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• 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...

Platelets are crucial regulators of tumor vascular homeostasis and continuously prevent tumor hemorrhage through secretion of their granules. However, the reason for tumor bleeding in the absence of platelets remains unknown. Tumors are associated with inflammation, a cause of hemorrhage in thrombocytopenia. Here, we investigated the role of the inflamed tumor microenvironment in the induction of tumor vessel injury in thrombocytopenic mice. Using s.c. injections of vascular endothelial growth factor or tumor necrosis factor-alpha combined with depletion of neutrophils, we demonstrate that enhancing the opening of endothelial cell junctions was not sufficient to cause bleeding in the absence of platelets; instead, induction of tissue hemorrhage in thrombocytopenia required recruitment of leukocytes. Immunohistology revealed that thrombocytopenia-induced tumor hemorrhage occurs at sites of macrophage and neutrophil accumulation. Mice deficient in beta2 or beta3 integrins, which have decreased neutrophil and/or macrophage infiltration in their tumor stroma, were protected from thrombocytopenia-induced tumor hemorrhage, indicating that, in the absence of platelets, stroma-infiltrating leukocytes induced tumor vessel injury. This injury was independent of reactive oxygen species generation and of complement activation, as suggested by the persistence of tumor hemorrhage in C3- and nicotinamide adenine dinucleotide phosphate oxidase-deficient thrombocytopenic mice. Our results show that platelets counteract tumor-associated inflammation and that the absence of this platelet function elicits vascular injuries by tumor-infiltrating innate immune cells.

There is endothelial activation and damage in hematopoietic stem cell transplantation (HSCT). The impact of the conditioning and type of HSCT on endothelial dysfunction in the early phases of HSCT has been evaluated. Plasma samples were obtained before and at different times after autologous and allogeneic HSCT with and without early complications. Changes in soluble markers of endothelial damage (VWF, ADAMTS-13, sVCAM-1, sICAM-1, and sTNFRI) were measured. There were changes in all markers evaluated that followed different patterns in auto and allo settings. For VWF and sTNRI, progressive increases from day Pre to day 14 and to day 21 were observed in the auto and the allo group, respectively. ADAMTS-13 activity correlated inversely with VWF levels. Levels of sVCAM-1 decreased until day 7, and raised significantly to day 14 and to day 21 in the auto and the allo HSCT, respectively. No significant changes were detected for sICAM-1. Our results confirm that there is endothelial damage at the early phases of HSCT, apparently induced by the consecutive effects of the conditioning, the proinflammatory agents used during transplantation, the translocation of endotoxins across the damaged gastrointestinal tract, and the engraftment. However, the comparative analysis between patients with and without complications suggests that none of these markers has diagnostic or prognostic value.

This study evaluated the relative impact of the intensity of the conditioning regimen and the alloreactivity in the endothelial dysfunction occurring after allogeneic hematopoietic stem cell transplantation (allo-HSCT). It involved a comparative analysis of the effect of incubating human umbilical vein endothelial cells (ECs) with serum samples from patients receiving autologous HSCT (auto-HSCT) or unrelated donor allo-HSCT. In both groups, blood samples were collected through a central line before conditioning (Pre), before transplantation (day 0), and at days 7, 14, and 21 after transplantation. Changes in the expression of EC receptors and adhesion proteins, adhesion of leukocytes and platelets under flow, and signaling pathways were analyzed. Endothelial activation and damage were observed in both groups, but with differing patterns. All markers of endothelial dysfunction demonstrated a progressive increase from day Pre to day 14 in the auto-HSCT group and exhibited 2 peaks of maximal expression (at days 0 and 21) in the allo-HSCT group. Both treatments induced a proinflammatory state (ie, expression of adhesion receptors, leukocyte adhesion, and p38 MAPK activation) and cell proliferation (ie, morphology and activation of ErK42/44). Prothrombotic changes (ie, von Willebrand factor expression and platelet adhesion) predominated after allo-HSCT, and a proapoptotic tendency (ie, activation of SAPK/JNK) was seen only in this group. These findings indicate that endothelial activation and damage after HSCT also occur in the autologous setting and affect macrovascular ECs. After the initial damage induced by the conditioning regimen, other factors, such as granulocyte colony-stimulating factor (G-CSF) toxicity, engraftment, and alloreactivity, may contribute to the endothelial damage seen during HSCT. Further studies are needed to explore the association between this endothelial damage and the vascular complications associated with HSCT.

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CalDAG-GEFI regulates neutrophil chemotaxis independent of integrin function by a mechanism that involves F-actin distribution and cell polarization. Chemotaxis and integrin activation are essential processes for neutrophil transmigration in response to injury. CalDAG-GEFI plays a key role in the activation of β1, β2, and β3 integrins in platelets and neutrophils by exchanging a GDP for a GTP on Rap1. Here, we explored the role of CalDAG-GEFI and Rap1b in integrin-independent neutrophil chemotaxis. In a transwell assay, CalDAG-GEFI−/− neutrophils had a 46% reduction in transmigration compared with WT in response to a low concentration of LTB4. Visualization of migrating neutrophils in the presence of 10 mM EDTA revealed that CalDAG-GEFI−/− neutrophils had abnormal chemotactic behavior compared with WT neutrophils, including reduced speed and directionality. Interestingly, Rap1b−/− neutrophils had a similar phenotype in this assay, suggesting that CalDAG-GEFI may be acting through Rap1b. We investigated whether the deficit in integrin-independent chemotaxis in CalDAG-GEFI−/− neutrophils could be explained by defective cytoskeleton rearrangement. Indeed, we found that CalDAG-GEFI−/− neutrophils had reduced formation of F-actin pseudopodia after LTB4 stimulation, suggesting that they have a defect in polarization. Overall, our studies show that CalDAG-GEFI helps regulate neutrophil chemotaxis, independent of its established role in integrin activation, through a mechanism that involves actin cytoskeleton and cellular polarization.