Cancer patients are at an increased risk of developing thromboembolic complications. Several mechanisms have been proposed to explain cancer-associated thrombosis including the release of tumor-derived extracellular vesicles and the activation of host vascular cells. It was proposed that neutrophil extracellular traps (NETs) contribute to the prothrombotic phenotype in cancer. In this study, we evaluated the possible cooperation between tumor-derived exosomes and NETs in cancer-associated thrombosis. Female BALB/c mice were orthotopically injected with 4T1 breast cancer cells. The tumor-bearing animals exhibited increased levels of plasma DNA and myeloperoxidase in addition to significantly increased numbers of circulating neutrophils. Mice were subjected to either Rose Bengal/laser-induced venous thrombosis or ferric chloride-induced arterial thrombosis models. The tumor-bearing mice exhibited accelerated thrombus formation in both models compared to tumor-free animals. Treatment with recombinant human DNase 1 reversed the prothrombotic phenotype of tumor-bearing mice in both models. Remarkably, 4T1-derived exosomes induced NET formation in neutrophils from mice treated with granulocyte colony-stimulating factor (G-CSF). In addition, tumor-derived exosomes interacted with NETs under static conditions. Accordingly, the intravenous administration of 4T1-derived exosomes into G-CSF-treated mice significantly accelerated venous thrombosis in vivo. Taken together, our observations suggest that tumor-derived exosomes and neutrophils may act cooperatively in the establishment of cancer-associated thrombosis.

The establishment of prothrombotic states during cancer progression is well reported but the precise mechanisms underlying this process remain elusive. A number of studies have implicated the presence of the clotting initiator protein, tissue factor (TF), in circulating tumor-derived extracellular vesicles (EVs) with thrombotic manifestations in certain cancer types. Tumor cells, as well as tumor-derived EVs, may activate and promote platelet aggregation by TF-dependent and independent pathways. Cancer cells and their secreted EVs may also facilitate the formation of neutrophil extracellular traps (NETs), which may contribute to thrombus development. Alternatively, the presence of polyphosphate (polyP) in tumor-derived EVs may promote thrombosis through a TF-independent route. We conclude that the contribution of EVs to cancer coagulopathy is quite complex, in which one or more mechanisms may take place in a certain cancer type. In this context, strategies that could attenuate the crosstalk between the proposed pro-hemostatic routes could potentially reduce cancer-associated thrombosis.

Invasion of mosquito salivary glands (SGs) by Plasmodium falciparum sporozoites is an essential step in the malaria life cycle. How infection modulates gene expression, and affects hematophagy remains unclear. Using Affimetrix chip microarray, we found that at least 43 genes are differentially expressed in the glands of Plasmodium falciparum-infected Anopheles gambiae mosquitoes. Among the upregulated genes, one codes for Agaphelin, a 58-amino acid protein containing a single Kazal domain with a Leu in the P1 position. Agaphelin displays high homology to orthologs present in Aedes sp and Culex sp salivary glands, indicating an evolutionarily expanded family. Kinetics and surface plasmon resonance experiments determined that chemically synthesized Agaphelin behaves as a slow and tight inhibitor of neutrophil elastase (K_D∼10 nM), but does not affect other enzymes, nor promotes vasodilation, or exhibit antimicrobial activity. TAXIscan chamber assay revealed that Agaphelin inhibits neutrophil chemotaxis toward fMLP, affecting several parameter associated with cell migration. In addition, Agaphelin reduces paw edema formation and accumulation of tissue myeloperoxidase triggered by injection of carrageenan in mice. Agaphelin also blocks elastase/cathepsin-mediated platelet aggregation, abrogates elastase-mediated cleavage of tissue factor pathway inhibitor, and attenuates neutrophil-induced coagulation. Notably, Agaphelin inhibits neutrophil extracellular traps (NETs) formation and prevents FeCl₃-induced arterial thrombosis, without impairing hemostasis. Blockade of neutrophil elastase emerges as a novel antihemostatic mechanism in hematophagy; it also supports the notion that neutrophils and the innate immune response are targets for antithrombotic therapy. In addition, Agaphelin is the first antihemostatic whose expression is induced by Plasmodium sp infection. These results suggest that an important interplay takes place in parasite-vector-host interactions. Malaria is transmitted by Plasmodium falciparum-infected Anopheles gambiae mosquitoes. Salivary gland contributes to the development of the parasite by creating a favorable environment for the infection and facilitating blood feeding and reproduction of the vector. However, the molecular mechanism by which the vector salivary gland modulates parasite/host interactions is not understood. We discovered that infection of the mosquito salivary gland upregulates several genes; among them, one codes for a protease inhibitor named Agaphelin. Notably, Agaphelin was found to exhibit multiple antihemostatic functions by targeting elastase. As a result, it inhibits platelet function which is required for blood to clot, and it prevents cleavage of TFPI, an anticoagulant that has recently been found to play a crucial role in thrombus formation in vivo. Agaphelin also attenuates neutrophils chemotaxis and the release of Neutrophil Extracellular Traps. These results provide evidence that neutrophils serve as a link between coagulation and the innate immune response. Agaphelin also exhibits anti-inflammatory and antithrombotic effects in vivo. Furthermore, Agaphelin did not promote bleeding, suggesting that targeting neutrophil exhibits potential therapeutic value. Altogether, these results highlight that the interplay between parasite, vector and host is a dynamic process that contributes and sustains the interface among Plasmodium, Anopheles and humans.

Recent studies demonstrate that interstitial inorganic phosphate is significantly elevated in the breast cancer microenvironment as compared to normal tissue. In addition it has been shown that breast cancer cells express high levels of the NaP_i-IIb carrier (SLC34A2), suggesting that this carrier may play a role in breast cancer progression. However, the biochemical behavior of inorganic phosphate (P_i) transporter in this cancer type remains elusive. In this work, we characterize the kinetic parameters of Pi transport in the aggressive human breast cancer cell line, MDA-MB-231, and correlated P_i transport with cell migration and adhesion. We determined the influence of sodium concentration, pH, metabolic inhibitors, as well as the affinity for inorganic phosphate in P_i transport. We observed that the inorganic phosphate is dependent on sodium transport (K_0,5 value = 21.98 mM for NaCl). Furthermore, the transport is modulated by different pH values and increasing concentrations of P_i, following the Michaelis-Menten kinetics (K_0,5 = 0.08 mM P_i). PFA, monensin, furosemide and ouabain inhibited P_i transport, cell migration and adhesion. Taken together, these results showed that the uptake of P_i in MDA-MB-231 cells is modulated by sodium and by regulatory mechanisms of intracellular sodium gradient. General Significance: Pi transport might be regarded as a potential target for therapy against tumor progression.