Characterization and Novel Purification of Recombinant Human Protein C from Three Mammalian Cell Lines

Yan, Su; Razzano, Pasquale; Chao, Y B; Walls, J D; Berg, David T.; McClure, Don B.; Grinnell, Brian W.

doi:10.1038/nbt0790-655

Cited by 129 publications

(95 citation statements)

References 30 publications

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“…PNGase F treatment of APC reduced the molecular mass of the APC doublet ϳ10 kDa (Fig. 1A), corresponding to the disappearance of the fully glycosylated APC heavy chain and the formation of lower molecular mass APC glycoforms upon N-linked glycan proteolysis (16,22). In keeping with previous reports, total APC deglycosylation could not be achieved without significant APC degradation and loss of function (16).…”

Section: Resultssupporting

confidence: 88%

Activated Protein C N-Linked Glycans Modulate Cytoprotective Signaling Function on Endothelial Cells

Áinle

O’Donnell

Johnson

et al. 2011

Journal of Biological Chemistry

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Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that limit clot formation, inhibit apoptosis, and protect vascular endothelial cell barrier integrity. In this study, the role of N-linked glycans in modulating APC endothelial cytoprotective signaling via endothelial cell protein C receptor/protease-activated receptor 1 (PAR1) was investigated. Enzymatic digestion of APC N-linked glycans (PNG-APC) decreased the APC concentration required to achieve half-maximal inhibition of thrombin-induced endothelial cell barrier permeability by 6-fold. Furthermore, PNG-APC exhibited increased protection against staurosporineinduced endothelial cell apoptosis when compared with untreated APC. To investigate the specific N-linked glycans responsible, recombinant APC variants were generated in which each N-linked glycan attachment site was eliminated. Of these, APC-N329Q was up to 5-fold more efficient in protecting endothelial barrier function when compared with wild type APC. Based on these findings, an APC variant (APC-L38D/ N329Q) was generated with minimal anticoagulant activity, but 5-fold enhanced endothelial barrier protective function and 30-fold improved anti-apoptotic function when compared with wild type APC. These data highlight the previously unidentified role of APC N-linked glycosylation in modulating endothelial cell protein C receptor-dependent cytoprotective signaling via PAR1. Furthermore, our data suggest that plasma ␤-protein C, characterized by aberrant N-linked glycosylation at Asn-329, may be particularly important for maintenance of APC cytoprotective functions in vivo.

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Section: Resultssupporting

confidence: 88%

Activated Protein C N-Linked Glycans Modulate Cytoprotective Signaling Function on Endothelial Cells

Áinle

O’Donnell

Johnson

et al. 2011

Journal of Biological Chemistry

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“…This protocol selected for properly γ-carboxylated protein S since under-carboxylated vitamin K-dependent proteins are not eluted by CaCl2. 32 A second anion exchange chromatography step was performed using a mono Q column (Amersham/Pharmacia). In this step, a NaCl gradient (0-400 mM) was used to purify the murine protein S. After this step the fractions of interest were pooled and dialyzed against 50 mM Tris, 100 mM NaCl, pH 7.4.…”

Section: Purification Of Recombinant Wild-type Murine Protein Smentioning

confidence: 99%

Species-specific anticoagulant and mitogenic activities of murine protein S

Fernández¹,

Heeb²,

Xu³

et al. 2009

Haematologica

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The online version of this article contains a supplementary appendix. BackgroundThe protein C pathway down-regulates thrombin generation and promotes cytoprotection during inflammation and stress. In preclinical studies using models of murine injury (e.g., sepsis and ischemic stroke), murine protein S may be required because of restrictive species specificity. Design and MethodsWe prepared and characterized recombinant murine protein S using novel coagulation assays, immunoassays, and cell proliferation assays. ResultsPurified murine protein S had good anticoagulant co-factor activity for murine activated protein C, but not for human activated protein C, in mouse or rat plasma. In human plasma, murine protein S was a poor co-factor for murine activated protein C and had no anticoagulant effect with human activated protein C, suggesting protein S species specificity for factor V in addition to activated protein C. We estimated that mouse plasma contains 22±1 µg/mL protein S and developed assays to measure activated protein C co-factor activity of the protein S in murine plasma. Activated protein C-independent anticoagulant activity of murine protein S was demonstrable and quantifiable in mouse plasma, and this activity was enhanced by exogenous murine protein S. Murine protein S promoted the proliferation of mouse and human smooth muscle cells. The potency of murine protein S was higher for mouse cells than for human cells and similarly, human protein S was more potent for human cells than for mouse cells. ConclusionsThe spectrum of bioactivities of recombinant murine protein S with mouse plasma and smooth muscle cells is similar to that of human protein S. However, in vitro and in vivo studies of the protein C pathway in murine disease models are more appropriately performed using murine protein S. This study extends previous observations regarding the remarkable species specificity of protein S to the mouse.

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“…2A). Recombinant proteins were expressed in the human fibroblast cell line HEK293 because this line has been shown to ␥-carboxylate properly a high percentage of expressed recombinant vitamin K-dependent protein (37). In a standard aPTT assay, plasma-derived FIX demonstrated a specific activity of 200 units/mg (1 unit equaling the FIX activity in 1 ml of normal plasma).…”

Section: Recombinant Fix Proteins and Activity In Plasma Clottingmentioning

confidence: 99%

The Factor IX γ-Carboxyglutamic Acid (Gla) Domain Is Involved in Interactions between Factor IX and Factor XIa

Aktimur

Gabriel

Gailani

et al. 2003

Journal of Biological Chemistry

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During hemostasis, factor IX is activated to factor IXa␤ by factor VIIa and factor XIa. The glutamic acidrich ␥-carboxyglutamic acid (Gla) domain of factor IX is involved in phospholipid binding and is required for activation by factor VIIa. In contrast, activation by factor XIa is not phospholipid-dependent, raising questions about the importance of the Gla for this reaction. We examined binding of factors IX and IXa␤ to factor XIa by surface plasmon resonance. Plasma factors IX and IXa␤ bind to factor XIa with K d values of 120 ؎ 11 nM and 110 ؎ 8 nM, respectively. Recombinant factor IX bound to factor XIa with a K d of 107 nM, whereas factor IX with a factor VII Gla domain (rFIX/VII-Gla) and factor IX expressed in the presence of warfarin (rFIX-des␥) did not bind. An anti-factor IX Gla monoclonal antibody was a potent inhibitor of factor IX binding to factor XIa (K i 34 nM) and activation by factor XIa (K i 33 nM). In activated partial thromboplastin time clotting assays, the specific activities of plasma and recombinant factor IX were comparable (200 and 150 units/mg), whereas rFIX/VIIGla activity was low (<2 units/mg). In contrast, recombinant factor IXa␤ and activated rFIX/VIIa-Gla had similar activities (80 and 60% of plasma factor IXa␤), indicating that both proteases activate factor X and that the poor activity of zymogen rFIX/VII-Gla was caused by a specific defect in activation by factor XIa. The data demonstrate that factor XIa binds with comparable affinity to factors IX and IXa␤ and that the interactions are dependent on the factor IX Gla domain.

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Characterization and Novel Purification of Recombinant Human Protein C from Three Mammalian Cell Lines

Cited by 129 publications

References 30 publications

Activated Protein C N-Linked Glycans Modulate Cytoprotective Signaling Function on Endothelial Cells

Activated Protein C N-Linked Glycans Modulate Cytoprotective Signaling Function on Endothelial Cells

Species-specific anticoagulant and mitogenic activities of murine protein S

The Factor IX γ-Carboxyglutamic Acid (Gla) Domain Is Involved in Interactions between Factor IX and Factor XIa

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