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.

IntroductionHeparin cofactor II (HCII) is a serine protease inhibitor (serpin) that inactivates thrombin rapidly in the presence of certain glycosaminoglycans (reviewed in ref. 1). HCII does not inhibit other proteases involved in coagulation or fibrinolysis. Thrombin stimulates platelet aggregation, promotes coagulation by cleavage of fibrinogen and activation of factors V, VIII, XI, and XIII, and inhibits fibrinolysis by activation of a plasma carboxypeptidase (2). Conversely, when thrombin binds to thrombomodulin on the surface of endothelial cells, it activates protein C, which inhibits further thrombin generation. Thrombin also engages in a variety of activities unrelated to hemostasis (3). For example, it causes proliferation of fibroblasts and other cells, induces monocyte chemotaxis, promotes adhesion of neutrophils to endothelial cells, and inhibits neurite outgrowth. HCII could potentially regulate the activity of thrombin in one or more of these diverse biological processes.The rate of inhibition of thrombin by HCII increases more than 1000-fold in the presence of heparin, heparan sulfate, or dermatan sulfate (4). HCII is unique among serpins in its ability to be stimulated by dermatan sulfate and binds to a minor subpopulation of dermatan sulfate oligosaccharides (5). By contrast, antithrombin binds to a specific pentasaccharide structure in heparin or heparan sulfate and is thereby stimulated to inhibit thrombin and other coagulation proteases (especially factors Xa and IXa) (1). The distribution of specific glycosaminoglycans on the cell surface or in the ECM may serve to localize protease inhibition by HCII and antithrombin to different sites. Cultured fibroblasts and vascular smooth muscle cells synthesize proteoglycans that stimulate inhibition of thrombin by HCII, but endothelial cells do not, suggesting that HCII may inhibit thrombin at sites outside the vascular lumen (6, 7).Although the physiologic function of HCII is unknown, the presence of thrombin-HCII complexes in human plasma indicates that HCII inhibits thrombin in vivo (8, 9). HCII is synthesized by the liver and circulates in human plasma at a concentration of about 1 µM (10). Turnover studies of labeled HCII in humans suggest that about 40% of the protein equilibrates with an extravascular compartment (11), but the tissue distribution of HCII has not been thoroughly investigated. HCII has been detected in the intima of normal human arteries, and the ability of dermatan sulfate in the arterial wall to stimulate HCII is decreased in atherosclerotic lesions (12, 13). During pregnancy, both the maternal and the fetal blood contain trace amounts of a dermatan sulfate proteoglycan that stimulates thrombin inhibition by HCII (14). The placenta is rich in der- Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate, heparan sulfate, or heparin. HCII has been proposed to regulate coagulation or to participate in processes such as inflammation, atherosclerosis, and wound repair. To in...

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Shedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initiator protein tissue factor (TF). On the other hand, tumour- and melanocyte-derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial thrombosis model, we observed that melanoma- but not melanocyte-derived MVs strongly accelerate thrombus formation in a TF-dependent manner, and accumulate at the site of vascular injury. Analysis of plasma obtained from melanoma-bearing mice showed the presence of MVs with a similar procoagulant pattern as compared to Tm1 MVs produced in vitro. Remarkably, flow-cytometric analysis demonstrated that 60% of ex vivo MVs are TF-positive and carry the melanoma-associated antigen, demonstrating its tumour origin. Altogether our data suggest that malignant transformation in melanocytes increases the production of procoagulant MVs, which may contribute for a variety of coagulation-related protumoural responses.

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. We previously reported that the time to thrombotic occlusion of the carotid artery after photochemical injury was shorter in HCII-deficient mice than in wildtype control animals. In this paper, we describe the antithrombotic activity of dermatan sulfate in wild-type and HCIIdeficient mice. Intravenous administration of porcine skin dermatan sulfate induced a dose-dependent prolongation of the carotid artery occlusion time in HCII ؉/؉ mice that was not observed in HCII ؊/؊ animals. Pharmacokinetic studies suggested that porcine skin dermatan sulfate expresses antithrombotic activity after being transferred from the plasma to sites in the vessel wall. Using invertebrate dermatan sulfate preparations, we showed that N-acetylgalactosamine-4-O-sulfate residues are required for the HCII-dependent antithrombotic effect. Furthermore, the invertebrate dermatan sulfates, which have higher charge densities than mammalian dermatan sulfate, slightly prolonged the thrombotic occlusion time of HCII ؊/؊ mice. These results indicate that HCII mediates the antithrombotic effect of porcine skin dermatan sulfate after injury to the carotid arterial endothelium in mice, whereas more highly charged dermatan sulfates possess weak antithrombotic activity independent of HCII. (Blood. 2004; 104:3965-3970)