scholarly journals Estimation of the distance between the divalent cation binding site of des-1-41-light chain-activated bovine plasma protein C and a nitroxide spin label attached to the active-site serine residue

1988 ◽  
Vol 251 (1) ◽  
pp. 229-236 ◽  
Author(s):  
K A W Hill ◽  
S A Steiner ◽  
F J Castellino
Keyword(s):  
Light Chain ◽  
Active Site ◽  
Spin Label ◽  
Protein C ◽  
Activated Protein C ◽  
Cation Binding ◽  
Serine Residue ◽  
Paramagnetic Effect ◽  
Cation Binding Site ◽  
Bovine Plasma

The paramagnetic effect of Mn2+ on the electron paramagnetic resonance spectrum of a nitroxide spin label covalently attached to the active-site serine residue of des-1-41-light chain bovine plasma-activated protein C, and situated at a distance of approximately 1.2 nm from this amino acid, has been utilized to estimate the distance on the enzyme surface between the single Mn2+ site and the free electron of the spin label. This distance has been found to be approx. 1.12 nm. A significant paramagnetic effect of Mn2+ on the spectrum of this same nitroxide spin label bound to activated protein C (APC) has been found. However, in this case distance calculations are complicated by the existence of a multiplicity of Mn2+ sites on APC. If it is assumed that a single Mn2+ site is responsible for the paramagnetic effect on the spectrum of the spin label, the interelectron distance on APC would be approx. 0.90 nm.

scholarly journals Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor

2003 ◽  
Vol 373 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Laurent O. MOSNIER ◽  
John H. GRIFFIN
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein ◽  
Protease Activated Receptor 1 ◽  
Induced Apoptosis ◽  
Apoptotic Effects ◽  
Dose Dependent

In a model of staurosporine-induced apoptosis using EAhy926 endothelial cells, inhibition of apoptosis by activated protein C was dose-dependent and required the enzyme's active site, implicating activated protein C-mediated proteolysis. Consistent with this implication, both protease-activated receptor-1 (PAR-1) and endothelial cell protein C receptor (EPCR) were required for the anti-apoptotic effects of activated protein C.

scholarly journals Contribution of factor VIII light-chain residues 2007–2016 to an activated protein C-interactive site

2013 ◽  
Vol 109 (02) ◽  
pp. 187-198 ◽  
Author(s):  
Masahiro Takeyama ◽  
Hironao Wakabayashi ◽  
Philip Fay
Keyword(s):  
Light Chain ◽  
Protein C ◽  
Activated Protein C ◽  
Fluid Phase ◽  
Fold Increase ◽  
Binding Assays ◽  
Dot Blot ◽  
Binding Studies ◽  
Dot Blot Assay ◽  
Dot Blotting

SummaryAlthough factor (F) VIIIa is inactivated by activated protein C (APC) through cleavages in the FVIII heavy chain-derived A1 (Arg336) and A2 subunits (Arg562), the FVIII light chain (LC) contributes to catalysis by binding the enzyme. ELISA-based binding assays showed that FVIII and FVIII LC bound to immobilised active site-modified APC (DEGRAPC) (apparent K d ~270 nM and 1.0 μM, respectively). Fluid-phase binding studies using fluorescence indicated an estimated K d of ~590 nM for acrylodan-labelled LC binding to DEGR-APC. Furthermore, FVIII LC effectively competed with FVIIIa in blocking APC-catalysed cleavage at Arg336 (K i = 709 nM). A binding site previously identified near the C-terminal end of the A3 domain (residues 2007–2016) of FVIII LC was subjected to Ala-scanning mutagenesis. FXa generation assays and western and dot blotting were employed to assess the contribution of these residues to FVIIIa interactions with APC. Virtually all variants tested showed reductions in the rates of APC-catalysed inactivation of the cofactor and cleavage at the primary inactivation site (Arg336), with maximal reductions in inactivation rates (~3-fold relative to WT) and cleavage rates (~3 to ~9-fold relative to WT) observed for the Met2010Ala, Ser2011Ala, and Leu2013Ala variants. Titration of FVIIIa substrate concentration monitoring cleavage by a dot blot assay indicated that these variants also showed ~3-fold increases relative to WT while a double mutant (Met2010Ala/Ser2011Ala) showed a >4-fold increase in K m. These results show a contribution of a number of residues within the 2007–2016 sequence, and in particular residues Met2010, Ser2011, and Leu2013 to an APC-interactive site.

scholarly journals Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 642-647 ◽  
Author(s):  
K Murakami ◽  
K Okajima ◽  
M Uchiba ◽  
M Johno ◽  
T Nakagaki ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Vascular Injury ◽  
Active Site ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Myeloperoxidase Activity ◽  
Granulocyte Elastase ◽  
Pulmonary Vascular Permeability ◽  
Elastase Inhibitor

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS- induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.

scholarly journals Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiao-Yan Zhao ◽  
Andreas Wilmen ◽  
Dongli Wang ◽  
Xinquan Wang ◽  
Maxine Bauzon ◽  
...  
Keyword(s):  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Inhibitory Antibody ◽  
Targeted Inhibition
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