During the last three decades, a growing body of clinical, basic science and animal model data has demonstrated that blood transfusions have important effects on the immune system. These effects include: dysregulation of inflammation and innate immunity leading to susceptibility to microbial infection, down-regulation of cellular (T and NK cell) host defenses against tumors, and enhanced B cell function that leads to alloimmunization to blood group, histocompatibility and other transfused antigens. Furthermore, transfusions alter the balance between hemostasis and thrombosis through inflammation, nitric oxide scavenging, altered rheologic properties of the blood, immune complex formation and, no doubt, several mechanisms not yet elucidated. The net effects are rarely beneficial to patients, unless they are in imminent danger of death due to exsanguination or life threatening anemia. These findings have led to appeals for more conservative transfusion practice, buttressed by randomized trials showing that patients do not benefit from aggressive transfusion practices. At the risk of hyperbole, one might suggest that if the 18th and 19th centuries were characterized by physicians unwittingly harming patients through venesection and bleeding, the 20th century was characterized by physicians unwittingly harming patients through current transfusion practices. In addition to the movement to more parsimonious use of blood transfusions, an effort has been made to reduce the toxic effects of blood transfusions through modifications such as leukoreduction and saline washing. More recently, there is early evidence that reducing the storage period of red cells transfused might be a strategy for minimizing adverse outcomes such as infection, thrombosis, organ failure and mortality in critically ill patients particularly at risk for these hypothesized effects. The present review will focus on two approaches, leukoreduction and saline washing, as means to reduce adverse transfusion outcomes.

Platelets are small anucleate blood cells derived from megakaryocytes. In addition to their pivotal roles in hemostasis, platelets are the smallest, yet most abundant, immune cells and regulate inflammation, immunity, and disease progression. Although platelets lack DNA, and thus no functional transcriptional activities, they are nonetheless rich sources of RNAs, possess an intact spliceosome, and are thus capable of synthesizing proteins. Previously, it was thought that platelet RNAs and translational machinery were remnants from the megakaryocyte. We now know that the initial description of platelets as “cellular fragments” is an antiquated notion, as mounting evidence suggests otherwise. Therefore, it is reasonable to hypothesize that platelet transcription factors are not vestigial remnants from megakaryocytes, but have important, if only partly understood functions. Proteins play multiple cellular roles to minimize energy expenditure for maximum cellular function; thus, the same can be expected for transcription factors. In fact, numerous transcription factors have non-genomic roles, both in platelets and in nucleated cells. Our lab and others have discovered the presence and non-genomic roles of transcription factors in platelets, such as the nuclear factor kappa β (NFκB) family of proteins and peroxisome proliferator-activated receptor gamma (PPARγ). In addition to numerous roles in regulating platelet activation, functional transcription factors can be transferred to vascular and immune cells through platelet microparticles. This method of transcellular delivery of key immune molecules may be a vital mechanism by which platelet transcription factors regulate inflammation and immunity. At the very least, platelets are an ideal model cell to dissect out the non-genomic roles of transcription factors in nucleated cells. There is abundant evidence to suggest that transcription factors in platelets play key roles in regulating inflammatory and hemostatic functions.

Pulmonary arterial hypertension (PAH) is a progressive disease that involves pathological remodeling, vasoconstriction, and thrombosis. Alterations in hemostasis, coagulation, and platelet activation are consistently observed in PAH patients. Microparticles derived from platelets, inflammatory cells, and the endothelium are an increasingly well-recognized signal in a variety of cardiovascular diseases, including PAH. This review will focus on the role of coagulation, thrombosis, platelet activation, and microparticles in the pathology and progression of PAH.

SUMMARY Background Antiplatelet therapy is a cornerstone of modern medical practice and is routinely employed to reduce the likelihood of myocardial infarction, thrombosis, and stroke. However, current antiplatelet therapies, such as aspirin, often have adverse side effects, including increased risk of bleeding, and some patients are relatively “aspirin-resistant”. Platelets are intimately involved in hemostasis and inflammation, and clinical consequences are associated with excessive or insufficient platelet activation. Objectives A major unmet need in the field of hematology is the development of new agents that safely prevent unwanted platelet activation in patients with underlying cardiovascular disease, while minimizing the risk of bleeding. Here, we investigate the potential of endogenously produced, specialized pro-resolving mediators (SPMs) as novel antiplatelet agents. SPMs are a recently discovered class of lipid-derived molecules that drive the resolution of inflammation, without being overtly immunosuppressive. Methods Human platelets were treated with lipoxin A4, resolvin D1, resolvin D2, 17-HDHA, or maresin 1 for 15 minutes, then were subjected to platelet function tests, including spreading, aggregation, and inflammatory mediator release. Results We show for the first time that human platelets express the SPM receptors, GPR32 and ALX. Furthermore, our data demonstrate that maresin 1 differentially regulates platelet hemostatic function, by enhancing platelet aggregation and spreading, while suppressing release of proinflammatory and pro-thrombotic mediators. Conclusions These data support the concept that SPMs differentially regulate platelet function and may represent a novel class of antiplatelet agents. SPMs also may play an important role in the resolution of inflammation in cardiovascular diseases.

Summary Stored platelets undergo biochemical, structural and functional changes that lead to decreased efficacy and safety of platelet transfusions. Not only do platelets acquire markers of activation during storage, but they also fail to respond normally to agonists post-storage. We hypothesized that resveratrol, a cardioprotective antioxidant, could act as a novel platelet storage additive to safely prevent unwanted platelet activation during storage, while simultaneously preserving normal haemostatic function. Human platelets treated with resveratrol and stored for five days released less thromboxane B2 and prostaglandin E2 compared to control platelets. Resveratrol preserved the ability of platelets to aggregate, spread and respond to thrombin, suggesting an improved ability to activate post-storage. Utilizing an in vitro model of transfusion and thromboelastography, clot strength was improved with resveratrol treatment compared to conventionally stored platelets. The mechanism of resveratrol’s beneficial actions on stored platelets was partly mediated through decreased platelet apoptosis in storage, resulting in a longer half-life following transfusion. Lastly, an in vivo mouse model of transfusion demonstrated that stored platelets are prothrombotic and that resveratrol delayed vessel occlusion time to a level similar to transfusion with fresh platelets. We show resveratrol has a dual ability to reduce unwanted platelet activation during storage, while preserving critical haemostatic function.

Vaccination has been the most effective way to prevent or reduce infectious diseases, examples include the eradication of smallpox, and attenuation of tetanus and measles. However, there is a large segment of the population that responds poorly to vaccines, in part because they are immunocompromised due to disease, age, or pharmacologic therapy and are unable to generate long-term protection. Specialized pro-resolving mediators (SPMs) are endogenously produced lipids that have potent pro-resolving and anti-inflammatory activities. Lipoxin B4 (LXB4) is a member of the lipoxin family, with its pro-resolving effects shown in allergic airway inflammation. However, its effects on the adaptive immune system, especially on human B cells are not known. Here, we investigated the effects of LXB4 on human B cells using cells from healthy donors and donors vaccinated against influenza virus in vitro. LXB4 promoted IgG antibody production in memory B cells, and also increased the number of IgG-secreting B cells. LXB4 enhanced expression of two key transcription factors involved in plasma cell differentiation, BLIMP1 and XBP1. Interestingly, LXB4 increased expression of cyclooxygenase-2 (COX2), an enzyme that is required for efficient B cell antibody production. The effects of LXB4 are at least partially COX2-dependent as COX2 inhibitors attenuated LXB4-stimulated BLIMP1 and Xpb-1 expression as well as IgG production. Thus, our study reveals for the first time that LXB4 boosts memory B cell activation through COX2 and suggests that LXB4 can serve as a new vaccine adjuvant.

BACKGROUND: Stored red cells release hemoglobin that leads to oxidative damage, which may contribute to thrombosis in susceptible transfusion recipients. Oxidative stress stimulates the generation of a new class of lipid mediators called F2-isoprostanes (F2-IsoPs) and isofurans (IsoFs) that influence cellular behavior. The present study investigated red cell-derived F2-IsoPs and IsoFs during storage, and their influence on human platelets. STUDY DESIGN AND METHODS: F2-IsoP and IsoF levels in red cell supernatants were measured by mass spectrometry during storage and after washing. The effects of stored supernatants, cell-free hemoglobin or a key F2-IsoP, 8-iso-PGF2α, on platelet function were examined in vitro. RESULTS: F2-IsoPs, IsoFs and hemoglobin accumulated in stored red cell supernatants. Pre-storage leukoreduction reduced supernatant F2-IsoPs and IsoFs levels, which increased again over storage time. Stored red cell supernatants and 8-iso-PGF2α induced platelet activation marker CD62P (P-selectin) expression and prothrombotic thromboxane A2 release. Cell-free hemoglobin did not alter platelet mediator release, but did inhibit platelet spreading. Post-storage red cell washing reduced F2-IsoP and IsoF levels up to twenty-four hours. CONCLUSIONS: F2-IsoPs and IsoFs are produced by stored red cells and induce adverse effects on platelet function in vitro, supporting a potential novel role for bioactive lipids in adverse transfusion outcomes. F2-IsoP and IsoF levels could be useful biomarkers for determining the suitability of blood components for transfusion. A novel finding is that cell-free hemoglobin inhibits platelet spreading and could adversely influence wound healing. Post-storage red cell washing minimizes harmful lipid mediators, and its use could potentially reduce transfusion complications.

There are 37 million people globally infected with the Human Immunodeficiency Virus (HIV). People living with HIV can achieve nearly normal lifespans due to the use of antiretroviral drugs (ARVs). However, people living with HIV experience chronic inflammation and increased risk for cardiovascular diseases (CVD) relative to uninfected people. While the cause for this risk is unclear, some ARVs have been associated with CVD, and it is speculated that some ARVs potentiate inflammation in infected individuals. Platelets are a critical link between inflammation and the development and progression of CVD, but the effects of ARVs on platelets are largely understudied. In this study, we examined the effects of ARVs on human platelet function in vitro. Our data show that the ARV ritonavir, a protease inhibitor, severely altered human platelet lipid mediator production (prostaglandin E2 and thromboxane) in both resting and activated platelets. Further characterization revealed that ritonavir altered measures of platelet hemostatic and thrombotic function that included significantly decreased platelet spreading, increased platelet aggregation, and trended toward increased clot strength. These data provide proof-of-principle that ARVs can directly dysregulate human platelets, possibly contributing to inflammation-related comorbidities. These data may provide mechanistic insight into the factors contributing to increased risk of CVD in people living with HIV, and may help guide future development of new HIV agents and ARV regimens that mitigate platelet dysregulation by ARVs.