4D intravital imaging identifies platelets as the predominant cellular procoagulant surface in a mouse hemostasis model.

Interplay between platelets, coagulation factors, endothelial cells (ECs) and fibrinolytic factors is necessary for effective hemostatic plug formation. This study describes a four-dimensional (4D) imaging platform to visualize and quantify hemostatic plug components in mice with high spatiotemporal resolution. Fibrin accumulation following laser-induced vascular injury was observed at the platelet plug-EC interface, controlled by the antagonistic balance between fibrin generation and breakdown. We observed less fibrin accumulation in mice expressing low levels of tissue factor (TFlow) or F12-/- mice compared to controls, whereas increased fibrin accumulation, including on the vasculature adjacent to the platelet plug, was observed in plasminogen-deficient mice or wild-type mice treated with tranexamic acid (TXA). Phosphatidylserine (PS), a membrane lipid critical for the assembly of coagulation factors, was first detected at the platelet plug-EC interface, followed by exposure across the endothelium. Impaired PS exposure resulted in a significant reduction in fibrin accumulation in cyclophilin D-/- mice. Adoptive transfer studies demonstrated a key role for PS exposure on platelets, and to a lesser degree on ECs, in fibrin accumulation during hemostatic plug formation. Together, these studies suggest that (1) platelets are the functionally dominant procoagulant cellular surface, and (2) plasmin is critical for limiting fibrin accumulation at the site of a forming hemostatic plug.

Using heparan sulfate octadecasaccharide (18-mer) as a multi-target agent to protect against sepsis.

Sepsis is a lethal syndrome manifested by an unregulated, overwhelming inflammation from the host in response to infection. Here, we exploit the use of a synthetic heparan sulfate octadecasaccharide (18-mer) to protect against sepsis. The 18-mer not only inhibits the pro-inflammatory activity of extracellular histone H3 and high mobility group box 1 (HMGB1), but also elicits the anti-inflammatory effect from apolipoprotein A-I (ApoA-I). We demonstrate that the 18-mer protects against sepsis-related injury and improves survival in cecal ligation and puncture mice and reduces inflammation in an endotoxemia mouse model. The 18-mer neutralizes the cytotoxic histone-3 (H3) through direct interaction with the protein. Furthermore, the 18-mer enlists the actions of ApoA-I to dissociate the complex of HMGB1 and lipopolysaccharide, a toxic complex contributing to cell death and tissue damage in sepsis. Our study provides strong evidence that the 18-mer mitigates inflammatory damage in sepsis by targeting numerous mediators, setting it apart from other potential therapies with a single target.

Factor XII contributes to thrombotic complications and vaso-occlusion in sickle cell disease.

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMß2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.

Host fibrinogen drives antimicrobial function in Staphylococcus aureus peritonitis through bacterial-mediated prothrombin activation.

The blood-clotting protein fibrinogen has been implicated in host defense following Staphylococcus aureus infection, but precise mechanisms of host protection and pathogen clearance remain undefined. Peritonitis caused by staphylococci species is a complication for patients with cirrhosis, indwelling catheters, or undergoing peritoneal dialysis. Here, we sought to characterize possible mechanisms of fibrin(ogen)-mediated antimicrobial responses. Wild-type (WT) (Fib+) mice rapidly cleared S. aureus following intraperitoneal infection with elimination of ∼99% of an initial inoculum within 15 min. In contrast, fibrinogen-deficient (Fib-) mice failed to clear the microbe. The genotype-dependent disparity in early clearance resulted in a significant difference in host mortality whereby Fib+ mice uniformly survived whereas Fib- mice exhibited high mortality rates within 24 h. Fibrin(ogen)-mediated bacterial clearance was dependent on (pro)thrombin procoagulant function, supporting a suspected role for fibrin polymerization in this mechanism. Unexpectedly, the primary host initiator of coagulation, tissue factor, was found to be dispensable for this antimicrobial activity. Rather, the bacteria-derived prothrombin activator vWbp was identified as the source of the thrombin-generating potential underlying fibrin(ogen)-dependent bacterial clearance. Mice failed to eliminate S. aureus deficient in vWbp, but clearance of these same microbes in WT mice was restored if active thrombin was administered to the peritoneal cavity. These studies establish that the thrombin/fibrinogen axis is fundamental to host antimicrobial defense, offer a possible explanation for the clinical observation that coagulase-negative staphylococci are a highly prominent infectious agent in peritonitis, and suggest caution against anticoagulants in individuals susceptible to peritoneal infections.

Targeting the AnxA1/Fpr2/ALX pathway regulates neutrophil function, promoting thromboinflammation resolution in sickle cell disease.

Neutrophils play a crucial role in the intertwined processes of thrombosis and inflammation. An altered neutrophil phenotype may contribute to inadequate resolution, which is known to be a major pathophysiological contributor of thromboinflammatory conditions such as sickle cell disease (SCD). The endogenous protein annexin A1 (AnxA1) facilitates inflammation resolution via formyl peptide receptors (FPRs). We sought to comprehensively elucidate the functional significance of targeting the neutrophil-dependent AnxA1/FPR2/ALX pathway in SCD. Administration of AnxA1 mimetic peptide AnxA1Ac2-26 ameliorated cerebral thrombotic responses in Sickle transgenic mice via regulation of the FPR2/ALX (a fundamental receptor involved in resolution) pathway. We found direct evidence that neutrophils with SCD phenotype play a key role in contributing to thromboinflammation. In addition, AnxA1Ac2-26 regulated activated SCD neutrophils through protein kinase B (Akt) and extracellular signal-regulated kinases (ERK1/2) to enable resolution. We present compelling conceptual evidence that targeting the AnxA1/FPR2/ALX pathway may provide new therapeutic possibilities against thromboinflammatory conditions such as SCD.

Plasmin-mediated cleavage of high-molecular-weight kininogen contributes to acetaminophen-induced acute liver failure.

Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor-dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK-/-) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK-/- mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.

Red blood cell microvesicles activate the contact system, leading to factor IX activation via 2 independent pathways.

Storage lesion-induced, red cell-derived microvesicles (RBC-MVs) propagate coagulation by supporting the assembly of the prothrombinase complex. It has also been reported that RBC-MVs initiate coagulation via the intrinsic pathway. To elucidate the mechanism(s) of RBC-MV-induced coagulation activation, the ability of storage lesion-induced RBC-MVs to activate each zymogen of the intrinsic pathway was assessed in a buffer system. Simultaneously, the thrombin generation (TG) assay was used to assess their ability to initiate coagulation in plasma. RBC-MVs directly activated factor XII (FXII) or prekallikrein, but not FXI or FIX. RBC-MVs initiated TG in normal pooled plasma and in FXII- or FXI-deficient plasma, but not in FIX-deficient plasma, suggesting an alternate pathway that bypasses both FXII and FXI. Interestingly, RBC-MVs generated FIXa in a prekallikrein-dependent manner. Similarly, purified kallikrein activated FIX in buffer and initiated TG in normal pooled plasma, as well as FXII- or FXI-deficient plasma, but not FIX-deficient plasma. Dual inhibition of FXIIa by corn trypsin inhibitor and kallikrein by soybean trypsin inhibitor was necessary for abolishing RBC-MV-induced TG in normal pooled plasma, whereas kallikrein inhibition alone was sufficient to abolish TG in FXII- or FXI-deficient plasma. Heating RBC-MVs at 60°C for 15 minutes or pretreatment with trypsin abolished TG, suggesting the presence of MV-associated proteins that are essential for contact activation. In summary, RBC-MVs activate both FXII and prekallikrein, leading to FIX activation by 2 independent pathways: the classic FXIIa-FXI-FIX pathway and direct kallikrein activation of FIX. These data suggest novel mechanisms by which RBC transfusion mediates inflammatory and/or thrombotic outcomes.

Thrombin activation of PAR-1 contributes to microvascular stasis in mouse models of sickle cell disease.

Vaso-occlusive crisis (VOC) is the primary cause of morbidity and hospitalization in sickle cell disease (SCD); however, only 4 therapies (hydroxyurea, l-glutamine, crizanlizumab, and voxeletor) are currently approved in SCD. These agents limit the duration, severity, and frequency of crises. Activation of coagulation is a hallmark of SCD. Studies in animal models of SCD have shown that coagulation contributes to the chronic inflammation and end-organ damage associated with the disease; however, it is unknown whether coagulation directly contributes to the microvascular stasis that causes VOC. Herein, we demonstrate that inhibition of tissue factor (TF) and the downstream coagulation proteases factor Xa and thrombin significantly attenuates heme-induced microvascular stasis in mouse models of VOC. Pharmacologic inhibition of the principal thrombin receptor, protease activated receptor-1 (PAR-1), as well as deficiency of PAR-1 in all nonhematopoietic cells, also reduces stasis in sickle mice. PAR-1 deficiency was associated with reduced endothelial von Willebrand factor expression, which has been shown to mediate microvascular stasis. In addition, TF inhibition reduces lung vaso-occlusion in sickle mice mediated by arteriolar neutrophil-platelet microemboli. In sum, these results suggest that prophylactic anticoagulation might attenuate the incidence of VOC.

Red blood cells modulate structure and dynamics of venous clot formation in sickle cell disease.

Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Erythrocyte sickling, the primary pathologic event in SCD, results in dramatic morphological changes in red blood cells (RBCs) because of polymerization of the abnormal hemoglobin. We used a mouse model of SCD and blood samples from sickle patients to determine if these changes affect the structure, properties, and dynamics of sickle clot formation. Sickling of RBCs and a significant increase in fibrin deposition were observed in venous thrombi formed in sickle mice. During ex vivo clot contraction, the number of RBCs extruded from sickle whole blood clots was significantly reduced compared with the number released from sickle cell trait and nonsickle clots in both mice and humans. Entrapment of sickled RBCs was largely factor XIIIa-independent and entirely mediated by the platelet-free cellular fraction of sickle blood. Inhibition of phosphatidylserine, but not administration of antisickling compounds, increased the number of RBCs released from sickle clots. Interestingly, whole blood, but not plasma clots from SCD patients, was more resistant to fibrinolysis, indicating that the cellular fraction of blood mediates resistance to tissue plasminogen activator. Sickle trait whole blood clots demonstrated an intermediate phenotype in response to tissue plasminogen activator. RBC exchange in SCD patients had a long-lasting effect on normalizing whole blood clot contraction. Furthermore, RBC exchange transiently reversed resistance of whole blood sickle clots to fibrinolysis, in part by decreasing platelet-derived PAI-1. These properties of sickle clots may explain the increased risk of venous thromboembolism observed in SCD.

A pilot study of the effect of rivaroxaban in sickle cell anemia.

INTRODUCTION: The contribution of coagulation activation to the pathogenesis of sickle cell disease (SCD) remains incompletely defined. We evaluated the efficacy and safety of rivaroxaban, an oral direct factor Xa inhibitor, in subjects with sickle cell anemia. MATERIALS AND METHODS: In this pilot, single-center, randomized, double-blind, placebo-controlled, crossover study, eligible subjects with sickle cell anemia received rivaroxaban or placebo. The effect of rivaroxaban on coagulation activation, endothelial activation, inflammation, and microvascular blood flow was evaluated. RESULTS: Fourteen patients (HbSS - 14; females - 9) with mean age of 38 ± 10.6 years were randomized to receive rivaroxaban 20 mg daily or placebo for 4 weeks and, following a 2-week washout phase, were "crossed-over" to the treatment arm opposite to which they were initially assigned. Mean adherence to treatment with rivaroxaban, assessed by pill counts, was 85.6% in the first treatment period and 93.6% in the second period. Treatment with rivaroxaban resulted in a decrease from baseline of thrombin-antithrombin complex versus placebo (-34.4 ug/L [95% CI: -69.4, 0.53] vs. 0.35 ug/L [95% CI: -3.8, 4.5], p = .08), but the difference was not statistically significant. No significant differences were observed in changes from baseline of D-dimer, inflammatory, and endothelial activation markers or measures of microvascular blood flow. Rivaroxaban was well tolerated. CONCLUSIONS: Rivaroxaban was safe but did not significantly decrease coagulation activation, endothelial activation, or inflammation. Rivaroxaban did not improve microvascular blood flow. Adequately powered studies are required to further evaluate the efficacy of rivaroxaban in SCD. Clinicaltrials.gov Identifier: NCT02072668.

Novel Role for the AnxA1-Fpr2/ALX Signaling Axis as a Key Regulator of Platelet Function to Promote Resolution of Inflammation.

BACKGROUND: Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role. METHODS: Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1-/-) mice. Fluorescence intravital microscopy was used to visualize cellular trafficking and to monitor light/dye-induced thrombosis. The mice were treated with vehicle, AnxA1 (3.3 mg/kg), WRW4 (1.8 mg/kg), or all 3, and the effect of AnxA1 was determined in vivo and in vitro. RESULTS: Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1-/- mice. AnxA1 administration regulated platelet function directly (eg, via reducing thromboxane B2 and modulating phosphatidylserine expression) to promote cerebral protection post-I/RI and act as an effective preventative strategy for stroke by reducing platelet activation, aggregate formation, and cerebral thrombosis, a prerequisite for ischemic stroke. To translate these findings into a clinical setting, we show that AnxA1 plasma levels are reduced in human and murine stroke and that AnxA1 is able to act on human platelets, suppressing classic thrombin-induced inside-out signaling events (eg, Akt activation, intracellular calcium release, and Ras-associated protein 1 [Rap1] expression) to decrease αIIbß3 activation without altering its surface expression. AnxA1 also selectively modifies cell surface determinants (eg, phosphatidylserine) to promote platelet phagocytosis by neutrophils, thereby driving active resolution. (n=5-13 mice/group or 7-10 humans/group.) Conclusions: AnxA1 affords protection by altering the platelet phenotype in cerebral I/RI from propathogenic to regulatory and reducing the propensity for platelets to aggregate and cause thrombosis by affecting integrin (αIIbß3) activation, a previously unknown phenomenon. Thus, our data reveal a novel multifaceted role for AnxA1 to act both as a therapeutic and a prophylactic drug via its ability to promote endogenous proresolving, antithromboinflammatory circuits in cerebral I/RI. Collectively, these results further advance our knowledge and understanding in the field of platelet and resolution biology.

In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps.

NETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins.

Elevated hematocrit enhances platelet accumulation following vascular injury.

Red blood cells (RBCs) demonstrate procoagulant properties in vitro, and elevated hematocrit is associated with reduced bleeding and increased thrombosis risk in humans. These observations suggest RBCs contribute to thrombus formation. However, effects of RBCs on thrombosis are difficult to assess because humans and mice with elevated hematocrit typically have coexisting pathologies. Using an experimental model of elevated hematocrit in healthy mice, we measured effects of hematocrit in 2 in vivo clot formation models. We also assessed thrombin generation, platelet-thrombus interactions, and platelet accumulation in thrombi ex vivo, in vitro, and in silico. Compared with controls, mice with elevated hematocrit (RBCHIGH) formed thrombi at a faster rate and had a shortened vessel occlusion time. Thrombi in control and RBCHIGH mice did not differ in size or fibrin content, and there was no difference in levels of circulating thrombin-antithrombin complexes. In vitro, increasing the hematocrit increased thrombin generation in the absence of platelets; however, this effect was reduced in the presence of platelets. In silico, direct numerical simulations of whole blood predicted elevated hematocrit increases the frequency and duration of interactions between platelets and a thrombus. When human whole blood was perfused over collagen at arterial shear rates, elevating the hematocrit increased the rate of platelet deposition and thrombus growth. These data suggest RBCs promote arterial thrombosis by enhancing platelet accumulation at the site of vessel injury. Maintaining a normal hematocrit may reduce arterial thrombosis risk in humans.

Protease-activated receptor 1 activation enhances doxorubicin-induced cardiotoxicity.

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.

It's "PT" for SCD!

In this issue of Blood, Arumugam et al report that long-term reduction of prothrombin (PT) expression results in diminished vascular inflammation, attenuation of multiple organ damage, and survival advantage in a mouse model of sickle cell disease (SCD).

A monocyte-TNF-endothelial activation axis in sickle transgenic mice: Therapeutic benefit from TNF blockade.

Elaboration of tumor necrosis factor (TNF) is a very early event in development of ischemia/reperfusion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S+SAntilles , and SS-BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally activated blood monocytes exerting an endothelial activating effect, dependent upon Egr-1 in both vessel wall and blood cells, and upon NFκB(p50) in a blood cell only. Involvement of TNF was identified by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an IL-1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion), tissue production of neuro-inflammatory mediators, endothelial activation (monitored by tissue factor and VCAM-1 expression), histopathologic liver injury, and three surrogate markers of pulmonary hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular mean systolic pressure). In aggregate, these studies identify a prominent-and possibly dominant-role for an abnormal monocyte-TNF-endothelial activation axis in the sickle context. Its presence, plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should be considered for human sickle cell anemia, a challenging but achievable translational research goal.

Protease-Activated Receptor 1 Contributes to Angiotensin II-Induced Cardiovascular Remodeling and Inflammation.

BACKGROUND: Angiotensin II (Ang II) plays an important role in cardiovascular disease. It also leads to the activation of coagulation. The coagulation protease thrombin induces cellular responses by activating protease-activated receptor 1 (PAR-1). We investigated whether PAR-1 contributes to Ang II-induced cardiovascular remodeling and inflammation. METHODS AND RESULTS: PAR-1+/+ (wild-type; WT) and PAR-1-/- mice were infused with Ang II (600 ng/kg/min) for up to 4 weeks. In WT mice, this dose of Ang II did not cause a significant increase in blood pressure but it did cause pathological changes in both the aorta and the heart. Ang II infusion resulted in vascular remodeling of the aorta, demonstrated by a significant increase in medial wall thickening and perivascular fibrosis. Importantly, both parameters were significantly attenuated by PAR-1 deficiency. Furthermore, perivascular fibrosis around coronary vessels was reduced in Ang II-treated PAR-1-/- mice compared to WT mice. In addition, PAR-1 deficiency significantly attenuated Ang II induction of inflammatory cytokines and profibrotic genes in the aortas compared to WT mice. Finally, PAR-1 deficiency had no effect on Ang II-induced heart hypertrophy. However, the heart function measured by fractional shortening was less impaired in PAR-1-/- mice than in WT mice. CONCLUSION: Our data indicate that PAR-1 plays a significant role in cardiovascular remodeling mediated by a blood pressure-independent action of Ang II.

Tissue factor expression provokes escape from tumor dormancy and leads to genomic alterations.

The coagulation system links immediate (hemostatic) and late (inflammatory, angiogenic) tissue responses to injury, a continuum that often is subverted in cancer. Here we provide evidence that tumor dormancy is influenced by tissue factor (TF), the cancer cell-associated initiator of the coagulation system and a signaling receptor. Thus, indolent human glioma cells deficient for TF remain viable but permanently dormant at the injection site for nearly a year, whereas the expression of TF leads to a step-wise transition to latent and overt tumor growth phases, a process that is preceded by recruitment of vascular (CD105(+)) and myeloid (CD11b(+) and F4/80(+)) cells. Importantly, the microenvironment orchestrated by TF expression drives permanent changes in the phenotype, gene-expression profile, DNA copy number, and DNA methylation state of the tumor cells that escape from dormancy. We postulate that procoagulant events in the tissue microenvironment (niche) may affect the fate of occult tumor cells, including their biological and genetic progression to initiate a full-blown malignancy.

Hepatocyte tissue factor contributes to the hypercoagulable state in a mouse model of chronic liver injury.

BACKGROUND & AIMS: Patients with chronic liver disease and cirrhosis have a dysregulated coagulation system and are prone to thrombosis. The basis for this hypercoagulable state is not completely understood. Tissue factor (TF) is the primary initiator of coagulation in vivo. Patients with cirrhosis have increased TF activity in white blood cells and circulating microparticles. The aim of our study was to determine the contribution of TF to the hypercoagulable state in a mouse model of chronic liver injury. METHODS: We measured levels of TF activity in the liver, white blood cells and circulating microparticles, and a marker of activation of coagulation (thrombin-antithrombin complexes (TATc)) in the plasma of mice subjected to bile duct ligation for 12days. We used wild-type mice, mice with a global TF deficiency (low TF mice), and mice deficient for TF in either myeloid cells (TF(flox/flox),LysMCre mice) or in hepatocytes (TF(flox/flox),AlbCre). RESULTS: Wild-type mice with liver injury had increased levels of white blood cell, microparticle TF activity and TATc compared to sham mice. Low TF mice and mice lacking TF in hepatocytes had reduced levels of TF in the liver and in microparticles and exhibited reduced activation of coagulation without a change in liver fibrosis. In contrast, mice lacking TF in myeloid cells had reduced white blood cell TF but no change in microparticle TF activity or TATc. CONCLUSIONS: Hepatocyte TF activates coagulation in a mouse model of chronic liver injury. TF may contribute to the hypercoagulable state associated with chronic liver diseases in patients.