Room-Temperature-Stable Recombinant Activated Coagulation Factor VII Recombinant: Chemical and Microbiologic Stability Over 24 Hours During Continuous In Vitro Infusion

AbstractBlood coagulation comprises a series of enzymatic reactions leading to thrombin generation and fibrin formation. This process is commonly illustrated in a waterfall-like manner, referred to as the coagulation cascade. In vivo, this “cascade” is initiated through the tissue factor (TF) pathway, once subendothelial TF is exposed and bound to coagulation factor VII (FVII) in blood. In vitro, a diminutive concentration of recombinant TF (rTF) is used as a clotting trigger in various global hemostasis assays such as the calibrated automated thrombogram, methods that assess fibrin turbidity and fibrin viscoelasticity tests such as rotational thromboelastometry. These assays aim to mimic in vivo global coagulation, and are useful in assessing hyper-/hypocoagulable disorders or monitoring therapies with hemostatic agents. An excess of rTF, a sufficient amount of negatively charged surfaces, various concentrations of exogenous thrombin, recombinant activated FVII, or recombinant activated FIXa are also used to initiate activation of specific sub-processes of the coagulation cascade in vitro. These approaches offer important information on certain specific coagulation pathways, while alterations in pro-/anticoagulants not participating in these pathways remain undetectable by these methods. Reviewing available data, we sought to enhance our knowledge of how choice of clotting trigger affects the outcome of hemostasis assays, and address the call for further investigations on this topic.

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The In Vitro Analysis of the Coagulation Mechanism of Activated Factor VII Using Thrombelastogram

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613300 ◽

2002 ◽

Vol 88 (11) ◽

pp. 768-772 ◽

Cited By ~ 25

Author(s):

Chiharu Kawaguchi ◽

Yae Hanesaka ◽

Akira Yoshioka ◽

Yukihiro Takahashi

Keyword(s):

Platelet Rich Plasma ◽

Coagulation Factor ◽

Factor Vii ◽

Factor V ◽

Coagulation Factor Vii ◽

Activated Factor Vii ◽

Coagulation Mechanism ◽

Vitro Analysis ◽

Hemostatic Effects

SummaryWe investigated the effects of addition of recombinant activated coagulation factor VII (rFVIIa) to coagulation factor-deficient plasma and whole blood, using thrombelastograms (TEGs). The addition of rFVIIa to factor II-or X-deficient plasma did not correct hemostatic parameters, whereas it produced partial responses in factor V-, VIII-or IX-deficient plasma and good responses in factor VII-, XI-or XII-deficient plasma. Furthermore, the addition of rFVIIa and platelets (30-100 X 103/µl) to platelet-poor plasma produced marked corrections, producing TEGs similar to those of platelet-rich plasma. These results indicate that factors II and X are essential for the hemostatic effects of rFVIIa, and that factors V and VIII promote these effects. We believe that TEGs are, at present, one of the most useful tools for evaluating in vitro hemostatic effects of rFVIIa.

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Glyco-engineered HEK 293-F cell lines for the production of therapeutic glycoproteins with human N-glycosylation and improved pharmacokinetics

Glycobiology ◽

10.1093/glycob/cwaa119 ◽

2021 ◽

Author(s):

Rico Uhler ◽

Ruth Popa-Wagner ◽

Mario Kröning ◽

Anja Brehm ◽

Paul Rennert ◽

...

Keyword(s):

Cell Line ◽

Therapeutic Proteins ◽

Coagulation Factor ◽

Factor Vii ◽

Hek 293 ◽

Knock Out ◽

Hek 293 Cells ◽

Coagulation Factor Vii ◽

293 Cells

Abstract N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4, and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties.

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111Indium Labelling of Recombinant Activated Coagulation Factor VII: In Vitro and Preliminary In Vivo Studies in Healthy Rats

International Journal of Molecular Imaging ◽

10.1155/2012/464810 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7

Author(s):

Amarnadh Nalla ◽

Inge Buch ◽

Maibritt Sigvardt ◽

Rasmus Poul Bodholdt ◽

Andreas Kjaer ◽

...

Keyword(s):

Coagulation Factor ◽

In Vivo Studies ◽

Factor Vii ◽

Acute Bleeding ◽

Coagulation Factor Vii ◽

Cardiac Region ◽

Vitro Binding ◽

Liver Region

The aim of this study is to investigate whether 111Indium-labelled recombinant FVIIa (rFVIIa) could be a potential radiopharmaceutical for localization of bleeding sources. DTPA-conjugated rFVIIa was radiolabelled with 111In chloride. In vitro binding efficiency of 111In-DTPA-rFVIIa to F1A2-Mab-sepharose was 99% in buffer, while it was 88–82% in serum. The binding efficiency of 111In-DTPA-rFVIIa to TF (1–209)-sepharose was 48% in buffer whereas 39%–36% in serum, respectively. In vivo experiment was conducted in healthy rats, and gamma camera images were taken immediately after iv. administration of 1.6–1.8 MBq 111In-DTPA-rFVIIa up to 120–130 min. Five min after administration of 111In-DTPA-rFVIIa, percentage of 111In activity was 6.0% in the cardiac region and 24.5% in the liver region. After 2 hours activity was decreased to 3.3% in heart while it had increased to 42.0% in the liver. The 111In-DTPA-rFVIIa might be a potential radiopharmaceutical for visualisation of tissues with significant TF expression such as acute bleeding lesions in the gastrointestinal tract.

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Blocking the Initiation of Coagulation by RNA Aptamers to Factor VIIa

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614126 ◽

2000 ◽

Vol 84 (11) ◽

pp. 841-848 ◽

Cited By ~ 53

Author(s):

H. Lyerly ◽

Jeffrey Lawson ◽

Christopher Rusconi ◽

Alice Yeh ◽

Bruce Sullenger

Keyword(s):

Human Plasma ◽

Tissue Factor ◽

In Vitro Selection ◽

Factor Viia ◽

Coagulation Factor ◽

Factor Vii ◽

Dependent Manner ◽

Factor X ◽

Coagulation Factor Vii

SummaryThe tissue factor/factor VIIa complex is thought to be the primary initiator of most physiologic blood coagulation events. Because of its proximal role in this process, we sought to generate new inhibitors of tissue factor/factor VIIa activity by targeting factor VIIa. We employed a combinatorial RNA library and in vitro selection methods to isolate a high affinity, nuclease-resistant RNA ligand that binds specifically to coagulation factor VII/VIIa. This RNA inhibits the tissue factordependent activation of factor X by factor VIIa. Kinetic analyses of the mechanism of action of this RNA suggest that it antagonizes factor VIIa activity by preventing formation of a functional factor VII/tissue factor complex. Furthermore, this RNA significantly prolongs the prothrombin time of human plasma in a dose dependent manner, and has an in vitro half-life of ∼15 h in human plasma. Thus, this RNA ligand represents a novel class of anticoagulant agents directed against factor VIIa.

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