Alternatively spliced human tissue factor (asHTF) is not pro-coagulant

2007 ◽  
Vol 97 (01) ◽  
pp. 11-14 ◽  
Author(s):  
Petra Censarek ◽  
Anett Bobbe ◽  
Maria Grandoch ◽  
Karsten Schrör ◽  
Artur-Aron Weber
Keyword(s):  
Smooth Muscle ◽  
Tissue Factor ◽  
Human Tissue ◽  
Thrombin Generation ◽  
Expression System ◽  
Hek293 Cells ◽  
Transfected Cells ◽  
Mammalian Expression ◽  
Coagulant Activity ◽  
Alternatively Spliced

SummaryIt has been proposed that alternatively-spliced human tissue factor (asHTF) is pro-coagulant. We have evaluated the function of asHTF in a mammalian expression system. Full-length human tissue factor (HTF) and asHTF were cloned from smooth muscle cells and over-expressed in HEK293 cells. As expected, a marked pro-coagulant activity (FX activation, thrombin generation) was observed on the surface, in lysates, and on microparticles from HTF transfected cells. In contrast, no pro-coagulant activity of as HTF was observed.

Alternatively spliced human tissue factor promotes tumor growth and angiogenesis in a pancreatic cancer tumor model

2007 ◽  
Vol 120 ◽  
pp. S13-S21 ◽  
Author(s):  
Jennifer E. Hobbs ◽  
Anaadriana Zakarija ◽  
Deborah L. Cundiff ◽  
Jennifer A. Doll ◽  
Emily Hymen ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Tissue Factor ◽  
Human Tissue ◽  
Tumor Model ◽  
Cancer Tumor ◽  
Alternatively Spliced

Abstract 1503: Knockdown of Mog1 Expression Reduces Sodium Currents by Reducing the Trafficking of Cardiac Sodium Channel Na V 1.5 to the Cell Membrane

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Susmita Chakrabarti ◽  
Sandro Yong ◽  
Shin Yoo ◽  
Ling Wu ◽  
Qing Kenneth Wang
Keyword(s):  
Sodium Channel ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Expression System ◽  
Heart Association ◽  
Hek293 Cells ◽  
Similar Reduction ◽  
Mammalian Expression ◽  
Ventricular Cells ◽  
Cardiac Sodium Channel

The cardiac sodium channel (Na v 1.5) plays a significant role in cardiac physiology and leads to cardiac arrhythmias and sudden death when mutated. Modulation of Na v 1.5 activity can also arise from changes to accessory subunits or proteins. Our laboratory has recently reported that MOG1, a small protein that is highly conserved from yeast to humans, is a co-factor of Na v 1.5. Increased MOG1 expression has been shown to increase Na v 1.5 current density. In adult mouse ventricular myocytes, these two proteins were found to be co-localized at the intercalated discs. Here, we further characterize the regulatory role of MOG1 using the RNA interference technique. Sodium current was recorded in voltage-clamp mode from a holding potential of −100 mV and activated to −20 mV. In 3-day old mouse neonatal ventricular cells transfected with siRNA against mouse MOG1 decreased sodium current densities (pA/pF) compared to control or scramble siRNA treated cells (−10.2±3.3, n=11 vs. −165±16, n=20 or −117.9±11.7, n=11). A similar reduction in sodium current was observed in mammalian expression system consisting of HEK293 cells stably expressing human Na v 1.5, by transfecting siRNAs against either human or mouse MOG1 (−41.7±8.3, n=7 or, −82.6±9.6, n=7 vs. −130.6±11.5, n=7; −111.5±8.5, n=7, respectively). Immunocytochemistry revealed that the expression of MOG1 and Na v 1.5 were decreased in both HEK and neonatal cells when compared to scramble siRNAs or control groups. These results show that MOG1 is an essential co-factor for Na v 1.5 by way of a channel trafficking. Such interactions between MOG1 and Na v 1.5 suggest that early localization of MOG1 on the membrane of neonatal cardiomyocytes may be necessary for proper localization and the distribution of Na v 1.5 during cardiac development. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Expression of Alternatively-Spliced Human Tissue Factor, but Not Full-Length Tissue Factor, Enhances Tumor Growth and Tumor-Associated Vasculature.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1746-1746
Author(s):  
Gerald A. Soff ◽  
Jennifer Hobbs ◽  
Emily Hyman ◽  
Deborah L. Cundiff
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Tissue Factor ◽  
Human Tissue ◽  
Primary Tumor ◽  
Conditioned Media ◽  
Full Length ◽  
Human Pancreatic Cancer ◽  
Primary Tumor Growth ◽  
Alternatively Spliced

Abstract It is well-established that cancer is associated with activation of the blood coagulation system, with associated thrombosis as a major cause of morbidity and mortality. Increased expression of Tissue Factor (TF) by cancer cells correlates with a more aggressive grade and clinical course. It is widely presumed that activation of coagulation facilitates cancer growth, and in mouse models, anticoagulation can reduce development of lung metastases. Yet primary tumors are not reduced in a fibrinogen knock-out mouse host, and most importantly, anticoagulation has not been shown to reduce tumor growth in cancer patients. We therefore studied the effect of expression of full-length Tissue Factor (FLTF) and alternatively-spliced human Tissue Factor (asHTF) in a mouse model of human pancreatic cancer. Due to the loss of exon 5, asHTF has a truncated extracellular domain with incomplete procoagulant activity. And due to a frame shift, exon 6 does not code for the transmembrane domain and cytoplasmic tail of FLTF, but codes for a novel peptide sequence. asHTF is soluble and of unknown function. We show that 5 of 6 human pancreatic cancer cell lines tested expressed both FLTF as well as asHTF. The MiaPaca-2 line did not express detectable mRNA or protein of either TF isoform. We generated mammalian expression vectors for both FLTF and asHTF, and established Miapaca-2 clones, stably expressing FLTF, asHTF, or control clones with an empty vector. As anticipated, conditioned media from all FLTF clones shortened the whole blood clotting times by approximately 75%. Conditioned media from control cells and asHTF expressing cells had no effect on clotting times. To evaluate the effect of the TF isoforms on primary tumor growth, 5 X 106 cells from three independent clones of stably transfected clones of FLTF, asHTF, or control clones were injected into the flanks of nude mice (4 mice per clone). At 31 days, the mice were sacrificed and tumor mass measured. Tumors grew in 10 of 12 control mice, but were small (mean tumors 90 mg, SEM 21 mg). Interestingly, FLTF was associated with reduced primary tumor growth; only 4 of 12 developed measurable tumors (mean tumors 10 mg, SEM 4 mg, p = 0.002). In contrast, asHTF expression was associated with enhanced tumor growth; 12 of 12 animals developed tumors (mean tumors 390 mg, SEM 102 mg, p=0.018). In animals with asHTF expressing tumors, circulating asHTF protein was observed in the plasma. The asHTF tumors had increased vascular density compared with controls, suggesting a role of asHTF promoting angiogenesis. In contrast to the prevailing paradigm, our data suggest that FLTF, with procoagulant activity, not only fails to promote primary tumor growth, but may actually inhibit tumor growth. In contrast, asHTF, may be the more important TF isoform in the enhancement of tumor growth.

SUSTAINED EXPRESSION OF FULL LENGTH AND VARIANT RECOMBINANT FACTOR VIII IN GENETICALLY ENGINEERED CELLS

1987 ◽  
Author(s):  
Nava Sarver ◽  
George A Ricca
Keyword(s):  
Factor Viii ◽  
Recombinant Dna ◽  
Expression System ◽  
Genetically Engineered ◽  
Full Length ◽  
Cdna Insert ◽  
Mammalian Expression ◽  
Coagulant Activity ◽  
Recombinant Fviii

A major effort is presently underway to provide factor VIII (FVIII) in a form free of viral pathogens via a recombinant DNA approach. We have constructed two chimeric FVIII cDNA vectors based on the bovine papillomavirus mammalian expression system. The first vector (FVIII) contained a full length FVIII cDNA; the second vector (AFVIII) contained a cDNA insert with an extensive deletion, corresponding to amino acid residues 747 to 1560 in the region encoding the "B" domain. This internal region is removed during activation of the parental FVIII molecule and is believed not to be required for coagulant activity. We have found that recombinant FVIII produced by stable cell lines harboring either the full length or the variant FVIII was capable of restoring coagulant activity to FVIII deficient plasma in. vitro. This expressed activity was neutralized by anti-FVIII antibodies. Similar to observations with FVIII derived from human plasma, the two recombinant FVIII forms were (i) inactivated by the chelating agent EDTA, (ii) demonstrated a biphasic response of an initial activation followed by a decay in activity when treated with thrombin, and (iii) presented the expected peptide banding pattern by western blot analyses. A higher percentage of ΔFVIII transformants were isolated expressing coagulant activity compared to transformants harboring the complete FVIII cDNA. Among the positive transformants isolated, those harboring ΔFVIII produced higher levels of coagulant activity than their full length counterparts. Comparable steady state levels of FVIII specific transcripts were detected in FVIII and ΔFVIII transformants indicating that the difference in expression levels is due to a post transcriptional event(s). Our study demonstrates the efficacy of a full length and an abridged recombinant FVIII produced by stably transformed cells in correcting coagulation deficiency in. vitro. It further suggests the potential usefulness of other molecular variants for efficient expression in genetically engineered cells.

Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment

2018 ◽  
Vol 118 (06) ◽  
pp. 990-1000 ◽  
Author(s):  
Yuanjie Yu ◽  
Anita Böing ◽  
Chi Hau ◽  
Najat Hajji ◽  
Wolfram Ruf ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Tissue Factor ◽  
Plasma Membranes ◽  
Low Density ◽  
Percoll Gradient ◽  
Protein Disulphide Isomerase ◽  
Coagulant Activity ◽  
Different Types ◽  
Detergent Resistant Membrane

Background Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored. Objective This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains. Methods Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined. Results Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density (ρ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI. Conclusion Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity.

scholarly journals Characterization of a Novel Human Factor VIIa Chimera with Increased Tissue Factor-Independent Activity for Emergency Hemostasis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3623-3623
Author(s):  
Ammon Fager ◽  
Maureane Hoffman ◽  
Dougald Monroe
Keyword(s):  
Tissue Factor ◽  
Research Funding ◽  
Protein C ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Hek293 Cells ◽  
Cell Protein ◽  
Acute Bleeding ◽  
Hemostatic Efficacy

While prophylactic treatment with emicizumab has shown remarkable efficacy in patients with hemophilia A, the treatment options for traumatic, perioperative, and breakthrough bleeding in hemophilia A or B patients with inhibitors remain extremely limited. Recombinant Factor VIIa (rFVIIa) is routinely used to promote hemostasis in hemophilia patients with inhibitors and is recommended as first line therapy for acute bleeding, especially for patients on emicizumab. In addition, rFVIIa has extensive off-label use for hemostasis in cardiovascular surgery, trauma, and intracranial hemorrhage. The hemostatic efficacy of rFVIIa depends on its ability to bind activated platelets and promote thrombin generation by activating Factor X (FX) in a tissue factor (TF)-independent manner. However, the use of rFVIIa requires frequent high doses at significant cost, and is limited by an inconsistent response. Therefore, there is a critical need for new strategies to treat acute bleeding in hemophilia patients with inhibitors and others requiring emergency hemostasis. We have previously shown that human platelets express endothelial cell protein C receptor which contributes to the platelet binding and activity of rFVIIa. Based on this work, we designed a novel FVIIa chimera (PC-FVIIa) with the potential for increased hemostatic efficacy and an enhanced safety profile compared to rFVIIa. The purpose of the current study was to characterize the in vitro activity of this chimera. A cDNA construct encoding the Gla and EGF1 domains of human Protein C along with the EGF2 and catalytic domains of human FVIIa was synthesized and cloned into HEK293 cells. Stable transfectants were selected and PC-FVIIa was purified from the media. Protein electrophoresis of eluates confirmed bands consistent with the expected molecular weight. Similar to rFVIIa, we found that autoactivation of PC-FVIIa readily occurs in the presence of calcium and phospholipid (15% PS/41% PC/44% PE). Autoactivation is rapidly accelerated by the addition of Factor Xa (FXa) and significantly impaired in the absence of either calcium or phospholipid. There was no significant difference between activated PC-FVIIa and rFVIIa in their ability to cleave a synthetic FVIIa substrate. As the Gla and EGF1 domains of rFVIIa are primarily responsible for binding TF, we hypothesized that PC-FVIIa would have little to no affinity for TF. Indeed, we found that the interaction between PC-FVIIa and TF is too weak to be measured in an assay designed to detect weak TF binding. We therefore determined the TF-independent activity of PC-FVIIa using FXa and thrombin generation assays. For some experiments, phospholipid vesicles were incubated with rFVIIa or PC-FVIIa. Plasma levels of FX were added and FXa generation was assessed using a chromogenic substrate. In these assays, the rate of FX activation by PC-FVIIa was significantly higher than that of rFVIIa (Figure 1). To determine the ability of PC-FVIIa to promote thrombin generation, we performed a modified calibrated automated thrombography assay in the absence of TF. Hemophilia A plasma was incubated with PC-FVIIa or rFVIIa in the presence of phospholipid. Subsequent thrombin generation was assessed by monitoring cleavage of a fluorogenic substrate. No appreciable thrombin generation was seen in plasma alone. Adding rFVIIa resulted in a shorter lag time than PC-FVIIa. However, PC-FVIIa led to significantly higher peak thrombin concentration and endogenous thrombin potential compared to rFVIIa. Finally, we used a prothrombinase detection system to determine the activity of PC-FVIIa on the surface of platelets activated with thrombin plus a collagen receptor agonist to generate highly procoagulant platelets. Once again, the rate of thrombin generation was significantly higher with PC-FVIIa as compared to rFVIIa, consistent with a higher rate of FX activation on the platelet surface. Taken together, these data suggest that the PC-FVIIa chimera has the potential for increased hemostatic efficacy compared to rFVIIa. Additional studies will characterize the in vivo activity of PC-FVIIa. However, the lack of affinity for TF represents a potential advantage for PC-FVIIa since long-term exposure to high levels of rFVIIa can lead to thrombosis in TF-rich tissues. As such, PC-FVIIa warrants further study as a potential therapeutic agent with unique characteristics compared to rFVIIa. Disclosures Fager: Otello Medical Inc.: Research Funding. Hoffman:Novo Nordisk A/S: Honoraria, Research Funding. Monroe:Novo Nordisk: Honoraria, Research Funding.

scholarly journals Activation of Protease-Activated Receptors 3 and 4 Accelerates Tissue Factor–Induced Thrombin Generation on the Surface of Vascular Smooth Muscle Cells

2010 ◽  
Vol 30 (12) ◽  
pp. 2587-2596 ◽  
Author(s):  
Param Vidwan ◽  
Alokkumar Pathak ◽  
Samar Sheth ◽  
Jianhua Huang ◽  
Dougald M. Monroe ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Tissue Factor ◽  
Vascular Smooth Muscle Cells ◽  
Thrombin Generation ◽  
Muscle Cells ◽  
Protease Activated Receptors

scholarly journals Evaluation of a Blood Coagulation Factor IX Variant That Functions Independently of Factor VIII As an Alternative Treatment for Hemophilia A

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1110-1110
Author(s):  
Viola J.F. Strijbis ◽  
Ka Lei Cheung ◽  
Pavlina Konstantinova ◽  
Ying Poi Liu ◽  
Sander J van Deventer ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Alternative Treatment ◽  
Active Site ◽  
Research Funding ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Activation Mechanism ◽  
Hek293 Cells ◽  
Synergistic Enhancement

The serine protease factor IXa (FIXa) serves an important role in coagulation by catalyzing the proteolytic activation of factor X (FX) together with its cofactor VIIIa (FVIIIa). Being a critical protease in coagulation, the FIXa structure has evolved to be subjected to strict regulatory mechanisms. While FIXa displays considerable structural homology with other coagulation serine proteases, its active site is uniquely controlled by the 99-loop that blocks access to the active site pocket. Cofactor-mediated interaction of FIXa with its substrate FX induces a conformational change that allows for active site engagement and substrate catalysis. Previously, the molecular constraints of the 99-loop were lifted due to specific modifications in both the 99-loop (K265A), the S1 active site subpocket (V181I, I383V), and the L6F substitution, thereby generating FIX-FIAV [Quade-Lyssy et al. J. Thromb. Haemost. 2014]. As a result, this variant is capable of functioning independently of factor VIII (FVIII). Moreover, FIX-FIAV was demonstrated to ameliorate the hemophilia A phenotype both in vitro and in vivo. To further evaluate its therapeutic potential, FIX-FIAV was stably expressed in HEK293 cells and purified by ion-exchange and hydrophobic interaction chromatography. Evaluation of the kinetics of tissue factor-factor VIIa (TF-FVIIa) activation of FIX-FIAV revealed kinetic parameters similar to those of human wild-type FIX(-WT). Analysis of FIX activation intermediates that are formed upon proteolysis by TF-FVIIa or factor XIa demonstrated prolonged formation of FIX-FIAVα, while no FIXa-WTα could be observed. This is consistent with delayed cleavage at position 180, likely resulting from the V181I substitution in FIX-FIAV. Given that the activation mechanism of FIX-FIAV is unperturbed, we next assessed the specific FVIII clotting activity and demonstrated that FIX-FIAV exhibited significant FVIII-like clotting activity (56 ± 4 U/mg) as opposed to FIX-WT (<13 U/mg). These values correlate with up to 28% of FVIII-independent activity for FIX-FIAV at FIX plasma levels (5 ug/mL), confirming that FIX-FIAV has the potential to enhance thrombin generation in FVIII deficiency. To validate this, tissue factor-initiated (0.5 or 1.0 pM) thrombin generation was assessed in FVIII-immunodepleted plasma, leading to a severely reduced thrombin peak (88% or 81% reduction, respectively) relative to conditions with 100% FVIII. Addition of FIX-FIAV (5 ug/mL) partially restored thrombin generation, demonstrated by an up to ~30% increase in both thrombin peak and endogenous thrombin potential. Evaluation of the FVIII-independent activity of FIX-FIAV in severe hemophilia A patient plasma with or without an inhibitor resulted in an up to 18% or 32% FVIII-like activity, respectively, demonstrating efficacy of FIX-FIAV in the presence of FVIII inhibitors. Although unlikely, it remains to be determined whether specific FVIII-inhibitors may impact FIX-FIAV function. Adding 100% FVIII or low- to mid-range therapeutic concentrations of the bispecific antibody emicizumab to FVIII-deficient plasma incubations with FIX-FIAV resulted in a synergistic enhancement of thrombin generation, demonstrated by a 9-fold increase in thrombin peak. This is consistent with the previously demonstrated hyperactivity of FIX-FIAV in a cofactor-dependent system. In contrast, no synergistic effect on thrombin generation was observed when combining FIX-FIAV with physiologically relevant concentrations of FEIBA or NovoSeven. Summarizing, FIX-FIAV is characterized by a preserved mechanism of activation in addition to being capable of sustaining therapeutic levels of coagulation activity in FVIII deficiency. This provides support for the use of FIX-FIAV as an alternative treatment for hemophilia A. Disclosures Strijbis: uniQure Biopharma B.V.: Research Funding. Konstantinova:uniQure Biopharma B.V.: Employment. Liu:uniQure Biopharma B.V.: Employment. van Deventer:uniQure Biopharma B.V.: Employment. Bos:uniQure Biopharma B.V.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Sign in / Sign up

Export Citation Format

Share Document