scholarly journals The effect of Arg306→ Ala and Arg506→ Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S

1997 ◽  
Vol 6 (9) ◽  
pp. 2016-2027 ◽  
Author(s):  
Jack O. Egan ◽  
Michael Kalafatis ◽  
Kenneth G. Mann
Keyword(s):  
Protein C ◽  
Human Factor ◽  
Activated Protein C ◽  
Protein S ◽  
Factor Va

A Novel Functional Assay of Protein C in Human Plasma and fts Comparison with Amidolytic and Anticoagulant Assays

1992 ◽  
Vol 67 (01) ◽  
pp. 046-049 ◽  
Author(s):  
H A Guglielmone ◽  
M A Vides
Keyword(s):  
Oral Anticoagulant ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Normal Subjects ◽  
Test Sample ◽  
Functional Assay ◽  
Fast Method ◽  
Factor Va ◽  
Protein C Activity

SummaryA simple and fast method for the quantitative determination of protein C activity in plasma is here described. The first step consists in the conversion of protein C in the test sample into activated protein C by means of an activator isolated from Southern Copperhead venom. Subsequently, the degradation of factor Va, in presence of protein C-deficient plasma, is measured by the prolongation of the prothrombin time which is proportional to the amount of protein C in the sample. The dose-response curve showed a linear relationship from 6 to 150% protein C activity and the inter- and intra-assay reproducibility was 3.5% and 5.6% respectively. In normal subjects, a mean of protein C level of 98 ± 15% of normal pooled plasma was found. Comparison with the anticoagulant assay in samples of patients with oral anticoagulant, liver cirrhosis, disseminated intravascular coagulation and severe preeclampsia revealed an excellent correlation (r = 0.94, p <0.001). Also, a similar correlation (r = 0.93, p <0.001) existed between amidolytic assay and the method here proposed for all the samples studied without including the oral anticoagulant group. These results allowed us to infer that this method evaluates the ability of protein C to interact with protein S, phospholipids, calcium ions and factor Va.

scholarly journals Binding of protein S to factor Va associated with inhibition of prothrombinase that is independent of activated protein C.

1993 ◽  
Vol 268 (4) ◽  
pp. 2872-2877
Author(s):  
M.J. Heeb ◽  
R.M. Mesters ◽  
G. Tans ◽  
J. Rosing ◽  
J.H. Griffin
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Factor Va

scholarly journals Importance of Protein S and Phospholipid for Activated Protein C-mediated Cleavages in Factor Va

2003 ◽  
Vol 278 (27) ◽  
pp. 24904-24911 ◽  
Author(s):  
Eva A. Norstrøm ◽  
Mårten Steen ◽  
Sinh Tran ◽  
Björn Dahlbäck
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Factor Va

PROTEIN S-C4BP COMPLEX STIMULATES ACTIVATED PROTEIN C-MEDIATED INACTIVATION AT R306 BUT INHIBITS INACTIVATION AT R506 IN FACTOR VA

2007 ◽  
Vol 5 ◽  
pp. P-M-082-P-M-082
Author(s):  
L. Maurissen ◽  
S. Thomassen ◽  
G. Nicolaes ◽  
B. Dahlbäck ◽  
G. Tans ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Factor Va

scholarly journals Neutral Glycosphingolipid-dependent Inactivation of Coagulation Factor Va by Activated Protein C and Protein S

2001 ◽  
Vol 277 (11) ◽  
pp. 8861-8865 ◽  
Author(s):  
Hiroshi Deguchi ◽  
José A. Fernández ◽  
John H. Griffin
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Protein S ◽  
Factor Va ◽  
Dependent Inactivation

Regulation Of Bovine Activated Protein C By Protein S: The Role Of The Cofactor Protein In Species Specificity

1981 ◽  
Author(s):  
F J Walker
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Species Specificity ◽  
Rabbit Plasma ◽  
Factor Va ◽  
Bovine Plasma ◽  
Species Specific

The anticoagulant activity of activated Protein C has been observed to be species specific. This could be due either to the inability of the bovine enzyme to recognize its substrate, Factor Va, in non-bovine plasmas, or the absence of cofactor-Protein S, a protein that has been shown to be necessary for the maximum expression of the anticoagulant activity of activated Protein C. Activated Protein C was found to be an effective inhibitor of Factor Xa-initiated clotting of bovine plasma, but without activity in either human or rabbit plasma. Human and rabbit plasma supplemented with bovine Protein S was sensitive to the anticoagulant activity of activated Protein C. Neither rabbit nor human plasma contained bovine activated Protein C cofactor activity as measured by the enhancement of bovine activated Protein C-catalyzed inactivation of Factor Va. However, bovine activated Protein C was able to inactivate both human and rabbit Factor Va. The inactivation of both of these proteins could be stimulated by the addition of bovine Protein S. These results indicate that the species specificity of bovine activated Protein C is due to the absence of a cofactor protein in non-bovine plasma that will interact with the bovine enzyme. Secondly, these findings further confirm that Protein S is required for the maximal expression of the anticoagulant activity of activated Protein C.

In Vivo Anti-Thrombotic Potency of Engineered Activated Protein C Variants.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2704-2704
Author(s):  
Laurent O. Mosnier ◽  
Jose A. Fernandez ◽  
Antonella Zampolli ◽  
Xia V. Yang ◽  
Zaverio M. Ruggeri ◽  
...  
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Dependent Manner ◽  
Thrombosis Model ◽  
Factor Va ◽  
Cofactor Activity

Abstract Activated protein C (APC) has both anticoagulant activity via inactivation of factors Va and VIIIa and cytoprotective activities on cells that include anti-apoptotic and anti-inflammatory activities, alterations of gene expression profiles and protection of endothelial barrier function. The relative importance of APC’s anticoagulant activity vs. APC’s direct cytoprotective effects on cells for reduction of mortality in severe sepsis patients and protective effects in animal injury models is not entirely clear. In this current study, genetically engineered APC variants with different activity spectra were tested for in vivo anti-thrombotic potency. Recently we made a non-anticoagulant APC variant, 5A-APC (RR229/230AA and KKK191-193AAA), that retains normal in vitro cytoprotective effects and an ability to reduce mortality in murine sepsis models (Kerschen et al, ASH2006, J Exper Med, 2007). In contrast to 5A-APC, mutation of E149 to A in APC increased anticoagulant activity in clotting assays while diminishing cytoprotective effects on cells. Murine APC variants, E149A-APC and 5A-APC (KKK192-194AAA + RR230/231AA) were used to determine in vivo anti-thrombotic potency in an acute carotid artery thrombosis model in mice, using FeCl3-induced injury. Under the conditions employed, first occlusion occurred within 3.5 min (mean: 171 sec; range 150-200 sec) in the absence of APC. Murine wild type (wt)-APC effectively delayed time to first occlusion in a dose-dependent manner (0 to 1.8 mg/kg wt-APC; mean: 561 sec; range 400-960 sec). The E149A-APC variant exhibited potent in vivo anti-thrombotic activity (1.8 mg/kg; mean: 1020 sec; range 540- >1600 sec) and was superior to wt-APC as evident by the absence of appreciable occlusion in 2/6 E149A-APC vs. 0/6 wt-APC treated animals. Thus E149A-APC was hyperactive in plasma clotting assays as well as hyperactive in an acute FeCl3-induced arterial thrombosis model. To test the hypothesis that an increased protein S cofactor activity contributed to its enhanced anticoagulant activity, E149A-APC anticoagulant activity was tested in normal and protein S deficient plasma. Compared to wt-APC, E149A-APC showed 3-fold increased anticoagulant activity in normal plasma but not in protein S deficient plasma. In studies with purified proteins, protein S concentrations required for half-maximal stimulation of factor Va inactivation by E149A-APC were 3-fold lower compared to wt-APC, whereas factor Va inactivation rates were indistinguishable in the absence of protein S. These data support our hypothesis that increased protein S cofactor activity is, at least partially, responsible for the observed hyper anticoagulant and anti-thrombotic potency in vitro and in vivo. In contrast to E149A-APC, 5A-APC was severely deficient in anti-thrombotic activity in vivo. Even at concentrations up to 8 mg/kg, 5A-APC (mean: 245 sec; range 172-300 sec) failed to delay significantly time to first occlusion compared to no APC. These data highlight important distinctions between structural requirements for APC’s anticoagulant, anti-thrombotic and cytoprotective functions. Engineered APC variants with differentially altered activities (e.g. cytoprotective vs. anticoagulant) may lead to safer or better therapeutic APC variants for a variety of indications including sepsis, ischemic stroke or other pathologies.

Extensive Venous and Arterial Thrombosis Associated With an Inhibitor to Activated Protein C

Blood ◽  
1999 ◽  
Vol 94 (3) ◽  
pp. 895-901 ◽  
Author(s):  
Ariella Zivelin ◽  
Sanford Gitel ◽  
John H. Griffin ◽  
Xiao Xu ◽  
Jose A. Fernandez ◽  
...  
Keyword(s):  
Protein C ◽  
Protein A ◽  
Activated Protein C ◽  
Protein S ◽  
Factor V ◽  
Cleavage Sites ◽  
Profound Change ◽  
Factor Va ◽  
Crossed Immunoelectrophoresis ◽  
Factor V Gene

Activated protein C resistance (APCR) in the absence of alterations in the factor V gene has been observed during pregnancy, in patients on oral contraceptives, in the presence of antiphospholipid antibodies, and in patients with ischemic stroke. We report a 49-year-old woman with recurrent major venous and arterial thromboses who displayed pronounced APCR, yet no changes in the activated protein C (APC) cleavage sites of factor V. The APCR values determined by four different assays were similar to those obtained in plasma from a homozygote for factor V Q506. Addition of IgG isolated from the patient’s serum to normal plasma lowered the APCR ratio from 2.4 to 1.6. Incubation of patient’s IgG with normal APC resulted in a profound change in the mobility of APC in crossed immunoelectrophoresis. APC was also shown to bind to patient’s IgG immobilized on a protein A agarose column. Factor Va inactivation by APC was inhibited by patient’s IgG, but not by control IgG in the presence or absence of either phospholipids or protein S. These results provide evidence for the existence of an acquired antibody against APC in the patient’s plasma, which gave rise to the APCR phenotype and was probably responsible for the major thrombotic events. We suggest that acquired APCR due to anti-APC antibodies be considered a potential cause for severe venous and arterial thromboses.

Anticoagulant Dysfunction of Human Arg352Trp-Activated Protein C Caused by Defective Factor Va Inactivation

2001 ◽  
Vol 85 (02) ◽  
pp. 274-279 ◽  
Author(s):  
Claudia Rintelen ◽  
Subramanian Yegneswaran ◽  
John Griffin
Keyword(s):  
Venous Thrombosis ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Protein S ◽  
Wild Type ◽  
Factor Va ◽  
Wild Type Protein ◽  
Factor Viiia

SummaryThe dysfunctional mutant R352W-protein C was found in two patients with venous thrombosis. The mutant R352A-protein C was constructed to define the contribution of charge/size of the residue at 352 on protein C (chymotrypsin numbering 187). Compared with wild type-protein C, R352W-protein C showed no difference in activation by thrombin·thrombomodulin or α-thrombin. However, R352W-activated protein C (APC) anticoagulant activity (aPTT assay) was reduced to ~65%. Although the catalytic efficiency of R352W-APC towards the oligopeptide substrate S-2366 was unperturbed, factor Va and R506Q-factor Va were not efficiently inactivated by R352W-APC compared with wild type-APC. R352A-APC showed reduced anticoagulant activity and reduced efficiency in factor Va inactivation and in factor VIIIa-inactivation in the presence of protein S. These observations suggest that the dysfunction of R352W-APC in factor Va inactivation may be one of the mechanisms leading to venous thrombosis in affected patients and that R352 plays an important role in the physiological functioning of APC.

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