The role of platelets in hemostasis is to produce a plug to arrest bleeding. During thrombocytopenia, spontaneous bleeding is seen in some patients but not in others; the reason for this is unknown. Here, we subjected thrombocytopenic mice to models of dermatitis, stroke, and lung inflammation. The mice showed massive hemorrhage that was limited to the area of inflammation and was not observed in uninflamed thrombocytopenic mice. Endotoxin-induced lung inflammation during thrombocytopenia triggered substantial intraalveolar hemorrhage leading to profound anemia and respiratory distress.By imaging the cutaneous Arthus reaction through a skin window, we observed in real time the loss of vascular integrity and the kinetics of skin hemorrhage in thrombocytopenic mice. Bleeding-observed mostly from venulesoccurred as early as 20 minutes after challenge, pointing to a continuous need for platelets to maintain vascular integrity in inflamed microcirculation. IntroductionInflammation and hemostasis are tightly intertwined. In particular, this is becoming very evident in platelet biology. While the classical role of platelets is to mediate hemostatic plug formation, it has been demonstrated that platelets also play an important role in inflammation. For example, previous studies show that platelets promote inflammatory responses in atherosclerosis, in hepatitis, and after cerebral ischemia. 1-4 Furthermore, early in inflammation prothrombotic functions of platelets are reduced, 5 and activated platelets are capable of up-regulating inflammatory molecules on the endothelium. 6,7 Recently, our group showed that in angiogenesis-which is strongly linked to inflammation 8 -platelets play an important role in preventing hemorrhage of sprouting vessels. 9 That platelets support vascular integrity during injury is well established. Early studies also demonstrated a supportive role for platelets during organ perfusion with buffers 10,11 and growthpromoting effects on endothelial cultures. 12 Whether platelets have a supportive role in inflamed microcirculation still remains experimentally unexplored.In humans, profound thrombocytopenia is found, for example, in patients suffering from idiopathic thrombocytopenic purpura. Interestingly, in the absence of injury, some patients bleed while others do not show spontaneous bleeding despite equally low platelet counts. 13 Thus, thrombocytopenia alone cannot explain this phenomenon and other, yet to be defined contributing factors are required to induce bleeding in thrombocytopenic patients, as suggested in a recent review. 14 As thrombocytopenia may lead to life-threatening bleedings, it is important to further understand the cofactors leading to hemorrhage.In the present study, we investigate the effects of inflammation on vascular integrity during thrombocytopenia. We challenged mice in 4 different inflammatory models and observed the affected blood vessels over time in the presence or absence of platelets. We show that thrombocytopenia rapidly induces massive bleeding in inflamed sk...

Key Points• NET formation is stimulated by platelet or soluble P-selectin.Neutrophil extracellular traps (NETs) can be released in the vasculature. In addition to trapping microbes, they promote inflammatory and thrombotic diseases. Considering that P-selectin induces prothrombotic and proinflammatory signaling, we studied the role of this selectin in NET formation. NET formation (NETosis) was induced by thrombin-activated platelets rosetting with neutrophils and was inhibited by anti-P-selectin aptamer or anti-P-selectin glycoprotein ligand-1 (PSGL-1) inhibitory antibody but was not induced by platelets from P-selectin 2/2 mice. Moreover, NETosis was also promoted by P-selectin-immunoglobulin fusion protein but not by control immunoglobulin. We isolated neutrophils from mice engineered to overproduce soluble P-selectin (P-selectin DCT/DCT mice). Although the levels of circulating DNA and nucleosomes (indicative of spontaneous NETosis)were normal in these mice, basal neutrophil histone citrullination and presence of P-selectin on circulating neutrophils were elevated. NET formation after stimulation with platelet activating factor, ionomycin, or phorbol 12-myristate 13-acetate was significantly enhanced, indicating that the P-selectin DCT/DCT neutrophils were primed for NETosis. In summary, P-selectin, cellular or soluble, through binding to PSGL-1, promotes NETosis, suggesting that this pathway is a potential therapeutic target for NET-related diseases. (Blood. 2015;126(2):242-246)

Key Points• During inflammation, serotonin released by platelets activates vessel wall promoting leukocyte adhesion and recruitment.• Absence of platelet serotonin improves survival after lipopolysaccharide-induced endotoxic shock.The majority of peripheral serotonin is stored in platelets, which secrete it on activation. Serotonin releases Weibel-Palade bodies (WPBs) and we asked whether absence of platelet serotonin affects neutrophil recruitment in inflammatory responses. Tryptophan hydroxylase (Tph)1-deficient mice, lacking non-neuronal serotonin, showed mild leukocytosis compared with wild-type (WT), primarily driven by an elevated neutrophil count. Despite this, 50% fewer leukocytes rolled on unstimulated mesenteric venous endothelium of Tph1 ؊/؊ mice. The velocity of rolling leukocytes was higher in Tph1 ؊/؊ mice, indicating fewer selectin-mediated interactions with endothelium. Stimulation of endothelium with histamine, a secretagogue of WPBs, or injection of serotonin normalized the rolling in Tph1 ؊/؊ mice. Diminished rolling in Tph1 ؊/؊ mice resulted in reduced firm adhesion of leukocytes after lipopolysaccharide treatment. Blocking platelet serotonin uptake with fluoxetine in WT mice reduced serum serotonin by > 80% and similarly reduced leukocyte rolling and adhesion. Four hours after inflammatory stimulation, neutrophil extravasation into lung, peritoneum, and skin wounds was reduced in Tph1 ؊/؊ mice, whereas in vitro neutrophil chemotaxis was independent of serotonin. Survival of lipopolysaccharide-induced endotoxic shock was improved in Tph1 ؊/؊ mice. In conclusion, platelet serotonin promotes the recruitment of neutrophils in acute inflammation, supporting an important role for platelet serotonin in innate immunity. (Blood. 2013;121(6):1008-1015) IntroductionPlatelets store serotonin in their dense granules at millimolar concentration and secrete it when they become activated. 1,2 This requires a complex mechanism of uptake, storage, and targeted release that is similar to that in neurons, with the exception that platelets are not stationary but circulate in high numbers throughout the vasculature. Platelets do not synthesize serotonin but incorporate and store serotonin that is synthesized in duodenal enterochromaffin cells and secreted into blood. Several different effects of non-neuronal serotonin have been unraveled in the past, including prohemostatic (on platelets and vascular smooth muscle cells), 3,4 mitogenic (on hepatocytes and pulmonary smooth muscle cells), 5,6 and immunomodulatory (on lymphocytes, monocytes, and smooth muscle cells) 7-9 functions. In vitro studies have shown that serotonin also activates the release of Weibel-Palade bodies (WPBs) from endothelial cells, which would promote leukocyte rolling via the WPB constituent P-selectin. 10,11 However, it is not clear whether serotonin influences neutrophil-endothelial interactions, a central step in early innate immune responses.We chose 2 approaches to study serotonin effects on leukocyte rolling and recruitment: genetic defi...

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related death. The biologic processes contributing to TRALI are poorly understood. All blood products can cause TRALI, and no specific treatment is available. A "2-event model" has been proposed as the trigger. The first event may include surgery, trauma, or infection; the second involves the transfusion of antileukocyte antibodies or bioactive lipids within the blood product. Together, these events induce neutrophil activation in the lungs, causing endothelial damage and capillary leakage. Neutrophils, in response to pathogens or under stress, can release their chromatin coated with granule contents, thus forming neutrophil extracellular traps (NETs). Although protective against infection, these NETs are injurious to tissue. Here we show that NET biomarkers are present in TRALI patients' blood and that NETs are produced in vitro by primed human neutrophils when challenged with anti-HNA-3a antibodies previously implicated in TRALI. NETs are found in alveoli of mice experiencing antibody-mediated TRALI. DNase 1 inhalation prevents their alveolar accumulation and improves arterial oxygen saturation even when administered 90 minutes after TRALI onset. We suggest that NETs form in the lungs during TRALI, contribute to the disease process, and thus could be targeted to prevent or treat TRALI. IntroductionTransfusion-related acute lung injury (TRALI) is a rare but serious complication of blood transfusion that occurs within 6 hours of transfusion and is characterized by hypoxemia, respiratory distress, and pulmonary infiltrates. 1 Over the years, prevention measures have resulted in a significant reduction in cases. However, TRALI is still the leading cause of transfusion-related mortality, and its prevalence is likely underestimated; one study suggested that more than 2% of cardiac surgery patients are affected. 2 Only supportive treatment is available to the patient, including mechanical ventilation and oxygen supplementation. Many of the severe cases have been linked to the presence of antineutrophil antibodies in the transfused product. 3,4 These antibodies bind to the recipients' neutrophils, activate them, and induce sequestration in the pulmonary capillaries, resulting in tissue injury. 5 Activated neutrophils can release neutrophil extracellular traps (NETs) 6 that are composed of DNA fibers decorated with histones and antimicrobial proteins 7 originally contained in the neutrophil granules. The structure and the composition of NETs allow them to trap and prevent the spread of pathogens and also to kill Gram-negative and Gram-positive bacteria, as well as yeast. 6 NET formation follows a specific pattern characterized by histone hypercitrullination, 8 chromatin decondensation, dissolution of the granular and nuclear membranes, and cytolysis. 9 Despite NETs' beneficial antimicrobial function, 6,10 their formation at the wrong time, in the wrong place, or in the wrong amount can have a negative effect on the host. NETs and their c...