Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps

Abstract

BackgroundThe hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis.MethodsUtilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. Platelet aggregation was assessed using collagen-activated impedance aggregometry. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Thromboelastograms (TEGs) were performed to assess hypercoagulability and changes associated with treatment. Correlative data and samples from a randomized clinical trial of preoperative gemcitabine/nab-paclitaxel with and without hydroxychloroquine were studied and the impact of treatment on venous thromboembolism (VTE) rate was evaluated.ResultsThe addition of NETs to whole blood stimulated platelet activation and aggregation. DNA and the receptor for advanced glycation end products (RAGE) were necessary for induction of NET associated platelet aggregation. PAD4 knockout tumor-burdened mice, unable to form NETs, had decreased aggregation and decreased circulating tissue factor. The NET inhibitor chloroquine reduces platelet aggregation, reduces circulating tissue factor and decreases hypercoagulability on TEG. Review of correlative data from patients treated on a randomized protocol of preoperative chemotherapy with and without hydroxychloroquine demonstrated a reduction in peri-operative VTE rate from 30 to 9.1% with hydroxychloroquine that neared statistical significance (p = 0.053) despite the trial not being designed to study VTE.ConclusionNETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine inhibits NETs and diminishes hypercoagulability. These findings support clinical study of chloroquine to lower rates of venous thromboembolism in patients with cancer.Trial registrationThis study reports correlative data from two clinical trials that registered with clinicaltrials.gov, NCT01128296 (May 21, 2010) and NCT01978184 (November 7, 2013).Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4584-2) contains supplementary material, which is available to authorized users.

Platelet‐derived extracellular vesicles released after trauma promote hemostasis and contribute to DVT in mice

“…This warrants further work in the future to further elucidate the antiplatelet mechanism of HCQ. HCQ is a well‐known as an anti‐autophagy agent and is also known to regulate NET formation, both of which have been implicated in DVT formation and could contribute to the observed effects in our model …”

“…Whether HCQ represents a potential VTE thromboprophylaxis strategy in addition to current standard of care warrants further clinical investigation. Importantly, however, HCQ is known to impact numerous cell types and cellular pathways in addition to platelets . Although HCQ markedly decreases PEV release from platelets, the mechanism of this effect is unknown.…”

“…Importantly, however, HCQ is known to impact numerous cell types and cellular pathways in addition to platelets. 10,64 Although HCQ markedly decreases PEV release from platelets, the mechanism of this effect is unknown. Interestingly, work on human platelets has shown HCQ decreases release of arachidonic acid and thromboxane generation in activated platelets.…”

“…HCQ is a well-known as an anti-autophagy agent and is also known to regulate NET formation, both of which have been implicated in DVT formation and could contribute to the observed effects in our model. 64 The present work has a number of important limitations, including the methodology for isolation and functional testing of PEVs. First, we isolated PEVs from whole blood samples by centrifugation techniques and characterized them via flow cytometry.…”

Covid-19 treatment update: follow the scientific evidence

“…There has been interest in the potential antithrombotic effects of chloroquine. While the contribution to its overall benefit may be modest and largely unknown in patients with Covid-19, chloroquine reduces neutrophil extracellular trap (NET) formation, platelet aggregation and circulating tissue factor in mice [7].…”