blood and blood components led to the development of ultraviolet (UV) light irradiation technologies known as pathogen reduction technologies (PRT) to enhance blood safety. While these PRTs demonstrate germicidal efficiency, it is generally accepted that these photoinactivation techniques have limitations as they employ treatment conditions shown to compromise the quality of the blood components. During ex vivo storage, platelets having mitochondria for energy production suffer most from the consequences of UV irradiation. Recently, application of visible violet-blue light in the 400-470 nm wavelength range has been identified as a relatively more compatible alternative to UV light. Hence, in this report, we evaluated 405 nm light-treated platelets to assess alterations in energy utilization by measuring different mitochondrial bioenergetic parameters, glycolytic flux, and reactive oxygen species (ROS). Furthermore, we employed untargeted data-independent acquisition mass spectrometry to characterize platelet proteomic differences in protein regulation after the light treatment. Overall, our analyses demonstrate that ex vivo treatment of human platelets with antimicrobial 405 nm violet-blue light leads to mitochondrial metabolic reprogramming to survive the treatment, and alters a fraction of platelet proteome.

The microbicidal potential of visible blue light in clinical medicine and public health.

Visible blue light of wavelengths in the 400-470 nm range has been observed to have microbicidal properties. A widely accepted hypothesis for the mechanism of microbial inactivation by visible blue light is that the light causes photoexcitation of either endogenous (present within the microbe) or, exogenous (present in the biological medium surrounding the microbe) photosensitizers such as porphyrins and flavins, which leads to the release of reactive oxygen species that subsequently manifests microbicidal activity. Some of the factors that have been observed to be associated with enhanced microbicidal action include increased duration of exposure, and either pre- or co-treatment with quinine hydrochloride. In case of bacteria, repetitive exposure to the blue light shows no significant evidence of resistance development. Additionally, visible blue light has exhibited the ability to inactivate fungal and viral pathogens and, multidrug-resistant bacteria as well as bacterial biofilms. Visible blue light has demonstrated efficacy in eliminating foodborne pathogens found on food surfaces and exposed surfaces in the food processing environment as well as in the decontamination of surfaces in the clinical environment to minimize the spread of nosocomial infections. We conclude from reviewing existing literature on the application of the blue light in clinical medicine and public health settings that this microbicidal light is emerging as a safer alternative to conventional ultraviolet light-based technologies in multiple settings. However, further comprehensive studies and thorough understanding of the mechanism of microbicidal action of this light in different scenarios is warranted to determine its place in human health and disease.

Visible 405 nm Violet-Blue Light Successfully Inactivates HIV-1 in Human Plasma.

Despite significant advances in ensuring the safety of the blood supply, there is continued risk of transfusion transmitted infections (TTIs) from newly emerging or re-emerging infections. Globally, several pathogen reduction technologies (PRTs) for blood safety have been in development as an alternative to traditional treatment methods. Despite broad spectrum antimicrobial efficacy, some of the approved ultraviolet (UV) light-based PRTs, understandably due to UV light-associated toxicities, fall short in preserving the full functional spectrum of the treated blood components. As a safer alternative to the UV-based microbicidal technologies, investigations into the use of violet-blue light in the region of 405 nm have been on the rise as these wavelengths do not impair the treated product at doses that demonstrate microbicidal activity. Recently, we have demonstrated that a 405 nm violet-blue light dose of 270 J/cm2 was sufficient for reducing bacteria and the parasite in plasma and platelets suspended in plasma while preserving the quality of the treated blood product stored for transfusion. Drawn from the previous experience, here we evaluated the virucidal potential of 405 nm violet-blue light dose of 270 J/cm2 on an important blood-borne enveloped virus, the human immunodeficiency virus 1 (HIV-1), in human plasma. Both test plasma (HIV-1 spiked and treated with various doses of 405 nm light) and control plasma (HIV-1 spiked, but not treated with the light) samples were cultured with HIV-1 permissive H9 cell line for up to 21 days to estimate the viral titers. Quantitative HIV-1 p24 antigen (HIV-1 p24) levels reflective of HIV-1 titers were measured for each light dose to assess virus infectivity. Our results demonstrate that a 405 nm light dose of 270 J/cm2 is also capable of 4-5 log HIV-1 reduction in plasma under the conditions tested. Overall, this study provides the first proof-of-concept that 405 nm violet-blue light successfully inactivates HIV-1 present in human plasma, thereby demonstrating its potential towards being an effective PRT for this blood component safety.

Violet-blue 405-nm Light-based Photoinactivation for Pathogen Reduction of Human Plasma Provides Broad Antibacterial Efficacy Without Visible Degradation of Plasma Proteins.

In transfusion medicine, bacterial contamination can occur in ex vivo stored blood plasma, and there are continued efforts to improve blood safety and reduce the risk of transfusion-transmitted infections. Visible 405-nm violet-blue light has demonstrated potential for in situ pathogen reduction in ex vivo stored plasma and platelet concentrates. This study investigates the broad-spectrum antibacterial efficacy and compatibility potential of 405-nm light for treatment of blood plasma. Human plasma seeded with bacteria at a range of densities (101 -103 , 104 -106 , 107 -108 CFU mL-1 ) was exposed to 360 J cm-2 405-nm light (1 h at 0.1 W cm-2 ), with this fixed dose selected based on the initial analysis of inactivation kinetics. One-dimensional protein mobility analysis and measurement of advanced oxidation protein products (AOPP) was conducted to evaluate compatibility of the antimicrobial dose with plasma proteins and, identify upper levels at which protein degradation can be detected. Broad-spectrum antibacterial efficacy was observed with a fixed treatment of 360 J cm-2 , with 98.9-100% inactivation achieved across all seeding densities for all organisms, except E. coli, which achieved 95.1-100% inactivation. At this dose (360 J cm-2 ), no signs of protein degradation occurred. Overall, 405-nm light shows promise for broad-spectrum bacterial inactivation in blood plasma, while preserving plasma protein integrity.

MiRNA-103b Downregulates ITGB3 and Mediates Apoptosis in Ex Vivo Stored Human Platelets.

BACKGROUND: Blood bank-stored human platelets are one of the life-saving transfusion products to prevent bleeding in multiple clinical settings. In ex vivo storage, platelets undergo apoptosis and it is highly desirable to prevent this process to preserve platelet quality. However, underlying mechanisms of apoptosis are not well understood in stored platelets. Integrin beta 3 (ITGB3) glycoprotein plays multiple roles in platelet physiological processes, and it was reported in other cell types that downregulation of ITGB3 induces apoptosis. Small noncoding regulatory RNAs known as microRNAs (miRNAs), some of which are abundant in platelets such as miR-103b that belong to miR-103 family of miRNAs, known to play key roles in platelet functions both in vivo and during storage; Cellular miR-103 downregulates certain genes in other cell types and promotes apoptosis. However, whether miR-103b can target and downregulate ITGB3 in stored platelets and such miRNA regulation promotes apoptosis is not known. Here, we tested this working hypothesis. OBJECTIVE: Our objective of this study is to validate the abundance of miR-103b in stored platelets and identify whether ITGB3 is a target of miR-103b for the downregulation and this interaction promotes apoptosis. METHODS: RT-qPCR validation of miR-103b was performed in 11 donor samples at 3 different storage time points. In-silico analysis was performed to identify predicted targets of the miR-103b. The miRNA and messenger RNA interactions were confirmed using different biochemical approaches such as qRT-PCR, western blotting and, suppression of luciferase reporter gene expression by ectopic expression of miR-103b in HeLa cells. Final validation of the functional role of miR-103b in ITGB3 downregulation and resulting induction of apoptosis was assessed in stored platelets by FACS analysis following ectopic expression of miR-103b. RESULTS: Using the Target Scan Vert algorithm, we identified several integrin subunit-encoding mRNAs as potential targets of miR-103b. While ITGB3 and ITGB6 were found to have two targeting sites for miR-103b, since ITGB3 is known to play a role in apoptosis, we chose this for further validation in this study. Ectopic expression of miR-103b decreased the luciferase reporter activity in HeLa cells and decreased ITGB3 mRNA and protein levels in platelets, concomitant with an increase in apoptosis. CONCLUSION: The results demonstrate that in stored platelets, miR-103b is highly expressed and can interact with and downregulate ITGB3 and promote apoptosis in stored platelets.

Analysis of the mechanism of damage produced by thiazole orange photoinactivation in apheresis platelets.

BACKGROUND: Pathogen Reduction Technologies (PRTs) are broad spectrum nucleic acid replication-blocking antimicrobial treatments designed to mitigate risk of infection from blood product transfusions. Thiazole Orange (TO), a photosensitizing nucleic acid dye, was previously shown to photoinactivate several types of bacterial and viral pathogens in RBC suspensions without adverse effects on function. In this report we extended TO treatment to platelet concentrates (PCs) to see whether it is compatible with in vitro platelet functions also, and thus, could serve as a candidate technology for further evaluation. MATERIAL AND METHODS: PCs were treated with TO, and an effective treatment dose for inactivation of Staphylococci was identified. Platelet function and physiology were then evaluated by various assays in vitro. RESULTS: Phototreatment of PCs yielded significant reduction (≥4-log) in Staphylococci at TO concentrations ≥20 µM. However, treatment with TO reduced aggregation response to collagen over time, and platelets became unresponsive by 24 hours post-treatment (from >80% at 1 h to 0% at 24 h). TO treatment also significantly increased CD62P expression (<1% CD62P+ for untreated and >50% for TO treated at 1 h) and induced apoptosis in platelets (<1% Annexin V+ for untreated and >50% for TO treated at 1 h) and damaged mitochondrial DNA. A mitochondria-targeted antioxidant and reactive oxygen species (ROS) scavenger Mito-Tempo mitigated these adverse effects. DISCUSSION: The results demonstrate that TO compromises mitochondria and perturbs internal signaling that activates platelets and triggers apoptosis. This study illustrates that protecting platelet mitochondria and its functions should be a fundamental consideration in selecting a PRT for transfusion units containing platelets, such as PCs.

Complete Inactivation of Blood Borne Pathogen Trypanosoma cruzi in Stored Human Platelet Concentrates and Plasma Treated With 405 nm Violet-Blue Light.

The introduction of pathogen reduction technologies (PRTs) to inactivate bacteria, viruses and parasites in donated blood components stored for transfusion adds to the existing arsenal toward reducing the risk of transfusion-transmitted infectious diseases (TTIDs). We have previously demonstrated that 405 nm violet-blue light effectively reduces blood-borne bacteria in stored human plasma and platelet concentrates. In this report, we investigated the microbicidal effect of 405 nm light on one important bloodborne parasite Trypanosoma cruzi that causes Chagas disease in humans. Our results demonstrated that a light irradiance at 15 mWcm-2 for 5 h, equivalent to 270 Jcm-2, effectively inactivated T. cruzi by over 9.0 Log10, in plasma and platelets that were evaluated by a MK2 cell infectivity assay. Giemsa stained T. cruzi infected MK2 cells showed that the light-treated parasites in plasma and platelets were deficient in infecting MK2 cells and did not differentiate further into intracellular amastigotes unlike the untreated parasites. The light-treated and untreated parasite samples were then evaluated for any residual infectivity by injecting the treated parasites into Swiss Webster mice, which did not develop infection even after the animals were immunosuppressed, further demonstrating that the light treatment was completely effective for inactivation of the parasite; the light-treated platelets had similar in vitro metabolic and biochemical indices to that of untreated platelets. Overall, these results provide a proof of concept toward developing 405 nm light treatment as a pathogen reduction technology (PRT) to enhance the safety of stored human plasma and platelet concentrates from bloodborne T. cruzi, which causes Chagas disease.

A Foundational Study for Normal F8-Containing Mouse Models for the miRNA Regulation of Hemophilia A: Identification and Analysis of Mouse miRNAs that Downregulate the Murine F8 Gene.

Hemophilia A (HA) is associated with defects in the F8 gene, encoding coagulation factor VIII (FVIII). Our previous studies show that F8-targeting micro RNAs (miRNAs), a group of small RNAs involved in gene regulation, can downregulate F8 expression causing HA in individuals with normal F8-genotypes and increased HA severity in patients with mutations in F8. Understanding the mechanistic underpinnings of human genetic diseases caused or modulated by miRNAs require a small animal model, such as a mouse model. Here, we report a foundational study to develop such a model system. We identified the mouse 3'untranslated region (3'UTR) on murine F8-mRNA (muF8-mRNA) that can bind to murine miRNAs. We then selected three miRNAs for evaluation: miR-208a, miR-351 and miR-125a. We first demonstrate that these three miRNAs directly target the 3'UTR of muF8-mRNA and reduce the expression of a reporter gene (luciferase) mRNA fused to the muF8-3' UTR in mammalian cells. Furthermore, in mouse cells that endogenously express the F8 gene and produce FVIII protein, the ectopic expression of these miRNAs downregulated F8-mRNA and FVIII protein. These results provide proof-of-concept and reagents as a foundation for using a normal F8-containing mouse as a model for the miRNA regulation of normal F8 in causing or aggravating the genetic disease HA.

Further Evidence That MicroRNAs Can Play a Role in Hemophilia A Disease Manifestation: F8 Gene Downregulation by miR-19b-3p and miR-186-5p.

Hemophilia A (HA) is a F8 gene mutational disorder resulting in deficiency or dysfunctional FVIII protein. However, surprisingly, in few cases, HA is manifested even without mutations in F8. To understand this anomaly, we recently sequenced microRNAs (miRNAs) of two patients with mild and moderate HA with no F8 gene mutations and selected two highly expressing miRNAs, miR-374b-5p and miR-30c-5p, from the pool to explain the FVIII deficiency that could be mediated by miRNA-based F8/FVIII suppression. In this report, an established orthogonal in vivo RNA-affinity purification approach was utilized to directly identify a group of F8-interacting miRNAs and we tested them for F8/FVIII suppression. From this pool, two miRNAs, miR-19b-3p and miR-186-5p, were found to be upregulated in a severe HA patient with a mutation in the F8 coding sequence and two HA patients without mutations in the F8 coding sequence were selected to demonstrate their role in F8 gene expression regulation in mammalian cells. Overall, these results provide further evidence for the hypothesis that by targeting the 3'UTR of F8, miRNAs can modulate FVIII protein levels. This mechanism could either be the primary cause of HA in patients who lack F8 mutations or control the severity of the disease in patients with F8 mutations.

Role of microRNAs in Hemophilia and Thrombosis in Humans.

MicroRNAs (miRNA) play an important role in gene expression at the posttranscriptional level by targeting the untranslated regions of messenger RNA (mRNAs). These small RNAs have been shown to control cellular physiological processes including cell differentiation and proliferation. Dysregulation of miRNAs have been associated with numerous diseases. In the past few years miRNAs have emerged as potential biopharmaceuticals and the first miRNA-based therapies have entered clinical trials. Our recent studies suggest that miRNAs may also play an important role in the pathology of genetic diseases that are currently considered to be solely due to mutations in the coding sequence. For instance, among hemophilia A patients there exist a small subset, with normal wildtype genes; i.e., lacking in mutations in the coding and non-coding regions of the F8 gene. Similarly, in many patients with missense mutations in the F8 gene, the genetic defect does not fully explain the severity of the disease. Dysregulation of miRNAs that target mRNAs encoding coagulation factors have been shown to disturb gene expression. Alterations in protein levels involved in the coagulation cascade mediated by miRNAs could lead to bleeding disorders or thrombosis. This review summarizes current knowledge on the role of miRNAs in hemophilia and thrombosis. Recognizing and understanding the functions of miRNAs by identifying their targets is important in identifying their roles in health and diseases. Successful basic research may result in the development and improvement of tools for diagnosis, risk evaluation or even new treatment strategies.

Clinical manifestation of hemophilia A in the absence of mutations in the F8 gene that encodes FVIII: role of microRNAs.

BACKGROUND: Hemophilia A (HA) is associated with mutations in the F8 gene that expresses factor VIII (FVIII). Unexpectedly, HA also manifests in a small subset of individuals with no mutations (exonic or intronic) in their F8 gene. MicroRNAs (miRNAs) cause translational interference, affecting protein quality and stoichiometry. Here, by analyzing miRNAs of two patients from this subset, we evaluated miRNA-based FVIII suppression as a testable hypothesis to explain FVIII deficiency in patients with HA with no F8 gene mutations. STUDY DESIGN AND METHODS: To test the hypothesis, miRNA sequencing from two patients with mild and moderate HA with no mutations in their F8 gene, followed by experimental verification, was used to identify a group of upregulated miRNAs in patients with HA compared to normal controls; with binding sites in the 3' untranslated region (UTR) of F8 messenger RNA (mRNA), a prerequisite for miRNA-based gene regulation. From this pool, miR-374b-5p and miR-30c-5p, known to be expressed in human liver, where FVIII is expressed, were subjected to extensive characterization. RESULTS: In two cell lines that constitutively express FVIII, we demonstrated that overexpression of miR-374b or miR-30c decreased FVIII expression, while an miR-30c inhibitor partially restored FVIII expression. CONCLUSION: These data support a role for microRNAs in fine-tuning F8 gene regulation. Based on our findings, our current model suggests that in HA cases where the F8 gene is normal and is predicted to express normal levels of FVIII, F8 mRNA 3' UTR targeting miRNAs may be responsible for a FVIII-deficiency phenotype clinically manifesting as HA.

MiR-181a Reduces Platelet Activation via the Inhibition of Endogenous RAP1B.

AIM: Since RAP1B is critical for platelet functions, including hemostasis, this study was conducted to identify RAP1B regulating microRNAs (miRNAs) in ex vivo stored platelets. BACKGROUND: Previous studies with platelets identified factors affecting RAP1B activity but regulatory miRNAs that affect RAP1B protein expression have not been reported. OBJECTIVE: To understand the functional significance of miRNA mediated regulation of RAP1B in stored platelets, using microRNA, miR-181a as an example. METHODS: A Tagged RNA Affinity approach (MS2-TRAP) was employed to identify miRNAs that bound to the 3` untranslated region (3`UTR) of the RAP1B mRNA in HeLa cells as an assay system. And subsequently, the mRNA 3'UTR:miRNA interactions were verified in platelets through the ectopic expression of miR-181a mimic and appropriate controls. The interaction of such miRNAs with RAP1B mRNA was also validated by qRT-PCR and Western analysis. RESULTS: Sixty-two miRNAs from MS2 assay were then compared with already known 171 platelet abundant miRNAs to identify a common set of miRNAs. This analysis yielded six miRNAs (miR- 30e, miR-155, miR-181a, miR-206, miR-208a and miR-454), which are also predicted to target RAP1B mRNA. From this pool, miR-181a was selected for further study since RAP1B harbors two binding sites for miR-181a in its 3'UTR. Ectopic expression of miR-181a mimic in platelets resulted in lowering the endogenous RAP1B at both mRNA and protein levels. Further, miR-181a ectopic expression reduced the surface expression of the platelet activation marker, P-selectin. CONCLUSION: MicroRNA-181a can target RAP1B and this interaction has the potential to regulate platelet activation during storage.

RAP1 Downregulation by miR-320c Reduces Platelet Activation in Ex-vivo Storage.

BACKGROUND: A small GTPase Protein, the Ras-related Protein 1 (RAP1), abundant in platelets is known to be activated following agonist-induced platelet activation, suggesting that RAP1 downregulation could, in turn, reduce platelet activation in storage. Our objective of this study is to identify RAP1 regulating miRNAs and their role in platelet activation during storage. METHODS: We applied MS2-TRAP (tagged RNA affinity purification) methodology to enrich miRNAs that target the 3' untranslated region (3'UTR) of RAP1 mRNA in two mammalian cell lines followed by miRNA identification by microarray of total RNA samples enriched for miRNAs. Data analyses were done using different bioinformatics approaches. The direct miR:RAP1 3'UTR interaction was confirmed by using a dual luciferase reporter gene expression system in a mammalian cell line. Subsequently, platelets were transfected with one selected miR to evaluate RAP1 downregulation by this miRNA and its effect on platelet activation. RESULTS: Six miRNAs (miR-320c, miR-181a, miR-3621, miR-489, miR-4791 and miR-4744) were identified to be enriched in the two cell lines tested. We randomly selected miR-320c for further evaluation. The luciferase reporter assay system confirmed the direct interaction of miR-320c with RAP1 3'UTR. Further, in platelets treated with miR-320c, RAP1 protein expression was decreased and concomitantly, platelet activation was also decreased. CONCLUSION: Overall, the results demonstrate that miRNA-based RAP1 downregulation in ex vivo stored platelets reduces platelet activation.

Non-ionizing 405 nm Light as a Potential Bactericidal Technology for Platelet Safety: Evaluation of in vitro Bacterial Inactivation and in vivo Platelet Recovery in Severe Combined Immunodeficient Mice.

Bacterial contamination of ex vivo stored platelets is a cause of transfusion-transmitted infection. Violet-blue 405 nm light has recently demonstrated efficacy in reducing the bacterial burden in blood plasma, and its operational benefits such as non-ionizing nature, penetrability, and non-requirement for photosensitizing agents, provide a unique opportunity to develop this treatment for in situ treatment of ex vivo stored platelets as a tool for bacterial reduction. Sealed bags of platelet concentrates, seeded with low-level Staphylococcus aureus contamination, were 405 nm light-treated (3-10 mWcm-2) up to 8 h. Antimicrobial efficacy and dose efficiency was evaluated by quantification of the post-treatment surviving bacterial contamination levels. Platelets treated with 10 mWcm-2 for 8 h were further evaluated for survival and recovery in severe combined immunodeficient (SCID) mice. Significant inactivation of bacteria in platelet concentrates was achieved using all irradiance levels, with 99.6-100% inactivation achieved by 8 h (P < 0.05). Analysis of applied dose demonstrated that lower irradiance levels generally resulted in significant decontamination at lower doses: 180 Jcm-2/10 mWcm-2 (P = 0.008) compared to 43.2 Jcm-2/3 mWcm-2 (P = 0.002). Additionally, the recovery of light-treated platelets, compared to non-treated platelets, in the murine model showed no significant differences (P = >0.05). This report paves the way for further comprehensive studies to test 405 nm light treatment as a bactericidal technology for stored platelets.

MicroRNA-223 Regulates Septin-2 and Septin-6 in Stored Platelets.

BACKGROUND: Septins have been identified to play important roles in platelets, but their regulation in platelets is unknown. Human platelet being an enucleated and terminally differentiated cell, mRNA downregulation by miRs is one of the posttranscriptional mechanisms operative in platelets. OBJECTIVE: Since platelets are known to have miR-223 in abundance, the objective of this study is to test whether a) platelet septins have miR-223 interacting target sites in their mRNA 3'UTRs, b) septin mRNAs and miR-223 form complexes with Argonaute 2 (AGO2) protein in platelets, which is the catalytic component of an RNA Induced Silencing Complex (RISC), c) a reporter gene with septin mRNA 3' untranslated region (UTR) is subjected to downregulation by miR-223 and d) anti-miR-223 can suppress miR-223 activity and enhance septin-2 expression in platelets. METHOD: Bioinformatics tools were used to screen mRNA 3'UTRs of septin-2 and septin-6 for miR- 223 target sites. Subsequently, platelet extracts were immunoprecipitated by AGO2 antibodies to identify that the two septin mRNAs and miR-223 were in complex with AGO2. A luciferase reporter chimeric- gene expression system was utilized to monitor miR-223 mediated downregulation luciferase gene containing the 3'UTR of either septin-2 or septin-6. Further, anti-miR-223 was utilized in platelets to directly demonstrate the role of miR-223 on the expression of septin-2. RESULTS: Our results demonstrate that in stored platelets a) septine-2 and septin-6 mRNAs have miR- 223 target sites, b) septin-2 and septin-6 are in complex with Ago-2, c) in luciferase reporter gene system, the interaction of miR-223 with 3' UTRs of septin-2 and septin-6 leads to downregulation of luciferase expression and d) anti-miR-223 downregulated miR-223 activity and thereby the expression of septin-2 is upregulated. CONCLUSION: The results demonstrate that like in nucleated cells, enucleated platelets also have miRbased mechanisms for the regulation of their septins.