Plasma Thrombin-antithrombin Levels Research Articles

Neutrophil Extracellular Traps (NETs) Thrombogenicity and Endothelial Cell Toxicity Are Ameliorated By Platelet Factor 4 (PF4): Implications to the Treatment of Sepsis

Sepsis, the dysregulated response to infection, is a leading cause of mortality worldwide for which there are no targeted treatments. Neutrophils play a crucial role in sepsis by releasing NETs, webs of DNA complexed with histones and antimicrobial proteins that capture pathogens. However, when NETs are degraded by circulating nucleases, they release NET-degradation products (NDPs) including cell-free (cf) DNA, histones and myeloperoxidase, which trigger thrombosis, induce complement activation and cause oxidative vascular damage. We propose a novel NET-directed therapy in sepsis, in which NETs are stabilized by the platelet chemokine PF4 (CXCL4), which enhances NET resistance to DNase degradation, promoting NDP sequestration and bacterial capture, improving sepsis outcomes. As NETs are traditionally considered to be prothrombotic, we investigated whether PF4-stabilization alters the thrombogenicity of NETs. We examined the effect of PF4 on both NET- and DNA-mediated thrombus formation and endothelial cell injury using NET and DNA fragments of different lengths. Human neutrophils were stimulated to release NETs and treated with mild DNase I (4U/mL) to liberate high-molecular-weight (hmw) NETs (>50kb), and then digested with restriction enzymes to generate low-molecular-weight (lmw) fragments of ~4kb and ~250bp. We also studied single-stranded (ss) DNA, generated by heating double stranded (ds) DNA fragments to 100°C. We assessed the effects of PF4 on NETs/dsDNA/ssDNA-induced thrombin and fibrin generation in normal and in factor (F) XII- and FXI-depleted plasma. PF4 significantly reduced plasma thrombin and fibrin generation induced by digested NET and DNA fragments and by ssDNA without impairing thrombolysis. PF4 did not exert any effects on DNA-induced fibrin generation in FXII- or FXI-depleted plasma, while supplementation with purified FXII or FXI restored the anticoagulant effects of PF4, suggesting that PF4 binding reduces the ability of cfDNA and NETs to activate the contact pathway of coagulation. We next studied how PF4 alters the ability of DNA to induce a prothrombotic phenotype in human umbilical vein endothelial cells (HUVECs) incubated with dsDNA or ssDNA fragments by measuring von Willebrand factor (VWF) release with immunofluorescence and by quantifying tissue factor (TF) expression with a chromogenic substrate assay. To study the mechanism of endothelial activation, HUVECs were treated with inhibitors of toll-like receptor (TLR) 9, a pattern recognition receptor that binds to dsDNA. Unlike intact NETs or hmwDNA, both digested dsDNA and ssDNA upregulated VWF release and TF expression by endothelial cells via a TLR9-dependent mechanism, which was prevented by the addition of PF4. To study these observations in vivo, hmwDNA was administered intravenously to wildtype (WT) and Cxcl4-/- mice and plasma thrombin-anti-thrombin (TAT) levels were measured 4h post infusion. Cxcl4-/- mice exhibited higher TAT levels as compared to WT mice. We also studied two distinct sepsis-like states: (1) WT mice exposed to lipopolysaccharide (LPS, IP; 35mg/kg) ± human (h)PF4 (IV; 40mg/kg), where plasma cfDNA and TAT levels were measured at 6h, and (2) the cecal slurry model of polymicrobial infection in endothelial-specific TLR9-/- mice, where plasma TAT levels were measured at 4h. In the LPS endotoxemia model (1), TAT and cfDNA levels were elevated at 6h post LPS challenge, which were prevented with the administration of hPF4. In the cecal slurry model (2), endothelial TLR9-/- mice exhibited lower TAT levels at 4h post challenge as compared to WT controls. In summary, while intact NETs help to contain infection, NET degradation liberates lmwDNA and ssDNA that activate the contact pathway and induce a prothrombotic phenotype in endothelial cells via activation of TLR9. PF4-stabilization of NETs decreases the risk of NET-mediated toxicity by (1) inhibiting DNase cleavage of intact NETs and (2) neutralizing released lmwDNA and ssDNA. NET stabilization by PF4 tips the balance, limiting NET- and NDP-induced thrombosis and vascular toxicity, while enhancing NET's ability to fight infection by microbial entrapment. These studies add support to our hypothesis that PF4 stabilization of NETs is protective in sepsis. Strategies that augment the beneficial effects of PF4 merit further investigation.

Parmodulin, an Allosteric Inhibitor of Protease Activated Receptor 1 (PAR1) Protects Sickle Cell Mice from Thromboinflammation and Endothelial Dysfunction

Protease activated receptor 1 (PAR1) couples the coagulation cascade to endothelial inflammation in many diseases, including sickle cell disease (SCD). PAR1 is a GPCR activated by proteolytic cleavage of an extracellular N-terminus, and demonstrates biased signaling. Activation of PAR1 by thrombin stimulates prothrombotic and proinflammatory signaling, whereas activation by activated protein C (APC) stimulates cytoprotective and anti-inflammatory pathways. The ideal PAR-1 targeted therapy would inhibit detrimental signaling while preserving beneficial signaling. 3K3A-APC is a signaling selective variant of APC that activates cytoprotective PAR1 signaling with minimal anticoagulant effects, demonstrated to have a beneficial role in stroke models. Parmodulin 2 (PM2) is a small molecule allosteric inhibitor of PAR1 that simultaneously blocks detrimental signaling and activate beneficial cytoprotective signaling similar to that induced by APC. We previously demonstrated that endogenous APC-PAR1 signaling contributes to thrombin generation and inflammation in a mouse model of SCD, whereas thrombin-PAR signaling enhances these pathways. We hypothesize that inducing cytoprotective PAR1 signaling with either 3K3A-APC and PM2 will reduce thrombo-inflammation in sickle cell mice at steady state. Townes wild type (HbAA) and sickle (HbSS) mice (male and female, 4 months old) were used in the studies. In the first experiment, HbAA and HbSS (n=4-5 per group) mice were treated daily with either saline (SAL) or 3K3A-APC (0.2mg/kg, BW) for 4 days. Samples were collected 1 hour after final dose. As previously demonstrated, plasma levels of thrombin-anti thrombin (TAT) complexes, the proinflammatory cytokine IL-6, high mobility group box 1 (HMGB1), and, soluble vascular cell adhesion molecule (sVCAM), were increased in HbSS mice compared to HbAA controls. Surprisingly 3K3A-APC treatment did reduce any of these parameters in HbSS mice. APC and its variant 3K3A-APC require binding to the endothelial protein C receptor (EPCR) to activate PAR1; it is known that SCD causes increased EPCR shedding. Indeed, we found that both SAL- and 3K3A-APC treated HbSS mice had elevated sEPCR levels compared to HbAA controls. Together, these data indicate that 3K3A-APC does not affect thrombo-inflammation in HbSS mice at steady state most likely due to by reduced ECPR expression on the endothelium. In the second experiment, HbAA and HbSS (male and female, 4 months old) mice (n=8-11 per group) were treated with vehicle (20% DMSO, 20% PEG800, 60% PBS) or PM2 (10 mg/kg, BW) every other day for 1 week. The last dose was administered on the 8th day 1 hour prior to collecting plasma samples. As expected, HbSS mice had elevated plasma TAT levels compared to HbAA mice (11.04 ± 1.4 vs 45.19 ± 6.3, p<0.05) which was significantly attenuated by PM2 treatment in SS mice (16.17 ± 2, p<0.05). Similar results were observed for IL-6 (AA: 2.06 ± 0.7 vs SS: 29.12 ± 9.6 vs SS+PM2: 5.83 ± 2.9; p<0.05); sVCAM (AA: 330.6 ± 31.5 vs SS: 571.4 ± 50.4 vs SS+PM2: 403.3 ± 41.22; p<0.05) and HMGB1 (AA: 11.46 ± 1.1 vs SS: 25.37 ± 1.5 vs SS+PM2: 19.12 ± 1.7; p<0.05). PM2 had no effect on markers of anemia in HbSS mice including RBC, hematocrit, and reticulocyte counts. In the present study ,3K3A-APC did not attenuate thrombin generation and cytokine production in HbSS mice; this might be due to the shedding of EPCR which is important co-receptor for the protective APC-PAR1 signaling. In contrast, PM2-mediated allosteric inhibition of thrombin/PAR1 and simultaneous induction of APC-like cytoprotective signaling attenuated levels of TAT, IL-6, HMGB1, and sVCAM in HbSS mice. Future studies towards evaluating the effect of PM2 on downstream targets of PAR1 signaling in sickle mice will provide more insight into the mechanism of this protective effect.

High Molecular Weight Kininogen but Not Factor XII Deficiency Attenuates Acetaminophen-Induced Liver Injury in Mice

Acetaminophen (APAP) toxicity is a common cause of acute liver failure (ALF). Dysregulation of coagulation is well documented in ALF patients. Recent reports indicate that small subset of ALF patients (~10%) exhibit spontaneous bleeding, while thrombotic complications are more frequent. Animal studies demonstrated that the inhibition of tissue factor-initiated thrombin generation attenuates APAP-induced liver injury in mice via both fibrin- and protease activated receptor 1-dependent mechanisms. Activation of FXII leads to two events: propagation of coagulation by activation of FXI (intrinsic pathway) and activation of plasma prekallikrein to kallikrein, with subsequent cleavage of high molecular weight kininogen (HK) into bradykinin (BK) and cleaved HK fragments (contact pathway). There is a growing interest in therapeutic targeting of FXII or FXI to prevent thrombosis, primarily because this goal may be attainable without incurring a significant bleeding risk. In this study, we evaluated if the intrinsic coagulation pathway contributes to the activation of coagulation and liver injury in a mouse model of APAP-induced ALF. We postulated that targeting FXII would attenuate coagulation and reduce liver injury without affecting hemostasis. In addition, FXII deficiency could provide a further protection by preventing activation of contact pathway and subsequent reduction of inflammatory response. We used 12 weeks old FXII, FXI, prekallkrein and HK deficient male mice and their respective WT controls. Mice were fasted for 16 hours followed by a single intraperitoneal injection of APAP (400 mg/kg) or sterile saline. Livers and plasma samples were collected 6 and 24 hours after injections. Hepatocellular necrosis was determined on liver sections stained with H&amp;E. Plasma levels of alanine transaminase (ALT- marker of liver injury), thrombin-antithrombin (TAT) complexes, plasmin-α2 antiplasmin (PAP) complexes and interleukin-6 were analyzed using commercially available assays. HK cleavage was determined using BK ELISA, Western blot, and mass spectrometry analysis. Consistently with previously published data, plasma levels of ALT, TAT and IL-6, as well as liver injury scores were significantly elevated in APAP-challenged mice compared to saline-injected WT mice at both 6 and 24 hours. Surprisingly, neither FXII, FXI, nor prekallikrein deficiency had statistically significant effects on any of these parameters in APAP-challenged mice at either time point. Interestingly, however, plasma levels of ALT were significantly attenuated in HK-/- mice (n=23-34) compared to HK+/+ (n=19-31) controls at both 6 (1278 ± 184 vs. 1850 ± 235 U/L; p&amp;lt;0.05) and 24 hours (1546 ± 302 vs. 3857 ± 493 U/L; p&amp;lt;0.01) after injection of APAP. The attenuation of liver injury was observed despite the lack of a significant effect on APAP-induced coagulation activation (plasma TAT levels [mean±SEM]: 72.9 ± 8.2 μg/L in HK+/+ vs. 70.8 ± 4.3 μg/L in HK-/-). HK deficiency also reduced plasma levels of IL-6 (75.8%; p&amp;lt;0.001), number of infiltrating neutrophils in liver (59.8%; p&amp;lt;0.001) and liver necrotic area (28.1%; p&amp;lt;0.001) 24 hours after APAP challenge. Importantly, the protective effects of HK deficiency were completely reversed by the injection of HK protein into APAP-treated HK-/- mice. It has been previously shown that plasminogen deficiency protects against APAP-induced liver injury. Indeed, APAP administration activated the fibrinolytic system in WT mice as demonstrated by increased plasma levels of active tissue plasminogen activator (19.5 fold; p&amp;lt;0.01) and PAP complexes (64.4 fold; p&amp;lt;0.01). Using Western blotting analysis, BK ELISA, and mass spectrometry analysis we demonstrated that plasmin efficiently cleaves HK in a buffer system as well as in mouse and human plasma. Importantly, plasmin mediated cleavage of HK resulted in the release of BK and generation of cleaved HK fragments. In summary, our data indicate that the extrinsic but not the intrinsic coagulation pathway drives the coagulation-mediated pathologies associated with APAP-induced liver injury in mice. HK contributes to the liver injury independently of thrombin generation. We propose that in APAP-challenged mice, HK cleavage and downstream hepatotoxicity, are mediated by plasmin rather than FXIIa-dependent generation of kallikrein. Disclosures McCrae: Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees. Key:Uniqure BV: Research Funding.

Increased perihematomal neuron autophagy and plasma thrombin-antithrombin levels in patients with intracerebral hemorrhage: An observational study.

Animal studies have demonstrated that autophagy was involved in neuronal damage after intracerebral hemorrhage (ICH). Several studies showed thrombin–antithrombin (TAT) plasma levels were elevated in patients with ICH. In this study, we aimed to evaluate if autophagy occurred in patients with ICH; and the relationship between the severity of brain injury and plasma TAT levels.A novel tissue harvesting device was used during hematoma removal surgery to collect loose fragments of tissue surrounding the affected brain area in 27 ICH patients with hematoma volumes of >30 mL in the basal ganglia. Control tissues were obtained from patients who underwent surgery for arteriovenous malformation (n = 25). Transmission electron microscopy (TEM) and immunohistochemistry for autophagy-related proteins were used to evaluate the ultrastructural and morphologic cellular characteristics; and the extent of autophagy in the recovered tissue specimens. Stroke severity was assessed by using the Glasgow Coma Scale (GCS) and the National Institutes of Health Stroke Scale (NIHSS). An enzyme-linked immunosorbent assay (ELISA) was used to measure plasma TAT levels.Transmission electron microscopy showed autophagosomes and autolysosomes exist in neurons surrounding the hematoma, but not in the control tissues. The number of cells containing autophagic vacuoles correlated with the severity of brain injury. Immunohistochemistry showed strong LC3, beclin 1, and cathepsin D staining in ICH tissue specimens. Plasma TAT levels correlated positively with autophagic cells and ICH severity (P < .01).Autophagy was induced in perihematomal neurons after ICH. Autophagy and plasma TAT levels correlated positively with severity of brain injury. These results suggest that autophagy and increased plasma TAT levels may contribute to the secondary damage in ICH patients.

Direct Amplification of Tissue Factor:Factor VIIa Procoagulant Activity by Bile Acids Drives Intrahepatic Coagulation.

Regulation of TF (tissue factor):FVIIa (coagulation factor VIIa) complex procoagulant activity is especially critical in tissues where plasma can contact TF-expressing cells. One example is the liver, where hepatocytes are routinely exposed to plasma because of the fenestrated sinusoidal endothelium. Although liver-associated TF contributes to coagulation, the mechanisms controlling the TF:FVIIa complex activity in this tissue are not known. Approach and Results: Common bile duct ligation in mice triggered rapid hepatocyte TF-dependent intrahepatic coagulation coincident with increased plasma bile acids, which occurred at a time before observable liver damage. Similarly, plasma TAT (thrombin-antithrombin) levels increased in cholestatic patients without concurrent hepatocellular injury. Pathologically relevant concentrations of the bile acid glycochenodeoxycholic acid rapidly increased hepatocyte TF-dependent procoagulant activity in vitro, independent of de novo TF synthesis and necrotic or apoptotic cell death. Glycochenodeoxycholic acid increased hepatocyte TF activity even in the presence of the phosphatidylserine-blocking protein lactadherin. Interestingly, glycochenodeoxycholic acid and taurochenodeoxycholic acid increased the procoagulant activity of the TF:FVIIa complex relipidated in unilamellar phosphatidylcholine vesicles, which was linked to an apparent decrease in the Km for FX (coagulation factor X). Notably, the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a bile acid structural analog, did not increase relipidated TF:FVIIa activity. Bile acids directly enhanced factor X activation by recombinant soluble TF:FVIIa complex but had no effect on FVIIa alone. The results indicate that bile acids directly accelerate TF:FVIIa-driven coagulation reactions, suggesting a novel mechanism whereby elevation in a physiological mediator can directly increase TF:FVIIa procoagulant activity.

Plasminogen Activator Inhibitor-1 Is Critical in Alcohol-Enhanced Acute Lung Injury in Mice.

Chronic alcohol exposure is a clinically important risk factor for the development of acute respiratory distress syndrome, the most severe form of acute lung injury (ALI). However, the mechanisms by which alcohol sensitizes the lung to development of this disease are poorly understood. We determined the role of the antifibrinolytic protein plasminogen activator inhibitor-1 (PAI-1) in alcohol enhancement of experimental endotoxin-induced ALI. Wild-type, PAI-1-/-, and integrin β3-/- mice were fed ethanol-containing Lieber-DeCarli liquid or a control diet for 6 weeks, followed by systemic LPS challenge. LPS administration triggered coagulation cascade activation as evidenced by increased plasma thrombin-antithrombin levels and pulmonary fibrin deposition. Ethanol-exposed animals showed enhanced PAI-1 expression and pulmonary fibrin deposition with coincident exaggeration of pulmonary inflammatory edematous injury. PAI-1 deficiency markedly reduced pulmonary fibrin deposition and greatly reduced inflammation and injury without impacting upstream coagulation. Interestingly, pulmonary platelet accumulation was effectively abolished by PAI-1 deficiency in ethanol/LPS-challenged mice. Moreover, mice lacking integrin αIIBβ3, the primary platelet receptor for fibrinogen, displayed a dramatic reduction in early inflammatory changes after ethanol/LPS challenge. These results indicate that the mechanism whereby alcohol exaggerates LPS-induced lung injury requires PAI-1-mediated pulmonary fibrin accumulation, and suggest a novel mechanism whereby alcohol contributes to inflammatory ALI by enhancing fibrinogen-platelet engagement.

From the Cover: Coagulation-Driven Hepatic Fibrosis Requires Protease Activated Receptor-1 (PAR-1) in a Mouse Model of TCDD-Elicited Steatohepatitis.

Emerging evidence supports a role for environmental chemical exposure in the pathology of non-alcoholic fatty liver disease (NAFLD), a disease process tightly linked to increased activity of the blood coagulation cascade. Exposure of C57BL/6 mice to the persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) recapitulates features of the NAFLD spectrum, including steatosis, hepatic injury, inflammation, and fibrosis. We assessed coagulation cascade activation, and determined the role of the thrombin receptor protease activated receptor-1 (PAR-1) in experimental TCDD-elicited NAFLD. Chronic exposure to TCDD (30 µg/kg every 4 days for 28 days) was associated with intrahepatic coagulation, indicated by increased plasma thrombin-antithrombin levels and hepatic fibrin(ogen) deposition. PAR-1 deficiency diminished TCDD-elicited body weight loss and relative liver weight was reduced in TCDD-exposed PAR-1-/- mice compared with TCDD-exposed wild-type mice. PAR-1 deficiency did not affect TCDD-induced hepatic steatosis or hepatocellular injury, as indicated by serum alanine aminotransferase activity. Despite a lack of effect on these 2 features of NAFLD pathology, PAR-1 deficiency was associated with a reduction in hepatic inflammation evident in liver histopathology, and reflected by a reduction in serum levels of the proinflammatory cytokine interleukin-6. Moreover, TCDD-driven hepatic collagen deposition was markedly reduced in PAR-1-deficient mice. These results indicate that experimental TCDD-elicited steatohepatitis is associated with coagulation cascade activation and PAR-1-driven hepatic inflammation and fibrosis.

Platelets and protease-activated receptor-4 contribute to acetaminophen-induced liver injury in mice

AbstractAcetaminophen (APAP)-induced liver injury in humans is associated with robust coagulation cascade activation and thrombocytopenia. However, it is not known whether coagulation-driven platelet activation participates in APAP hepatotoxicity. Here, we found that APAP overdose in mice caused liver damage accompanied by significant thrombocytopenia and accumulation of platelets in the liver. These changes were attenuated by administration of the direct thrombin inhibitor lepirudin. Platelet depletion with an anti-CD41 antibody also significantly reduced APAP-mediated liver injury and thrombin generation, indicated by the concentration of thrombin-antithrombin (TAT) complexes in plasma. Compared with APAP-treated wild-type mice, biomarkers of hepatocellular and endothelial damage, plasma TAT concentration, and hepatic platelet accumulation were reduced in mice lacking protease-activated receptor (PAR)-4, which mediates thrombin signaling in mouse platelets. However, selective hematopoietic cell PAR-4 deficiency did not affect APAP-induced liver injury or plasma TAT levels. These results suggest that interconnections between coagulation and hepatic platelet accumulation promote APAP-induced liver injury, independent of platelet PAR-4 signaling. Moreover, the results highlight a potential contribution of nonhematopoietic cell PAR-4 signaling to APAP hepatotoxicity.

Abstract 607: Kininogen Regulates Thrombin Generation in a Mouse Model of Sickle Cell Disease

Sickle cell disease (SCD) is associated with activation of coagulation and vascular inflammation. We previously demonstrated that, in mouse models of SCD, tissue factor (TF) expressed on leukocytes activates coagulation and contributes to inflammation via microvascular thrombosis, whereas non-coagulant form of TF expressed by endothelial cells mediates factor Xa (FXa)-PAR2 signalling and contributes to inflammation via IL-6 expression. We also showed that both rivaroxaban and dabigatran, oral inhibitors of FXa and thrombin, attenuated the hypercoagulable state in sickle mice. It has been proposed that anticoagulants that target the intrinsic coagulation pathway may be associated with a lower risk of bleeding compared to targeting the extrinsic or common coagulation pathways. Therefore, we tested if the intrinsic pathway contributes to the activation of coagulation in sickle cell mice. Bone marrow from sickle (SS) and non-sickle (AA) Townes mice was transplanted into wild-type (WT) (n=18 AA, 15 SS), FXII-/- (n=14 AA, 22 SS), or FXI-/- (n=21 AA, 23 SS) recipients (all on C57Bl/6 genetic background). In addition, bone marrow from AA or SS mice was transplanted into high molecular weight kininogen (HK)+/+ (n=17 AA, 16 SS) or HK-/- (n=10 AA, 19 SS) mice. All mice were used for experiments 5 months after BMT. Thrombin generation, measured by plasma thrombin anti-thrombin (TAT) levels, was increased in WT mice injected with SS bone marrow (WT/BMSS) compared to WT/BMAA mice (4.4±0.7 vs 2.6±0.3 ng/mL, p&lt;0.05, mean ± S.E.M), however neither FXII nor FXI deficiency affected this parameter. Consistent with these data, inhibition of FXIIa-dependent activation of FXI with 14E11 antibody also did not reduce plasma TAT levels in SS Townes mice. Interestingly, elevated plasma TAT levels observed in HK+/+/BMSS mice was significantly reduced in HK-/-/BMSS mice (4.8±0.5 versus 3.48±0.2 ng/mL, p&lt;0.05). These data indicate that HK, but not FXII and FXI, contributes to thrombin generation in SCD at steady state of disease. In vitro, HK fragments induce TF expression on monocytes via activation of CD11b/18. We are now investigating if inhibition of HK fragments-induced TF expression on monocytes may attenuate the hypercoagulabIe state in SCD mice without impacting hemostasis.

Thrombin inhibition with dabigatran protects against high-fat diet-induced fatty liver disease in mice.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome. Robust coagulation cascade activation is common in obese patients with NAFLD. We identified a critical temporal relationship between thrombin generation and the manifestation of hepatic steatosis, inflammation, and injury in C57BL/6J mice fed a high-fat diet (HFD) for 1, 2, and 3 months. Mice fed a HFD exhibited dramatic increases in hepatocellular injury and inflammation over time. Hepatic fibrin deposition preceded an increase in serum alanine aminotransferase, and the most dramatic changes in liver histopathology occurred in conjunction with a detectable increase in plasma thrombin-antithrombin levels at 3 months. To directly determine whether thrombin activity promotes NAFLD pathogenesis, mice were fed a HFD and simultaneously treated with the direct thrombin inhibitor dabigatran etexilate for 3 months. Notably, dabigatran treatment significantly reduced hepatic fibrin deposition, hepatic inflammation, hepatocellular injury, and steatosis in mice fed a HFD. Of interest, dabigatran treatment also significantly attenuated HFD-induced body weight gain. Gene expression analysis suggested that thrombin potentially drives NAFLD pathogenesis by altering the expression of genes associated with lipid metabolism and bile acid synthesis. Collectively, the results suggest that thrombin activity is central to HFD-induced body weight gain, liver injury, and inflammation and provide the proof-of-principle evidence that pharmacological thrombin inhibition could be effective in limiting NAFLD and associated pathologies.

Heme Induces Systemic Activation of Coagulation in Vivo in a Tissue Factor-Dependent Manner

Abstract 820Heme is released from red blood cells and hemoproteins during several pathological states, including bacterial infections, ischemia-reperfusion and hemolytic anemias. An excess of free heme is toxic, causing oxidative stress that activates endothelial cells and the inflammatory response. Heme has been reported to increase the expression of tissue factor (TF) on endothelial cells in vitro. TF is the primary initiator of the coagulation cascade. We tested the hypothesis that heme induces TF-dependent activation of coagulation in vivo. Eight week old C57 Bl/6 mice were given a bolus injection (0.035ml/g i.v.) of hemin ranging from 0 – 35 μmols/kg, and plasma was collected 6 hours after treatment. Coagulation activation was assessed by measuring plasma thrombin-antithrombin (TAT) levels. Administration of 17.5 and 35 μmols/kg of hemin significantly increased TAT from 5.3 ± 0.7 ng/L to 9.7 ± 1.3 ng/L (p<0.001) and 13.8 ± 1.9 ng/L (p<0.0001), respectively. Mice were treated with 35 μmols/kg of hemin in all further experiments. In a time course study, plasma was collected after 1, 3 and 6 hrs. Plasma TAT levels were significantly increased as early as 1 hour after treatment (14.1 ± 2.4 ng/L compared to 6.46 ± 0.6 ng/L, p<0.0001), and remained elevated at 3 (12.5 ± 0.6 ng/L, p<0.0001) and 6 hours (14.5 ± 1.0 ng/L, p<0.0001). To determine the role of TF in hemin-induced activation of coagulation, mice were pretreated with the rat anti-mouse TF antibody (1H1) or IgG control (single i.p. injection; 20mg/kg). Hemin significantly increased plasma TAT levels in IgG treated mice (4.6 ± 1.3 ng/Lto 9.6 ± 1.3 ng/L, p<0.0001). Importantly, inhibition of TF with 1H1 significantly attenuated this increase (6.8 ± 0.4 ng/L, p<0.01 compared to IgG/ hemin group). Moreover, plasma TAT levels were also significantly reduced in hemin injected low TF mice (expressing 1–2% of normal TF levels) compared to control mice injected with hemin (p<0.01). These data indicate that heme-induced activation of coagulation is TF-dependent. Next, we used TF flox/flox, Tie-2 Cre mice to determine the effect of TF gene deletion in all hematopoietic and endothelial cells on hemin-induced activation of coagulation. Interestingly, plasma TAT levels in TF flox/flox, Tie-2 Cre mice (11.9 ± 2.7 ng/L) were not different from that observed in control TF flox/flox mice 6 hours after hemin injection (11.6 ± 3.2 ng/L), indicating that TF expressed by other cell types contributes to the activation of coagulation. It has been previously shown that heme induces vascular damage and increases vascular permeability which could expose perivascular TF. Therefore perivascular cells are the likely source of TF that may contribute to the activation of coagulation in hemin treated mice.In addition to the activation of coagulation, hemin (35 μmols/kg) increased total number of white cells (2.3 fold, p = 0.000032), monocytes (1.75, p=0.07) and neutrophils (3.5 fold, p=0.0002) as well as reduced platelet counts (0.86 fold, p=0.003) 6 hours after injection. Furthermore, plasma levels of the proinflamatory cytokine interleukin-6 were also increased (8.5-fold, p=0.002). However, inhibition of TF with 1H1 antibody did not affect any of these parameters.Our data indicate that heme-induced activation of coagulation is TF-dependent whereas heme-induced inflammation is independent of TF. Disclosures:No relevant conflicts of interest to declare.

Biochemical predictors of cardiac rhythm at 1year follow-up in patients with non-valvular atrial fibrillation.

C-reactive protein (CRP) is increased in patients with atrial fibrillation (AF) and it might predict the lack of sinus rhythm maintenance in the long term follow-up. Scarce data have been reported relating endothelial dysfunction and/or haemostatic or platelet markers to sinus rhythm maintenance in AF patients. We aimed to determine whether some biochemical markers including soluble thrombomodulin (sTM), soluble P-selectin, thrombin-antithrombin (TAT) complex and CRP levels could predict sinus rhythm maintenance at 1year follow-up in non-valvular AF patients. 130 patients (70 males, mean age 67±13years) with newly diagnosed AF naïve of antithrombotic or antiplatelet therapy were studied. Baseline CRP, P-selectin, sTM and TAT levels were compared to those of 20 matched-healthy subjects in sinus rhythm. AF patients had significantly higher plasma CRP (10.5±2.2 vs 3.25±0.3mg/l, p=0.001), P-selectin (219±141 vs 126ng/ml, p=0.01), and TAT (54±237 vs 2.7±3.3ng/l, p=0.001) levels compared with controls. Soluble TM levels, although higher than controls, did not reach statistical significance. Multivariate regression analyses showed that elevated CRP (OR=4.8, p=0.02) and sTM (OR=1.05, p=0.04) were the only two predictors of lack of sinus rhythm at 1year follow-up. An altered inflammatory, haemostatic, endothelial and platelet environment exists in newly diagnosed AF patients. CRP and sTM levels might be used as biochemical markers to predict the status of cardiac rhythm at 1year follow-up in some AF patients.

Lung Cancer Chemotherapy Agents Increase Procoagulant Activity In Vitro and In Vivo

Abstract 35 Introduction:Cancer patients have an elevated risk for thrombosis, with venous thromboembolism (VTE) being the second leading cause of death in cancer patients. The risk of VTE is further increased in patients undergoing standard chemotherapy. However, the mechanism(s) by which chemotherapy increases the risk for thrombosis in lung cancer patients is not well understood. Previous studies have shown that treatment of endothelial cells with chemotherapy agents (such as cisplatin, gemcitabine, and paclitaxel) promotes coagulation through an increase in tissue factor (TF) procoagulant activity. Objective:The purpose of this study was to determine in vitro and in vivo procoagulant effects of anti-lung cancer therapy agents cisplatin, carboplatin, paclitaxel, gemcitabine and bevacizumab. We studied the effects of these agents individually as well as in combinations (cisplatin/gemcitabine and carboplatin/paclitaxel) on cell surface TF activity, TF antigen levels, and phosphatidylserine (PS) exposure levels. We also examined the effects of lung cancer chemotherapy agents on thrombin generation in healthy mice. Methods:Human umbilical vein endothelial cells (HUVECs), blood monocytes, and A549 cells were treated for 24 hours with clinically relevant concentrations of chemotherapy agents alone as well as in combination. Cell surface TF activity was measured by factor Xa (FXa) generation in the presence or absence of factor VIIa and CaCl2 on drug-treated cells. To demonstrate that FXa generation was TF-dependent, Xa generation assays were performed on chemotherapy-treated cells incubated with a TF-inhibitory antibody. Cell surface TF antigen and PS levels were measured by flow cytometry. Healthy Balb/C mice received intraperitoneal injections of lung cancer chemotherapy agents alone or in combination. Blood was collected 24 hours post-chemotherapy by carotid artery cannulation. Plasma levels of thrombin-antithrombin (TAT) complex were measured by ELISA. Results:Treatment of all cell lines with single agent and combination chemotherapy significantly increased FXa generation on HUVEC, A459 cells and monocytes. To confirm that chemotherapy-induced FXa generation was TF dependent, FXa generation was repeated in the presence or absence of factor VIIa, an essential component of the extrinsic tenase complex. We also performed the FXa generation assay in the presence or absence of a TF inhibiting antibody HTF-1. Absence of factor VIIa as well as pre-incubation of cells with HTF-1 resulted in ablation of FXa generation, confirming that generation of FXa is due to cell surface TF activity. Through flow cytometry methods we determined that cell surface TF antigen levels remain unchanged on chemotherapy treated HUVEC and A459 cells. In contrast to HUVEC and A549 cells, monocytes treated with high-dose cisplatin alone or in combination with gemcitabine, expressed a significant increase in TF antigen levels. Measurement of cell surface PS exposure levels revealed a significant increase in PS levels only on HUVEC treated with high-dose cisplatin and gemcitabine combinations. However, inhibition of PS with Annexin V on HUVEC treated with high-dose cisplatin/gemcitabine combination did not decrease FXa generation. Furthermore, inhibition of protein disulfide isomerase (PDI) on A549 cells treated with cisplatin/gemcitabine combination but not carboplatin/paclitaxel combination resulted in moderately diminished levels of FXa. Our in vivo results demonstrated that treatment of healthy mice with bevacizumab, paclitaxel and carboplatin moderately increased plasma TAT levels in healthy mice. Conclusions:Our studies are the first to explore the effects of lung cancer chemotherapy agents on monocyte and A549 cell TF activity levels, as well as the first to investigate the mechanisms by which lung cancer agents may promote TF decryption on these cell lines. We are also the first to report in vivo increases in thrombin generation in response to treatment of healthy mice with anti-lung cancer agents. These studies reveal potential mechanisms by which lung cancer chemotherapy induce a hypercoagulable state. Disclosures:No relevant conflicts of interest to declare.

Protease-Activated Receptor 1 and Hematopoietic Cell Tissue Factor Are Required for Hepatic Steatosis in Mice Fed a Western Diet

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome and contributes to increased risk of cardiovascular disease and liver-related morbidity and mortality. Indeed, obese patients with metabolic syndrome generate greater amounts of thrombin, an indication of coagulation cascade activation. However, the role of the coagulation cascade in Western diet-induced NAFLD has not been investigated. Using an established mouse model of Western diet-induced NAFLD, we tested whether the thrombin receptor protease-activated receptor 1 (PAR-1) and hematopoietic cell-derived tissue factor (TF) contribute to hepatic steatosis. In association with hepatic steatosis, plasma thrombin-antithrombin levels and hepatic fibrin deposition increased significantly in C57Bl/6J mice fed a Western diet for 3 months. PAR-1 deficiency reduced hepatic inflammation, particularly monocyte chemoattractant protein-1 expression and macrophage accumulation. In addition, PAR-1 deficiency was associated with reduced steatosis in mice fed a Western diet, including reduced liver triglyceride accumulation and CD36 expression. Similar to PAR-1 deficiency, hematopoietic cell TF deficiency was associated with reduced inflammation and reduced steatosis in livers of low-density lipoprotein receptor-deficient mice fed a Western diet. Moreover, hematopoietic cell TF deficiency reduced hepatic fibrin deposition. These studies indicate that PAR-1 and hematopoietic cell TF are required for liver inflammation and steatosis in mice fed a Western diet.

Hematopoietic and nonhematopoietic cell tissue factor activates the coagulation cascade in endotoxemic mice

Tissue factor (TF) is the primary activator of the coagulation cascade. During endotoxemia, TF expression leads to disseminated intravascular coagulation. However, the relative contribution of TF expression by different cell types to the activation of coagulation has not been defined. In this study, we investigated the effect of either a selective inhibition of TF expression or cell type-specific deletion of the TF gene (F3) on activation of coagulation in a mouse model of endotoxemia. We found that inhibition of TF on either hematopoietic or nonhematopoietic cells reduced plasma thrombin-antithrombin (TAT) levels 8 hours after administration of bacterial lipopolysaccharide (LPS). In addition, plasma TAT levels were significantly reduced in endotoxemic mice lacking the TF gene in either myeloid cells (TF(flox/flox),LysM(Cre) mice) or in both endothelial cells (ECs) and hematopoietic cells (TF(flox/flox),Tie-2(Cre) mice). However, deletion of the TF gene in ECs alone had no effect on LPS-induced plasma TAT levels. Similar results were observed in mice lacking TF in vascular smooth muscle cells. Finally, we found that mouse platelets do not express TF pre-mRNA or mRNA. Our data demonstrate that in a mouse model of endotoxemia activation of the coagulation cascade is initiated by TF expressed by myeloid cells and an unidentified nonhematopoietic cell type(s).

Efficacy and Safety of Continuous Expression of An Improved Variant of FVIIa On Murine Hemostasis with or without Inhibitors to Human Factor IX.

Abstract 2466Poster Board II-443Recombinant human activated Factor VII (rhFVIIa) is extensively used in the management of hemophilic inhibitor patients. As an alternative, using an adeno-associated viral (AAV) vector as a delivery vehicle for an engineered transgene for activated canine FVII (cFVIIa), our large animal studies in hemophilia A and B dogs demonstrated effective, long-term hemostasis and elimination of the bleeding episodes. However, the relatively high vector doses required for efficacy underscores the need for improvements in the delivery vector and/or the transgene itself. To address this, we decided to investigate whether changes in the catalytic domain of FVIIa can improve its coagulant activity, thus resulting in lowering of the effective vector dose. Using the murine version of our engineered FVIIa (mFVIIa) as a backbone, we generated a variant with four amino-acid substitutions in the catalytic domain of murine FVIIa (L305V/A314E/K337A/I374Y, mFVIIa-VEAY), based on an existing enhanced activity human FVIIa variant. As we have previously described (Amer. Soc. Hematol. Meeting 2006, #3276; Amer. Soc. Gene Cell Ther. Meeting 2008, #127), purified mFVIIa-VEAY exhibited 6-7 fold higher intrinsic coagulant activity than mFVIIa. The hemostatic properties relative to mFVIIa were determined following AAV-mediated gene delivery in hemophilia A (HA) mice. Administration of AAV-mFVIIa-VEAY at 40-100 fold lower vector dose (1.2 - 3E10 vector genomes [vg]/mouse) than AAV-mFVIIa (1.2E12 vg/mouse, High dose) resulted in ∼10-fold lower expression of the mFVIIa-VEAY (based on RNA transcript levels), but was sufficient to result in long-term (>12 weeks) normalization of the hemophilic activated partial thromboplastin time (aPTT), similar to that seen with high dose AAV-mFVIIa (P>0.05). In addition, it improved hemostasis similar to that seen with AAV-mFVIIa following in vivo hemostatic challenges (tail clip assay and ferric chloride carotid artery injury). As an extension of these studies, we have now investigated the ability of low-dose mFVIIa-VEAY gene delivery to improve hemostasis in the presence of inhibitory antibodies to human Factor IX (hFIX). Using an adjuvant, we developed a protocol for generating persistent (>3 months, ongoing) inhibitory antibodies to human Factor IX (hFIX) in hemophilia B (HB) mice. Using the low dose of 3E10 vg/mouse, we administered AAV-mFVIIa-VEAY in HB mice with high titer (7-30 Bethesda units [BU]) to hFIX. In agreement with our previous results, following gene transfer, we observed normalization of the hemophilic aPTT at the expression plateau (4 weeks [ongoing]; P>0.05 vs. wildtype mice; P<0.05 vs. untreated HA mice with similar titers of inhibitory antibodies to hFIX). However, despite the clear efficacy observed in hemophilia mice following low-dose mFVIIa-VEAY gene delivery, our previous observations with mFVIIa-VEAY overexpression (following a 1.2E12 vg/mouse of administered AAV [High dose]) in HA mice indicated a 70% reduction in survival within 6 weeks post vector administration. This coincided with a time-dependent increase in plasma thrombin-antithrombin levels that peaked at 4 weeks post AAV infusion (∼70 ng/ml) and was not observed in untreated HA mice (∼ 20ng/ml, P>0.05), HA mice treated with low-dose mFVIIa-VEAY (∼40ng/ml, P>0.05) or HA mice treated with AAV-mFVIIa (∼35ng/ml, P<0.05). Further extending these studies, histological examination from organs of deceased mice revealed thrombi in the heart as well as gross loss of lung structure. Immunofluoresence microscopy demostrated fibrin deposition in the lung parenchyma, suggesting a compromise of the lung vascular bed in the deceased mice. The identical experiment using hemostatically normal mice resulted in 100% mortality within 6 weeks with similar histological findings compared to HA mice following high dose AAV-mFVIIa-VEAY administration. Overall, our results using a high activity mFVIIa variant, demonstrate similar efficacy to mFVIIa but at a substantially reduced vector dose/expression, in a gene transfer setting for hemophilia with or without inhibitors. This variant thus serves as a potential candidate that can lower the effective vector dose in FVIIa gene-based studies in large animal models of hemophilia. However, the increased mortality observed in mice expressing high levels of mFVIIa-VEAY, warrants further investigation into the long-term safety of coagulation proteases with enhanced activity. Disclosures:High:Novo Nordisk: Grant Review Panel.

Prior simvastatin treatment is associated with reduced thrombin generation and platelet activation in patients with acute ST-segment elevation myocardial infarction

Background It has been reported that statin therapy produces additional effects including impaired activation of blood coagulation. It is not clear whether statins can affect hemostasis in patients with acute coronary syndrome. The aim of this study was to investigate the effect of prior statin treatment on thrombin generation and platelet activation in patients with ST-segment elevation myocardial infarction (STEMI). Methods We studied 53 consecutive STEMI patients admitted within 12 hours of pain onset, including 19 treated with simvastatin (40 mg/d) (simvastatin group) at least one month prior to STEMI, and 34 not receiving any statins (no-statin group) on admission. Thrombin-antithrombin (TAT) complexes generation and soluble CD40 ligand (sCD40L) release were determined in 60-second blood samples collected at the site of microvascular injury. Results There were no significant intergroup differences with respect to clinical and laboratory variables, including plasma TAT and sCD40L levels, except lower total cholesterol and low-density lipoprotein cholesterol in the simvastatin group. The mean maximum rate of TAT generation was 47.5% lower (p = 0.0002) and sCD40L release 33.3% lower (p = 0.0006) in the simvastatin group. Total amounts of TAT (p < 0.0001) and sCD40L (p = 0.002) collected within 5 minutes of bleeding were lower in simvastatin-pretreated patients. By multivariate regression analysis, variables describing local TAT and sCD40L profiles in the whole group were independently associated with simvastatin pretreatment (p < 0.0001), but not with cholesterol, platelet count or troponin levels. Conclusions Prior simvastatin use is associated with lower thrombin generation and platelet activation following vascular injury in the early phase of STEMI.

Tissue factor-dependent coagulation contributes to α-naphthylisothiocyanate-induced cholestatic liver injury in mice

Separation of concentrated bile acids from hepatic parenchymal cells is a key function of the bile duct epithelial cells (BDECs) that form intrahepatic bile ducts. Using coimmunostaining, we found that tissue factor (TF), the principal activator of coagulation, colocalized with cytokeratin 19, a marker of BDECs in the adult mouse liver. BDEC injury induced by xenobiotics such as alpha-naphthylisothiocyanate (ANIT) causes cholestasis, inflammation, and hepatocellular injury. We tested the hypothesis that acute ANIT-induced cholestatic hepatitis is associated with TF-dependent activation of coagulation and determined the role of TF in ANIT hepatotoxicity. Treatment of mice with ANIT (60 mg/kg) caused multifocal hepatic necrosis and significantly increased serum biomarkers of cholestasis and hepatic parenchymal cell injury. ANIT treatment also significantly increased liver TF expression and activity. ANIT-induced activation of the coagulation cascade was shown by increased plasma thrombin-antithrombin levels and significant deposition of fibrin within the necrotic foci. ANIT-induced coagulation and liver injury were reduced in low-TF mice, which express 1% of normal TF levels. The results indicate that ANIT-induced liver injury is accompanied by TF-dependent activation of the coagulation cascade and that TF contributes to the progression of injury during acute cholestatic hepatitis.

Effects of adjuvant chemotherapy on the protein C anticoagulant pathway in patients with early stage breast cancer

8563 Background: Although chemotherapy treatment is associated with an increased risk of thrombosis, the pathogenic mechanisms are poorly understood. We have previously shown that treatment of endothelial cells with adriamycin and epirubicin impairs the protein C anticoagulant pathway by downregulating EPCR (endothelial protein C receptor), an endothelial receptor required for the conversion of protein C to the anticoagulant enzyme activated protein C (APC). In this study, we examined the effects of adriamycin- and epirubicin-containing chemotherapy on the protein C anticoagulant pathway in early stage breast cancer patients. Methods: We collected blood samples from 20 patients patients with early stage breast cancer undergoing adjuvant CEF (cyclophosphamide, epirubicin and 5-fluorouracil) or CMF (cyclophosphamide, methotrexate and 5-fluorouracil) chemotherapy on days 1, 2 and 8 for the first 2 cycles of treatment. Markers of protein C generation (protein C, thrombin-antithrombin (TAT) complexes, and APC) were measured on these days. Results: Plasma protein C levels were significantly lower at cycle 2 day 8 (0.88 ± 0.14 U/ml) compared to pre-chemotherapy levels (1.07 ± 0.23 U/ml) (p=0.0036). Plasma TAT levels were significantly higher at cycle 2 day 8 (2.87 ± 0.79 μg/L) compared to pre-chemotherapy levels (2.01 ± 1.24 μg/L) (p=0.02). Based on the APC levels, the patients appear to fall into two categories in terms of their ability to generate APC. The first group (n=16) has elevated APC levels that parallel the elevated TAT levels, whereas the second group (n=4) has low APC levels despite elevated TAT levels. Conclusions: Impaired ability to generate APC in a subpopulation of patients may reflect chemotherapy-induced impairment of the protein C pathway through the downregulation of EPCR. This pilot study provides new mechanistic insights into the prothrombotic effects of chemotherapy agents. No significant financial relationships to disclose.

Plasma levels of thrombin-antithrombin complexes predict preterm premature rupture of the fetal membranes

Decidual hemorrhage (abruption) is strongly associated with preterm premature rupture of fetal membranes (PPROM). Moreover, thrombin enhances decidual matrix metalloproteinase (MMP) expression, and MMP has been strongly linked to PPROM. The current study sought to determine whether increased thrombin activation, as assessed by circulating maternal plasma thrombin-antithrombin (TAT) complexes, predicted subsequent PPROM. We conducted a nested, case-control study of plasma TAT levels, measured by sensitive immunoassay, among 27 women with a singleton preterm birth preceded by PPROM and 54 matched, term controls. Receiver operating characteristic curve analysis was performed to identify the optimal TAT cut-off level predicting PPROM. Mean gestational age at delivery in cases was 33.3 weeks, compared to 39.7 weeks in controls (p < 0.001). Compared with controls, women with PPROM had increased median plasma TAT levels in both the second trimester (5.1 microg/l (range 2.2-26.3 microg/l) vs. 3.2 microg/l (range 1.3-7.3 microg/l); p = 0.001) and third trimester (7.0 microg/l (range 2.6-85.8 microg/dl) vs. 4.8 microg/l (range 1.7-15.4 microg/dl); p = 0.01). In the PPROM group, 16.0% of the women exhibited bleeding during the pregnancy, while the corresponding value among controls was 3.6% (p = 0.07). In the second trimester, the odds ratio for PPROM with a TAT level of > 3.9 microg/l was 6.0 (95% CI 1.67-21.1). This value predicted PPROM with a sensitivity of 88%, specificity of 68% and positive and negative predictive values of 82% and 97%, respectively. Second-trimester elevated plasma TAT concentrations are predictive of subsequent PPROM. These data provide further evidence that PPROM is associated with decidual thrombin activation.