scholarly journals The effect of divalent cations on the amidolytic activity of bovine plasma activated protein C and des-1-41-light chain activated protein C.

1987 ◽  
Vol 262 (20) ◽  
pp. 9581-9586 ◽  
Author(s):  
K A Hill ◽  
L M Kroon ◽  
F J Castellino
Keyword(s):  
Light Chain ◽  
Protein C ◽  
Divalent Cations ◽  
Activated Protein C ◽  
Amidolytic Activity ◽  
Bovine Plasma

scholarly journals A comparison between activated protein C and des-1-41-light chain- activated protein C in reactions with type 1 plasminogen activator inhibitor

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 173-181
Author(s):  
CL Gladson ◽  
RR Schleef ◽  
BR Binder ◽  
DJ Loskutoff ◽  
JH Griffin
Keyword(s):  
Plasminogen Activator ◽  
Light Chain ◽  
Protein C ◽  
Activated Protein C ◽  
Plasminogen Activator Inhibitor ◽  
Amidolytic Activity ◽  
Gla Domain ◽  
Activator Inhibitor ◽  
Pai 1

This study investigates the role of the gamma-carboxyglutamic acid (gla) containing domain of activated protein C in interactions with both platelet-derived and purified type 1 plasminogen activator inhibitor (PAI-1). The activity of human platelet PAI-1 was neutralized to the same extent by bovine activated protein C and bovine des-1–41- light chain-activated protein C. Both forms of activated protein C formed SDS-stable, divalent-cation independent complexes with platelet PAI-1, as demonstrated by immunoblotting using antibodies directed to either protein C or PAI-1. Since activated protein C neutralized PAI-1, the potential inhibition of the enzyme by PAI-1 was studied. Purified PAI-1 inhibited the amidolytic activity of bovine-activated protein C and bovine des-1–41-light chain-activated protein C with a k2 of 2.85 X 10(4) M-1 sec-1 for both proteins. These data suggest that the gla domain of activated protein C is not required for neutralization of PAI- 1 activity, for complex formation with PAI-1, or for inhibition of the amidolytic activity of activated protein C by PAI-1.

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 A comparison between activated protein C and des-1-41-light chain- activated protein C in reactions with type 1 plasminogen activator inhibitor

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 173-181 ◽  
Author(s):  
CL Gladson ◽  
RR Schleef ◽  
BR Binder ◽  
DJ Loskutoff ◽  
JH Griffin
Keyword(s):  
Plasminogen Activator ◽  
Light Chain ◽  
Protein C ◽  
Activated Protein C ◽  
Plasminogen Activator Inhibitor ◽  
Amidolytic Activity ◽  
Gla Domain ◽  
Activator Inhibitor ◽  
Pai 1

Abstract This study investigates the role of the gamma-carboxyglutamic acid (gla) containing domain of activated protein C in interactions with both platelet-derived and purified type 1 plasminogen activator inhibitor (PAI-1). The activity of human platelet PAI-1 was neutralized to the same extent by bovine activated protein C and bovine des-1–41- light chain-activated protein C. Both forms of activated protein C formed SDS-stable, divalent-cation independent complexes with platelet PAI-1, as demonstrated by immunoblotting using antibodies directed to either protein C or PAI-1. Since activated protein C neutralized PAI-1, the potential inhibition of the enzyme by PAI-1 was studied. Purified PAI-1 inhibited the amidolytic activity of bovine-activated protein C and bovine des-1–41-light chain-activated protein C with a k2 of 2.85 X 10(4) M-1 sec-1 for both proteins. These data suggest that the gla domain of activated protein C is not required for neutralization of PAI- 1 activity, for complex formation with PAI-1, or for inhibition of the amidolytic activity of activated protein C by PAI-1.

scholarly journals Inhibition and complexation of activated protein C by two major inhibitors in plasma

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 446-454 ◽  
Author(s):  
MJ Heeb ◽  
F Espana ◽  
JH Griffin
Keyword(s):  
Molecular Weight ◽  
Column Chromatography ◽  
Protein C ◽  
Activated Protein C ◽  
Plasma Formation ◽  
The Other ◽  
Amidolytic Activity

Abstract To determine the major physiologic inhibitors of activated protein C (APC), plasma was incubated with APC or with Protac C and subjected to immunoblotting. APC:inhibitor complexes gave two major bands reacting with antiprotein C antibodies when immunoblotted on nondenaturing gels, and additional minor bands that varied between serum and plasma. Formation of one of the two major bands of APC:inhibitor complex, but not the other, was stimulated by heparin and only this band reacted with antibodies to the previously described APC inhibitor that is here designated PCI-1. Plasma immunodepleted of PCI-1 formed complexes with APC as visualized with antiprotein C but not anti-PCI-1 antibodies, and exhibited heparin-independent inhibition of APC activity, providing evidence for the existence of a second major physiologic APC inhibitor, PCI-2. Formation of APC:PCI-2 complexes in PCI-1-depleted plasma paralleled inhibition of APC amidolytic activity. PCI-2 was separated from PCI-1 and partially purified using column chromatography. PCI-2 formed inactive complexes of approximately 110,000 molecular weight (mol wt) with APC suggesting PCI-2 has an approximate mol wt of 50,000. Thus, inhibition of APC in plasma involves two major distinct 50,000 mol wt inhibitors, the heparin-dependent PCI-1 and the heparin- independent PCI-2.

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.

C6PS Regulates the Inactivation of Factor Va by Activated Protein C.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1023-1023 ◽  
Author(s):  
Rinku Majumder ◽  
JinMing Wang ◽  
Barry R. Lentz
Keyword(s):  
Fluorescence Anisotropy ◽  
Binding Sites ◽  
Protein C ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Water Soluble ◽  
Amidolytic Activity ◽  
Factor Va ◽  
Partial Inactivation ◽  
Cofactor Activity

Abstract Protein C is a vitamin K-dependent, anti-thrombotic protein that is proteolytically cleaved by thrombin to produce the active serine protease, activated protein C (APC). APC inactivates co-factors Va and VIIIa, leading to down-regulation of thrombin generation. Factor Va requires phosphatidylserine (PS) for full cofactor activity. APC inactivates bovine factor Va by catalyzing cleavage in its heavy-chain at Arg505, Arg 662 and Arg306. The cleavage at Arg 306 is stimulated by PS-containing membranes. In this paper, we use water-soluble 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS) to ask whether a membrane or molecular PS regulates inactivation of factor Va by APC. Synthetic substrate titration suggested that APC has two Ca2+-requiring binding sites for C6PS: one site increased the APC amidolytic activity (Kdeff ~ 1.3 μM) while the other site decreased it (Kdeff ~ 2 μM) in the presence of 2 mM Ca2+. The effect of C6PS on APC amidolytic activity was PS-specific, with C6PE having no effect. However, titration of both intrinsic fluorescence and DEGR ([5-(dimethylamino)-l-naphthalenesulfonyl] glutamylycylarginyl chloromethyl )-labeled APC fluorescence showed only one Ca2+-requiring C6PS binding site (Kdeff ~ 0.8 μM). The fluorescence anisotropy of DEGR-APC in the presence of 200 μM C6PS revealed C6PS-dependent 1:1 binding to both factor Va isoforms (Va1or Va2) with Kdeff of 1.13x10-9 M (Va1) and 0.3x10-9 M (Va2). The inactivation of factors Va1/Va2 by APC was also promoted by C6PS. In the presence of 200 μM C6PS to saturate sites on both APC and factor Va, both factors Va1/Va2 were fully inactivated by APC (indicating cleavage at Arg 306). However, in the absence of C6PS or presence of only 4 μM C6PS (sufficient to saturate sites only on APC), only partial inactivation (48–52%) of factor Va1/Va2 was observed. These results suggest that PS binding to APC may have some effect on cleavage at Arg505 or Arg 662 but that PS binding to factor Va was needed to promote cleavage at Arg 306. Supported by USPHS grant HL072827 to BRL.

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