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Activation of the intrinsic pathway of coagulation contributes to the pathogenesis of arterial and venous thrombosis. Critical insights into the involvement of intrinsic pathway factors have been derived from the study of gene-specific knockout animals and targeted inhibitors. Importantly, preclinical studies have indicated that targeting components of this pathway, including FXI (factor XI), FXII, and PKK (prekallikrein), reduces thrombosis with no significant effect on protective hemostatic pathways. This review highlights the advances made from studying the intrinsic pathway using gene-specific knockout animals and inhibitors in models of arterial and venous thrombosis. Development of inhibitors of activated FXI and FXII may reduce thrombosis with minimal increases in bleeding compared with current anticoagulant drugs.

Activation of the blood coagulation cascade leads to fibrin deposition and platelet activation that are required for hemostasis. However, aberrant activation of coagulation can lead to thrombosis. Thrombi can cause tissue ischemia, and fibrin degradation products and activated platelets can enhance inflammation. In addition, coagulation proteases activate cells by cleavage of PARs (protease-activated receptors), including PAR1 and PAR2. Direct oral anticoagulants have recently been developed to specifically inhibit the coagulation proteases FXa (factor Xa) and thrombin. Administration of these inhibitors to wild-type mice can be used to determine the roles of FXa and thrombin in different inflammatory diseases. These results can be compared with the phenotypes of mice with deficiencies of either Par1 (F2r) or Par2 (F2rl1). However, inhibition of coagulation proteases will have effects beyond reducing PAR signaling, and a deficiency of PARs will abolish signaling from all proteases that activate these receptors. We will summarize studies that examine the roles of coagulation proteases, particularly FXa and thrombin, and PARs in different mouse models of inflammatory disease. Targeting FXa and thrombin or PARs may reduce inflammatory diseases in humans.

Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

Background: Hypoxia within acute venous thrombi is thought to drive resolution through stabilisation of hypoxia inducible factor 1 alpha (HIF1α). Prolyl hydroxylase domain (PHD) isoforms are critical regulators of HIF1α stability. Non-selective inhibition of PHD isoforms with l-mimosine has been shown to increase HIF1α stabilisation and promote thrombus resolution., Objective: The aim of this study was to investigate the therapeutic potential of PHD inhibition in venous thrombus resolution., Methods: Thrombosis was induced in the inferior vena cava of mice using a combination of flow restriction and endothelial activation. Gene and protein expression of PHD isoforms in the resolving thrombus was measured by RT-PCR and immunohistochemistry. Thrombus resolution was quantified in mice treated with pan PHD inhibitors AKB-4924 and JNJ-42041935 or inducible all-cell Phd2 knockouts by micro-computed tomography, 3D high frequency ultrasound or endpoint histology., Results: Resolving venous thrombi demonstrated significant temporal gene expression profiles for PHD2 and PHD3 (P < 0.05), but not for PHD1. PHD isoform protein expression was localised to early and late inflammatory cell infiltrates. Treatment with selective pan PHD inhibitors, AKB-4924 and JNJ-42041935, enhanced thrombus neovascularisation (P < 0.05), but had no significant effect on overall thrombus resolution. Thrombus resolution or its markers, macrophage accumulation and neovascularisation, did not differ significantly in inducible all-cell homozygous Phd2 knockouts compared with littermate controls (P > 0.05)., Conclusions: This data suggests that PHD-mediated thrombus neovascularisation has a limited role in the resolution of venous thrombi. Directly targeting angiogenesis alone may not be a viable therapeutic strategy to enhance venous thrombus resolution., (Copyright © 2018. Published by Elsevier Ltd.)

Objective: The anti-cancer anthracycline drug Doxorubicin (Dox) causes cardiotoxicity. We investigated the role of protease-activated receptor 1 (PAR-1) in Dox-induced cardiotoxicity., Methods and Results: In vitro experiments revealed that PAR-1 enhanced Dox-induced mitochondrial dysfunction, reactive oxygen species and cell death of cardiac myocytes and cardiac fibroblasts. The contribution of PAR-1 to Dox-induced cardiotoxicity was investigated by subjecting PAR-1 -/- mice and PAR-1 +/+ mice to acute and chronic exposure to Dox. Heart function was measured by echocardiography. PAR-1 -/- mice exhibited significant less cardiac injury and dysfunction compared to PAR-1 +/+ mice after acute and chronic Dox administration. PAR-1 -/- mice had reduced levels of nitrotyrosine, apoptosis and inflammation in their heart compared to PAR-1 +/+ mice. Furthermore, inhibition of PAR-1 in wild-type mice with vorapaxar significantly reduced the acute Dox-induced cardiotoxicity., Conclusion: Our results indicate that activation of PAR-1 contributes to Dox-induced cardiotoxicity. Inhibition of PAR-1 may be a new approach to reduce Dox-induced cardiotoxicity in cancer patients., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Rivaroxaban selectively inhibits factor Xa (FXa), which plays a central role in blood coagulation. In addition, FXa activates protease-activated receptor-2 (PAR-2). We have shown that PAR-2 -/- mice exhibit less cardiac dysfunction after cardiac injury., Material and Methods: Wild-type (WT) and PAR-2 -/- mice were subjected to left anterior descending artery (LAD) ligation to induce cardiac injury and heart failure. Mice received either placebo or rivaroxaban chow either starting at the time of surgery or 3 days after surgery and continued up to 28 days. Cardiac function was measured by echocardiography pre-surgery and 3, 7 and 28 days after LAD ligation. We also measured anticoagulation, intravascular thrombi, infarct size, cardiac hypertrophy and inflammation at various times., Results: Rivaroxaban increased the prothrombin time and inhibited the formation of intravascular thrombi in mice subjected to LAD ligation. WT mice receiving rivaroxaban immediately after surgery had similar infarct sizes at day 1 as controls but exhibited significantly less impairment of cardiac function at day 3 and beyond compared to the placebo group. Rivaroxaban also inhibited the expansion of the infarct at day 28. Rivaroxaban did not significantly affect the expression of inflammatory mediators or a neutrophil marker at day 2 after LAD ligation. Delaying the start of rivaroxaban administration until 3 days after surgery failed to preserve cardiac function. In addition, rivaroxaban did not reduce cardiac dysfunction in PAR-2 -/- mice., Conclusions: Early administration of rivaroxaban preserves cardiac function in mice after LAD ligation., (Copyright © 2018. Published by Elsevier Ltd.)

Tissue factor (TF) is the high-affinity receptor and cofactor for factor (F)VII/VIIa. The TF-FVIIa complex is the primary initiator of blood coagulation and plays an essential role in hemostasis. TF is expressed on perivascular cells and epithelial cells at organ and body surfaces where it forms a hemostatic barrier. TF also provides additional hemostatic protection to vital organs, such as the brain, lung, and heart. Under pathological conditions, TF can trigger both arterial and venous thrombosis. For instance, atherosclerotic plaques contain high levels of TF on macrophage foam cells and microvesicles that drives thrombus formation after plaque rupture. In sepsis, inducible TF expression on monocytes leads to disseminated intravascular coagulation. In cancer patients, tumors release TF-positive microvesicles into the circulation that may contribute to venous thrombosis. TF also has nonhemostatic roles. For instance, TF-dependent activation of the coagulation cascade generates coagulation proteases, such as FVIIa, FXa, and thrombin, which induce signaling in a variety of cells by cleavage of protease-activated receptors. This review will focus on the roles of TF in protective hemostasis and pathological thrombosis., (© 2018 American Heart Association, Inc.)

Thiol isomerases such as protein-disulfide isomerase (PDI) direct disulfide rearrangements required for proper folding of nascent proteins synthesized in the endoplasmic reticulum. Identifying PDI substrates is challenging because PDI catalyzes conformational changes that cannot be easily monitored ( e.g. compared with proteolytic cleavage or amino acid phosphorylation); PDI has multiple substrates; and it can catalyze either oxidation, reduction, or isomerization of substrates. Kinetic-based substrate trapping wherein the active site motif CGHC is modified to CGHA to stabilize a PDI-substrate intermediate is effective in identifying some substrates. A limitation of this approach, however, is that it captures only substrates that are reduced by PDI, whereas many substrates are oxidized by PDI. By manipulating the highly conserved -GH- residues in the CGHC active site of PDI, we created PDI variants with a slowed reaction rate toward substrates. The prolonged intermediate state allowed us to identify protein substrates that have biased affinities for either oxidation or reduction by PDI. Because extracellular PDI is critical for thrombus formation but its extracellular substrates are not known, we evaluated the ability of these bidirectional trapping PDI variants to trap proteins released from platelets and on the platelet surface. Trapped proteins were identified by mass spectroscopy. Of the trapped substrate proteins identified by mass spectroscopy, five proteins, cathepsin G, glutaredoxin-1, thioredoxin, GP1b, and fibrinogen, showed a bias for oxidation, whereas annexin V, heparanase, ERp57, kallekrein-14, serpin B6, tetranectin, and collagen VI showed a bias for reduction. These bidirectional trapping variants will enable more comprehensive identification of thiol isomerase substrates and better elucidation of their cellular functions., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Deep vein thrombosis and common complications, including pulmonary embolism and post-thrombotic syndrome, represent a major source of morbidity and mortality worldwide. Experimental models of venous thrombosis have provided considerable insight into the cellular and molecular mechanisms that regulate thrombus formation and subsequent resolution. Here, we critically appraise the ex vivo and in vivo techniques used to assess venous thrombosis in these models. Particular attention is paid to imaging modalities, including magnetic resonance imaging, micro-computed tomography, and high-frequency ultrasound that facilitate longitudinal assessment of thrombus size and composition., (© 2015 American Heart Association, Inc.)

Introduction: The assessment of thrombus size following treatments directed at preventing thrombosis or enhancing its resolution has generally relied on physical or histological methods. This cross-sectional design imposes the need for increased numbers of animals for experiments. Micro-computed tomography (microCT) has been used to detect the presence of venous thrombus in experimental models but has yet to be used in a quantitative manner. In this study, we investigate the use of contrast-enhanced microCT for the longitudinal assessment of experimental venous thrombus resolution., Materials and Methods: Thrombi induced by stenosis of the inferior vena cava in mice were imaged by contrast-enhanced microCT at 1, 7 and 14 days post-induction (n=18). Thrombus volumes were determined longitudinally by segmentation and 3D volume reconstruction of microCT scans and by standard end-point histological analysis at day 14. An additional group of thrombi were analysed solely by histology at 1, 7 and 14 days post-induction (n=15)., Results: IVC resident thrombus was readily detectable by contrast-enhanced microCT. MicroCT-derived measurements of thrombus volume correlated well with time-matched histological analyses (ICC=0.75, P<0.01). Thrombus volumes measured by microCT were significantly greater than those derived from histological analysis (P<0.001). Intra- and inter-observer analyses were highly correlated (ICC=0.99 and 0.91 respectively, P<0.0001). Further histological analysis revealed noticeable levels of contrast agent extravasation into the thrombus that was associated with the presence of neovascular channels, macrophages and intracellular iron deposits., Conclusion: Contrast-enhanced microCT represents a reliable and reproducible method for the longitudinal assessment of venous thrombus resolution providing powerful paired data., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Surgery is associated with an increased risk of venous thromboembolic events (VTE) including deep vein thrombosis and pulmonary embolism. Although the current treatment regiments such as mechanical manipulation and administration of pharmacological prophylaxis significantly reduced the incidence of postsurgical VTE, they remain a major cause of postoperative morbidity and mortality worldwide. The pathophysiology of venous thrombosis traditionally emphasizes the series of factors that constitute Virchow triad of factors. However, inflammation can also be a part of this by giving rise to a hypercoagulable state and endothelial damage. The inflammatory response after surgery, which is initiated by a cytokine "storm" and occurs within hours of surgery, creates a prothrombotic environment that is further accentuated by several cellular processes including neutrophil extracellular traps formation, platelet activation, and the generation of tissue factor-bearing microparticles. Although such inflammatory markers are elevated in undergoing surgery, the precise mechanism by which they give rise to venous thrombosis is poorly understood. Here, we discuss the potential mechanisms linking inflammation to thrombosis, and highlight strategies that may minimize surgical inflammation and reduce the incidence of postoperative VTE., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Savanna ecosystems comprise 22% of the global terrestrial surface and 25% of Australia (almost 1.9 million km2) and provide significant ecosystem services through carbon and water cycles and the maintenance of biodiversity. The current structure, composition and distribution of Australian savannas have coevolved with fire, yet remain driven by the dynamic constraints of their bioclimatic niche. Fire in Australian savannas influences both the biophysical and biogeochemical processes at multiple scales from leaf to landscape. Here, we present the latest emission estimates from Australian savanna biomass burning and their contribution to global greenhouse gas budgets. We then review our understanding of the impacts of fire on ecosystem function and local surface water and heat balances, which in turn influence regional climate. We show how savanna fires are coupled to the global climate through the carbon cycle and fire regimes. We present new research that climate change is likely to alter the structure and function of savannas through shifts in moisture availability and increases in atmospheric carbon dioxide, in turn altering fire regimes with further feedbacks to climate. We explore opportunities to reduce net greenhouse gas emissions from savanna ecosystems through changes in savanna fire management., (© 2014 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.)

Introduction: The formation of new vascular channels within and around venous thrombus contributes to its resolution. Neovascularisation arising from the surrounding vein may facilitate this process. Treatment of cancer patients with anti-angiogenic agents can lead to increased incidence of venous thromboembolic events, but the effect of these agents on the processes that govern thrombus resolution are unclear. The aim of this study was to determine the effect of anti-angiogenic treatment with 2-methoxyestradiol (2ME) on (i) angiogenic response in the thrombosed vein and (ii) venous thrombus resolution., Materials and Methods: Venous thrombus was induced in the inferior vena cava (IVC) of 36 adult male BALB/C mice. Thrombosed mice received either the anti-angiogenic agent, 2ME (150 mg/kg/day, i/p), or vehicle control (n=18/group). In the thrombosed IVC of both groups: hypoxia-inducible factor (HIF) 1α, and its angiogenic targets, vascular endothelial growth factor (VEGF) and placental growth factor (PLGF), were quantified using enzyme-linked immunosorbent assays at days 1 and 10 post-thrombus induction (n=6/group); and inflammatory cell content, cell proliferation, and vein recanalisation were quantified using immunostaining and image analysis at day 10 (n=6/group)., Results: In the IVC of mice treated with 2ME compared with control: HIF1α (P<0.005 and P<0.02), VEGF (P<0.005 and P<0.02), and PLGF levels (P<0.01 and P<0.001) were reduced at days 1 and 10 post-thrombus induction respectively, and macrophage content (P<0.005), neutrophil content (P<0.01), vein recanalistion (P<0.05), and thrombus resolution (P<0.001) were also reduced at day 10., Conclusions: Anti-angiogenic treatment with 2ME supressed the HIF1-mediated angiogenic drive in local vein wall and attenuated venous thrombus resolution. The potential pro-thrombotic effect of anti-angiogenic agents should be carefully considered when managing venous thromboembolic events in cancer patients., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: Deep venous thrombosis is a major health problem. Thrombolytic therapies are effective in recanalizing the veins and preventing post-thrombotic complications, but there is no consensus on selection criteria. The aim of this study was to investigate a fibrin-specific MRI contrast agent (EP-2104R) for the accurate quantification of thrombus' fibrin content in vivo and for the identification of thrombus suitable for thrombolysis., Approach and Results: Venous thrombosis was induced in the inferior vena cava of 8- to 10-week-old male BALB/C mice and MRI performed 2, 4, 7, 10, 14, and 21 days later. Eighteen mice were scanned at each time point pre and 2 hours post injection of EP-2104R (8.0 μmol/kg) with 12 mice at each time point used to correlate fibrin contrast uptake with thrombus' histological stage and fibrin content. Six mice at each time point were immediately subjected to intravascular thrombolytic therapy (10 mg/kg of tissue-type plasminogen activator). Mice were imaged to assess response to lytic therapy 24 hours after thrombolytic treatment. Two mice at each time point were scanned post injection of 0.2 mmol/kg of Gd-DTPA (gadolinium with diethylenetriaminepentacetate, Magnevist, Schering AG, Berlin, Germany) for control purpose. Contrast uptake was correlated positively with the fibrin content of the thrombus measured by Western blotting (R(2)=0.889; P<0.001). Thrombus relaxation rate (R1) post contrast and the change in visualized thrombus size on late gadolinium enhancement inversion recovery MRI pre-EP-2104R and post-EP-2104R injection were the best predictors for successful thrombolysis (area under the curve, 0.989 [95% confidence interval, 0.97-1.00] and 0.994 [95% confidence interval, 0.98-1.00] respectively)., Conclusions: MRI with a fibrin-specific contrast agent accurately estimates thrombus fibrin content in vivo and identifies thrombi that are amenable for thrombolysis., (© 2014 American Heart Association, Inc.)

Objective: Venous thromboembolism is a common complication in patients with cancer, resulting in significant morbidity and mortality. Clinical studies suggest that the incidence of venous thromboembolic events increased after treatment of these patients with antiangiogenic agents. Thrombi resolve through a process of remodeling, involving the formation of microvascular channels within the thrombus. Our aim was to determine whether inhibiting angiogenesis affects venous thrombus resolution., Approach and Results: Thrombus was induced in the inferior vena cava of mice. These mice were treated with axitinib (50 mg/kg per day), 2-methoxyestradiol (2ME, 150 mg/kg per day), or vehicle control. Thrombus size, recanalization, neovascularization, inflammatory cell content, and collagen content were assessed after axitinib (days 3, 10, 17) and 2ME (day 10 only) treatment (n=6/group). Axitinib treatment resulted in reduced thrombus resolution (P<0.002) and vein recanalization (P<0.001) compared with vehicle-treated controls. This was associated with inhibition of organization as seen through reduced thrombus neovascularization (P<0.0001) and collagen (P<0.0001) content, as well as reduced macrophage accumulation in the thrombus (P<0.001). Treatment with a second antiangiogenic agent, 2ME, mirrored these findings, with a similar order of magnitude of effect of treatment over vehicle control in all of the parameters measured, with the exception of neutrophil content, which was significantly reduced after 2ME treatment but not affected by axitinib., Conclusions: Antiangiogenic therapy (using axitinib and 2ME) inhibits the resolution of venous thrombi, which could lead to persistent venous obstruction and the possibility of thrombus extension. This potential prolongation of venous occlusion by antiangiogenic agents should therefore be taken into consideration in trials of these agents and when managing the complications of venous thromboembolic events in patients with cancer.

Designed, green infrastructures are becoming a customary feature of the urban landscape. Sustainable technologies for stormwater management, and biofilters in particular, are increasingly used to reduce stormwater runoff volumes and peaks as well as improve the water quality of runoff discharged into urban water bodies. Although a lot of research has been devoted to these technologies, their effect in terms of greenhouse gas fluxes in urban areas has not been yet investigated. We present the first study aimed at quantifying greenhouse gas fluxes between the soil of stormwater biofilters and the atmosphere. N2O, CH4, and CO2 were measured periodically over a year in two operational vegetated biofiltration cells at Monash University in Melbourne, Australia. One cell had a saturated zone at the bottom, and compost and hardwood mulch added to the sandy loam filter media. The other cell had no saturated zone and was composed of sandy loam. Similar sedges were planted in both cells. The biofilter soil was a small N2O source and a sink for CH4 for most measurement events, with occasional large emissions of both N2O and CH4 under very wet conditions. Average N2O fluxes from the cell with the saturated zone were almost five-fold greater (65.6 μg N2O-N m(-2) h(-1)) than from the other cell (13.7 μg N2O-N m(-2) h(-1)), with peaks up to 1100 μg N2O-N m(-2) h(-1). These N2O fluxes are of similar magnitude to those measured in other urban soils, but with larger peak emissions. The CH4 sink strength of the cell with the saturated zone (-3.8 μg CH4-C m(-2) h(-1)) was lower than the other cell (-18.3 μg CH4-C m(-2) h(-1)). Both cells of the biofilter appeared to take up CH4 at similar rates to other urban lawn systems; however, the biofilter cells displayed occasional large CH4 emissions following inflow events, which were not seen in other urban systems. CO2 fluxes increased with soil temperature in both cells, and in the cell without the saturated zone CO2 fluxes decreased as soil moisture increased. Other studies of CO2 fluxes from urban soils have found both similar and larger CO2 emissions than those measured in the biofilter. The results of this study suggest that the greenhouse gas footprint of stormwater treatment warrant consideration in the planning and implementation of engineered green infrastructures., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The magnetic resonance longitudinal relaxation time (T1) changes with thrombus age in humans. In this study, we investigate the possible mechanisms that give rise to the T1 signal in venous thrombi and whether changes in T1 relaxation time are informative of the susceptibility to lysis., Methods and Results: Venous thrombosis was induced in the vena cava of BALB/C mice, and temporal changes in T1 relaxation time correlated with thrombus composition. The mean T1 relaxation time of thrombus was shortest at 7 days following thrombus induction and returned to that of blood as the thrombus resolved. T1 relaxation time was related to thrombus methemoglobin formation and further processing. Studies in inducible nitric oxide synthase (iNOS(-/-))-deficient mice revealed that inducible nitric oxide synthase mediates oxidation of erythrocyte lysis-derived iron to paramagnetic Fe3+, which causes thrombus T1 relaxation time shortening. Studies using chemokine receptor-2-deficient mice (Ccr2(-/-)) revealed that the return of the T1 signal to that of blood is regulated by removal of Fe3+ by macrophages that accumulate in the thrombus during its resolution. Quantification of T1 relaxation time was a good predictor of successful thrombolysis with a cutoff point of <747 ms having a sensitivity and specificity to predict successful lysis of 83% and 94%, respectively., Conclusions: The source of the T1 signal in the thrombus results from the oxidation of iron (released from the lysis of trapped erythrocytes in the thrombus) to its paramagnetic Fe3+ form. Quantification of T1 relaxation time appears to be a good predictor of the success of thrombolysis.

A third of patients with critical limb ischemia (CLI) will eventually require limb amputation. Therapeutic neovascularization using unselected mononuclear cells to salvage ischemic limbs has produced modest results. The TIE2-expressing monocytes/macrophages (TEMs) are a myeloid cell subset known to be highly angiogenic in tumours. This study aimed to examine the kinetics of TEMs in patients with CLI and whether these cells promote neovascularization of the ischemic limb. Here we show that there are 10-fold more circulating TEMs in CLI patients, and removal of ischemia reduces their numbers to normal levels. TEM numbers in ischemic muscle are two-fold greater than normoxic muscle from the same patient. TEMs from patients with CLI display greater proangiogenic activity than TIE2-negative monocytes in vitro. Using a mouse model of hindlimb ischemia, lentiviral-based Tie2 knockdown in TEMs impaired recovery from ischemia, whereas delivery of mouse macrophages overexpressing TIE2, or human TEMs isolated from CLI patients, rescued limb ischemia. These data suggest that enhancing TEM recruitment to the ischemic muscle may have the potential to improve limb neovascularization in CLI patients., (Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)