Carla Carbo

Inflammation induces hemorrhage in thrombocytopenia

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 intra-alveolar 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 venules-occurred as early as 20 minutes after challenge, pointing to a continuous need for platelets to maintain vascular integrity in inflamed microcirculation. Inflammatory hemorrhage was not seen in genetically engineered mice lacking major platelet adhesion receptors or their activators (alphaIIbbeta3, glycoprotein Ibalpha [GPIbalpha], GPVI, and calcium and diacylglycerol-regulated guanine nucleotide exchange factor I [CalDAG-GEFI]), thus indicating that firm platelet adhesion was not necessary for their supporting role. While platelets were previously shown to promote endothelial activation and recruitment of inflammatory cells, they also appear indispensable to maintain vascular integrity in inflamed tissue. Based on our observations, we propose that inflammation may cause life-threatening hemorrhage during thrombocytopenia.

Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice.

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.

Innate Immune Cells Induce Hemorrhage in Tumors during Thrombocytopenia

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.

Endothelial Dysfunction after Hematopoietic Stem Cell Transplantation: Role of the Conditioning Regimen and the Type of Transplantation

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.

Integrin-independent role of CalDAG-GEFI in neutrophil chemotaxis.

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.

Petechial bleeding after sunburn in a patient with mild thrombocytopenia

There is growing evidence pointing to key role of platelets in maintaining vascular integrity during inflammation [1]. Using different mouse models of inflammation, we recently showed that inflammation induces hemorrhage in thrombocytopenic animals [2]. Mice with low platelet counts (<2.5% of normal) challenged with different inflammatory stimuli developed bleeding only in areas of inflammation, but not in areas without inflammation, and the bleeding could be rescued by transfusion of only 10% of platelets. Mice with a normal platelet count did not exhibit any bleeding, despite similar degrees of inflammation. Here, we report a case of petechial bleeding due to acute inflammation secondary to sunburn in a patient with mild thrombocytopenia. A 31-year-old man with mild chronic immune thrombocytopenic purpura (ITP) developed localized petechial bleeding in areas of inflamed skin after a first-degree sunburn. The patient was first diagnosed with ITP at the age of 21 with a platelet count of 82,000 platelets/lL. Since then, his platelet count has oscillated between 54,000 and 110,000 platelets/lL. He has never had petechiae, epistaxis, gingivorrhagia, or other spontaneous bleeding and has never received treatment for ITP. Three weeks prior to the index sunburn, the patient’s platelet count was 68,000 platelets/lL. The patient exposed himself to direct sunlight on a Caribbean beach. He applied sunscreen unevenly to his lower legs, leaving the medial and anterior aspects of his shins unprotected. He sunbathed, and 7 hr later developed a first-degree sunburn that was limited to the areas to which he had not adequately applied sunscreen. Interestingly, a clear petechial rash developed specifically in the sunburned skin, but not in adjacent skin that had not been sunburned. There had been no anteceding trauma, and the patient was not taking any medications. The image on the left shows petechiae and sunburned skin on the anterior and medial aspect of the patient’s lower leg, while the posterior aspect is free of both sunburn and petechiae(Image 1). The image on the right shows a more detailed image of the petechiae. Sun exposure has been reported to induce petechiae in patients taking photosensitizing drugs, such as ciprofloxacin, even in the presence of a normal platelet count [3]. It is intriguing that the patient we describe developed petechial bleeding despite having only mild thrombocytopenia. We wonder whether the propensity for bleeding in deep organs, such as the liver or heart, is similarly increased in patients with mild thrombocytopenia in diseases characterized by an inflammatory response, such as hepatitis or myocarditis. This case recapitulates in humans our previous observations in mouse models of inflammation and highlights the importance of a normal platelet count to prevent bleeding in the setting of inflammation.