Effects Of FXa Research Articles

Coagulation Factor Xa Induces Proinflammatory Responses in Cardiac Fibroblasts via Activation of Protease-Activated Receptor-1.

Coagulation factor (F) Xa induces proinflammatory responses through activation of protease-activated receptors (PARs). However, the effect of FXa on cardiac fibroblasts (CFs) and the contribution of PARs in FXa-induced cellular signalling in CF has not been fully characterised. To answer these questions, human and rat CFs were incubated with FXa (or TRAP-14, PAR-1 agonist). Gene expression of pro-fibrotic and proinflammatory markers was determined by qRT-PCR after 4 and 24 h. Gene silencing of F2R (PAR-1) and F2RL1 (PAR-2) was achieved using siRNA. MCP-1 protein levels were measured by ELISA of FXa-conditioned media at 24 h. Cell proliferation was assessed after 24 h of incubation with FXa ± SCH79797 (PAR-1 antagonist). In rat CFs, FXa induced upregulation of Ccl2 (MCP-1; >30-fold at 4 h in atrial and ventricular CF) and Il6 (IL-6; ±7-fold at 4 h in ventricular CF). Increased MCP-1 protein levels were detected in FXa-conditioned media at 24 h. In human CF, FXa upregulated the gene expression of CCL2 (>3-fold) and IL6 (>4-fold) at 4 h. Silencing of F2R (PAR-1 gene), but not F2RL1 (PAR-2 gene), downregulated this effect. Selective activation of PAR-1 by TRAP-14 increased CCL2 and IL6 gene expression; this was prevented by F2R (PAR-1 gene) knockdown. Moreover, SCH79797 decreased FXa-induced proliferation after 24 h. In conclusion, our study shows that FXa induces overexpression of proinflammatory genes in human CFs via PAR-1, which was found to be the most abundant PARs isoform in this cell type.

Activated clotting factor X mediates mitochondrial alterations and inflammatory responses via protease-activated receptor signaling in alveolar epithelial cells

There is growing evidence for the contribution of the activated coagulation factor X (FXa) in the development of chronic inflammatory lung diseases. Therefore, we aimed to investigate effects of exogenous FXa on mitochondrial and metabolic function as well as the induction of inflammatory molecules in type II alveolar epithelial cells. Effects of FXa on epithelial cells were investigated in A549 cell line. Activation of extracellular signal-regulated kinase (ERK) and induction of inflammatory molecules were examined by immunoblot and gene expression analysis. Mitochondrial function was assessed by the measurement of oxygen consumption during maximal oxidative phosphorylation and quantitative determination of cardiolipin oxidation. Apoptosis was tested using a caspase 3 antibody. Metabolic activity and lactate dehydrogenase assay were applied for the detection of cellular viability.FXa activated ERK1/2 and induced an increase in the expression of pro-inflammatory cytokines, which was prevented by an inhibitor of FXa, edoxaban, or an inhibitor of protease-activated receptor 1, vorapaxar. Exposure to FXa caused mitochondrial alteration with restricted capacity for ATP generation, which was effectively prevented by edoxaban, vorapaxar and GB83 (inhibitor of protease-activated receptor 2). Of note, exposure to FXa did not initiate apoptosis in epithelial cells.FXa-dependent pro-inflammatory state and impairment of mitochondria did not reach the level of significance in lung epithelial cells. However, these effects might limit regenerative potency of lung epithelial cells, particular under clinical circumstances where lung injury causes exposure to clotting factors.

Abstract 594: Characterization of Anti-thrombotic and Anti-inflammatory Properties of New Synthetic, Protamine Reversible Low Molecular Weight Heparin

Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders; however, lack of full reversibility with protamine and clearance by kidney limit treatment options. Recently we have developed a chemoenzymatic method to synthesize a new synthetic dodecasaccharide (12mer) with high anti-factor Xa (FXa) activity which was protamine reversible in vitro and in vivo. In the current study, we further characterized the pharmacokinetic, anti-thrombotic and anti-inflammatory properties of this compound using various mouse models. First, time course (0.5, 1, 2, 4, 8 hours, n=4) and dose response (0.5, 1, 1.5, 2.0 mg/kg, n=4) studies in C57Bl/6J mice showed that FXa activity was inhibited by 12mer in vivo and the half-life of the compound injected s.c. at the dose of 1.5 mg/kg was 3 hours. 12mer was cleared from the circulation by 8 hours. Furthermore, fibrin accumulation observed in the femoral vein 60 minutes after electrolytic injury (2.99 ± 1.16; p<0.05; normalized relative fluorescent intensity to control mouse, mean ± SD), was similarly reduced by 12mer (0.6 mg/kg, 1.63± 0.48), UFH (150 U/kg, 1.26 ± 0.35) and enoxaparin (1 mg/kg, 1.39 ± 0.85, p<0.05 for all). Importantly, protamine reversed the anti-thrombotic effect of 12mer and returned fibrin accumulation to normal levels (2.69 ± 0.87; p < 0.05 vs.12mer alone). Finally, we investigated if 12mer can inhibit thrombotic and inflammatory effect of FXa, previously demonstrated by us in the Berkeley mouse model of sickle cell disease. Sickle mice received subcutaneous injection of saline (n=6) or 12-mer (n=6; 2.0 mg/kg) every 8 hours for 7 days. Compared to saline, 12mer reduced plasma levels of thrombin anti-thrombin complexes (8.2±1.4 vs 4.4±1.3 ng/mL; p=0.07, mean ± S.E.M), IL-6 (5.5±1.5 vs 1.1±0.4 pg/mL, p<0.05) and sVCAM1 (1.1±0.2 vs 0.6±0.07 μg/mL, p< 0.05) in sickle mice. Together, our data demonstrates that chemoenzymatic synthesis offers a promising approach toward obtaining new, protamine-reversible LMWHs with excellent anti-coagulant and anti-inflammatory properties.

Factor Xa inhibits HMGB1-induced septic responses in human umbilical vein endothelial cells and in mice.

Nuclear DNA-binding protein high mobility group box 1 (HMGB1) acts as a late mediator of severe vascular inflammatory conditions, such as sepsis. Activated factor X (FXa) is an important player in the coagulation cascade responsible for thrombin generation, and it influences cell signalling in various cell types by activating protease-activated receptors (PARs). However, the effect of FXa on HMGB1-induced inflammatory response has not been studied. First, we addressed this issue by monitoring the effects of post-treatment with FXa on lipopolysaccharide (LPS)- and cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Post-treatment with FXa was found to suppress LPS-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangements. FXa also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice. In addition, FXa inhibited the production of tumour necrosis factor-α and interleukin (IL)-1β. FXa also facilitated the downregulation of CLP-induced release of HMGB1, production of IL-6, and mortality. Collectively, these results suggest that FXa may be regarded as a candidate therapeutic agent for treating vascular inflammatory diseases by inhibiting the HMGB1 signalling pathway.

Activated Blood Coagulation Factor X (FXa) Induces Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells

To determine the transcriptional regulation of the blood coagulation factor X (FX) in cultured human retinal pigment epithelial (RPE) cells, and whether the effects of FXa on the chemotaxis and expression of angiogenic growth factors are mediated by autocrine growth factor signaling. Alterations in gene expression and secretion of growth factors were determined by real-time RT-PCR and ELISA, respectively. Cellular proliferation and chemotaxis were investigated with a bromodeoxyuridine immunoassay and a Boyden chamber assay, respectively. The gene expression of FX in RPE cells was increased by hypoxia and prostaglandin E(2), and decreased by blood serum, FXa, thrombin, transforming growth factor beta (TGF-β1), and platelet-derived growth factor (PDGF). The serum-induced downregulation of FX was mediated by thrombin and TGF-β signaling. FXa induced chemotaxis of RPE cells via activation of the p38 mitogen-activated protein kinase signal transduction pathway. FXa also induced expression of vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF), as well as release of VEGF, bFGF, and TGF-β1 from RPE cells. The stimulatory effects of FXa on the expression of growth factors and secretion of VEGF were prevented by inhibition of the TGF-β activin receptor-like kinase, but not by the thrombin inhibitor hirudin. FXa induced phosphorylation of ERK1/2, p38, and Akt proteins. FXa induces chemotaxis of RPE cells, as well as expression and release of angiogenic growth factors from RPE cells, including VEGF. The effects of FXa on the expression and secretion of VEGF are mediated by autocrine/paracrine TGF-β signaling.

Coagulation Factor Xa inhibits cancer cell migration via LIMK1-mediated cofilin inactivation

Previously, we showed that activated coagulation factor X (FXa) inhibits migration of breast, lung and colon cancer cells. We showed that the effect of FXa on migration was protease-activated receptor (PAR)-1-dependent, but the subsequent cellular signaling routes remained elusive. In the current manuscript, we show that both the Rho/ROCK and Src/FAK/paxillin pathways are required for FXa-mediated inhibition of breast cancer cell migration. FXa induced pronounced stress fiber formation that was partially inhibited by pre-treatment with specific ROCK or Src inhibitors. Downstream of Rho/ROCK and Src/FAK/paxillin, FXa induced myosin light chain phosphorylation and LIMK1 activation resulting in cofilin inactivation. Knocking-down LIMK1 expression abolished FXa-induced inhibition of cell invasion. Our results reveal that FXa-mediated sustained cofilin inactivation leads to stabilization of actin filaments incompatible with migration. Overall we confirm that, beyond its role in blood coagulation, FXa plays a key role in cell migration and we unravel a new mechanism of PAR-1-mediated inhibition of migration via Rho and Src dependent pathways.

Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

Tissue factor (TF) assembled with activated factor VII (FVIIa) initiates the coagulation cascade. We recently showed that TF was essential for FVIIa-induced vascular endothelial growth factor (VEGF) production by human fibroblasts. We investigated whether this production resulted from TF activation by its binding to FVIIa or from the production of clotting factors activated downstream. Incubation of fibroblasts with a plasma-derived FVIIa concentrate induced the generation of activated factor X (FXa) and thrombin and the secretion of VEGF, which was inhibited by hirudin and FXa inhibitors. By contrast, the addition of recombinant FVIIa to fibroblasts did not induce VEGF secretion unless factor X was present. Moreover, thrombin and FXa induced VEGF secretion and VEGF mRNA accumulation, which were blocked by hirudin and FXa inhibitors, respectively. The effect of thrombin was mediated by its specific receptor, protease-activated receptor-1; in contrast, the effect of FXa did not appear to involve effector cell protease receptor-1, because it was not affected by an anti-effector cell protease receptor-1 antibody. An increase in intracellular calcium with the calcium ionophore A23187 or intracellular calcium chelation by BAPTA-AM had no effect on either basal or FXa-induced VEGF secretion, suggesting that the calcium signaling pathway was not sufficient to induce VEGF secretion. Finally, FVIIa, by itself, had no effect on mitogen-activated protein (MAP) kinase activation, contrary to thrombin and FXa, which activate the p44/p42 MAP kinase pathway, as shown by the blocking effect of PD 98059 and by Western blotting of activated MAP kinases. These findings indicate that FVIIa protease induction of VEGF expression is mediated by thrombin and FXa generated in response to FVIIa binding to TF-expressing fibroblasts; they also exclude a direct signaling involving MAP kinase activation via the intracellular domain of TF when expressed by these cells.