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Haematologica ; 105(1): 218-225, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31048354


Pancreatic cancer is associated with a high incidence of venous thromboembolism. Neutrophils have been shown to contribute to thrombosis in part by releasing neutrophil extracellular traps (NET). A recent study showed that increased plasma levels of the NET biomarker, citrullinated histone H3 (H3Cit), are associated with venous thromboembolism in patients with pancreatic and lung cancer but not in those with other types of cancer, including breast cancer. In this study, we examined the contribution of neutrophils and NET to venous thrombosis in nude mice bearing human pancreatic tumors. We found that tumor-bearing mice had increased circulating neutrophil counts and levels of granulocyte-colony stimulating factor, neutrophil elastase, H3Cit and cell-free DNA compared with controls. In addition, thrombi from tumor-bearing mice contained increased levels of the neutrophil marker Ly6G, as well as higher levels of H3Cit and cell-free DNA. Thrombi from tumor-bearing mice also had denser fibrin with thinner fibers consistent with increased thrombin generation. Importantly, either neutrophil depletion or administration of DNase I reduced the thrombus size in tumor-bearing but not in control mice. Our results, together with clinical data, suggest that neutrophils and NET contribute to venous thrombosis in patients with pancreatic cancer.

Thromb Res ; 175: 46-52, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30703701


INTRODUCTION: The ultrastructure and cellular composition of thrombi has a profound effect on the outcome of acute ischemic stroke (AIS), coronary (CAD) and peripheral artery disease (PAD). Activated neutrophils release a web-like structure composed mainly of DNA and citrullinated histones, called neutrophil extracellular traps (NET) that modify the stability and lysability of fibrin. Here, we investigated the NET-related structural features of thrombi retrieved from different arterial localizations and their interrelations with routinely available clinical data. PATIENTS AND METHODS: Thrombi extracted from AIS (n = 78), CAD (n = 66) or PAD (n = 64) patients were processed for scanning electron microscopy, (immune)stained for fibrin, citrullinated histone H3 (cH3) and extracellular DNA. Fibrin fiber diameter, cellular components, DNA and cH3 were measured and analyzed in relation to clinical parameters. RESULTS: DNA was least present in AIS thrombi showing a 2.5-fold lower DNA/fibrin ratio than PAD, whereas cH3 antigen was unvaryingly present at all locations. The NET content of thrombi correlated parabolically with systemic inflammatory markers and positively with patients' age. The median platelet content was lower in PAD (2.2%) than in either AIS (3.9%) or CAD (3.1%) and thrombi from smokers contained less platelets than non-smokers. Fibrin fibers were significantly thicker in male patients with CAD (median fiber diameter 76.3 nm) compared to AIS (64.1 nm) or PAD (62.1 nm) and their diameter correlated parabolically with systemic inflammatory markers. CONCLUSIONS: The observed NET-related variations in thrombus structure shed light on novel determinants of thrombus stability that eventually affect both the spontaneous progress and therapeutic outcome of ischemic arterial diseases.

Armadilhas Extracelulares/metabolismo , Isquemia/sangue , Neutrófilos/metabolismo , Doença Arterial Periférica/sangue , Trombose/sangue , Feminino , Humanos , Isquemia/patologia , Masculino , Doença Arterial Periférica/patologia , Trombose/patologia
Front Immunol ; 10: 2967, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31921206


Staphylococcus aureus causes localized infections or invasive diseases (abscesses or endocarditis). One of its virulence factors is staphylocoagulase (SCG), which binds prothrombin to form a complex with thrombin-like proteolytic activity and leads to uncontrolled fibrin generation at sites of bacterial inoculation. The aim of this study was to characterize the formation, structure, mechanical properties and lysis of SCG-generated clots. Recombinant SCG was expressed in Escherichia coli, purified and the amidolytic activity of its complexes with human prothrombin (SCG-PT) and thrombin (SCG-T) was determined using human thrombin as a reference. Fibrin clots were prepared from purified fibrinogen and human plasma using thrombin, SCG-PT or SCG-T as a coagulase. The kinetics of clot formation and lysis by tissue-type plasminogen activator (tPA) were monitored with turbidimetric assays. Fibrin ultrastructure was examined with scanning electron microscopy and small-angle X-ray scattering (SAXS). Fibrin clot porosity was characterized with fluid permeation assays, whereas the viscoelastic properties and mechanical stability were evaluated with oscillation rheometry. Compared to thrombin, the amidolytic and clotting activity of SCG-PT was 1.6- to 2.5-fold lower on a molar basis. SCG-T had equivalent amidolytic, but reduced clotting activity both on pure fibrinogen (1.6-fold), and in plasma (1.3-fold). The SCG-PT and SCG-T generated fibrin with thicker fibers (10-60% increase in median diameter) than thrombin due to increased number of fibrin protofibrils per fiber cross-section. According to the fluid permeability of the clots SCG-PT and SCG-T promoted the formation of more porous structures. The shear stress resistance in the pure fibrin and plasma clots generated by SCG-PT was significantly lower than in the thrombin clots (243.8 ± 22.0 Pa shear stress was sufficient for disassembly of SCG-PT fibrin vs. 937.3 ± 65.6 Pa in thrombin clots). The tPA-mediated lysis of both pure fibrin and plasma clots produced by SCG-PT or SCG-T was accelerated compared to thrombin, resulting in up to a 2.1-fold increase in tPA potency. Our results indicate that SCG generates a thrombus scaffold with a structure characterized by impaired mechanical stability and increased lytic susceptibility. This proneness to clot disintegration could have implications in the septic embolism from endocardial bacterial vegetation.