Factor V as Anticoagulant Cofactor for Activated Protein C in Factor Va Inactivation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3075-3075
Author(s):  
Thomas J Cramer ◽  
John H. Griffin ◽  
Andrew J. Gale
Keyword(s):  
Protein C ◽  
Time Course ◽  
Activated Protein C ◽  
Protein S ◽  
Anticoagulant Effect ◽  
Factor V ◽  
Dependent Manner ◽  
Experimental Conditions ◽  
Anticoagulant Function ◽  
Apc Protein

Abstract Factor V (FV) is a cofactor that promotes inactivation of activated factor VIII (FVIIIa) by the activated protein C and protein S complex (APC/protein S). Cleavage in FV at Arg506 is required for proteolytic inactivation of FVa, but also for the anticoagulant function of FV as cofactor for APC in the inactivation of FVIIIa. This is demonstrated by the well known FVLeiden mutant with Arg506 mutated to glutamine (Q506), causing APC resistance due to both impaired sensitivity of Q506FVa to APC and reduced cofactor activity of Q506FV for APC inactivation of FVIIIa. However, FVIIIa loses activity rapidly due to dissociation of the A2 domain, and this may be the primary mechanism of FVIIIa inactivation. Thus, we question whether the APC-mediated inactivation of FVIIIa is relevant to the FVLeiden thrombophilic phenotype. Rather, we hypothesized that FV can function as an anticoagulant cofactor for the APC/protein S complex in the inactivation of activated FV (FVa). To test this hypothesis, we designed a coagulation assay initiated by tissue factor that was sensitive to FV but was insensitive to FVIII. FV was titrated into FV deficient plasma and clotting times were measured in absence and presence of APC to determine an APC sensitivity ratio (APCsr). An increase in the APCsr was observed as the level of FV was increased, suggesting an anticoagulant function of FV. Similar titrations were done with Q506FV, showing no increase in clotting time when APC was present and an APCsr of 1.0 in the presence of Q506 FV. Control experiments confirmed that this clotting assay was insensitive to the presence or absence of FVIII; thus, these assays were reflecting FVa inactivation. The potential anticoagulant effect of FV as cofactor for APC in FVa inactivation was further investigated by monitoring proteolysis of purified FVa by APC over time using SDS PAGE. Recombinant purified FVa was labeled with a fluorescent dye, and then subjected to proteolysis by APC/protein S in the absence or presence of FV in a time course. The resulting FVa fragments seen on SDS gels reflected the known cleavages at Arg306 and Arg506, and the FVa cleavage products were quantified by digital fluorescent scanning of the gel. FV stimulated a small but statistically insignificant increase in the rate of FVa cleavage by APC/protein S. Thus, in our experimental conditions, we found a significant anticoagulant effect of FV in clotting assays that were sensitive to FV but not sensitive to FVIII whereas in purified reaction mixtures there was not a significant enhancement by FV of APC proteolysis of FVa. These data contrasting FV’s apparent APC-cofactor activities between plasma and purified reaction mixtures lead us to speculate that other factors or mechanisms present in plasma also contribute to the anticoagulant function of APC in a FV dependent manner.

The anticoagulant function of coagulation factor V

2012 ◽  
Vol 107 (01) ◽  
pp. 15-21 ◽  
Author(s):  
Thomas J. Cramer ◽  
Andrew J. Gale
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Protein S ◽  
Factor V ◽  
Phospholipid Membrane ◽  
Terminal Part ◽  
Coagulation Factor V ◽  
Cofactor Activity ◽  
Anticoagulant Function

SummaryAlmost two decades ago an anticoagulant function of factor V (FV) was discovered, as an anticoagulant cofactor for activated protein C (APC). A natural mutant of FV in which the R506 inactivation site was mutated to Gln (FVLeiden) was inactivated slower by APC, but also could not function as anticoagulant cofactor for APC in the inactivation of activated factor VIII (FVIIIa). This mutation is prevalent in populations of Caucasian descent, and increases the chance of thrombotic events in carriers. Characterisation of the FV anticoagulant effect has elucidated multiple properties of the anticoagulant function of FV: 1) Cleavage of FV at position 506 by APC is required for anticoagulant function. 2) The C-terminal part of the FV B domain is required and the B domain must have an intact connection with the A3 domain of FV. 3) FV must be bound to a negatively charged phospholipid membrane. 4) Protein S also needs to be present. 5) FV acts as a cofactor for inactivation of both FVa and FVIIIa. 6) The prothrombotic function of FVLeiden is a function of both reduced APC cofactor activity and resistance of FVa to APC inactivation. However, detailed structural and mechanistic properties remain to be further explored.

C4b-binding Protein Inhibits the Factor V-dependent but not the Factor V-independent Cofactor Activity of Protein S in the Activated Protein C-mediated Inactivation of Factor VIIIa

2001 ◽  
Vol 85 (05) ◽  
pp. 761-765 ◽  
Author(s):  
Robbert van de Poel ◽  
Joost Meijers ◽  
Bonno Bouma
Keyword(s):  
Protein C ◽  
Binding Protein ◽  
Activated Protein C ◽  
Protein S ◽  
Coagulation Factors ◽  
Factor V ◽  
Factor Viiia ◽  
S Factor ◽  
Cofactor Activity ◽  
Apc Protein

SummaryActivated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein S levels, C4BP is an important regulator of protein S cofactor activity. In the factor VIIIa inactivation, protein S and factor V act as synergistic cofactors to APC. We investigated the effect of C4BP on both the factor V-independent and factor V-dependent cofactor activity of protein S in the factor VIIIa inactivation using a purified system. Protein S increased the APC-mediated inactivation of factor VIIIa to 60% and in synergy with protein S, factor V at equi-molar concentrations increased this effect further to 90%. The protein S/factor V synergistic effect was inhibited by preincubation of protein S and factor V with a four-fold molar excess of C4BP. However, C4BP did not inhibit the factor V-independent protein S cofactor activity in the purified system whereas it inhibited the cofactor activity in plasma. We conclude that C4BP-bound protein S retains its cofactor activity to APC in the factor VIIIa inactivation.

Synergistic Cofactor Function of Factor V and Protein S to Activated Protein C in the Inactivation of the Factor Villa – Factor IXa Complex

1997 ◽  
Vol 78 (03) ◽  
pp. 1030-1036 ◽  
Author(s):  
Lei Shea ◽  
Xuhua He ◽  
Björn Dahlbäck
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Species Specificity ◽  
Factor V ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Cofactor Activity ◽  
Apc Protein ◽  
Bovine Factor

SummaryHuman factor V has been shown not only to be a precursor to procoagulant factor Va but also to express anticoagulant properties. Thus, factor V was recently found to potentiate the effect of protein S as cofactor to activated protein C (APC) in the inactivation of the factor VIIIa-factor IXa complex. The purpose of this study was to determine whether the APC-cofactor function of factor V was also expressed in the bovine protein C system and to elucidate the molecular background for the species specificity of APC. For this purpose, the effects of protein S and factor V on APC-mediated inactivation of factor VIIIa were studied using purified APC, protein S and factor V of human and bovine.origin. The factor VIIIa investigated here was part of a Xase complex (i.e. factor IXa, factor VIIIa, phospholipid and calcium) and the APC-mediated inhibition of factor VIIIa was monitored by the ability of the Xase complex to activate factor X. Synergistic APC-cofactor function of factor V and protein S was demonstrated in the bovine system. The effect of bovine APC was potentiated by bovine protein S but not by human protein S, whereas both human or bovine protein S stimulated the function of human APC. Factor V did not express species specificity in its APC-cofactor activity even though bovine factor V was more potent than its human counterpart. Recombinant human/bovine protein S chimeras were used to demonstrate that the thrombin sensitive region and first epidermal growth factor-like module of protein S determine the species specificity of the APC-protein S interaction. In conclusion, both human and bovine factor V were found to express APC-cofactor activity which depends on the presence of protein S. The species specificity of APC was shown to be caused by the interaction between APC and protein S.

scholarly journals Increased prothrombin activation in protein S-deficient plasma under flow conditions on endothelial cell matrix: an independent anticoagulant function of protein S in plasma

Blood ◽  
1995 ◽  
Vol 85 (7) ◽  
pp. 1815-1821 ◽  
Author(s):  
C van't Veer ◽  
TM Hackeng ◽  
D Biesbroeck ◽  
JJ Sixma ◽  
BN Bouma
Keyword(s):  
Endothelial Cell ◽  
Tissue Factor ◽  
Protein C ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Protein S ◽  
Flow Conditions ◽  
Cell Matrix ◽  
Anticoagulant Function ◽  
Prothrombin Activation

Protein S is a vitamin K-dependent nonenzymatic coagulation factor involved in the regulation of activated protein C (aPC). In this study, we report an aPC-independent anticoagulant function of protein S in plasma under flow conditions. Plasma, anticoagulated with low-molecular-weight heparin allowing tissue factor-dependent prothrombin activation, was perfused at a wall shear rate of 100 s-1 over tissue factor containing matrices of stimulated endothelial cells placed in a perfusion chamber. Fractions were collected in time at the outlet and prothrombin activation was determined by measuring the activation fragment F1+2 of prothrombin. In normal plasma, a time-dependent prothrombin activation was detected by the generation of fragment1+2. Prothrombin activation had ceased after 12 minutes perfusion, independent of the amount of tissue factor present in the matrix. Depletion of protein S from plasma or inhibition of protein S in plasma by monoclonal antibodies induced a 5- to 25-fold increase of prothrombin activation on the procoagulant endothelial cell matrix. A prolonged prothrombin activation was detected in protein S-depleted plasma up to 20 minutes after onset of the thrombin generation. The increased prothrombin activation in protein S-depleted plasma could not be explained by the absence of the cofactor function of protein S for aPC because depletion of protein C from plasma did not result in increased prothrombin activation. These data provide further evidence for a strong anticoagulant function of protein S in plasma independent from activated protein C.

Cleavage of Factor V at Arg 506 by Activated Protein C and the Expression of Anticoagulant Activity of Factor V

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2552-2558 ◽  
Author(s):  
Elisabeth Thorelli ◽  
Randal J. Kaufman ◽  
Björn Dahlbäck
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Factor V ◽  
Wild Type ◽  
Apc Resistance ◽  
S 100 ◽  
Negative Effect ◽  
Cofactor Activity

Activated protein C (APC) inhibits coagulation by cleaving and inactivating procoagulant factor Va (FVa) and factor VIIIa (FVIIIa). FV, in addition to being the precursor of FVa, has anticoagulant properties; functioning in synergy with protein S as a cofactor of APC in the inhibition of the FVIIIa-factor IXa (FIXa) complex. FV:Q506 isolated from an individual homozygous for APC-resistance is less efficient as an APC-cofactor than normal FV (FV:R506). To investigate the importance of the three APC cleavage sites in FV (Arg-306, Arg-506, and Arg-679) for expression of its APC-cofactor activity, four recombinant FV mutants (FV:Q306, FV:Q306/Q506, FV:Q506, and FV:Q679) were tested. FV mutants with Gln (Q) at position 506 instead of Arg (R) were found to be poor APC-cofactors, whereas Arg to Gln mutations at positions 306 or 679 had no negative effect on the APC-cofactor activity of FV. The loss of APC-cofactor activity as a result of the Arg-506 to Gln mutation suggested that APC-cleavage at Arg-506 in FV is important for the ability of FV to function as an APC-cofactor. Using Western blotting, it was shown that both wild-type FV and mutant FV was cleaved by APC during the FVIIIa inhibition. At optimum concentrations of wild-type FV (11 nmol/L) and protein S (100 nmol/L), FVIIIa was found to be highly sensitive to APC with maximum inhibition occurring at less than 1 nmol/L APC. FV:Q506 was inactive as an APC-cofactor at APC-concentrations ≤ 1 nmol/L and only partially active at higher APC concentrations. Our results show that increased expression of FV anticoagulant activity correlates with APC-mediated cleavage at Arg-506 in FV, but not with cleavage at Arg-306 nor at Arg-679.

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.

Identification of a Protein S-Interactive Site on the Factor VIII A2 Domain.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2684-2684
Author(s):  
Masahiro Takeyama ◽  
Keiji Nogami ◽  
Tetsuhiro Soeda ◽  
Akira Yoshioka ◽  
Midori Shima
Keyword(s):  
Factor Viii ◽  
Activated Protein C ◽  
Protein S ◽  
Cross Linking ◽  
Factor V ◽  
Dependent Manner ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Mutant Forms ◽  
A2 Domain

Abstract Protein S functions as a cofactor of activated protein C that inactivates factor VIII(a) and factor V(a). We recently have reported a new regulatory mechanism that protein S interacted with both the A2 and A3 domains in factor VIII, and consequently this cofactor directly impaired the factor Xase complex by competing the interaction of factor IXa to factor VIIIa (Blood2006; 108, 487a). Since factor IXa blocked the binding of A2 subunit to protein S, we attempted several approaches to localize the protein S-interactive site(s) on the factor VIII A2 domain. An anti-A2 monoclonal antibody (mAb413) with the 484–509 epitope, recognizing a factor IXa-interactive site on the A2, inhibited the A2 binding to immobilized protein S up to approximately 90% in a dose-dependent manner in a surface plasmon resonance-based assay. Furthermore, ELISA-based assay showed that a synthetic peptide corresponding to residues 484–509 directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484–509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. The cross-linked product formed with EDC was consistent with 1:1 stoichiometry of reactants, suggesting specificity in the 484–509 peptide and protein S interaction. This cross-linking formation was blocked by the addition of the 484–497 peptide, whilst not by the 498–509 peptide, supporting the presence of protein S-interactive site within residues 484–497. Furthermore, N-terminal sequence analysis of the 484–509 peptide-protein S product showed that three sequential basic residues (S488, R489 and R490) could not be detected, supporting that three residues participate in cross-link formation. To confirm the significance of these residues in A2 domain for protein S-binding, the mutant forms of the A2 domain, converted to alanine, were expressed in baculovirus system and purified. Compared with wild type A2 (Kd: ∼9 nM), each binding affinity of S488A, R489A, or R490A A2 mutant for protein S was decreased by 4∼5-fold (32, 40 and 40 nM, respectively). These results indicate that the 484–509 region in the factor VIII A2 domain, and in particular a cluster of basic amino acids at residues 488–490, contributes to a unique protein S-interactive site.

Protein S and factor V in regulation of coagulation on platelet microparticles by activated protein C

2014 ◽  
Vol 134 (1) ◽  
pp. 144-152 ◽  
Author(s):  
Sofia Somajo ◽  
Ruzica Livaja Koshiar ◽  
Eva Norström ◽  
Björn Dahlbäck
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Factor V ◽  
Platelet Microparticles

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.

Sign in / Sign up

Export Citation Format

Share Document