The platelets’ perspective to pathogen reduction technologies

A wide variety of clinical conditions, associated with low circulating platelet counts, require platelet transfusion in order to normalize hemostatic function. Although single-donor apheresis platelets bear the lowest risk of transfusion-transmitted infections, pathogen reduction technologies (PRT) are being implemented worldwide to reduce this risk further through inactivation of known, emergent and as yet to be discovered nucleic acid-based pathogens. Human blood platelets are now known to harbor a diverse transcriptome, important to their function and comprised of >5000 protein-coding messenger RNAs and different classes of non-coding RNAs, including microRNAs. Our appreciation of the nucleic acid-dependent functions of platelets is likely to increase. On the other hand, the side effects of PRT on platelet function are underappreciated. Recent evidences suggest that PRT may compromise platelets’ responsiveness to agonists, and induce platelet activation. For instance, platelets have the propensity to release proinflammatory microparticles (MPs) upon activation, and the possibility that PRT may enhance the production of platelet MPs in platelet concentrates (PCs) appears likely. With this in mind, it would be timely and appropriate to investigate other means to inactivate pathogens more specifically, or to modify the currently available PRT so to better preserve the platelet function and improve the safety of PCs; platelets’ perspective to PRT deserves to be considered

Peculiarities of studying the effects of pathogen reduction technologies on platelets

The transfusion of platelet concentrates (PCs) is mainly used for treatment of thrombocytopenic, trauma or surgery patients. The integrity and safety of these platelet preparations, however, is compromised by the presence of pathogens, such as viruses, bacteria and parasites. The transfer of allogeneic donor leukocytes contaminating PCs can also potentially cause adverse reactions in recipients. These considerations prompted the development and implementation of pathogen reduction technologies (PRT), which are based on chemically induced cross‐linking and inactivation of nucleic acids. While the incumbent PRT may provide some protection against transfusion‐transmitted infections, they are ineffective against infectious prions and may not inactivate other emerging pathogens. In addition, the safety of PRT concerning platelet viability and function has been questioned in several reports. Recent studies suggest that PRT, such as Intercept, may adversely affect the messenger RNA (mRNA) and microRNA content of platelets, as well as their functional integrity, which may compromise the clinical benefits of PRT. Here, we will discuss about the peculiarities of studying the effects of PRT on platelets, which will need to be taken into account in future studies aimed to characterize further, and polish, the rugged side of this otherwise useful and potentially important approach in transfusion medicine

Differential Expression Analysis by RNA-Seq Reveals Perturbations in the Platelet mRNA Transcriptome Triggered by Pathogen Reduction Systems

Platelet concentrates (PCs) are prepared at blood banks for transfusion to patients in certain clinical conditions associated with a low platelet count. To prevent transfusion-transmitted infections via PCs, different pathogen reduction (PR) systems have been developed that inactivate the nucleic acids of contaminating pathogens by chemical cross-linking, a mechanism that may also affect platelets nucleic acids. We previously reported that treatment of stored platelets with the PR system Intercept significantly reduced the level of half of the microRNAs that were monitored, induced platelet activation and compromised the platelet response to physiological agonists. Using genome-wide differential expression (DE) RNA sequencing (RNA-Seq), we now report that Intercept markedly perturbs the mRNA transcriptome of human platelets and alters the expression level of greater than800 mRNAs (Pless than0.05) compared to other PR systems and control platelets. Of these, 400 genes were deregulated with DE corresponding to fold changes (FC) greater than= 2. At the p-value less than 0.001, as many as 147 genes were deregulated by greater than= 2-fold in Intercept-treated platelets, compared to none in the other groups. Finally, integrated analysis combining expression data for microRNA (miRNA) and mRNA, and involving prediction of miRNA-mRNA interactions, disclosed several positive and inverse correlations between miRNAs and mRNAs in stored platelets. In conclusion, this study demonstrates that Intercept markedly deregulates the platelet mRNA transcriptome, concomitant with reduced levels of mRNA-regulatory miRNAs. These findings should enlighten authorities worldwide when considering the implementation of PR systems, that target nucleic acids and are not specific to pathogens, for the management of blood products.Funding Agencies|Regional Council of Ostergotland, Sweden [LIO-353011]; Canadian Blood Services/Canadian Institutes of Health Research (CIHR) Blood Utilization and Conservation Initiative via Health Canada [293933]</p

Differential Expression Analysis by RNA-Seq Reveals Perturbations in the Platelet mRNA Transcriptome Triggered by Pathogen Reduction Systems

Platelet concentrates (PCs) are prepared at blood banks for transfusion to patients in certain clinical conditions associated with a low platelet count. To prevent transfusion-transmitted infections via PCs, different pathogen reduction (PR) systems have been developed that inactivate the nucleic acids of contaminating pathogens by chemical cross-linking, a mechanism that may also affect platelets' nucleic acids. We previously reported that treatment of stored platelets with the PR system Intercept significantly reduced the level of half of the microRNAs that were monitored, induced platelet activation and compromised the platelet response to physiological agonists. Using genome-wide differential expression (DE) RNA sequencing (RNA-Seq), we now report that Intercept markedly perturbs the mRNA transcriptome of human platelets and alters the expression level of &gt;800 mRNAs (P&lt;0.05) compared to other PR systems and control platelets. Of these, 400 genes were deregulated with DE corresponding to fold changes (FC) &gt;= 2. At the p-value &lt; 0.001, as many as 147 genes were deregulated by &gt;= 2-fold in Intercept-treated platelets, compared to none in the other groups. Finally, integrated analysis combining expression data for microRNA (miRNA) and mRNA, and involving prediction of miRNA-mRNA interactions, disclosed several positive and inverse correlations between miRNAs and mRNAs in stored platelets. In conclusion, this study demonstrates that Intercept markedly deregulates the platelet mRNA transcriptome, concomitant with reduced levels of mRNA-regulatory miRNAs. These findings should enlighten authorities worldwide when considering the implementation of PR systems, that target nucleic acids and are not specific to pathogens, for the management of blood products.This work was supported by grant no. LIO-353011 from the Regional Council of Ostergotland, Sweden (to A.O.), and grant no. 293933 from the Canadian Blood Services/Canadian Institutes of Health Research (CIHR) Blood Utilization and Conservation Initiative via Health Canada (to W.E.H and P.P.).</p

Platelet Pathogen Reduction Technologies Alter the MicroRNA Profile of Platelet-Derived Microparticles

Despite improvements in donor screening and increasing efforts to avoid contamination and the spread of pathogens in clinical platelet concentrates (PCs), the risks of transfusion-transmitted infections remain important. Relying on an ultraviolet photo activation system, pathogen reduction technologies (PRTs), such as Intercept and Mirasol, utilize amotosalen, and riboflavin (vitamin B2), respectively, to mediate inactivation of pathogen nucleic acids. Although they are expected to increase the safety and prolong the shelf life of clinical PCs, these PRTs might affect the quality and function of platelets, as recently reported. Upon activation, platelets release microparticles (MPs), which are involved in intercellular communications and regulation of gene expression, thereby mediating critical cellular functions. Here, we have used small RNA sequencing (RNA-Seq) to document the effect of PRT treatment on the microRNA profiles of platelets and derived MPs. PRT treatment did not affect the microRNA profile of platelets. However, we observed a specific loading of certain microRNAs into platelet MPs, which was impaired by treatment with Intercept or its Additive solution (SSP+). Whereas, Intercept had an impact on the microRNA profile of platelet-derived MPs, Mirasol did not impact the microRNA profile of platelets and derived MPs, compared to non-treated control. Considering that platelet MPs are able to transfer their microRNA content to recipient cells, and that this content may exert biological activities, those findings suggest that PRT treatment of clinical PCs may modify the bioactivity of the platelets and MPs to be transfused and argue for further investigations into PRT-induced changes in clinical PC content and function.Funding Agencies|Fonds de Recherche du Quebec-Sante (FRQ-S) [262093]; Canadian Blood Services (CBS)/Canadian Institutes of Health Research (CIHR) Partnership-Blood Utilization and Conservation Initiative, via Health Canada [286,777, 327,364]</p

Venn diagram depicting the overlap of detected mRNAs in this study (Osman et al.) with that of Londin et al. [17] are shown.

<p>Genes detected above the threshold >1/10,000 of ß-actin expression (this study) or ≥1/10,000 of ß-actin expression (Londin et al.) were compared.</p

The fold changes (in log2) relative to the control group and the corresponding adjusted p-values (padj) are depicted for the pathogen reduction treatment groups.

<p>Blue and red horizontal dotted lines indicate padj thresholds at 0.05 and 0.001, respectively.</p

Functional annotation clusters generated by DAVID tools for the most significant (padj<0.05) differentially expressed (FC≥2) mRNAs in platelets treated with the Intercept pathogen reduction system.

<p>Functional annotation clusters generated by DAVID tools for the most significant (padj<0.05) differentially expressed (FC≥2) mRNAs in platelets treated with the Intercept pathogen reduction system.</p

Thirteen (13) platelet mRNAs that were found to be downregulated by more than 4 fold (p-value < 0.001) upon treatment with Intercept, ranked in order of DE significance by padj values.

<p>FC = Fold change; pval = p-value; padj = adjusted p-value</p><p>Thirteen (13) platelet mRNAs that were found to be downregulated by more than 4 fold (p-value < 0.001) upon treatment with Intercept, ranked in order of DE significance by padj values.</p