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

Abstract 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.

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.

Impaired APC cofactor activity of factor V plays a major role in the APC resistance associated with the factor V Leiden (R506Q) and R2 (H1299R) mutations

Blood ◽  
2004 ◽  
Vol 103 (11) ◽  
pp. 4173-4179 ◽  
Author(s):  
Elisabetta Castoldi ◽  
Jeroen M. Brugge ◽  
Gerry A. F. Nicolaes ◽  
Domenico Girelli ◽  
Guido Tans ◽  
...  
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Gene Mutations ◽  
Factor V Leiden ◽  
Activated Partial Thromboplastin Time ◽  
Factor V ◽  
Thrombotic Risk ◽  
Apc Resistance ◽  
Cofactor Activity

Abstract Activated protein C (APC) resistance is a major risk factor for venous thrombosis. Factor V (FV) gene mutations like FVLeiden (R506Q) and FVR2 (H1299R) may cause APC resistance either by reducing the susceptibility of FVa to APC-mediated inactivation or by interfering with the cofactor activity of FV in APC-catalyzed FVIIIa inactivation. We quantified the APC cofactor activity expressed by FVLeiden and FVR2 and determined the relative contributions of reduced susceptibility and impaired APC cofactor activity to the APC resistance associated with these mutations. Plasmas containing varying concentrations of normal FV, FVLeiden, or FVR2 were assayed with an APC resistance assay that specifically measures the APC cofactor activity of FV in FVIIIa inactivation, and with the activated partial thromboplastin time (aPTT)-based assay, which probes both the susceptibility and APC cofactor components. FVR2 expressed 73% of the APC cofactor activity of normal FV, whereas FVLeiden exhibited no cofactor activity in FVIIIa inactivation. Poor susceptibility to APC and impaired APC cofactor activity contributed equally to FVLeiden-associated APC resistance, whereas FVR2-associated APC resistance was entirely due to the reduced APC cofactor activity of FVR2. Thrombin generation assays confirmed the importance of the anticoagulant activity of FV and indicated that FVLeiden homozygotes are exposed to a higher thrombotic risk than heterozygotes because their plasma lacks normal FV acting as an anticoagulant protein.

scholarly journals Coagulation Factor V Mediates Inhibition of Tissue Factor Signaling By Activated Protein C

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 216-216
Author(s):  
Hartmut Weiler ◽  
Hai-Po Liang ◽  
Edward J Kerschen ◽  
Alireza Rezaie ◽  
Jose A. Fernandez ◽  
...  
Keyword(s):  
Cell Signaling ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Protein S ◽  
Survival Advantage ◽  
Factor V ◽  
Apc Resistance

Abstract BACKGROUND: The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. The naturally occurring R506Q Leiden polymorphism in fV largely abrogates the anticoagulant functions of aPC by rendering fVa partially refractory to aPC proteolysis, but also by preventing the formation of the anticoagulant cofactor form of fV. Among patients enrolled in the placebo arm of the PROWESS sepsis trial, heterozygous fV Leiden carriers showed significantly reduced mortality 1, and a similar survival advantage of heterozygous Leiden carriers was documented in mice harboring the fV R504Q mutation (equivalent to the human R506Q mutation) that were challenged with endotoxin1, gram-positive (S.aureus), or gram-negative infection (Y.pestis)2. The objective of the current study was to examine how aPC-resistance of fV Leiden modulates responsiveness to sepsis therapy with aPC in mice. RESULTS: In murine sepsis models of S.aureus-induced septic peritonitis, aPC-resistance of endogenous fV R504Q prevents marked disease stage-specific deleterious effects associated with aPC's anticoagulant activity, but also abrogated the mortality-reducing benefits of therapy with the signaling-selective 5A-aPC variant that only exerts minimal anticoagulant activity towards activated fVa. In mice homozygous for the R504Q mutation (fVQQ mice), 5A-aPC failed to suppress inflammatory gene expression in the presence of fVR504Q. This finding was reproduced in an in vitro culture model of murine RAW cells and bone marrow-derived dendritic cells, in which thrombosis and thrombin generation play no role. Gene expression analyses and functional in vitro studies of LPS-induced inflammatory cell signaling showed that fV, as well as protein S were required for the aPC-mediated suppression of inflammatory tissue factor-PAR2 signaling3. Structure-function analyses of recombinant variants of aPC and fV showed that this anti-inflammatory cofactor function of protein S and fV involved the same structural features that underlie their accessory role for aPC's anticoagulant function, but did not involve the degradation of activated fVa or fVIIIa. CONCLUSION: These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection. This cofactor function is structurally related, but mechanistically distinct from the anticoagulant cofactor activities of protein S and fV. APC-resistance of fV thus emerges as a response modifier of the endogenous host response to infection, as well as the outcome of sepsis therapy with normal APC and signaling-selective variants thereof. REFERENCES 1. Kerlin BA, Yan SB, Isermann BH, et al. Survival advantage associated with heterozygous factor V Leiden mutation in patients with severe sepsis and in mouse endotoxemia. Blood. 2003;102(9):3085-3092. 2. Kerschen E, Hernandez I, Zogg M, Maas M, Weiler H. Survival advantage of heterozygous factor V Leiden carriers in murine sepsis. J Thromb Haemost. 2015;13(6):1073-1080. 3. Liang HP, Kerschen EJ, Hernandez I, et al. EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice. Blood. 2015. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.

The Second Laminin G-type Domain of Protein S Is Indispensable for Expression of Full Cofactor Activity in Activated Protein C-catalysed Inactivation of Factor Va and Factor VIIIa

2000 ◽  
Vol 84 (08) ◽  
pp. 271-277 ◽  
Author(s):  
Petra Evenäs ◽  
Pablo García de Frutos ◽  
Gerry Nicolaes ◽  
Björn Dahlbäck
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Factor V ◽  
Factor Va ◽  
Factor Viiia ◽  
Dependent Protein ◽  
Cofactor Activity ◽  
The Individual

SummaryVitamin K-dependent protein S is a cofactor to the anticoagulant serine protease activated protein C (APC) in the proteolytic inactivation of the procoagulant, activated factor V (FVa) and factor VIII (FVIIIa). In the FVa degradation, protein S selectively accelerates the cleavage at Arg306, having no effect on the Arg506 cleavage. In the FVIIIa inactivation, the APC-cofactor activity of protein S is synergistically potentiated by FV, which thus has the capacity to function both as a pro- and an anticoagulant protein. The SHBG-like region of protein S, containing two laminin G-type domains, is required for the combined action of protein S and FV. To elucidate whether both G domains in protein S are needed for expression of APC-cofactor activities, chimeras of human protein S were created in which the individual G domains were replaced by the corresponding domain of the homologous Gas6, which in itself has no anticoagulant activity. In a plasmabased assay, chimera I (G1 from Gas6) was as efficient as wild-type recombinant protein S, whereas chimera II (G2 from Gas6) was less effective. The synergistic cofactor activity with FV in the inactivation of FVIIIa was lost by the replacement of the G2 domain in protein S (chimera II). However, chimera I did not exert full APC-cofactor activity in the FVIIIa degradation, indicating involvement of both G domains or the entire SHBG-like region in this reaction. Chimera I was fully active in the degradation of FVa in contrast to chimera II, which exhibited reduced cofactor activity compared to protein S. In conclusion, by using protein S-Gas6 chimeric proteins, we have identified the G2 domain of protein S to be indispensable for an efficient inactivation of both FVIIIa and FVa, whereas the G1 domain was found not to be of direct importance in the FVa-inactivation experiments.

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.

scholarly journals Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V

Blood ◽  
1995 ◽  
Vol 85 (12) ◽  
pp. 3405-3411 ◽  
Author(s):  
MJ Heeb ◽  
Y Kojima ◽  
JS Greengard ◽  
JH Griffin
Keyword(s):  
Protein C ◽  
Molecular Mechanisms ◽  
Activated Protein C ◽  
Factor V ◽  
Activated Protein C Resistance ◽  
Sequence Analyses ◽  
Coagulation Assays ◽  
Apc Resistance ◽  
Initial Cleavage ◽  
Cofactor Activity

Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity. In purified reaction mixtures, Gln506-FVa in comparison to normal FVa showed reduced susceptibility to APC, because it was inactivated approximately 10-fold slower than normal Arg506-FVa. It was previously reported that inactivation of normal FVa by APC involves an initial cleavage at Arg506 followed by phospholipid-dependent cleavage at Arg306. Immunoblot and amino acid sequence analyses showed that the 102-kD heavy chain of Gln506-FVa was cleaved at Arg306 during inactivation by APC in a phospholipid-dependent reaction. This reduced but measurable susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance is a mild risk factor for thrombosis because APC can inactivate both normal FVa and variant Gln506-FVa. In summary, this study shows that purified Gln506-FV can account for APC resistance of plasma because Gln506-FVa, whether generated by thrombin or FXa, is relatively resistant to APC.

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.

scholarly journals Exacerbated venous thromboembolism in mice carrying a protein S K196E mutation

Blood ◽  
2015 ◽  
Vol 126 (19) ◽  
pp. 2247-2253 ◽  
Author(s):  
Fumiaki Banno ◽  
Toshiyuki Kita ◽  
José A. Fernández ◽  
Hiroji Yanamoto ◽  
Yuko Tashima ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Venous Thrombosis ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Protein S Deficiency ◽  
Wild Type ◽  
S Deficiency ◽  
Key Points ◽  
Cofactor Activity

Key Points A protein S-K196E mutation reduced its activated protein C cofactor activity in recombinant murine protein S-K196E and in K196E mutant mice. Mice carrying a protein S-K196E mutation or heterozygous protein S deficiency were more vulnerable to venous thrombosis than wild-type mice.

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.

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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