Characterization of platelet functionality in pediatric patients with kaposiform hemangioendothelioma / Kasabach-Merritt phenomenon

Background. Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy commonly associated with Kasabach-Merritt phenomenon (KMP) that includes thrombocytopenia and coagulation dysfunction. Platelet receptor CLEC-2 -tumor cell podoplanin interaction is considered the key mechanism of thrombocytopenia in KMP, however, the effect of long-term exposure to podoplanin on platelet function is unknown. Procedure. Here we examined blood samples from six patients with KHE and one KMP. Platelet calcium signaling and functional responses to conventional activation and CLEC-2 stimulation were analyzed by continuous and endpoint live cell flow cytometry. Platelet aggregation in response to ADP or rhodocytin was analyzed by low-angle light scattering approach (LaSca). Additionally, ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Results. We demonstrate that in KHE/KMP platelet functional responses to strong stimulation were on the lower boundary of age-matched normal ranges, while calcium mobilization and fibrinogen binding upon stimulation with ADP alone were significantly lower than control values. Platelet di-aggregate formation in response to ADP was also diminished in most of the patients. Formation of platelet aggregates in collagen-coated parallel plate flow chambers was also noticeably lower than in the age-matched control group. Calcium mobilization in response to CLEC-2 stimulation was unaltered in the patients and could be blocked by low-molecular-weight inhibitors, 2CP and HB125. Conclusions. While platelet responsiveness in KHE/KMP is moderately altered, their CLEC-2 receptors remain functional and respond to inhibition. Therefore, our findings suggest that CLEC-2-targeting molecules are new potential agents in therapeutic management of this life-threatening condition.

Characterization of platelet functionality in pediatric patients with kaposiform hemangioendothelioma / Kasabach-Merritt phenomenon

Background. Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy commonly associated with Kasabach-Merritt phenomenon (KMP) that includes thrombocytopenia and coagulation dysfunction. Platelet receptor CLEC-2 -tumor cell podoplanin interaction is considered the key mechanism of thrombocytopenia in KMP, however, the effect of long-term exposure to podoplanin on platelet function is unknown. Procedure. Here we examined blood samples from 7 patients with KHE/KMP. Platelet calcium signaling and functional responses to conventional activation and CLEC-2 stimulation were analyzed by continuous and endpoint live cell flow cytometry. Platelet aggregation in response to ADP or rhodocytin was analyzed by low-angle light scattering approach (LaSca). Additionally, ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Results. We demonstrate that in KHE/KMP platelet functional responses to strong stimulation were on the lower boundary of age-matched normal ranges, while calcium mobilization and fibrinogen binding upon stimulation with ADP alone were significantly lower than control values. Platelet di-aggregate formation in response to ADP was also diminished in most of the patients. Formation of platelet aggregates in collagen-coated parallel plate flow chambers was also noticeably lower than in the age-matched control group. Calcium mobilization in response to CLEC-2 stimulation was unaltered in the patients and could be blocked by low-molecular-weight inhibitors, 2CP and HB125. Conclusions. While platelet responsiveness in KHE/KMP is moderately altered, platelet CLEC-2 receptors remain functional and respond to inhibition. Therefore, our findings suggest that CLEC-2-targeting molecules are new potential agents in therapeutic management of this life-threatening condition.

Dimerized Translationally Controlled Tumor Protein-Binding Peptide 2 Attenuates Systemic Anaphylactic Reactions Through Direct Suppression of Mast Cell Degranulation

Dimerized translationally controlled tumor protein (dTCTP) amplifies allergic responses through activation of several types of immune cells and release of inflammatory mediators. In particular, dTCTP plays an important role in histamine release by triggering mast cells and has been proposed as a target in the treatment of allergic diseases. dTCTP-binding peptide 2 (dTBP2) is known to attenuate severe allergic rhinitis and asthma through inhibition of dTCTP activity on airway epithelial cells and T cells; however, it is unclear whether dTBP2 affects mast cell function and mast cell disease. In this study, we explored the effects of dTBP2 on mast cell degranulation and allergen-induced anaphylactic reactions. We found that bacterial product lipopolysaccharide increased the expression of dTCTP in mast cells and rapidly released dTCTP by the mast cell stimulator compound 48/80. Interestingly, the released dTCTP further promoted mast cell degranulation in an autocrine activation manner and increased calcium mobilization in mast cells, which is essential for degranulation. Furthermore, dTBP2 directly and dose-dependently inhibited in vitro mast cell degranulation enhanced by compound 48/80, suggesting a direct and potent anti-anaphylactic activity of dTBP2. dTBP2 also significantly suppressed the dTCTP-induced degranulation and histamine release through inhibition of the p38 MAPK signaling pathway and suppression of lysosomal expansion and calcium mobilization in mast cells. More importantly, in vivo administration of dTBP2 decreased mortality and significantly attenuated histamine release and inflammatory cytokine production in compound 48/80-induced systemic anaphylactic reactions. These results suggest that dTBP2 is beneficial for the control of anaphylaxis with increased dTCTP.

Synthesis and Anti-HIV Activity of a Novel Series of Isoquinoline-Based CXCR4 Antagonists

An expansion of the structure–activity relationship study of CXCR4 antagonists led to the synthesis of a series of isoquinolines, bearing a tetrahydroquinoline or a 3-methylpyridinyl moiety as head group. All compounds were investigated for CXCR4 affinity and antagonism in competition binding and calcium mobilization assays, respectively. In addition, the anti-HIV activity of all analogues was determined. All compounds showed excellent activity, with compound 24c being the most promising one, since it displayed consistently low nanomolar activity in the various assays.

Synergistic inhibition of platelet function by NADPH oxidase inhibitors and clopidogrel

Previous studies have shown platelets play an important role in prothrombotic complications due to several factors such as hyperglycemia, oxidative stress, and hypercholesterolemia, which affected platelets reactivity. Platelets activation involves ADP stimulation via P2Y12 receptor, whereas reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide (H2O2) produced by NADPH oxidase (Nox) act as the second messenger, which involved in platelets activation and may contribute to thrombus formation. The aim of the present study was to investigate the influence of Nox on the purinergic receptor (P2Y12 receptor) in activation of human platelets function stimulated by platelets agonist. This research explored the effects of Nox inhibitors and clopidogrel either alone or in combination, on various agonist-stimulated human platelets, including platelets aggregation and adhesion measured by modified LTA, expression of platelets activation markers, and calcium mobilization using flow cytometry, and ROS formation, NADPH oxidase activity, as well as cAMP levels by chemiluminescence assay. Taken together, findings from these experiments suggest that the combination of clopidogrel and Nox inhibitors synergistically reduced platelets aggregation, platelets adhesion, and expression of platelets activation marker during late activation, ROS formation, NADPH oxidase activity, calcium mobilization and increased cAMP levels in vitro. This combination showed that P2Y12receptor reactivity was influenced by the activation of NADPH oxidase. Thus, these data demonstrated a potential combination therapy to reduce the risk of thrombosis formation.