Platelet FcγRIIA-induced serotonin release exacerbates the severity of Transfusion-Related Acute Lung Injury in mice

Transfusion-related acute lung injury (TRALI) remains a major cause of transfusion-related fatalities. The mechanism of human antibody-mediated TRALI, especially the involvement of the Fcγ receptors, is not clearly established. Contrary to mice, human platelets are unique in their expression of the FcγRIIA/CD32A receptor, suggesting that our understanding of the pathogenesis of antibody-mediated TRALI is partial, as the current murine models incompletely recapitulate the human immunology. We evaluated the role of FcγRIIA/CD32A in TRALI using a humanized mouse model expressing the FcγRIIA/CD32A receptor. When challenged with a recombinant chimeric human immunoglobulin G1/mouse anti–major histocompatibility complex class I monoclonal antibody, these mice exhibited exacerbated alveolar edema and higher mortality compared with wild-type (WT) mice. Unlike in WT mice, monocytes/macrophages in CD32A(+) mice were accessory for TRALI initiation, indicating the decisive contribution of another cell type. Platelet activation was dramatically increased in CD32A(+) animals, resulting in their increased consumption and massive release of their granule contents. Platelet depletion prevented the exacerbation of TRALI in CD32A(+) mice but did not affect TRALI in WT animals. By blocking platelet serotonin uptake with fluoxetine, we showed that the severity of TRALI in CD32A(+) mice resulted from the serotonin released by the activated platelets. Furthermore, inhibition of 5-hydroxytryptamine 2A serotonin receptor with sarpogrelate, before or after the induction of TRALI, abolished the aggravation of lung edema in CD32A(+) mice. Our findings show that platelet FcγRIIA/CD32A activation exacerbates antibody-mediated TRALI and provide a rationale for designing prophylactic and therapeutic strategies targeting the serotonin pathway to attenuate TRALI in patients

The ATP-gated P2X1 ion channel contributes to the severity of antibody-mediated Transfusion-Related Acute Lung Injury in mice

The biological responses that control the development of Transfusion-Related Acute Lung Injury (TRALI), a serious post-transfusion respiratory syndrome, still need to be clarified. Since extracellular nucleotides and their P2 receptors participate in inflammatory processes as well as in cellular responses to stress, we investigated the role of the ATP-gated P2X1 cation channel in antibody-mediated TRALI. The effects of NF449, a selective P2X1 receptor (P2RX1) antagonist, were analyzed in a mouse two-hit model of TRALI. Mice were primed with lipopolysaccharide (LPS) and 24 h later challenged by administrating an anti-MHC I antibody. The selective P2RX1 antagonist NF449 was administrated before the administration of LPS and/or the anti-MHC I antibody. When given before antibody administration, NF449 improved survival while maximal protection was achieved when NF449 was also administrated before the sensitization step. Under this later condition, protein contents in bronchoalveolar lavages were dramatically reduced. Cell depletion experiments indicated that monocytes/macrophages, but not neutrophils, contribute to this effect. In addition, the reduced lung periarteriolar interstitial edemas in NF449-treated mice suggested that P2RX1 from arteriolar smooth muscle cells could represent a target of NF449. Accordingly, inhibition of TRPC6, another cation channel expressed by smooth muscle cells, also reduced TRALI-associated pulmonary interstitial and alveolar edemas. These data strongly suggest that cation channels like P2RX1 or TRPC6 participate to TRALI pathological responses

Residual platelet ADP reactivity after clopidogrel treatment is dependent on activation of both the unblocked P2Y1 and the P2Y12 receptor and is correlated with protein expression of P2Y12

Two ADP receptors have been identified on human platelets: P2Y1 and P2Y12. The P2Y12 receptor blocker clopidogrel is widely used to reduce the risks in acute coronary syndromes, but, currently, there is no P2Y1 blocker in clinical use. Evidence for variable responses to clopidogrel has been described in several reports. The mechanistic explanation for this phenomenon is not fully understood. The aim of this study was to examine mechanisms responsible for variability of 2MeS-ADP, a stable ADP analogue, induced platelet reactivity in clopidogrel-treated patients. Platelet reactivity was assessed by flow cytometry measurements of P-selectin (CD62P) and activated GpIIb/IIIa complex (PAC-1). Residual 2MeS-ADP activation via the P2Y12 and P2Y1 receptors was determined by co-incubation with the selective antagonists AR-C69931 and MRS2179 in vitro. P2Y1 and P2Y12 receptor expression on both RNA and protein level were determined, as well as the P2Y12 H1 or H2 haplotypes. Our data suggest that the residual platelet activation of 2MeS-ADP after clopidogrel treatment is partly due to an inadequate antagonistic effect of clopidogrel on the P2Y12 receptor and partly due to activation of the P2Y1 receptor, which is unaffected by clopidogrel. Moreover, a correlation between increased P2Y12 protein expression on platelets and decreased response to clopidogrel was noticed, r2=0.43 (P<0.05). No correlation was found between P2Y12 mRNA levels and clopidogrel resistance, indicating post-transcriptional mechanisms. To achieve additional ADP inhibition in platelets, antagonists directed at the P2Y1 receptor could be more promising than the development of more potent P2Y12 receptor antagonists

Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

Retinoids, vitamin A derivatives, are important regulators of the growth and differentiation of skin cells. Although retinoids are therapeutically used for several skin ailments, little is known about their effects on P2 receptors, known to be involved in various functions in the skin. DNA array analysis showed that treatment of normal human epidermal keratinocytes (NHEKs) with all-trans-retinoic acid (ATRA), an agonist to RAR (retinoic acid receptor), enhanced the expression of mRNA for the P2Y2 receptor, a metabotropic P2 receptor that is known to be involved in the proliferation of the epidermis. The expression of other P2 receptors in NHEKs was not affected by ATRA. ATRA increased the mRNA for the P2Y2 receptor in a concentration-dependent fashion (1 nM to 1 μM). Am80, a synthesized agonist to RAR, showed a similar enhancement, whereas 9-cis-retinoic acid (9-cisRA), an agonist to RXR (retinoid X receptor), enhanced P2Y2 gene expression to a lesser extent. Ca2+ imaging analysis showed that ATRA also increased the function of P2Y2 receptors in NHEKs. Retinoids are known to enhance the turnover of the epidermis by increasing both proliferation and terminal differentiation. The DNA microarray analysis also revealed that ATRA upregulates various genes involved in the differentiation of NHEKs. Our present results suggest that retinoids, at least in part, exert their proliferative effects by upregulating P2Y2 receptors in NHEKs. This effect of retinoids may be closely related to their therapeutic effect against various ailments or aging events in skins such as over-keratinization, pigmentation and re-modeling

Long-term (trophic) purinergic signalling: purinoceptors control cell proliferation, differentiation and death

The purinergic signalling system, which uses purines and pyrimidines as chemical transmitters, and purinoceptors as effectors, is deeply rooted in evolution and development and is a pivotal factor in cell communication. The ATP and its derivatives function as a 'danger signal' in the most primitive forms of life. Purinoceptors are extraordinarily widely distributed in all cell types and tissues and they are involved in the regulation of an even more extraordinary number of biological processes. In addition to fast purinergic signalling in neurotransmission, neuromodulation and secretion, there is long-term (trophic) purinergic signalling involving cell proliferation, differentiation, motility and death in the development and regeneration of most systems of the body. In this article, we focus on the latter in the immune/defence system, in stratified epithelia in visceral organs and skin, embryological development, bone formation and resorption, as well as in cancer. Cell Death and Disease (2010) 1, e9; doi:10.1038/cddis.2009.11; published online 14 January 201

The P2X1 receptor and platelet function

Extracellular nucleotides are ubiquitous signalling molecules, acting via the P2 class of surface receptors. Platelets express three P2 receptor subtypes, ADP-dependent P2Y1 and P2Y12 G-protein-coupled receptors and the ATP-gated P2X1 non-selective cation channel. Platelet P2X1 receptors can generate significant increases in intracellular Ca2+, leading to shape change, movement of secretory granules and low levels of αIIbβ3 integrin activation. P2X1 can also synergise with several other receptors to amplify signalling and functional events in the platelet. In particular, activation of P2X1 receptors by ATP released from dense granules amplifies the aggregation responses to low levels of the major agonists, collagen and thrombin. In vivo studies using transgenic murine models show that P2X1 receptors amplify localised thrombosis following damage of small arteries and arterioles and also contribute to thromboembolism induced by intravenous co-injection of collagen and adrenaline. In vitro, under flow conditions, P2X1 receptors contribute more to aggregate formation on collagen-coated surfaces as the shear rate is increased, which may explain their greater contribution to localised thrombosis in arterioles compared to venules within in vivo models. Since shear increases substantially near sites of stenosis, anti-P2X1 therapy represents a potential means of reducing thrombotic events at atherosclerotic plaques

The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation

Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation

The calcium activated nucleotidases: A diverse family of soluble and membrane associated nucleotide hydrolyzing enzymes

It has long been known that the salivary glands of hematophagous (blood-feeding) arthropods secrete soluble apyrases, which are potent nucleotide hydrolyzing enzymes capable of hydrolyzing extracellular ATP and ADP, the latter being a major agonist contributing to platelet aggregation. Only recently, however, has the identification of proteins homologous to these apyrases been reported in non-blood-feeding organisms such as rodents and humans. In this review, we present an overview of the diverse family of apyrases first described in the blood-feeding arthropods, including the identification and characterization of the soluble and membrane-bound vertebrate enzymes homologous to these arthropod apyrases. We also describe the enzymatic properties and nucleotide specificities of the expressed enzymes, and insights gained into the structure and function of this calcium activated nucleotidase (CAN) family from biophysical, mutagenesis and crystallography studies. The potential therapeutic value of these proteins is also discussed