Tissue factor–positive tumor microvesicles activate platelets and enhance thrombosis in mice

Abstract

Background Cancer patients have an approximately four-fold increased risk of venous thromboembolism (VTE) compared with the general population, and cancer patients with VTE have reduced survival. Tumor cells constitutively release small membrane vesicles called microvesicles (MVs) that may contribute to thrombosis in cancer patients. Clinical studies have shown that levels of circulating tumor-derived, tissue factor-positive (TF+) MVs in pancreatic cancer patients are associated with VTE. Objectives We tested the hypothesis that TF+ tumor-derived MVs (TMVs) activate platelets in vitro and in mice. Materials and Methods We selected two human pancreatic adenocarcinoma cell lines expressing high (BxPc-3) and low (L3.6pl) levels of TF as models to study the effect of TF+ TMVs on platelets and thrombosis. Results and Conclusions We found that both types of TF+ TMVs activated human platelets and induced aggregation in vitro in a TF- and thrombin-dependent manner. Further, injection of BxPc-3 TF+ TMVs triggered platelet activation in vivo and enhanced thrombosis in two mouse models of venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF+ TMV-enhanced thrombosis was reduced in Par4-deficient mice and in wild-type mice treated with clopidogrel, suggesting that platelet activation was required for enhanced thrombosis. These studies suggest that TF+ TMV-induced platelet activation contributes to thrombosis in cancer patients.

Mouse models of cancer-associated thrombosis

“…Interestingly, colorectal cancer cell lines with mutations in both K- ras and p53 , such as the HCT116 subline 379.2, express higher levels of tissue factor (TF) than those with mutations in only K- ras , such as HCT116 [11]. There is also a wide range of TF expression in the different human pancreatic cell lines; HPAF-II, HPAC and BxPc-3 express high levels of TF, L3.6pl express medium levels, PANC-1 expresses low levels and MIAPaCa-2 do not express TF [12–14]. The A549 epithelial-like lung cancer cell line was isolated from human cancerous lung tissue [15, 16].…”

“…The most popular are those involving the infrarenal vena cava (IVC). An advantage of the IVC is that high frequency ultrasonography can be used for non-invasive longitudinal monitoring of thrombus formation [14, 27, 28]. Clot formation is typically analyzed between 0–48 hours.…”

“…High levels of TF are expressed by a variety of tumors, particularly pancreatic tumors, as well as human and mouse pancreatic cancer cell lines [12–14, 46, 47]. In pancreatic cancer patients increased levels of microvesicle (MV) TF activity are associated with VTE [48, 49].…”

Emerging anticoagulant strategies

“…Thus, venous thrombosis is attenuated in mice rendered thrombocytopenic, 8 in those deficient in von Willebrand factor, 9 or in those treated with antiplatelet agents. 10 With venous flow restriction, endothelial cells become activated and attract platelets and neutrophils to form heterotypic aggregates. Activated neutrophils release neutrophil extracellular traps (NETs), lattices composed of DNA and histones, to which additional platelets adhere ( Figure 1).…”

Cancer‐associated thrombosis: The search for the holy grail continues

“…As EVs typically contain similar membrane‐bound proteins as their mother cell, EVs can possess procoagulant activity that may contribute to VTE. Preclinical mouse models have demonstrated that TF + EVs are being shed from pancreatic cancer cells into the bloodstream, mediating platelet activation and thrombus formation 26, 27, 28. Unfortunately, a relationship between circulating TF + EVs and VTE in a clinical setting was only established in pancreatic cancer patients, while no correlation was found in other moderate‐to‐high‐risk groups such as brain, colorectal, or lung cancer patients 29, 30.…”