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Histologic analysis of gastrocnemius muscles. PBS, nonpreconditioned, or preconditioned ECFC (npECFC or pECFC, respectively) were infused intravenously in normoglycemic and type 2 diabetic (T2D) mice 5 h after ischemia-inducing surgery. Histologic analysis of gastrocnemius muscles, stained with hematoxylin and eosin (H/E) or Masson’s trichrome, was performed after 14 days postischemia in normoglycemic and type 2 diabetic (T2D) mice (n = 6 per group). Original magnification, 100×. Scale bar = 20 μm. (DOCX 1573 kb)

ECFC adhesion under proinflammatory and high glucose conditions. Nonpreconditioned or preconditioned ECFC (npECFC or pECFC, respectively) were incubated with high glucose, TNFα, or their combination, and seeded onto fibronectin, collagen, or TNFα-activated HUVEC. After (A) 30 min or (B) 2 h, the number of adherent cells was counted. Results are expressed as percentage of nonpreconditioned ECFC (n = 5). *p

ECFC cell death induced by histones or MSU is not affected under high glucose conditions. Nonpreconditioned ECFC (npECFC) were incubated with histones (A) or MSU crystals (B) at the indicated concentrations, in the presence or absence of high glucose, and cell death was analyzed after 24 h (n = 3–5). (DOCX 135 kb)

Además de ser elementos clave en la hemostasia y trombosis, las plaquetas tienen un rol preponderante en la respuesta inflamatoria e inmune asociada con su capacidad para reconocer patógenos a través de la expresión de los receptores tipo Toll, la secreción de una amplia variedad de citoquinas, quemoquinas y factores de crecimiento almacenados en sus gránulos y por la expresión de moléculas de adhesión que permiten la interacción con otras células vasculares. Como parte de la primera línea de defensa, los neutrófilos controlan la infección por fagocitosis, liberación de proteínas antimicrobianas durante la degranulación o a través de la formación de estructuras tipo red, conocidas como trampas extracelulares de neutrófilos (NETs). Estas están formadas por cromatina, proteasas y proteínas antimicrobianas cuya función principal es atrapar y eliminar bacterias, virus y hongos, impidiendo su diseminación. Además de microorganismos, la formación de NETs es gatillada por moléculas proinflamatorias y plaquetas. Su formación descontrolada puede ocasionar daño tisular y es considerada un mecanismo patogénico de eventos protrombóticos, inflamatorios y autoinmunes. En esta revisión se discute el rol de las plaquetas en la formación de NETs y se destacan los mediadores, estímulos y mecanismos moleculares participantes de este fenómeno, en humanos y modelos murinos. In addition to being key elements in hemostasis and thrombosis, platelets have an important role in inflammatory and innate immune responses. This activity is associated with their capability to recognize pathogens through the expression of TLRs, the secretion of a wide variety of cytokines, chemokines and growth factors stored within their granules and cell adhesion molecule expresssion that enable interaction with other vascular cells. As part of the first line of defense, neutrophils control invading pathogens by phagocytosis, the release of antimicrobial proteins during degranulation or through the formation of web-like structures known as neutrophil extracellular traps (NETs). NETs are formed by chromatin, proteases and antimicrobial proteins and their main function is to trap and kill bacteria, virus and fungi, thus avoiding their dissemination. Besides microorganisms, NETs formation is also triggered by proinflammatory molecules, and platelets. The uncontrolled formation of NETs might exert tissue damage and has been involved as a pathogenic mechanism of autoimmune and prothrombotic events. In this review, the role of platelets in NET generation is discussed, highlighting the mediators, stimuli and molecular mechanisms involved in this phenomenon, both in human and murine models. Além de serem elementos chave na hemostasia e trombose, as plaquetas têm um papel preponderante na resposta inflamatória e imune associada a sua capacidade para reconhecer patógenos através da expressão dos receptores tipo Toll, a secreção de uma ampla variedade de citocinas, quemocinas e fatores de crescimento armazenados em seus grânulos e pela expressão de moléculas de adesão que permitem a interação com outras células vasculares. Como parte da primeira linha de defesa, os neutrófilos controlam a infecção por fagocitose, liberação de proteínas antimicrobianas durante a degranulação ou através da formação de estruturas tipo rede, conhecidas como armadilhas extracelulares de neutrófilos (NETs). Elas estão formadas por cromatina, proteases e proteínas antimicrobianas cuja função principal é prender e eliminar bactérias, vírus e fungos, impedindo sua disseminação. Além de microorganismos, a formação de NETs é disparada por moléculas pró-inflamatórias e plaquetas. Sua formação descontrolada pode provocar dano tissular e é considerada um mecanismo patogê- nico de eventos pró-trombóticos, inflamatórios e autoimunes. Nesta revisão é discutido o papel das plaquetas na formação de NETs, destacando os mediadores, estímulos e mecanismos moleculares participantes deste fenômeno, em humanos e modelos murídeos. Fil: Carestia, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Kaufman, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

En condiciones fisiológicas, la activación de las plaquetas circulantes es limitada de manera constitutiva a través de la liberación, a partir del endotelio vascular, de potentes inhibidores de la función plaquetaria tales como el óxido nítrico y prostaciclina. Frente a una injuria vascular, las plaquetas se adhieren firmemente a proteínas expuestas del subendotelio permitiendo la formación del trombo y la detención de la hemorragia. Sin embargo, durante las últimas dos décadas numerosas evidencias revelaron que, además, en ausencia de un aparente daño morfológico pero en situaciones inflamatorias, las plaquetas son capaces de interactuar con el endotelio debido tanto a la disminución de los mecanismos fisiológicos inhibitorios como a un aumento en la expresión de moléculas de adhesión en la superficie de las células endoteliales activadas. Estos descubrimientos no sólo han creado un nuevo paradigma en la biología vascular sino también han abierto un novedoso y extenso campo de estudio asociado a las consecuencias fisio-patológicas de la interacción entre las plaquetas y el endotelio. Fil: Etulain, Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

A major function of platelets is keeping the vascular system intact. Platelet activation at sites of vascular injury leads to the formation of a hemostatic plug. Activation of platelets is therefore crucial for normal hemostasis; however, uncontrolled platelet activation may also lead to the formation of occlusive thrombi that can cause ischemic events. Although they are essential for proper hemostasis, platelet function extends to physiologic processes such as tissue repair, wound remodeling and antimicrobial host defense, or pathologic conditions such as thrombosis, atherosclerosis, chronic inflammatory diseases and cancer. Platelets can be activated by soluble molecules including thrombin, thromboxane A2 (TXA2), adenosine diphosphate (ADP), serotonin or by adhesive extracellular matrix (ECM) proteins such as von Willebrand factor (vWF) and collagen. Here we describe recent advances in the activation of platelets by non-canonical platelet agonists such as galectins. By acting either in soluble or immobilized form, these glycan-binding proteins trigger all platelet activation responses through modulation of discrete signaling pathways. We also offer new hypotheses and some speculations about the role of platelet-galectin interactions not only in hemostasis and thrombosis but also in inflammation and related diseases such as atherosclerosis and cancer.

A major function of platelets is keeping the vascular system intact. Platelet activation at sites of vascular injury leads to the formation of a hemostatic plug. Activation of platelets is therefore crucial for normal hemostasis; however, uncontrolled platelet activation may also lead to the formation of occlusive thrombi that can cause ischemic events. Although they are essential for proper hemostasis, platelet function extends to physiologic processes such as tissue repair, wound remodeling and antimicrobial host defense, or pathologic conditions such as thrombosis, atherosclerosis, chronic inflammatory diseases and cancer. Platelets can be activated by soluble molecules including thrombin, thromboxane A2 (TXA2), adenosine diphosphate (ADP), serotonin or by adhesive extracellular matrix (ECM) proteins such as von Willebrand factor (vWF) and collagen. Here we describe recent advances in the activation of platelets by non-canonical platelet agonists such as galectins. By acting either in soluble or immobilized form, these glycan-binding proteins trigger all platelet activation responses through modulation of discrete signaling pathways. We also offer new hypotheses and some speculations about the role of platelet-galectin interactions not only in hemostasis and thrombosis but also in inflammation and related diseases such as atherosclerosis and cancer. Fil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Background and Purpose Platelets are major players in every step of vessel development through the local delivery of angiogenesis-modulating factors, including the pro-angiogenic protein VEGF and the anti-angiogenic endostatin. Although thrombin is a potent agonist and is highly elevated in angiogenesis-related diseases, studies regarding its action on the release of platelet angiogenic factors are scarce and controversial. Herein, we have investigated the role of thrombin not only in VEGF and endostatin release but also in net platelet angiogenic activity. Experimental Approach Human platelets were stimulated with thrombin in the presence of the various inhibitors of the signalling pathways involved in platelet activation. Supernatants/releasates were used to determine the levels of angiogenic molecules and to induce angiogenic responses. Key Results We found that thrombin induced the secretion of both VEGF and endostatin; however, the overall effect of the releasates was pro-angiogenic as they promoted tubule-like formation and increased the proliferation of endothelial cells. Both responses were only slightly suppressed in the presence of a VEGF receptor-neutralizing antibody. Pharmacological studies revealed that while inhibitors of PKC, p38, ERK1/2, Src kinases or PI3K/Akt exerted only partial inhibitory effects, aspirin fully blocked the pro-angiogenic activity of the releasate. Conclusions and Implications In contrast to current belief, platelet pro-angiogenic responses are independent of VEGF and appear to be the result of the combined action of several molecules. Moreover, our data reinforce the notion that aspirin is a good candidate for a therapeutic agent to treat angiogenesis-related diseases. Fil: Etulain, Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Fondevila, C.. Clínica Bazterrica. Servicio de Hematología; Argentina Fil: Negrotto, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

La acidosis es una característica del microambiente inflamatorio presente en situaciones de injuria vascular, tumor, aterosclerosis y enfermedades inflamatorias pulmonares. Las plaquetas además de ser elementos claves en la hemostasia y la trombosis, también juegan un rol importante en estas patologías. En estudios previos mostramos que el descenso del pH apaga las funciones hemostáticas de las plaquetas y promueve respuestas inflamatorias mediadas por la expresión de P-selectina en la superficie plaquetaria. En este trabajo estudiamos los mecanismos moleculares involucrados en estos procesos. Mientras la fosforilación de algunas moléculas involucradas en la activación plaquetaria (Src, Akt y ERK) fue significativamente inhibida en acidosis, la activación de p38 o NFκB no fue modificada o aumentó respectivamente, con el descenso del pH. El incremento en la expresión de P-selectina observado en acidosis se mantuvo en presencia de inhibidores de Src (PP1), PI3K/Akt (Ly294002) y ERK (U0126). Este fenómeno tampoco fue modificado por la incubación individual de las plaquetas con bloqueantes de p38 (SB203580) o NFκB (Ro1069920), pero sí disminuyó significativamente ante la presencia simultánea de ambos inhibidores. En concordancia con el aumento en la expresión de P-selectin, la formación de agregados plaquetas-leucocitos, así como la migración de leucocitos en presencia de plaquetas, aumentó en acidosis y ambos fenómenos fueron revertidos por la presencia conjunta de SB203580 y Ro1069920. Estos resultados indican que las respuestas inflamatorias mediadas por plaquetas en condiciones de acidosis requieren la activación simultánea de p38 y NFκB y plantean la posibilidad de nuevos blancos terapéuticos para el tratamiento de la inflamación. Fil: Etulain, Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Carestia, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Rivadeneyra, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Fondevila Sancet, Juan Carlos. Clínica Bazterrica. Servicio de Hematología; Argentina Fil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Fil: Pacienza, Natalia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; Argentina Fil: D'Atri, Lina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; Argentina Fil: Pozner, Roberto Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; Argentina Fil: Negrotto, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; Argentina Fil: Malaver Marín, Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; Argentina Fil: Torres, Oscar. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; Argentina Fil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; Argentina

Background and purpose: Oncocalyxone A (OncoA) has a concentration- dependent anti-platelet activity. The present study aimed to further understand the mechanisms related to this effect. Experimental approach: Human platelet aggregation was measured by means of a turbidimetric method. OncoA (32-256 μM) was tested against several platelet-aggregating agents, such as adenosine diphosphate (ADP), collagen, arachidonic acid (AA), ristocetin and thrombin. Key results: OncoA completely inhibited platelet aggregation with a calculated mean inhibitory concentration (IC 50-μM) of 122 for ADP, 161 for collagen, 159 for AA, 169 for ristocetin and 85 for thrombin. The anti-aggregatory activity of OncoA was not inhibited by 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ). OncoA, at a concentration that caused no significant anti-aggregatory activity, potentiated sodium nitroprusside (SNP) anti-aggregatory activity (18.8±2.9%-SNP vs 85.0±8.2%-SNP+OncoA). The levels of nitric oxide (NO) or cAMP were not altered by OncoA while cGMP levels were increased more than 10-fold by OncoA in resting or ADP-activated platelets. Flow cytometry revealed that OncoA does not interact with receptors for fibrinogen, collagen or P-selectin. Nevertheless, OncoA decreased the binding of antibodies to GP Ibα, a glycoprotein that is related both to von Willebrand factor and to thrombin-induced platelet aggregation. Conclusion and implications: OncoA showed anti-aggregatory activity in platelets that was associated with increased cGMP levels, not dependent on NO and with blocking GP Ibα glycoprotein. This new mechanism has the prospect of leading to new anti-thrombotic drugs. © 2008 Nature Publishing Group All rights reserved. Fil: Ferreira, M. A. D.. Universidade Estadual do Ceará; Brasil Fil: Do Nascimento, N. R. F.. Universidade Estadual do Ceará; Brasil Fil: De Sousa, C. M.. Universidade Estadual do Ceará; Brasil Fil: Pessoa, O. D. L.. Universidade Estadual do Ceará; Brasil Fil: De Lemos, T. L. G.. Universidade Estadual do Ceará; Brasil Fil: Ventura, J. S.. Butantan Institute; Brasil Fil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Chudzinski Tavassi, A. M.. Butantan Institute; Brasil

We have previously demonstrated that nitric oxide (NO) triggers CD34+-derived megakaryocyte apoptosis. We here show that prostacyclin (PGI2) inhibits PAPA/NO-induced megakaryocyte death detected by fluorescent microscopy and flow cytometry.The cAMP-specific phosphodiesterase inhibitor, Ro 20-1724, and the permeable analog dibutyryl-cAMP also delayed apoptosis. PGI2 effect was fully prevented when adenylyl cyclase activity was suppressed by SQ 22536, and partially reversed by the permeable protein kinase A inhibitor PKI 14-22 amide. ELISA showed that while both PGI2 and NO alone or synergistically raised cAMP, only NO was able to increase intracellular cGMP levels.Treatment of megakaryocytes with PGI2 abolished both basal and NO-raised cGMP levels. Addition of 8-pCPT-cGMP or activation of soluble guanylyl cyclase by BAY 41-2272 induced cell death in a concentration-dependent manner, and ODQ, an inhibitor of guanylyl cyclase, prevented both PAPA/NO- or BAY 41-2272-induced apoptosis. Specific cGMP phosphodiesterase inhibition by Zaprinast or suppression of adenylyl cyclase by SQ 22536 enhanced the PAPA/NO proapoptotic effect.PGI2 completely inhibited NO-mediated generation and the increased activity of the cleaved form of caspase-3.In conclusion, our results demonstrate that contrary to their well-known direct and synergistic inhibitory effects on platelets, PGI2 and NO regulate opposite megakaryocyte survival responses through a delicate balance between intracellular cyclic nucleotide levels and caspase-3 activity control.

In this issue of Blood, Loria and colleagues present new aspects on the role of platelets in protecting against both lethal hemorrhagic diathesis and virus replication in viral hemorrhagic fever (VHF), using a murine model of lymphocytic choriomeningitis virus (LMCV) infection1. In this issue of Blood, Loria and colleagues present new aspects on the role of platelets in protecting against both lethal hemorrhagic diathesis and virus replication in viral hemorrhagic fever (VHF), using a murine model of lymphocytic choriomeningitis virus (LMCV) infection1. Fil: Gomez, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Gomez, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina