Venous Thrombosis In Mice Research Articles

Critical role for platelet Ral GTPases in regulating venous thrombosis in mice

Deep vein thrombosis (DVT) is a major cause of morbidity and mortality globally. While its pathophysiology is complex, increasing evidence suggests a more prominent role for platelets than previously suspected. Genetic deletion of Ral GTPases, RalA and RalB, conditionally in mouse platelets (RalAB DKO), results in a near complete defect in P-selectin externalisation upon activation, while other platelet activation responses and arterial thrombosis are preserved. Given the critical role of P-selectin in mediating platelet-neutrophil interaction and thromboinflammation, we sought to investigate whether platelet Rals would also play critical roles in venous thrombosis, a thromboinflammatory disease, using RalAB DKO mice. DVT was induced by surgical partial ligation of the caudal (inferior) vena cava for 24-h or 48-h before venous thrombi were assessed by histology and immunofluorescence microscopy. RalAB DKO mice show a reduction in venous thrombus formation after 24-h, and near complete ablation of venous thrombosis by 48-h post IVC ligation. Immunofluorescence microscopy revealed that cross sections of thrombi from wildtype mice consist of an organised scaffolded structure of platelets surrounding leukocytes/neutrophils producing NETs to stabilize and propagate the thrombus. This organised structure was absent in platelet-specific conditional RalAB DKO thrombi. In vitro analysis of platelet-mediated NET formation was also significantly reduced when platelets lacked RalAB or when platelets were treated with the Ral inhibitor RBC8. We identify platelet Rals as novel, potentially critical regulators of venous thrombus stability, through their ability to regulate neutrophil NET formation via platelet P-selectin.

Extracellular histones: a unifying mechanism driving platelet-dependent extracellular vesicle release and thrombus formation in COVID-19

BackgroundCOVID-19 can cause profound inflammation and coagulopathy, and while many mechanisms have been proposed, there is no known common pathway leading to a prothrombotic state. ObjectivesFrom the beginning of the COVID-19 pandemic, elevated levels of extracellular histones have been found in plasma of patients infected with SARS-CoV-2. We hypothesized that platelet activation triggered by extracellular histones might represent a unifying mechanism leading to increased thrombin generation and thrombosis. MethodsWe utilized blood samples collected from an early clinical trial of hospitalized COVID-19 patients (NCT04360824) and recruited healthy subjects as controls. Using plasma samples, we measured the procoagulant and prothrombotic potential of circulating extracellular histones and extracellular vesicles (EVs). Platelet prothrombotic activity was assessed via thrombin generation potential and platelet thrombus growth. Circulating EVs were assessed for thrombin generation potential in vitro in plasma and enhancement of thrombotic susceptibility in vivo in mice. ResultsCompared with controls, COVID-19 patients had elevated plasma levels of citrullinated histone H3, cell-free DNA, nucleosomes, and EVs. Plasma from COVID-19 patients promoted platelet activation, platelet-dependent thrombin generation, thrombus growth under venous shear stress, and release of platelet-derived EVs. These prothrombotic effects of COVID-19 plasma were inhibited by an RNA aptamer that neutralizes both free and DNA-bound histones. EVs isolated from COVID-19 plasma enhanced thrombin generation in vitro and potentiated venous thrombosis in mice in vivo. ConclusionWe conclude that extracellular histones and procoagulant EVs drive the prothrombotic state in COVID-19 and that histone-targeted therapy may prove beneficial.

Mucin 1 and venous thrombosis in tumor-bearing mice and patients with cancer

IntroductionMucins released from epithelial tumors have been proposed to play a role in cancer-associated thrombosis. Mucin1 (MUC1) is a transmembrane mucin that is overexpressed in a variety of human malignancies, including breast and pancreatic cancer. We analyzed the association of MUC1 and venous thrombosis in a mouse tumor model and in patients with cancer. Materials and methodsWe used a human pancreatic cancer cell line HPAF-II that expresses a high level of MUC1. We grew HPAF-II tumors in the pancreas of Crl:NU-Foxn1nu male mice. MUC1 in plasma and extracellular vesicles (EVs) isolated from plasma was measured using an enzyme-linked immunosorbent assay. MUC1 in EVs and venous thrombi from tumor-bearing mice was assessed by western blotting. We measured MUC1 in plasma from healthy controls and patients with stomach, colorectal or pancreatic cancer with or without venous thromboembolism. Results and discussionMUC1 was detected in the plasma of mice bearing HPAF-II tumors and was associated with EVs. MUC1 was present in venous thrombi from mice bearing HFAP-II tumors. Recombinant MUC1 did not induce platelet aggregation. Levels of MUC1 were higher in patients with pancreatic cancer compared with healthy controls. In contrast to the mouse model, MUC1 was present in EV-free plasma in samples from healthy controls and patients with cancer. There was no significant difference in the levels of MUC1 in cancer patients with or without VTE. Our data did not find any evidence that MUC1 contributed to VTE in patients with cancer.

Platelet-Derived TGF-β1 Promotes Deep Vein Thrombosis.

Transforming growth factor-β1 (TGF-β1) modulates multiple cellular functions during development and tissue homeostasis. A large amount of TGF-β1 is stored in platelet α-granules and released upon platelet activation. Whether platelet-derived TGF-β1 plays a role in venous thrombosis remains unclear. This study intends to assess the role of platelet-derived TGF-β1 in the development of venous thrombosis in mice. TGF-β1flox/flox and platelet-specific TGF-β1-/- mice were utilized to assess platelet function in vitro, arterial thrombosis induced by FeCl3, tail bleeding time, prothrombin time (PT), activated partial thromboplastin time (APTT), and deep vein thrombosis induced through ligation of the inferior vena cava (IVC). The IVC sample was collected to measure accumulation of neutrophils, monocytes, and the formation of neutrophil extracellular traps (NETs) by immunofluorescence staining. TGF-β1 deficiency in platelets did not affect the number of circulating platelets, platelet aggregation, adenosine triphosphate release, and integrin αIIbβ3 activation. Meanwhile, TGF-β1 deficiency did not alter the arterial thrombus formation, hemostasis, and coagulation time (PT and APTT), but significantly impaired venous thrombus formation, inhibited the recruitment and accumulation of neutrophils and monocytes in thrombi, as well as reduced formation of NETs and platelet-neutrophil complex. In addition, adoptive transfer of TGF-β1flox/flox platelets to TGF-β1-/- mice rescued the impaired venous thrombus formation, recruitment of leukocytes and monocytes, as well as the NETs formation. In conclusion, platelet-derived TGF-β1 positively modulates venous thrombus formation in mice, indicating that targeting TGF-β1 might be a novel approach for treating venous thrombosis without increasing the risk of bleeding.

Role of circulating mitochondria in venous thrombosis in glioblastoma

BackgroundMany patients with glioblastoma multiforme (GBM) develop deep venous thrombosis or pulmonary emboli. Cell-free circulating mitochondria increase after brain injury and are associated with coagulopathy. ObjectivesThis study evaluated whether mitochondria play a role in the GBM-induced hypercoagulable state. MethodsWe examined the correlation between cell-free circulating mitochondria and venous thrombosis in patients with GBM and the impact of mitochondria on venous thrombosis in mice with inferior vena cava stenosis. ResultsUsing plasma samples of 82 patients with GBM, we found that patients with GBM had a higher number of mitochondria in their plasma (GBM with venous thromboembolism [VTE],: 2.8 × 107 mitochondria/mL; GBM without VTE, 1.9 × 107 mitochondria/mL) than that in healthy control subjects (n = 17) (0.3 × 107 mitochondria/mL). Interestingly, patients with GBM and VTE (n = 41) had a higher mitochondria concentration than patients with GBM without VTE (n = 41). In a murine model of inferior vena cava stenosis, intravenous delivery of mitochondria resulted in an increased rate of venous thrombosis compared with that in controls (70% and 28%, respectively). Mitochondria-induced venous thrombi were neutrophil-rich and contained more platelets than those in control thrombi. Furthermore, as mitochondria are the only source of cardiolipin in circulation, we compared the concentration of anticardiolipin immunoglobulin G in plasma samples of patients with GBM and found a higher concentration in patients with VTE (optical density, 0.69 ± 0.04) than in those without VTE (optical density, 0.51 ± 0.04). ConclusionWe concluded that mitochondria might play a role in the GBM-induced hypercoagulable state. We propose that quantifying circulating mitochondria or anticardiolipin antibody concentrations in patients with GBM might identify patients at increased risk of VTE.

SOD2 in platelets: with age comes responsibility

SOD2 in platelets: with age comes responsibility

Both G protein–coupled and immunoreceptor tyrosine-based activation motif receptors mediate venous thrombosis in mice

AbstractPlatelets are critical in hemostasis and a major contributor to arterial thrombosis (AT). (Pre)clinical studies suggest platelets also contribute to venous thrombosis (VT), but the mechanisms are largely unknown. We hypothesized that in VT, platelets use signaling machinery distinct from AT. Here we aimed to characterize the contributions of platelet G protein–coupled (GPCR) and immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling to VT. Wild-type (WT) and transgenic mice were treated with inhibitors to selectively inhibit platelet-signaling pathways: ITAM-CLEC2 (Clec2mKO), glycoprotein VI (JAQ1 antibody), and Bruton's tyrosine kinase (ibrutinib); GPCR-cyclooxygenase 1 (aspirin); and P2Y12 (clopidogrel). VT was induced by inferior vena cava stenosis. Thrombin generation in platelet-rich plasma and whole-blood clot formation were studied ex vivo. Intravital microscopy was used to study platelet–leukocyte interactions after flow restriction. Thrombus weights were reduced in WT mice treated with high-dose aspirin + clopidogrel (dual antiplatelet therapy [DAPT]) but not in mice treated with either inhibitor alone or low-dose DAPT. Similarly, thrombus weights were reduced in mice with impaired ITAM signaling (Clec2mKO + JAQ1; WT + ibrutinib) but not in Clec2mKO or WT + JAQ1 mice. Both aspirin and clopidogrel, but not ibrutinib, protected mice from FeCl3-induced AT. Thrombin generation and clot formation were normal in blood from high-dose DAPT- or ibrutinib-treated mice; however, platelet adhesion and platelet–neutrophil aggregate formation at the vein wall were reduced in mice treated with high-dose DAPT or ibrutinib. In summary, VT initiation requires platelet activation via GPCRs and ITAM receptors. Strong inhibition of either signaling pathway reduces VT in mice.

Genetic deletion of platelet PAR4 results in reduced thrombosis and impaired hemostatic plug stability

BackgroundProtease‐activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes, and lung epithelial cells. It is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and reduced thrombosis. ObjectiveWe aimed to generate a mouse line with a megakaryocyte/platelet‐specific deletion of PAR4 (PAR4fl/fl;PF4Cre+) and use the mouse line to investigate the role of platelet PAR4 in hemostasis and thrombosis in mice. MethodsPlatelets from PAR4fl/fl;PF4Cre+ were characterized in vitro. Arterial and venous thrombosis was analyzed. Hemostatic plug formation was analyzed using a saphenous vein laser injury model in mice with global or megakaryocyte/platelet‐specific deletion of PAR4 or wild‐type mice treated with thrombin or glycoprotein VI (GPVI) inhibitors. ResultsPAR4fl/fl;PF4Cre+ platelets were unresponsive to thrombin or specific PAR4 stimulation but not to other agonists. PAR4−/− and PAR4fl/fl;PF4Cre+ mice both exhibited a similar reduction in arterial thrombosis compared to their respective controls. More importantly, we show for the first time that platelet PAR4 is critical for venous thrombosis in mice. In addition, PAR4−/− mice and PAR4fl/fl;PF4Cre+ mice exhibited a similar impairment in hemostatic plug stability in a saphenous vein laser injury model. Inhibition of thrombin in wild‐type mice gave a similar phenotype. Combined PAR4 deficiency on platelets with GPVI inhibition did not impair hemostatic plug formation but further reduced plug stability. ConclusionWe generated a novel PAR4fl/fl;PF4Cre+ mouse line. We used this mouse line to show that PAR4 signaling in platelets is critical for arterial and venous thrombosis and hemostatic plug stability.

Podoplanin promotes tumor growth, platelet aggregation, and venous thrombosis in murine models of ovarian cancer

BackgroundPodoplanin (PDPN) is a sialylated membrane glycoprotein that binds to C‐type lectin‐like receptor 2 on platelets resulting in platelet activation. PDPN is expressed on lymphatic endothelial cells, perivascular fibroblasts/pericytes, cancer cells, cancer‐associated fibroblasts, and tumor stromal cells. PDPN's expression on malignant epithelial cells plays a role in metastasis. Furthermore, the expression of PDPN in brain tumors (high‐grade gliomas) was found to correlate with an increased risk of venous thrombosis. ObjectiveWe examined the expression of PDPN and its role in tumor progression and venous thrombosis in ovarian cancer. MethodsWe used mouse models of ovarian cancer and venous thrombosis. ResultsOvarian cancer cells express PDPN and release PDPN‐rich extracellular vesicles (EVs), and cisplatin and topotecan (chemotherapies commonly used in ovarian cancer) increase the expression of podoplanin in cancer cells. The expression of PDPN in ovarian cancer cells promotes tumor growth in a murine model of ovarian cancer and that knockdown of PDPN gene expression results in smaller primary tumors. Both PDPN‐expressing ovarian cancer cells and their EVs cause platelet aggregation. In a mouse model of venous thrombosis, PDPN‐expressing EVs released from HeyA8 ovarian cancer cells produce more frequent thrombosis than PDPN‐negative EVs derived from PDPN‐knockdown HeyA8 cells. Blood clots induced by PDPN‐positive EVs contain more platelets than those in blood clots induced by PDPN‐negative EVs. ConclusionsIn summary, our findings demonstrate that the expression of PDPN by ovarian cancer cells promotes tumor growth and venous thrombosis in mice.

The Small Gtpase Rap1 in Platelets Is Critical for Arterial but Not Venous Thrombosis in Mice

Background: Platelets and their main adhesion receptors, integrins, are critical in hemostasis and arterial thrombosis, i.e., in situations involving severe insult to the vasculature and elevated shear stress, respectively. We recently demonstrated that integrin activation under both of these conditions depends on the small GTPase Rap1 directly activating the integrin adapter protein, Talin1. Our studies further suggested that the Rap1-talin1 axis is less important for platelet function at sites of inflammation, i.e., in situations of mild endothelial insult and low shear stress.Aim: To investigate the contribution of the platelet Rap1-Talin1-integrin signaling axis in the pathogenesis of venous thrombosis (VT).Methods: The following mice with documented deficiencies in platelets were subjected to the inferior vena cava (IVC) stenosis VT model: Rap1amKO (Rap1a fl/fl pf4-Cre+), Rap1bmKO (Rap1b fl/fl pf4-Cre+), or both isoforms (Rap1a fl/flRap1b fl/fl pf4-Cre+ [Rap1dKO]), and Talin1mKO (Tln1 fl/flpf4-Cre+). Thrombus weight was determined 48 hours after flow restriction. Clot contraction and tissue plasminogen activator (tPA)-mediated lysis of whole blood clots were studied ex vivo to characterize effects on clot consolidation and stability .Results: Compared to control mice, thrombus weight was markedly reduced in Talin1mKO mice (13.1±2 and 2.2±1.2 mg, p<0.05), but not Rap1dKO or single knockout mice. Ex vivo clot contraction was significantly impaired in whole blood from Talin1mKO (clot weight: control 13.35±3.4 mg vs. 34.63±4.2 mg), Rap1amKO (control 18.0±3.9 mg vs. 25.6±6.0 mg), Rap1bmKO (control 18.0±3.9 vs 23.0±6.6 mg) and in Rap1dKO (control 18.03±3.9 mg vs. 30.02±4.6 mg). Clot weights were not significantly different between Rap1dKO and Talin1mKO mice. However, ex vivo clot lysis assays revealed that compared to controls, clots formed in whole blood from Talin1mKO mice lysed faster, whereas clots from Rap1dKO did not.Conclusions: Platelet Talin1 is critical for VT in mice, and this role is partially independent of its best-known upstream regulator, Rap1. Future studies will identify the alternative mechanism allowing for Rap1-independent Talin1 signaling and platelet function in VT and how these signaling affect various phases of VT. DisclosuresWolberg: Takeda: Research Funding; Bristol Myers Squibb: Research Funding; CSL Behring: Consultancy.

Tissue Factor and Extracellular Vesicles: Activation of Coagulation and Impact on Survival in Cancer

Simple SummaryThe tissue factor (TF)-factor VIIa complex is the major physiological initiator of blood coagulation. Tumors express TF and release TF-positive extracellular vesicles (EVs) into the circulation, and this is associated with the activation of coagulation. Circulating levels of EVTF activity may be a useful biomarker to identify patients at risk for thrombosis. Tumor TF and TF-positive EVs are also associated with reduced survival.Tissue factor (TF) is a transmembrane glycoprotein that functions as a receptor for FVII/FVIIa and initiates the extrinsic coagulation pathway. Tumors and cancer cells express TF that can be released in the form of TF positive (TF+) extracellular vesicles (EVs). In this review, we summarize the studies of tumor TF and TF + EVs, and their association with activation of coagulation and survival in cancer patients. We also summarize the role of tumor-derived TF + EVs in venous thrombosis in mouse models. Levels of tumor TF and TF + EVs are associated with venous thromboembolism in pancreatic cancer patients. In addition, levels of EVTF activity are associated with disseminated intravascular coagulation in cancer patients. Furthermore, tumor-derived TF + EVs enhance venous thrombosis in mice. Tumor TF and TF + EVs are also associated with worse survival in cancer patients, particularly in pancreatic cancer patients. These studies indicate that EVTF activity could be used as a biomarker to identify pancreatic cancer patients at risk for venous thrombosis and cancer patients at risk for disseminated intravascular coagulation. EVTF activity may also be a useful prognostic biomarker in cancer patients.

DNAse-dependent, NET-independent pathway of thrombus formation in vivo

The contribution of NETs (neutrophil extracellular traps) to thrombus formation has been intensively documented in both arterial and venous thrombosis in mice. We previously demonstrated that adenosine triphosphate (ATP)-activated neutrophils play a key role in initiating the tissue factor-dependent activation of the coagulation cascade, leading to thrombus formation following laser-induced injury. Here, we investigated the contribution of NETs to thrombus formation in a laser-induced injury model. In vivo, treatment of mice with DNase-I significantly inhibited the accumulation of polymorphonuclear neutrophils at the site of injury, neutrophil elastase secretion, and platelet thrombus formation within seconds following injury. Surprisingly, electron microscopy of the thrombus revealed that neutrophils present at the site of laser-induced injury did not form NETs. In vitro, ATP, the main neutrophil agonist present at the site of laser-induced injury, induced the overexpression of PAD4 and CitH3 but not NETosis. However, compared to no treatment, the addition of DNase-I was sufficient to cleave ATP and adenosine diphosphate (ADP) in adenosine. Human and mouse platelet aggregation by ADP and neutrophil activation by ATP were also significantly reduced in the presence of DNase-I. We conclude that following laser-induced injury, neutrophils but not NETs are involved in thrombus formation. Treatment with DNase-I induces the hydrolysis of ATP and ADP, leading to the generation of adenosine and the inhibition of thrombus formation in vivo.

Neutrophil extracellular traps and inflammasomes cooperatively promote venous thrombosis in mice

AbstractDeep vein thrombosis (DVT) is linked to local inflammation. A role for both neutrophil extracellular traps (NETs) and the assembly of inflammasomes (leading to caspase-1–dependent interleukin-1β activation) in the development of DVT was recently suggested. However, no link between these 2 processes in the setting of thrombosis has been investigated. Here, we demonstrate that stimulation of neutrophils induced simultaneous formation of NETs and active caspase-1. Caspase-1 was largely associated with NETs, suggesting that secreted active caspase-1 requires NETs as an adhesive surface. NETs and their components, histones, promoted robust caspase-1 activation in platelets with the strongest effect exerted by histones 3/4. Murine DVT thrombi contained active caspase-1, which peaked at 6 hours when compared with 48-hour thrombi. Platelets constituted more than one-half of cells containing active caspase-1 in dissociated thrombi. Using intravital microscopy, we identified colocalized NETs and caspase-1 as well as platelet recruitment at the site of thrombosis. Pharmacological inhibition of caspase-1 strongly reduced DVT in mice, and thrombi that still formed contained no citrullinated histone 3, a marker of NETs. Taken together, these data demonstrate a cross-talk between NETs and inflammasomes both in vitro and in the DVT setting. This may be an important mechanism supporting thrombosis in veins.

Role of tissue-factor bearing extracellular vesicles released from ovarian cancer cells in platelet aggregation in vitro and venous thrombosis in mice

ObjectiveVenous thromboembolism (VTE) contributes to morbidity and mortality in women with ovarian cancer. Underlying mechanisms of venous thrombosis in ovarian cancer are not well-understood. The aim of this study was to identify the potential role of tissue factor (TF)-bearing extracellular vesicles (EVs) originated from cancer cells in venous thrombosis in ovarian cancer models. MethodsWe examined the expression of TF on EVs generated by ovarian cancer cells and the effect of TF-positive EVs on platelet aggregation. Furthermore, we performed TF-knockdown or induced TF-overexpression in ovarian cancer cell lines and examined the effects of EVs obtained from these cells on platelet aggregation. We examined the effect of TF-bearing EVs originated from ovarian cancer cells on venous thrombosis in a mouse model of inferior vena cava (IVC) stenosis. ResultsTF was expressed in several ovarian cancer cell lines. EVs derived from ovarian cancer cell lines expressing TF promoted platelet aggregation. TF-knockdown in TF-high CAOV3 and OVCAR8 ovarian cancer cells delayed platelet aggregation induced by their EVs in vitro. Conversely, TF overexpression in TF-low A2780 and HeyA8 cells shortened platelet aggregation time induced by their EVs. EVs from TF-overexpressing A2780 ​cells enhanced thrombosis formation in the IVC stenosis model and resulted in a larger clot burden as compared to EVs from A2780 control cells. ConclusionsTF expression in ovarian cancer cell-derived EVs promoted platelet aggregation and thrombosis in preclinical models. These findings may have implications for reducing VTE rates in women with ovarian cancer.

Model‐dependent contributions of FXII and FXI to venous thrombosis in mice

BackgroundThe intrinsic pathway factors (F) XII and FXI have been shown to contribute to thrombosis in animal models. We assessed the role of FXII and FXI in venous thrombosis in three distinct mouse models. MethodsVenous thrombosis was assessed in mice genetically deficient for either FXII or FXI. Three models were used: the inferior vena cava (IVC) stasis, IVC stenosis, and femoral vein electrolytic injury models. ResultsIn the IVC stasis model, FXII and FXI deficiency did not affect the size of thrombi but their absence was associated with decreased levels of fibrin(ogen) and an increased level of the neutrophil extracellular trap marker citrullinated histone H3. In contrast, a deficiency of either FXII or FXI resulted in a significant and equivalent reduction in thrombus weight and incidence of thrombus formation in the IVC stenosis model. Thrombi formed in the IVC stenosis model contained significantly higher levels of citrullinated histone H3 compared with the thrombi formed in the IVC stasis model. Deletion of either FXII or FXI also resulted in a significant and equivalent reduction in both fibrin and platelet accumulation in the femoral vein electrolytic injury model. ConclusionsCollectively, these data indicate that FXII and FXI contribute to the size of venous thrombosis in models with blood flow and thrombus composition in a stasis model. This study also demonstrates the importance of using multiple mouse models to assess the role of a given protein in venous thrombosis.

Update from the laboratory: mechanistic studies of pathways of cancer-associated venous thrombosis using mouse models.

Cancer patients have an increased risk of venous thromboembolism (VTE). The rate of VTE varies with cancer type, with pancreatic cancer having one of the highest rates, suggesting that there are cancer type-specific mechanisms of VTE. Risk assessment scores, such as the Khorana score, have been developed to identify ambulatory cancer patients at high risk of VTE. However, the Khorana score performed poorly in discriminating pancreatic cancer patients at risk of VTE. Currently, thromboprophylaxis is not recommended for cancer outpatients. Recent clinical trials showed that factor Xa (FXa) inhibitors reduced VTE in high-risk cancer patients but also increased major bleeding. Understanding the mechanisms of cancer-associated thrombosis should lead to the development of safer antithrombotic drugs. Mouse models can be used to study the role of different prothrombotic pathways in cancer-associated thrombosis. Human and mouse studies support the notion that 2 prothrombotic pathways contribute to VTE in pancreatic cancer patients: tumor-derived, tissue factor-positive (TF+) extracellular vesicles (EVs), and neutrophils and neutrophil extracellular traps (NETs). In pancreatic cancer patients, elevated levels of plasma EVTF activity and citrullinated histone H3 (H3Cit), a NET biomarker, are independently associated with VTE. We observed increased levels of circulating tumor-derived TF+ EVs, neutrophils, cell-free DNA, and H3Cit in nude mice bearing human pancreatic tumors. Importantly, inhibition of tumor-derived human TF, depletion of neutrophils, or administration of DNAse I to degrade cell-free DNA (including NETs) reduced venous thrombosis in tumor-bearing mice. These studies demonstrate that tumor-derived TF+ EVs, neutrophils, and cell-free DNA contribute to venous thrombosis in a mouse model of pancreatic cancer.

Neutrophils and neutrophil extracellular traps enhance venous thrombosis in mice bearing human pancreatic tumors.

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.

Adenosine receptor agonism protects against NETosis and thrombosis in antiphospholipid syndrome

Potentiation of neutrophil extracellular trap (NET) release is one mechanism by which antiphospholipid antibodies (aPL Abs) effect thrombotic events in patients with antiphospholipid syndrome (APS). Surface adenosine receptors trigger cyclic AMP (cAMP) formation in neutrophils, and this mechanism has been proposed to regulate NETosis in some contexts. Here we report that selective agonism of the adenosine A2A receptor (CGS21680) suppresses aPL Ab-mediated NETosis in protein kinase A-dependent fashion. CGS21680 also reduces thrombosis in the inferior vena cavae of both control mice and mice administered aPL Abs. The antithrombotic medication dipyridamole is known to potentiate adenosine signaling by increasing extracellular concentrations of adenosine and interfering with the breakdown of cAMP. Like CGS21680, dipyridamole suppresses aPL Ab-mediated NETosis via the adenosine A2A receptor and mitigates venous thrombosis in mice. In summary, these data suggest an anti-inflammatory therapeutic paradigm in APS, which may extend to thrombotic disease in the general population.

Unexpected role of natural killer cell‐derived interferon‐γ as a driver of NETosis and DVT

Unexpected role of natural killer cell‐derived interferon‐γ as a driver of NETosis and DVT

Platelet-specific deletion of SNAP23 ablates granule secretion, substantially inhibiting arterial and venous thrombosis in mice

AbstractPlatelet secretion is central to physiological and pathophysiological platelet function. SNAP23 has long been implicated as being a principal SNARE protein regulating platelet granule secretion, although this has not been definitively demonstrated in genetic models. Here, using a platelet-specific conditional SNAP23 knockout mouse, we show that absence of SNAP23 results in complete ablation of dense granule, α granule, and lysosomal secretion. Measured granule cargo content and granule numbers were normal, suggesting SNAP23 regulates fusion of granules with the extracellular membrane, rather than granule loading or formation. A macrothrombocytopenia was also observed, which, combined with ablation of secretion, resulted in a pronounced bleeding defect in a tail bleed assay and almost complete ablation of arterial and venous thrombosis. The macrothrombocytopenia was not due to reduced megakaryopoiesis but instead likely was due to the increased loss of platelets through bleeding, consistent with an increase in platelet total RNA content indicating a greater number of reticulated platelets. The data definitively show SNAP23 to be critical for granule release of any kind from platelets, irrespective of stimulus, and this is the first single gene to be shown to be universally essential for exocytosis in platelets.