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Blood platelets can interact with bacteria, possibly leading to platelet activation, cytokine and microparticle release and immune signalling. Besides, bacteria can also affect the platelet RNA content. We investigated the impact of non-pathogenic K12 and pathogenic O18:K1 Escherichia (E.) coli strains on platelet activation, RNA expression patterns, and selected proteins. Depending on bacteria concentration, contact of platelets with E. coli K12 lead to an increase of P-selectin (24–51.3%), CD63 (15.9–24.3%), PAC-1 (3.8–14.9%) and bound fibrinogen (22.4–39%) on the surface. E. coli O18:K1 did not affect these markers. Sequencing analysis of total RNA showed that E. coli K12 caused a significant concentration change of 103 spliced mRNAs, of which 74 decreased. For the RNAs of HMBS (logFC = +5.73), ATP2C1 (logFC = −3.13) and LRCH4 (logFC = −4.07) changes were detectable by thromboSeq and Tuxedo pipelines. By Western blot we observed the conversion of HMBS protein from a 47 kDA to 40 kDa product by E. coli K12, O18:K1 and by purified lipopolysaccharide. While ATP2C1 protein was released from platelets, E. coli either reduced the secretion or broke down the released protein making it undetectable by antibodies. Our results demonstrate that different E. coli strains influence activation, RNA and protein levels differently which may affect platelet-bacteria crosstalk.

Purpose: Vascular adhesion protein (VAP)-1 is an adhesion molecule elucidated as a mediator of the leukocyte recruitment cascade. The purpose of this study was to investigate the role of VAP-1 in ocular inflammatory neovascularization using a mouse laser-induced choroidal neovascularization (CNV) model. Methods: CNV was induced with 532 nm laser irradiation in C57BL/6 mice, and production of VAP-1 protein in the retinal pigment epithelium (RPE) choroid during CNV formation was examined. CNV animals were treated with the specific VAP-1 inhibitor U-V002 or vehicle solution, and the volume of CNV tissue was evaluated with volumetric measurements. Macrophage infiltration into the CNV lesions was evaluated using two different techniques, flatmount staining and real-time polymerase chain reaction (PCR) for F4/80. The protein levels of intercellular adhesion molecule (ICAM)-1, monocyte chemoattractant protein (MCP)-1, P-selectin, and vascular endothelial growth factor (VEGF) in the RPE-choroid were measured with enzyme-linked immunosorbent assay (ELISA). Results: VAP-1 inhibition significantly suppressed CNV formation in a dose-dependent manner and reduced macrophage infiltration into CNV lesions. Furthermore, VAP-1 blockade decreased the expression of ICAM-1 and MCP-1, both of which play a pivotal role in macrophage recruitment. Conclusions: Our data suggest VAP-1 has an important role during ocular inflammatory neovascularization through leukocyte recruitment. VAP-1 inhibition may be a novel and potent therapeutic strategy in treating CNV formation.

© 2015 Elsevier Inc. All rights reserved., Reported in vitro data implicated soluble CD40 ligand (sCD40L) in endothelial dysfunction and angiogenesis. However, whether sCD40L could exert that influence in endothelial dysfunction and angiogenesis after injury in acute myocardial infarction (AMI) patients remains unclear. In the present study, we evaluated the association of sCD40L with markers of platelet activation, endothelial, and vascular function during a recovery period early after AMI. To achieve this goal, the time changes of soluble, platelet-bound, and microparticle-bound CD40L levels over 1 month were assessed in AMI patients and correlated with endothelial nitric oxide synthase (eNOS) polymorphisms, vascular endothelial growth factor (VEGF) concentrations, and platelet expression of P-selectin (CD62P). The association of soluble form, platelet-bound, and microparticle-bound CD40L with CD62P expression on platelets, a marker of platelet activation, was also assessed to evaluate the role of CD40L in the thrombosis, whereas the association with eNOS and VEGF was to evaluate the role of CD40L in vascular dysfunction. This work shows for the first time that time changes of sCD40L over 1 month after myocardial infarct onset were associated with G894T eNOS polymorphism and with the VEGF concentrations, but not to the platelet CD62P expression. These results indicate that, in terms of AMI pathophysiology, the sCD40L cannot be consider just as being involved in thrombosis and inflammation but also as having a relevant role in vascular and endothelial dysfunction., The work was financially supported by Fundação para a Ciência e Tencnologia (SFRM/BPD/6308/2009) and by Liga dos Amigos do Hospital de Santa Marta.

Thrombocytopenia is associated with bleeding risk. However, in thrombocytopenic patients platelet count does not correlate with bleeding risk and other factors are thus likely to contribute to this risk. The present review presents currently available platelet-related markers available on automated haematology analysers and commonly used methods for testing platelet function. The test principles, advantages and disadvantages of each test are described. We also evaluate the current literature regarding the clinical utility of the test for prediction of bleeding in thrombocytopenia in haematological and oncological diseases. We find that several platelet-related markers are available but information about the clinical utility in thrombocytopenia is limited. Studies support that mean platelet volume (MPV) can aid diagnosing the cause of thrombocytopenia and low MPV may be associated with bleeding in thrombocytopenia. Flow cytometry, platelet aggregometry and platelet secretion tests are used to diagnose specific platelet function defects. The flow cytometric activation marker P-selectin and surface coverage by the Cone and Plate[let] analyser™ predict bleeding in selected thrombocytopenic populations. To fully uncover the clinical utility of platelet-related tests, information about the prevalence of platelet function defects in thrombocytopenic conditions is required. Finally, knowledge of the performance in thrombocytopenic samples from patients is essential. Thrombocytopenia is associated with bleeding risk. However, in thrombocytopenic patients platelet count does not correlate with bleeding risk and other factors are thus likely to contribute to this risk. The present review presents currently available platelet-related markers available on automated haematology analysers and commonly used methods for testing platelet function. The test principles, advantages and disadvantages of each test are described. We also evaluate the current literature regarding the clinical utility of the test for prediction of bleeding in thrombocytopenia in haematological and oncological diseases. We find that several platelet-related markers are available but information about the clinical utility in thrombocytopenia is limited. Studies support that mean platelet volume (MPV) can aid diagnosing the cause of thrombocytopenia and low MPV may be associated with bleeding in thrombocytopenia. Flow cytometry, platelet aggregometry and platelet secretion tests are used to diagnose specific platelet function defects. The flow cytometric activation marker P-selectin and surface coverage by the Cone and Plate[let] analyser(™) predict bleeding in selected thrombocytopenic populations. To fully uncover the clinical utility of platelet-related tests, information about the prevalence of platelet function defects in thrombocytopenic conditions is required. Finally, knowledge of the performance in thrombocytopenic samples from patients is essential. This article is protected by copyright. All rights reserved.