3.4.21.69: Protein C (activated)
This is an abbreviated version!
For detailed information about Protein C (activated), go to the full flat file.
Word Map on EC 3.4.21.69
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3.4.21.69
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thrombosis
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venous
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leiden
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sepsis
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endothelial
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antithrombin
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thromboembolism
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thrombophilia
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clot
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thrombomodulin
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platelet
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procoagulant
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heparin
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arterial
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vein
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bleeding
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plasminogen
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hypercoagulable
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thromboplastin
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lupus
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fibrinogen
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fibrinolysis
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hemostatic
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viiia
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epcr
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intravascular
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contraceptive
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antiphospholipid
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antithrombotic
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prothrombotic
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fibrin
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k-dependent
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anticardiolipin
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d-dimers
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coagulopathy
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embolism
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prothrombinase
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amidolytic
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thrombin-antithrombin
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haemostasis
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par1
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profibrinolytic
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pharmacology
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goal-directed
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diagnostics
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drug development
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gamma-carboxyglutamic
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deep-vein
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time-based
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tafi
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hypofibrinolysis
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thromboprophylaxis
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medicine
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protac
- 3.4.21.69
- thrombosis
- venous
- leiden
- sepsis
- endothelial
- antithrombin
- thromboembolism
- thrombophilia
- clot
- thrombomodulin
- platelet
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procoagulant
- heparin
- arterial
- vein
-
bleeding
- plasminogen
-
hypercoagulable
- thromboplastin
-
lupus
- fibrinogen
-
fibrinolysis
-
hemostatic
- viiia
- epcr
-
intravascular
-
contraceptive
-
antiphospholipid
-
antithrombotic
-
prothrombotic
- fibrin
-
k-dependent
-
anticardiolipin
-
d-dimers
- coagulopathy
- embolism
- prothrombinase
-
amidolytic
-
thrombin-antithrombin
-
haemostasis
- par1
-
profibrinolytic
- pharmacology
-
goal-directed
- diagnostics
- drug development
-
gamma-carboxyglutamic
-
deep-vein
-
time-based
- tafi
-
hypofibrinolysis
-
thromboprophylaxis
- medicine
- protac
Reaction
degradation of blood coagulation factors Va and VIIIa =
Synonyms
Activated blood coagulation factor XIV, Activated protein C, anticoagulant activated protein C, anticoagulant protein C/protein S system, anticoagulant serine protease-activated protein C, anticoagulant-activated protein C, APC, Autoprothrombin II-A, Autoprothrombin IIA, Blood coagulation factor XIV, Blood-coagulation factor XIV, activated, Blood-coagulation factor XIVa, ghrelin endopeptidase, GSAPC, hAPC, PROC, Protein Ca, rhAPC
ECTree
3.4.21.69 Protein C (activated)Advanced search results
results (
results (Engineering
Engineering on EC 3.4.21.69 - Protein C (activated)
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
D167F/D172G
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at saturating Ca2+ concentrations, the activation rates of the mutant and wild-type protein C by the thrombin-TM complex are comparable, but the mutant requires four-fold higher Ca2+ concentrations than wild-type APC to achieve half-maximal activation rates. When only thrombin is present, Ca2+ is not able to influence the activation of the D167F/D172G mutant, though Ca2+ effectively inhibits activation of wild-type protein C by thrombin
D222E
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mutation of the Arg-Gly-Asp sequence abolishes both integrin binding and inhibition of neutrophil migration
D35T/D36A
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site-directed mutagenesis, the mutant shows slightly increased factor Va proteolysis compared to the wild-type enzyme, the activation by protein S is reduced
D36A/L38D/A39V
E149A
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the cytoprotective effects of the APC mutant are severely diminished, despite a normal cleavage of PAR-1 and normal binding to EPCR. E149A-APC expresses only 6% of the anti-apoptotic activity of wild-type APC in a staurosporine-induced apoptosis model in endothelial cells and was unable to down-regulate IL-6 release in lipopolysaccharide treated U937 monocytes
E167A
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site-directed mutagenesis, the surface loop residue mutation eliminates the cytoprotective signaling properties of APC without affecting its anticoagulant activity, inability of E167A to exhibit significant protective activity in response to TNF-alpha-induced inflammatory events in endothelial cells
E16D
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site-directed mutagenesis, the mutation causes aberrant Ca2+ binding and Gla domain misfolding
E170A
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site-directed mutagenesis, the surface loop residue mutation eliminates the cytoprotective signaling properties of APC without affecting its anticoagulant activity, inability of E170A to exhibit significant protective activity in response to TNF-alpha-induced inflammatory events in endothelial cells
E20A/V34M
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the mutation is associated with thrombotic complications, despite the fact that carriers of these mutations have normal protein C antigen levels and APC amidolytic activity
E357Q
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E357 is involved in binding of macromolecular substrates. Engineered E357Q-APC shows two to threefold improved FVa inactivation, but slightly reduced anticoagulant activity in plasma compared to wild-type APC
E7D
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the mutation is associated with thrombotic complications, despite the fact that carriers of these mutations have normal protein C antigen levels and APC amidolytic activity
G216D
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a naturally occuring mutation, the mutant shows impaired protease activity, while preserving the overall protein fold. Superposition of the integrin binding motifs in wild-type and mutant forms suggests that the interaction with integrin can still occur and thus the mutant is likely to retain its antiseptic function related to the neutrophyl integrin binding
H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y
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naturally occuring Gla-domain mutant, the mutant enzyme shows a higher anticoagulant effect compared to the wild-type enzyme, the combination with the B148 mutation in the serine protease domain even enhances the effect, overview
K174E
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site-directed mutagenesis, the activation rate of the mutant by thrombin is 12fold faster than that observed for wild-type protein C in the presence of Ca2+, and unchanged in the absence of Ca2+. Thrombin does not stimulate activation of the protein C variant
K191A
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Km- and kcat-value similar to wild-type, minor contribution to interaction with thrombin-thrombomodulin
K191A/K192A/K193A
K192A
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Km- and kcat-value similar to wild-type, major contribution to interaction with thrombin-thrombomodulin
K217A
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Km- and kcat-value similar to wild-type, minor contribution to interaction with thrombin-thrombomodulin
K218A
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Km- and kcat-value similar to wild-type, minor contribution to interaction with thrombin-thrombomodulin
L38D
site-directed mutagenesis, creation of an APC variant with individual amino acid substitutions within this region, L38D is the main source of lost anti-coagulant activity. Despite this, APC-L38D retains the ability to mediate PAR1-dependent signalling properties on endothelial cells
L8Q
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mutant variants L8Q and R9H show reduced affinity for EPCR and can contribute to the reduced anticoagulant activity
N329Q
N33S/V34S/D35T/D36A/L38D/A39V
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site-directed mutagenesis, the mutant shows slightly increased factor Va proteolysis compared to the wild-type enzyme, no activation by protein S
P168V
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at saturating Ca2+ concentrations, the activation rates of the mutant and wild-type protein C by the thrombin-TM complex are comparable, but the mutant requires four-fold higher Ca2+ concentrations than wild-type APC to achieve half-maximal activation rates. When only thrombin is present, Ca2+ is not able to influence the activation of the P168V mutant, though Ca2+ effectively inhibits activation of wild-type protein C by thrombin
R177E
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site-directed mutagenesis, the activation rate of the mutant by thrombin is 12fold faster than that observed for wild-type protein C in the presence of Ca2+, and unchanged in the absence of Ca2+. Thrombin does not stimulate activation of the protein C variant
R178E
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site-directed mutagenesis, the activation rate of the mutant by thrombin is 12fold faster than that observed for wild-type protein C in the presence of Ca2+, and unchanged in the absence of Ca2+. Thrombin does not stimulate activation of the protein C variant
R222C/D237C
site-directed mutagenesis, manipulation of the APC serine protease domain via the introduction of a new disulfide bridge is found to disproportionately inhibit APC anti-coagulant activity compared with EPCR-PAR1 signalling function
R229A
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Km- and kcat-value similar to wild-type, major contribution to interaction with thrombin-thrombomodulin
R229A/R230A/K191A/K192A/K193A
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site-directed mutagenesis, construction of an APC protease domain mutant, 5A-APC, the mutant has minimal anticoagulant activity but normal cytoprotective activities that are dependent on endothelial protein C receptor and protease-activated receptor-1 as compared to the wild-tpe enzyme, activation of thrombin activable fibrinolysis inhibitor is essentially unaffected by 5A-APC due to its low anticoagulant activity, a 1000fold higher concentration of 5A-APC is required to give a factor Va inactivation pattern similar to that of recombinant wild-type APC
R230A
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Km- and kcat-value similar to wild-type, major contribution to interaction with thrombin-thrombomodulin
R312A
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Km- and kcat-value similar to wild-type, minor contribution to interaction with thrombin-thrombomodulin
R67C/R82C
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site-directed mutagenesis, construction of a protein C variant in which an engineered disulfide bond between two beta-sheets stabilizes the functionally critical Ca2+-binding 70-80 loop of the molecule. The 70-80 loop of this mutant no longer binds Ca2+, and the activation of the mutant by thrombin is enhanced 60-80fold independently of thrombomodulin, the anticoagulant activity of the activated protein C mutant is nearly eliminated. The endothelial protein C receptor- and protease activated receptor-1-dependent protective signaling properties of the mutant are minimally altered compared to the wild-type enzyme. The mutant loses its ability to interact with the procoagulant cofactors but not with the protective signaling molecules. The binding of EPCR is 2fold reduced compared to the wild-type enzyme
R9H
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mutant variants L8Q and R9H show reduced affinity for EPCR and can contribute to the reduced anticoagulant activity
S11G/S12N
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site-directed mutagenesis, the mutant shows slightly increased factor Va proteolysis compared to the wild-type enzyme
S190A
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Km- and kcat-value similar to wild-type, minor contribution to interaction with thrombin-thrombomodulin
S360A
S360C
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site-directed mutagenesis, the active site residue mutant shows no amidolytic activity
W231A
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Km- and kcat-value similar to wild-type, minor contribution to interaction with thrombin-thrombomodulin
K191A/K192A/K193A
engineering of APC by site-directed mutagenesis provided a signaling selective APC mutant with 3 Lys residues replaced by 3 Ala residues, 3K3A-APC, that lacks over 90% anticoagulant activity but retains normal cell signaling activities. The 3K3A-APC mutant exerts multiple potent neuroprotective activities, which require the G-protein-coupled receptor, protease activated receptor 1 (PAR1). Potent neuroprotection in murine ischemic stroke models is linked to 3K3A-APC-induced signaling that arises due to APC's cleavage in protease activated receptor 1 at a noncanonical Arg46 site
N329Q
the recombinant APC variant APCN329Q mimics the glycosylation pattern of the endogenous plasma APC-beta glycoform and exhibits significantly enhances PAR1-dependent cytoprotective activity on endothelial cells compared with wild-type APC, determination of the molecular basis for superior APC-beta cytoprotective signaling
R506Q
site-directed mutagenesis, the Leiden mutation, abrogates the anti-inflammatory cofactor function of factor V for activated protein C
additional information
D36A/L38D/A39V
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site-directed mutagenesis, the mutant shows no activation by protein S
D36A/L38D/A39V
site-directed mutagenesis, substitution of APC amino acid residues within the C-terminal end of the gamma-carboxyglutamic acid (Gla) domain with those of prothrombin result in an APC variant (APC-36A/L38D/A39V) with diminished capacity to inhibit thrombin generation. FVa degradation by this APC variant is normal in the absence of protein S, but completely defective when FVa degradation is dependent upon protein S cofactor activity
K191A/K192A/K193A
site-directed mutagenesis, mutant APC-K191A/K192A/K193A (APC-3K3A) is assessed in a phase II clinical trial as an adjunctive neuroprotective therapy in combination with t-PA (EC 3.4.21.68) postischaemic stroke
K191A/K192A/K193A
site-directed mutagenesis, the mutant is engineered to reduce APC-associated bleeding risk while retaining normal cell signaling activity. Enzyme mutant 3K3A-APC stimulates neuronal production by human neural stem/progenitor cells (NSCs) in vitro via a PAR1-PAR3-sphingosine-1-phosphate receptor 1-Akt pathway. Effects of late post-ischemic 3K3A-APC treatment on the in vivo production of neurons from transplanted NSCs in mice, and the effects of this combination therapy on long-term neurological recovery and restoration of disrupted neural circuitry in the post-ischemic murine brain, functional integration of NSCs into the host neuronal circuitry, overview
N329Q
site-directed mutagenesis, recombinant APC variant in which this N-linked glycosylation sequon is disrupted possessing markedly enhanced PAR1-dependent cytoprotective activity on endothelial cells. PAR1-dependent maintenance of endothelial cell barrier integrity is also achieved at approximately 5fold lower APC-N329Q concentration than wild-type APC. APC-N329Q also blocks staurosporine-induced endothelial cell apoptosis at about 20fold lower APC-N329Q than occurs when wild-type APC is used
N329Q
the recombinant APC variant mimics the glycosylation pattern of the endogenous plasma APC-beta glycoform and exhibits 4fold enhanced PAR1-dependent cytoprotective activity on endothelial cells compared to wild-type APC, determination of the molecular basis for superior APC-beta cytoprotective signaling
S360A
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site-directed mutagenesis, catalytic site residue mutation, mutant thrombin W215A/E217A induces protective signaling only in the presence of wild type protein C but not mutant protein C S360A
S360A
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site-directed mutagenesis, the active site residue mutant shows no amidolytic activity
S360A
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the active site mutant lacks proteolytic activity but exhibits anticoagulant activity. APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) binding to FVa is critically dependent upon the presence of Arg506 and not Arg306. Inhibition of FVIIIa activity by APC(S360A) is 100fold less efficient than inhibition of FVa. Binding of APC(S360A) to FVa is almost completely dependent on Arg506 interacting with APC(S360A) to form a nonproductive Michaelis complex. No binding to the substrate FVa Gln506 mutant
additional information
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enzyme variant with seven point-mutations in Gla domain, enhanced affinity for negatively charged phospholipid and increased anticoagulantic potential. Degradation of Factor Va yields similar fragments from wild-type and mutant, mutant is more efficient in absence and presence of protein S
additional information
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replacement of autolysis loop of protein by the corresponding loop of factor X. Mutant is activated by thrombin with 5fold higher rate in presence of Ca2+, Ca2+-affinity of mutant is impaired 3fold and anticoagulation function of mutant is improved 4-5fold in the absence, but not in the presence of protein S. Plasma half-life of mutant is markedly shortened
additional information
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substitution of the autolysis loop of APC with the corresponding loop of trypsin has no influence on the proteolytic activity of the protease towards factor Va, however, this substitution increases the reactivity of APC with plasma inhibitors so that the mutant exhibits no anticoagulant activity in plasma, the mutant APC cannot interact with protein S as cofactor, overview
additional information
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a thrombin derivative containing the Gla-domain of APC recapitulates all protective effects of APC with a 20 to 50fold higher efficacy
additional information
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proteolysis at Arg336 occurs 25-fold faster than at Arg562. Replacing residues flanking Arg336 en bloc with the corresponding residues surrounding Arg562 markedly reduces the rate of cleavage at Arg336
additional information
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single mutations of Gla domnain residues at positions 6, 7, 16, 20, 26 or 29 result in APC variants with less than 10% of the anticoagulant activity of wild-type APC
additional information
APC variants specifically designed to possess divergent anti-coagulant and signalling properties are created by ablation of a substrate-binding exosite for FVa on the surface of the APC serine protease domain (APC-R229A/R230A, APC-K191A/K192A/K193A and a combination of these mutations, termed 5A-APC). These variants signal normally via EPCR-PAR1 on endothelial cells, but possess severely attenuated ability to degrade FVa and thus inhibit coagulation. In animal models of endotoxemia, APC-5A exhibits comparable ability to protect mice from LPS-induced lethality as wild-type APC, illustrating the limited requirement for APC anti-coagulant function to reduce LPS-mediated mortality, at least in mice. These mutant variants are also shown to be equally protective in rodent models of ischaemic stroke and APC-K191A/K192A/K193A (APC-3K3A) is assessed in a phase II clinical trial as an adjunctive neuroprotective therapy in combination with t-PA (EC 3.4.21.68) postischaemic stroke. Bio-engineering approaches to modify APC for maximum therapeutic activity, overview. Methods to improve recombinant APC as a plausible therapeutic agent focuse on the removal of unwanted anti-coagulant activity, either by disruption of FVa substrate recognition in the serine protease domain or reduced sensitivity to its obligate anti-coagulant co-factor protein S by modification of the N-terminal Gla domain. Generation of APC variants with diminished interaction with its inhibitor alpha1-anti-trypsin and therefore longer plasma half-life. APC variants with enhanced ability to initiate cytoprotective signalling at lower APC concentrations via modification of specific N-linked glycan sequons are described
additional information
engineering of APC by site-directed mutagenesis provides a signaling selective APC mutant with 3 Lys residues replaced by 3 Ala residues, 3K3A-APC, that lacks over 90% anticoagulant activity but retains normal cell signaling activities. The 3K3A-APC mutant exerts multiple potent neuroprotective activities, which require the G-protein-coupled receptor, protease activated receptor 1 (PAR1)
additional information
-
engineering of APC by site-directed mutagenesis provides a signaling selective APC mutant with 3 Lys residues replaced by 3 Ala residues, 3K3A-APC, that lacks over 90% anticoagulant activity but retains normal cell signaling activities. The 3K3A-APC mutant exerts multiple potent neuroprotective activities, which require the G-protein-coupled receptor, protease activated receptor 1 (PAR1)
additional information
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knockout mice show perinatal lethality, reduced enzyme levels permit birth and growth but lead to thrombosis and inflammation with an early onset
additional information
a signaling-selective 5A-aPC variant lacks deleterious effects upon early administration
additional information
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a signaling-selective 5A-aPC variant lacks deleterious effects upon early administration
additional information
human APC variants specifically designed to possess divergent anti-coagulant and signalling properties are created by ablation of a substrate-binding exosite for FVa on the surface of the APC serine protease domain (APC-R229A/R230A, APC-K191A/K192A/K193A and a combination of these mutations, termed 5A-APC). These variants signal normally via EPCR-PAR1 on endothelial cells, but possess severely attenuated ability to degrade FVa and thus inhibit coagulation. In animal models of endotoxemia, APC-5A exhibits comparable ability to protect mice from LPS-induced lethality as wild-type APC, illustrating the limited requirement for APC anti-coagulant function to reduce LPS-mediated mortality, at least in mice. These mutant variants are also shown to be equally protective in rodent models of ischaemic stroke and APC-K191A/K192A/K193A (APC-3K3A) is assessed in a phase II clinical trial as an adjunctive neuroprotective therapy in combination with t-PA (EC 3.4.21.68) postischaemic stroke
additional information
neuroprotective activity of a murine APC variant with limited anticoagulant activity (mAPCPS) is compared with an identical APC variant except for the absence of glycosylation at the APC-beta sequon (mAPCPS/N329Q). mAPCPS/N329Q limits cerebral ischemic injury and reduces brain lesion volume significantly more effectively than mAPCPS
additional information
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neuroprotective activity of a murine APC variant with limited anticoagulant activity (mAPCPS) is compared with an identical APC variant except for the absence of glycosylation at the APC-beta sequon (mAPCPS/N329Q). mAPCPS/N329Q limits cerebral ischemic injury and reduces brain lesion volume significantly more effectively than mAPCPS
