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Literature summary for 3.4.21.69 extracted from
- Engineering activated protein C to maximize therapeutic efficacy (2015), Biochem. Soc. Trans., 43, 691-695 .
Activating Compound
| Activating Compound | Comment | Organism | Structure |
|---|---|---|---|
| endothelial cell protein C receptor | activation of protein C is potentiated by the presence of the endothelial cell protein C receptor (EPCR), which binds protein C with high affinity and increases the rate of protein C activation by about 20fold in vivo | Homo sapiens |
Application
| Application | Comment | Organism |
|---|---|---|
| diagnostics | endogenous protein C levels positively correlate with a positive outcome in patients with severe sepsis | Homo sapiens |
| medicine | possible effects of APC administration may include attenuation of the proinflammatory cytokine storm, re-balancing dysregulated haemostasis or degradation of cytotoxic extracellular histones that circulate during sepsis. Worldwide evaluation in severe sepsis (PROWESS) study demonstrated that recombinant APC (Xigris) administration reduces the relative risk of death compared with patients treated with a placebo (28 day mortality rate of 24.7% in individuals treated with recombinant APC, compared with 30.8% treated with placebo), prompting food and drug administration (FDA) approval for the application of Xigris in the treatment of severe sepsis. Subsequent concerns as to the efficacy and safety profile of Xigris prompted the PROWESS SHOCK trial that, in contrast with the original PROWESS study, indicated a lack of efficacy for Xigris in reducing the risk of death in severe sepsis patients, resulting in market withdrawal. The broad cytoprotective functions of APC on multiple cell types mean APC continues to lend itself to therapeutic applications, overview | Homo sapiens |
| medicine | protective effect of recombinant APC administration in animals subject to Escherichia coli-induced sepsis. Possible effects of APC administration may include attenuation of the proinflammatory cytokine storm, re-balancing dysregulated haemostasis or degradation of cytotoxic extracellular histones that circulate during sepsis. Successful pre-clinical animal studies indicate that the neuroprotective effects of recombinant APC do not require anti-coagulant activity for therapeutic benefit to be achieved | Bos taurus |
| medicine | protective effect of recombinant APC administration in animals subject to Escherichia coli-induced sepsis. Possible effects of APC administration may include attenuation of the proinflammatory cytokine storm, re-balancing dysregulated haemostasis or degradation of cytotoxic extracellular histones that circulate during sepsis. Successful pre-clinical animal studies indicate that the neuroprotective effects of recombinant APC do not require anti-coagulant activity for therapeutic benefit to be achieved. The bleeding risk associated with the use of recombinant APC in the treatment of severe sepsis and apparent lack of requirement for APC anti-coagulant function for protective activity in murine endotoxemia and stroke models prompted the development of APC variants with selectively diminished anti-coagulant activity. APC variants that possess limited anti-coagulant function but normal cytoprotective signalling activity represent a potentially safer alternative to recombinant wild-type APC in disease settings in which APC has been shown to be protective | Mus musculus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| 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 | Homo sapiens |
| 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 | Homo sapiens |
| 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 | Homo sapiens |
| 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 | Homo sapiens |
| 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 | Mus musculus |
| 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 | Homo sapiens |
| 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 | Homo sapiens |
| R229A/R230A | site-directed mutagenesis | Homo sapiens |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| alpha1-anti-trypsin | APC inhibition by its principal plasma inhibitor alpha1-anti-trypsin | Homo sapiens | |
| alpha1-antitrypsin | APC anti-coagulant activity is eventually inhibited by the action of the serpins alpha1-antitrypsin and protein C inhibitor, which irreversibly bind and inactivate APC prior to clearance | Bos taurus | |
| alpha1-antitrypsin | APC anti-coagulant activity is eventually inhibited by the action of the serpins alpha1-antitrypsin and protein C inhibitor, which irreversibly bind and inactivate APC prior to clearance | Mus musculus | |
| Protein C inhibitor | APC anti-coagulant activity is eventually inhibited by the action of the serpins alpha1-antitrypsin and protein C inhibitor, which irreversibly bind and inactivate APC prior to clearance | Bos taurus | |
| Protein C inhibitor | APC anti-coagulant activity is eventually inhibited by the action of the serpins alpha1-antitrypsin and protein C inhibitor, which irreversibly bind and inactivate APC prior to clearance | Mus musculus |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| Factor Va + H2O | Homo sapiens | - |
? | - |
? |  |
| Factor Va + H2O | Bos taurus | - |
? | - |
? | |
| Factor Va + H2O | Mus musculus | - |
? | - |
? | |
| Factor VIIIa + H2O | Homo sapiens | - |
? | - |
? | |
| Factor VIIIa + H2O | Bos taurus | - |
? | - |
? | |
| Factor VIIIa + H2O | Mus musculus | - |
? | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Bos taurus | P00745 | - |
- |
| Homo sapiens | P04070 | - |
- |
| Mus musculus | P33587 | - |
- |
Posttranslational Modification
| Posttranslational Modification | Comment | Organism |
|---|---|---|
| glycoprotein | - |
Bos taurus |
| glycoprotein | - |
Mus musculus |
| glycoprotein | N-linked glycan attachment site that is occupied by a complex sialyated glycan chain in about 70% of plasma protein C (termed protein C-alpha), but not in the remaining protein C pool, which is termed protein C-beta | Homo sapiens |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| macrophage | - |
Mus musculus | - |
| marrow cell | - |
Homo sapiens | - |
| monocyte | - |
Homo sapiens | - |
| U-937 cell | - |
Homo sapiens | - |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| Factor Va + H2O | - |
Homo sapiens | ? | - |
? | |
| Factor Va + H2O | - |
Bos taurus | ? | - |
? | |
| Factor Va + H2O | - |
Mus musculus | ? | - |
? | |
| Factor VIIIa + H2O | - |
Homo sapiens | ? | - |
? | |
| Factor VIIIa + H2O | - |
Bos taurus | ? | - |
? | |
| Factor VIIIa + H2O | - |
Mus musculus | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| Activated protein C | - |
Homo sapiens |
| Activated protein C | - |
Bos taurus |
| Activated protein C | - |
Mus musculus |
| anticoagulant-activated protein C | - |
Homo sapiens |
| anticoagulant-activated protein C | - |
Bos taurus |
| anticoagulant-activated protein C | - |
Mus musculus |
| APC | - |
Homo sapiens |
| APC | - |
Bos taurus |
| APC | - |
Mus musculus |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| protein S | - |
Homo sapiens | |
| protein S | - |
Bos taurus | |
| protein S | - |
Mus musculus |
General Information
| General Information | Comment | Organism |
|---|---|---|
| malfunction | Mac-1 inhibition prevents APC attenuation of pro-inflammatory cytokine release from lipopolysaccharide (LPS)-stimulated murine macrophages. Furthermore, APC administration does not significantly reduce mortality in Mac-1 deficient endotoxemic mice, suggestive of an important role for Mac-1-dependent PAR1 activation on myeloid cells for the ability of APC to limit mortality in murine endotoxemia | Mus musculus |
| physiological function | the anticoagulant-activated protein C (APC) acts not solely as a crucial regulator of thrombus formation following vascular injury, but also as a potent signalling enzyme with important functions in the control of both acute and chronic inflammatory disease | Bos taurus |
| physiological function | the anticoagulant-activated protein C (APC) acts not solely as a crucial regulator of thrombus formation following vascular injury, but also as a potent signalling enzyme with important functions in the control of both acute and chronic inflammatory disease. Distinct from its crucial role in regulating blood coagulation, APC initiates cell signalling via engagement with multiple cell surface receptors. Although APC triggers an array of signalling pathways via distinct receptor interactions on different cell types, APC signalling is invariably cytoprotective, conferring protection against exposure to pro-inflammatory and/or pro-apoptotic agents. Prototypical APC signalling on endothelial cells involves initial binding to EPCR that in turn facilitates proteolysis and activation of protease-activated receptor 1 (PAR1). APC binding to EPCR is a pre-requisite for PAR3 activation by APC, which mediates similar protective signalling outputs as PAR1 activation. The role of EPCR as an APC co-receptor for PAR1 signalling can also be assumed by the integrin alphaMbeta2 (macrophage-1 antigen, Mac-1) | Mus musculus |
| physiological function | the anticoagulant-activated protein C (APC) acts not solely as a crucial regulator of thrombus formation following vascular injury, but also as a potent signalling enzyme with important functions in the control of both acute and chronic inflammatory disease. The proteolytic inactivation of FVa/FVIIIa by APC with protein S slows further thrombin generation and thus acts as a negative feedback loop to inhibit thrombus growth. Distinct from its crucial role in regulating blood coagulation, APC initiates cell signalling via engagement with multiple cell surface receptors. Although APC triggers an array of signalling pathways via distinct receptor interactions on different cell types, APC signalling is invariably cytoprotective, conferring protection against exposure to pro-inflammatory and/or pro-apoptotic agents. Prototypical APC signalling on endothelial cells involves initial binding to EPCR that in turn facilitates proteolysis and activation of protease-activated receptor 1 (PAR1). APC binding to EPCR is a pre-requisite for PAR3 activation by APC, which mediates similar protective signalling outputs as PAR1 activation. APC can also promote anti-inflammatory signalling on myeloid cells in a PAR-independent manner. APC binds apolipoprotein E receptor 2 (ApoER2) to trigger Dab1 phosphorylation and activate phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase 3beta (GSK3beta)-dependent signalling in monocytic U-937 cells. APC might interact concurrently with other APC receptors on monocytes | Homo sapiens |
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