Elisabetta Liverani

LPS-induced systemic inflammation is more severe in P2Y12 null mice

Thienopyridines are a class of antiplatelet drugs that are metabolized in the liver to several metabolites, of which only one active metabolite can irreversibly antagonize the platelet P2Y12 receptor. Possible effects of these drugs and the role of activated platelets in inflammatory responses have also been investigated in a variety of animal models, demonstrating that thienopyridines could alter inflammation. However, it is not clear whether it is caused only by the P2Y12 antagonism or whether off‐target effects of other metabolites also intervene. To address this question, we investigated P2Y12 KO mice during a LPS‐induced model of systemic inflammation, and we treated these KO mice with a thienopyridine drug (clopidogrel). Contrary to the reported effects of clopidogrel, numbers of circulating WBCs and plasma levels of cytokines were increased in LPS‐exposed KO mice compared with WT in this inflammation model. Moreover, both spleen and bone marrow show an increase in cell content, suggesting a role for P2Y12 in regulation of bone marrow and spleen cellular composition. Finally, the injury was more severe in the lungs of KO mice compared with WT. Interestingly, clopidogrel treatments also exerted protective effects in KO mice, suggesting off‐target effects for this drug. In conclusion, the P2Y12 receptor plays an important role during LPS‐induced inflammation, and this signaling pathway may be involved in regulating cell content in spleen and bone marrow during LPS systemic inflammation. Furthermore, clopidogrel may have effects that are independent of P2Y12 receptor blockade.

Prasugrel Metabolites Inhibit Neutrophil Functions

Clopidogrel and prasugrel belong to a thienopyridine class of oral antiplatelet drugs that, after having been metabolized in the liver, can inhibit platelet function by irreversibly antagonizing the P2Y12 receptor. Furthermore, thienopyridines influence numerous inflammatory conditions, but their effects on neutrophils have not been evaluated, despite the important role of these cells in inflammation. Therefore, we investigated the effect of prasugrel metabolites on neutrophils to further clarify the role of thienopyridines in inflammation. Interestingly, a prasugrel metabolite mixture, produced in vitro using rat liver microsomes, significantly inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)- and platelet-activating factor (PAF)-induced neutrophil activation. More specifically, prasugrel metabolites inhibited neutrophil transmigration, CD16 surface expression, and neutrophil-platelet aggregation. Moreover, prasugrel metabolite pretreatment also significantly decreased fMLP- or PAF-induced extracellular-signal–regulated kinase phosphorylation as well as calcium mobilization. To determine the target of prasugrel in neutrophils, the role of both P2Y12 and P2Y13 receptors was studied using specific reversible antagonists, AR-C69931MX and MRS2211, respectively. Neither antagonist had any direct effect on the agonist-induced neutrophil functional responses. Our findings indicate that prasugrel metabolites may directly target neutrophils and inhibit their activation, suggesting a possible explanation for their anti-inflammatory effects previously observed. However, these metabolites do not act through either the P2Y12 or P2Y13 receptor in neutrophils.

P2Y12 Receptor Modulates Sepsis-Induced Inflammation

Objective—Platelets modulate hemostasis and immune responses via interactions with immune cells through secretion of immunemodulators and cell–cell interactions. The P2Y12 receptor mediates ADP-induced aggregation and secretion in platelets. Approach and Results—Using a mouse model of intra-abdominal sepsis and acute lung injury, we investigated the role of the P2Y12 receptor in neutrophil migration and lung inflammation in P2Y12 null mice and in mice pretreated with the P2Y12 antagonist clopidogrel. Our data show a decrease in circulating white blood cells and a decrease in platelet activation and platelet–leukocyte interactions in treated mice compared with untreated mice. Additionally, lung injury and platelet sequestration were diminished in clopidogrel-treated mice compared with their untreated septic littermates. Similar results were observed in P2Y12 null mice: platelet activation and platelet–leukocyte aggregates were decreased in septic P2Y12 null mice compared with wild-type mice. P2Y12 null mice were refractory to lung injury compared with wild-type mice. Finally, to evaluate P2Y12-independent effects of clopidogrel, we pretreated P2Y12 null mice. Interestingly, the number of circulating neutrophils was reduced in treated septic P2Y12 null mice, suggesting neutrophils as a target for clopidogrel pleiotropic effects. No difference was observed in P2Y1 null mice during sepsis, indicating that the P2Y12 receptor is responsible for the effects. Conclusions—P2Y12 null mice are refractory to sepsis-induced lung injury, suggesting a key role for activated platelets and the P2Y12 receptor during sepsis.

The Role of P2Y 12 Receptor and Activated Platelets During Inflammation

Platelets play an important role not only during thrombosis, but also in modulating immune responses through their interaction with immune cells and by releasing inflammatory mediators upon activation. The P2Y12 receptor is a Gi-coupled receptor that not only regulates ADP-induced aggregation but can also dramatically potentiate secretion, when platelets are activated by other stimuli. Considering the importance of P2Y12 receptor in platelet function, a class of antiplatelet drugs, thienopyridines, have been designed and successfully used to prevent thrombosis. This review will focus on the role of activated platelets in inflammation and the effects that P2Y12 antagonism exerts on the inflammatory process. A change in platelet functions was noted in patients treated with thienopyridines during inflammatory conditions, suggesting that platelets may modulate the inflammatory response. Further experiments in a variety of animal models of diseases, such as sepsis, rheumatoid arthritis, myocardial infarction, pancreatitis and pulmonary inflammation have also demonstrated that activated platelets influence the inflammatory state. Platelets can secrete inflammatory modulators in a P2Y12-dependent manner, and, as a result, directly alter the inflammatory response. P2Y12 receptor may also be expressed in other cells of the immune system, indicating that thienopyridines could directly influence the immune system rather than only through platelets. Overall the results obtained to date strongly support the notion that activated platelets significantly contribute to the inflammatory process and that antagonizing P2Y12 receptor can influence the immune response.

Members of the novel UBASH3/STS/TULA family of cellular regulators suppress T-cell-driven inflammatory responses in vivo.

The UBASH3/STS/TULA family consists of two members sharing substantial homology and a similar multi‐domain architecture, which includes a C‐terminal histidine phosphatase domain capable of dephosphorylating phosphotyrosine‐containing substrates. TULA‐family proteins act as downregulators of receptor‐induced activation in several cell types, including T cells and platelets. Deletion of both family members in mice has been shown to result in hyperresponsiveness of T cells to T‐cell receptor (TCR)/CD3 complex engagement, but little is known about the biological consequences of double knockout (dKO) and especially of either single KO (sKO). We elucidated the biological consequences of the lack of TULA‐family proteins in dKO and TULA and TULA‐2 sKO animals. In order to do so, we examined immune responses in Trinitrobenzene sulfonic acid (TNBS)‐induced colitis, a mouse model of human inflammatory bowel disease, which is characterized by the involvement of multiple cell types, of which T cells have a crucial role, in the development of a pathological inflammatory condition. Our data indicate that TNBS treatment upregulates T‐cell responses in all KO mice studied to a significantly higher degree than in wild‐type mice. Although the lack of either TULA‐family member exacerbates inflammation and T‐cell responses in a specific fashion, the lack of both TULA and TULA‐2 in dKO exerts a higher effect than the lack of a single family member in TULA and TULA‐2 sKO. Analysis of T‐cell responses and TCR‐mediated signaling argues that the proteins investigated affect T‐cell signaling by regulating phosphorylation of Zap‐70, a key protein tyrosine kinase.

Erosive arthritis and hepatic granuloma formation induced by peptidoglycan polysaccharide in rats is aggravated by prasugrel treatment.

Administration of the thienopyridine P2Y12 receptor antagonist, clopidogrel, increased the erosive arthritis induced by peptidoglycan polysaccharide (PG-PS) in rats or by injection of the arthritogenic K/BxN serum in mice. To determine if the detrimental effects are caused exclusively by clopidogrel, we evaluated prasugrel, a third-generation thienopyridine pro-drug, that contrary to clopidogrel is mostly metabolized into its active metabolite in the intestine. Prasugrel effects were examined on the PG-PS-induced arthritis rat model. Erosive arthritis was induced in Lewis rats followed by treatment with prasugrel for 21 days. Prasugrel treated arthritic animals showed a significant increase in the inflammatory response, compared with untreated arthritic rats, in terms of augmented macroscopic joint diameter associated with significant signs of inflammation, histomorphometric measurements of the hind joints and elevated platelet number. Moreover, fibrosis at the pannus, assessed by immunofluorescence of connective tissue growth factor, was increased in arthritic rats treated with prasugrel. In addition to the arthritic manifestations, hepatomegaly, liver granulomas and giant cell formation were observed after PG-PS induction and even more after prasugrel exposure. Cytokine plasma levels of IL-1 beta, IL-6, MIP1 alpha, MCP1, IL-17 and RANTES were increased in arthritis-induced animals. IL-10 plasma levels were significantly decreased in animals treated with prasugrel. Overall, prasugrel enhances inflammation in joints and liver of this animal model. Since prasugrel metabolites inhibit neutrophil function ex-vivo and the effects of both clopidogrel and prasugrel metabolites on platelets are identical, we conclude that the thienopyridines metabolites might exert non-platelet effects on other immune cells to aggravate inflammation.

Role of Protein Kinase C-delta in regulating platelet activation and platelet-leukocyte interaction during sepsis

Sepsis is characterized by an intense systemic inflammatory response activating a cascade of proinflammatory events resulting in leukocyte dysregulation and host tissue damage. The lung is particularly susceptible to systemic inflammation, leading to acute lung injury. Key to inflammation-induced lung damage is the excessive migration of neutrophils across the vascular endothelium. The mechanisms which regulate neutrophil activation and migration in sepsis are not well defined but there is growing evidence that platelets are actively involved and play a key role in microvascular permeability and neutrophil-mediated organ damage. We previously identified PKC-delta (PKCδ) as a critical regulator of the inflammatory response in sepsis and demonstrated PKCδ inhibition was lung protective. However, the role of PKCδ in sepsis-induced platelet activation and platelet-leukocyte interactions is not known. In this study, rats underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Following surgeries, a PKCδ inhibitor (200μg/kg) or vehicle (PBS) was administered intra-tracheally. At 24 hours post-surgeries, lung tissue, BAL fluid, and blood samples were collected. While sepsis caused thrombocytopenia, the remaining circulating platelets were activated as demonstrated by increased p-selectin expression, elevated plasma PF4, and enhanced platelet-leukocyte aggregate formation compared to Sham animals. Platelet activation was associated with increased platelet PKCδ activity. Inhibition of PKCδ attenuated sepsis-induced platelet activation, secretion and aggregate formation. Sepsis-induced thrombocytopenia was also significantly reduced and circulating platelet numbers were similar to sham animals. In the lung, sepsis induced significant influx of platelets and neutrophils and the development of lung injury. Administration of the PKCδ inhibitor decreased platelet and neutrophil influx, and was lung protective. Thus, PKCδ inhibition modulated platelet activity both locally and systemically, decreased neutrophil influx into the lung, and was lung protective. We demonstrate for the first time that PKCδ plays an important role in platelet activation and platelet-neutrophil interaction during sepsis.