Host-Derived Molecules as Novel Chagas Disease Biomarkers: Hypercoagulability Markers in Plasma

The most severe clinical symptomatology of Chagas disease affects ~30% of those chronically infected with the Trypanosoma cruzi parasite. The pathogenic mechanisms that lead to life-threatening heart and gut tissue disruptions occur "silently" for a longtime in a majority of cases. As a result, despite there are several serological and molecular methods available to diagnose the infection in its acute and chronic stages, diagnosis is often achieved only after the onset of clinical symptoms in the chronic phase of the disease. Furthermore, although there are two drugs to treat it, the assessment of their performance is impractical with current parasite-derived diagnostics, and therapeutic efficacy cannot be acknowledged in a timely manner.In this chapter we present two procedures to measure host-derived molecules as surrogates of therapeutic response against chronic T. cruzi infection. Their outputs relate to the generation and activity of thrombin, a major component of the blood coagulation cascade. This is due to the fact that a hypercoagulability state has been described to occur in chronic Chagas disease patients and revert after treatment with benznidazole

Steady plasma concentration of unfractionated heparin reduces infarct volume and prevents inflammatory damage after transient focal cerebral ischemia in the rat

Unfractionated heparin (UH) decreases the extent of infarction after transient focal brain ischemia in the rat and abridges neuroinflammatory damage in patients with acute stroke. This study was aimed at assessing whether controlled and steady heparinemia in plasma can reduce infarct volume and exert neuroprotective effects after ischemia. Infarct volume was measured at 24 and 7 days following a 1-hr intraluminal middle cerebral artery (MCA) occlusion in rats treated with UH or with vehicle. After testing several UH administration protocols, we choose to give a bolus of 200 U/kg, which was started 3 hr after the occlusion, followed by a 24-hr intraperitoneal perfusion of 70 U/kg/hr, which maintained a 24-hr steady plasma heparinemia (0.3-0.6 U/ml) and caused no CNS or systemic bleeding. In addition, plasma IL-10 concentration was measured by ELISA, endothelial VCAM-1 expression was evaluated by i.v. injection of a 125I-labeled monoclonal antibody against VCAM-1, and brain hemeoxygenase-1 (HO-1) expression was determined by Western blot. UH-treated rats showed smaller infarctions than rats treated with vehicle, as well as higher IL-10 plasma levels and HO-1 brain expression and lower endothelial VCAM-1 induction. The study shows that a stable plasma concentration of UH given at nonhemorrhagic doses reduces infarct volume after ischemia-reperfusion in the rat. It also shows that UH prevented the induction of cell adhesion molecules in the cerebral vasculature and increased the expression of molecules with antiinflammatory and prosurvival properties. These findings support further testing of the clinical value of parenteral, adjusted, high-dose UH in patients with acute stroke. © 2004 Wiley-Liss, Inc.Contract grant sponsor: Fondo de Investigaciones Sanitarias of the Spanish Ministry of Health; Contract grant number: FIS 01/1150; Contract grant sponsor: Comisión Interministerial de Ciencia y Tecnología (CICYT); Contract grant number: SAF02-01963. A.Ce. has been partially supported by scholarships from the IDIBAPS and the Fundacio´ Privada from the Catalan Society of Neurology and C.J. by Red CIEN IDIBAPS-ISCIII RTIC C03/06Peer Reviewe

Contribution of the mRNA binding protein CPEB4 in platelet biology through translational control

Trabajo presentado en el European Congress on Thrombosis and Haemostasis (ECTH), celebrado en Glasgow (Reino Unido), del 2 al 4 de octubre de 2019Platelets are anucleated cells, lacking the mechanism of nuclear transcription. However, there are increasing data showing that platelets have post-transcriptional mechanisms that allow them to regulate the expression of a specific set of proteins, modulating platelet activation. Among the components of platelets, the abundant presence of messenger RNA (mRNA) is striking. This mRNA repertoire is specific and can be translated into proteins, however the mechanisms responsible for the translation of mRNA and its biological implications are unknown. Among these regulatory mechanisms, CPEBs (Cytoplasmic Polyadenylation Element Binding proteins) are a family of proteins that control mRNA translation by binding to the 3'UTR region and regulating the length of the poly (A) tail. These proteins were originally discovered to regulate the translation of maternal RNAs during embryonic development, but it is now known that they also regulate up to 20% of the transcriptome in the adult organism. AIMS The main objective of the project is to analyze the role of the CPEB4 protein in hemostasis and platelet functions. Physiological role of CPEB4 in platelets. Role does CPEB4 play in thrombosis and vascular pathology Molecular mechanism underlying the functions of CPEB4 in platelets. METHODS We have generated conditional knockout mice in order to study the role of CPEB4 in platelets (mouse Pf4-Cre:CPEB4lox/lox). Using in vitro (platelet activation and aggregation tests), in vivo (tail bleeding, TPO levels, megakaryocytopoiesis analysis, experimental pulmonary embolism) and high throughput (proteomics) strategies we have analyzed the functional effects and underlying molecular mechanism of CPEB4 absence in platelets. RESULTS CPEB4 is readily detected in both human and mouse platelet extracts. Animals with a platelet-specific deficiency in CPEB4 show a normal platelet counts in blood, but increased concentration of TPO in plasma, and increased megakaryocyte area and number in the bone marrow. These results suggest an increased platelet turn-over in the absence of CPEB4 in vivo. Moreover, CPEB4-deficient platelets have a tendency to lower activation and aggregation. In animal models of hemostasis and venous and arterial thrombosis, platelet CPEB4-deficient animals showed slightly increased bleeding times and less tendency to thrombus formation. Proteomic data comparing platelets WT or lacking CPEB4 suggest mechanisms of action of this regulatory pathway and possible relevant targets. SUMMARY/CONCLUSION Control of RNA translation by CPEB4 represents a novel regulatory mechanism of platelet function. The absence of CPEB4 reduces platelet activation by thrombin. Mice deficient in CPEB4 in platelets showed less thrombosis and increased bleeding. These results underscore the importance of the mRNA pool in platelet biology.Fundació Marató de TV3; SET

Control de la traducción por la proteína de unión A mRNA CPEB4 en la biología plaquetar

Trabajo presentado en el LXIII Congreso Nacional de la SEHH (Sociedad Española de Hematología y Hemoterapia) y XXXVII Congreso Nacional de la SETH (Sociedad Española de Trombosis y Hemostasia), celebrado en Pamplona (España) del 14 al 16 de octubre de 2021