Sherry L. Spinelli

15-deoxy-Δ12,14-PGJ2 enhances platelet production from megakaryocytes

Thrombocytopenia is a critical problem that occurs in many hematologic diseases, as well as after cancer therapy and radiation exposure. Platelet transfusion is the most commonly used therapy but has limitations of alloimmunization, availability, and expense. Thus, the development of safe, small, molecules to enhance platelet production would be advantageous for the treatment of thrombocytopenia. Herein, we report that an important lipid mediator and a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand called 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), increases Meg-01 maturation and platelet production. 15d-PGJ(2) also promotes platelet formation from culture-derived mouse and human megakaryocytes and accelerates platelet recovery after in vivo radiation-induced bone marrow injury. Interestingly, the platelet-enhancing effects of 15d-PGJ(2) in Meg-01 cells are independent of PPARgamma, but dependent on reactive oxygen species (ROS) accumulation; treatment with antioxidants such as glutathione ethyl ester (GSH-EE); or N-acetylcysteine (NAC) attenuate 15d-PGJ(2)-induced platelet production. Collectively, these data support the concept that megakaryocyte redox status plays an important role in platelet generation and that small electrophilic molecules may have clinical efficacy for improving platelet numbers in thrombocytopenic patients.

The platelet as an immune cell--CD40 ligand and transfusion immunomodulation

The discovery that platelets possess cell membrane, cytoplasmic, and secreted forms of the co-stimulatory molecule CD40 ligand (CD40L, also known as CD154) has led to a revolution in the view of this anucleate, differentiated cell fragment, previously thought only to be involved in blood clotting (hemostasis). During the last decade, it has become clear that platelets function in innate and adaptive immunity and possess pro-inflammatory, as well as pro-thrombotic properties. They interact not only with other platelets and endothelial cells, but also with lymphocytes, dendritic cells, and structural cells such as fibroblasts. Soluble forms of CD40L (sCD40L) in the human circulation are almost entirely derived from platelets. Elevated levels of CD40L are associated with clinically important conditions, such as vascular disease, abnormal clotting (thrombosis), lung injury, and autoimmune disease. Each year millions of platelet transfusions are given to patients that contain large amounts of sCD40L. sCD40L in the supernatant of stored platelets can induce cytokines, chemokines, and lipid mediators by activating CD40 bearing cells. Increased levels of sCD40L in transfused blood are associated with transfusion-related acute lung injury, a potentially fatal complication, as well as more common, milder transfusion reactions such as fever and rigors. These effects come under the rubric of transfusion immunomodulation, which postulates that transfusion recipient biology, particularly immune function, is dramatically altered by transfusion of stored allogeneic blood.

Peroxisome proliferator-activated receptor γ and retinoid X receptor transcription factors are released from activated human platelets and shed in microparticles

Peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligands are important regulators of lipid metabolism, inflammation, and diabetes. We previously demonstrated that anucleate human platelets express the transcription factor PPARgamma and that PPARgamma ligands blunt platelet activation. To further understand the nature of PPARgamma in platelets, we determined the platelet PPARgamma isoform(s) and investigated the fate of PPARgamma following platelet activation. Our studies demonstrated that human platelets contain only the PPARgamma1 isoform and after activation with thrombin, TRAP, ADP or collagen PPARgamma is released from internal stores. PPARgamma release was blocked by a cytoskeleton inhibitor, Latrunculin A. Platelet-released PPARgamma was complexed with the retinoid X receptor (RXR) and retained its ability to bind DNA. Interestingly, the released PPARgamma and RXR were microparticle associated and the released PPARgamma/RXR complex retained DNA-binding ability. Additionally, a monocytic cell line, THP-1, is capable of internalizing PMPs. Further investigation following treatment of these cells with the PPARgamma agonist, rosiglitazone and PMPs revealed a possible transcellular mechanism to attenuate THP-1 activation. These new findings are the first to demonstrate transcription factor release from platelets, revealing the complex spectrum of proteins expressed and expelled from platelets, and suggests that platelet PPARgamma has an undiscovered role in human biology.

Platelets and Megakaryocytes Contain Functional Nuclear Factor-κB

Objective—To investigate the presence and role of NF-&kgr;B proteins in megakaryocytes and platelets. The nuclear factor-&kgr;B (NF-&kgr;B) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-&kgr;B family members, including the regulatory inhibitor-&kgr;B (I-&kgr;B) and I-&kgr; kinase (IKK) molecules. Methods and Results—Anucleate platelets exposed to NF-&kgr;B inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-&kgr;B inhibition diminished lamellapodia formation, decreased clot retraction times, and reduced thrombus stability. Moreover, inhibition of I-&kgr;B-&agr; phosphorylation (BAY-11-7082) reverted fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-&bgr; or I-&kgr;B-&agr; protein to BAY inhibitor–treated platelets partially restored platelet spreading in I-&kgr;B-&agr; inhibited platelets, and addition of active IKK-&bgr; increased endogenous I-&kgr;B-&agr; phosphorylation levels. Conclusion—These novel findings support a crucial and nonclassical role for the NF-&kgr;B family in modulating platelet function and reveal that platelets are sensitive to NF-&kgr;B inhibitors. As NF-&kgr;B inhibitors are being developed as antiinflammatory and anticancer agents, they may have unintended effects on platelets. On the basis of these data, NF-&kgr;B is also identified as a new target to dampen unwanted platelet activation.

Release of biologically active CD154 during collection and storage of platelet concentrates prepared for transfusion

Summary.  Background:  Millions of platelet transfusions are given each year. Transfusion reactions occur in as many as 30% of patients receiving unmodified platelet transfusions. The cause of some transfusion reactions remains unclear. The current paradigm suggests that platelet concentrates (PC) contain proinflammatory mediators that are released by white blood cells during collection, processing and storage. CD154 (CD40 ligand, CD40L) is a potent inflammatory mediator, normally sequestered inside the resting platelet, that is known to translocate to the platelet membrane and be shed into plasma in response to agonist activation. We hypothesized that platelet‐soluble CD154 (sCD154) is ‘spontaneously’ released by transfused platelets and plays a major role in transfusion reactions.

Washing red blood cells and platelets transfused in cardiac surgery reduces postoperative inflammation and number of transfusions: results of a prospective, randomized, controlled clinical trial.

Objectives: Children undergoing cardiac surgery with cardiopulmonary bypass are susceptible to additional inflammatory and immunogenic insults from blood transfusions. We hypothesize that washing red blood cells and platelets transfused to these patients will reduce postoperative transfusion-related immune modulation and inflammation. Design: Prospective, randomized, controlled clinical trial. Setting: University hospital pediatric cardiac intensive care unit. Patients: Children from birth to 17 yrs undergoing cardiac surgery with cardiopulmonary bypass. Interventions: Children were randomized to an unwashed or washed red blood cells and platelet transfusion protocol for their surgery and postoperative care. All blood was leuko-reduced, irradiated, and ABO identical. Plasma was obtained for laboratory analysis preoperatively, immediately, and 6 and 12 hrs after cardiopulmonary bypass. Primary outcome was the 12-hr postcardiopulmonary bypass interleukin-6-to-interleukin-10 ratio. Secondary measures were interleukin levels, C-reactive protein, and clinical outcomes. Measurements and Main Results: One hundred sixty-two subjects were studied, 81 per group. Thirty-four subjects (17 per group) did not receive any blood transfusions. Storage duration of blood products was similar between groups. Among transfused subjects, the 12-hr interleukin ratio was significantly lower in the washed group (3.8 vs. 4.8; p = .04) secondary to lower interleukin-6 levels (after cardiopulmonary bypass: 65 vs.100 pg/mL, p = .06; 6 hrs: 89 vs.152 pg/mL, p = .02; 12 hrs: 84 vs.122 pg/mL, p = .09). Postoperative C-reactive protein was lower in subjects receiving washed blood (38 vs. 43 mg/L; p = .03). There was a numerical, but not statistically significant, decrease in total blood product transfusions (203 vs. 260) and mortality (2 vs. 6 deaths) in the washed group compared to the unwashed group. Conclusions: Washed blood transfusions in cardiac surgery reduced inflammatory biomarkers, number of transfusions, donor exposures, and were associated with a nonsignificant trend toward reduced mortality. A larger study powered to test for clinical outcomes is needed to determine whether these laboratory findings are clinically significant.

Transfusion immunomodulation - the case for leukoreduced and (perhaps) washed transfusions

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.

New frontiers in transfusion biology: identification and significance of mediators of morbidity and mortality in stored red blood cells

Red blood cell (RBC) transfusions are associated with inflammation and thrombosis, both arterial and venous, the mechanisms of which are not understood. Although a necessary life‐saving procedure in modern medicine, transfusions have rarely been subjected to modern assessments of efficacy and safety, including randomized trials. Storage of RBCs induces changes, including the release of free hemoglobin and the accumulation of biologically active soluble mediators and microparticles. These mediators likely play a direct role in the inflammatory and prothrombotic properties of RBC transfusions. Methods such as leukoreduction, washing of RBCs, and rejuvenation may improve the quality of RBC transfusions.

Platelets as a Novel Target for PPARγ Ligands

Peroxisome proliferator-activated receptor γ (PPARγ) is an important transcription factor for lipid and glucose metabolism. Currently, the PPARγ ligands rosiglitazone and pioglitazone are used for the treatment of type 2 diabetes mellitus because they are potent insulin sensitizers. Recently, PPARγ has emerged as an important anti-inflammatory factor. Platelets, anucleate cells involved in hemostasis, have also been implicated as key contributors to inflammation, because they produce many pro-inflammatory and pro-atherogenic mediators when activated. Surprisingly, it was discovered recently that platelets contain PPARγ and that PPARγ ligands, both natural and synthetic, inhibit platelet activation and release of bioactive mediators. In particular, release of soluble CD40 ligand (sCD40L) and thromboxane (TXA2) was inhibited by PPARγ ligands in thrombin-activated platelets. CD40L signaling induces pro-inflammatory processes in many cell types, and increased blood levels of sCD40L are closely associated with inflammation, diabetes, and cardiovascular disease. Targeting platelet PPARγ will, therefore, be an important treatment strategy for the attenuation of chronic inflammatory processes and prevention of thrombus formation.

A highly specific phosphatase that acts on ADP-ribose 1″-phosphate, a metabolite of tRNA splicing in Saccharomyces cerevisiae

One molecule of ADP-ribose 1″,2″-cyclic phosphate (Appr>p) is formed during each of the approximately 500 000 tRNA splicing events per Saccharomyces cerevisiae generation. The metabolism of Appr>p remains poorly defined. A cyclic phosphodiesterase (Cpd1p) has been shown to convert Appr>p to ADP-ribose-1″-phosphate (Appr1p). We used a biochemical genomics approach to identify two yeast phosphatases that can convert Appr1p to ADP-ribose: the product of ORF YBR022w (now Poa1p), which is completely unrelated to other known phosphatases; and Hal2p, a known 3′-phosphatase of 5′,3′-pAp. Poa1p is highly specific for Appr1p, and thus likely acts on this molecule in vivo. Poa1 has a relatively low KM for Appr1p (2.8 μM) and a modest kcat (1.7 min−1), but no detectable activity on several other substrates. Furthermore, Poa1p is strongly inhibited by ADP-ribose (KI, 17 μM), modestly inhibited by other nucleotides containing an ADP-ribose moiety and not inhibited at all by other tested molecules. In contrast, Hal2p is much more active on pAp than on Appr1p, and several other tested molecules were Hal2p substrates or inhibitors. poa1-Δ mutants have no obvious growth defect at different temperatures in rich media, and analysis of yeast extracts suggests that ∼90% of Appr1p processing activity originates from Poa1p.