Protein C, also known asautoprothrombin IIA andblood coagulation factor XIV,[5]: 6822 [6] is azymogen, that is, an inactive enzyme. The activated form plays an important role in regulatinganticoagulation,inflammation, andcell death and maintaining thepermeability of blood vessel walls in humans and other animals.Activated protein C (APC) performs these operations primarily byproteolytically inactivating proteinsFactor Va andFactor VIIIa. APC is classified as aserine protease since it contains aresidue ofserine in itsactive site.[7]: 35 In humans, protein C is encoded by thePROCgene, which is found onchromosome 2.[8]
The zymogenic form of protein C is avitamin K-dependentglycoprotein that circulates inblood plasma. Its structure is that of a two-chain polypeptide consisting of a light chain and a heavy chain connected by adisulfide bond.[8]: 4673 The protein C zymogen is activated when it binds tothrombin, another protein heavily involved in coagulation, and protein C's activation is greatly promoted by the presence ofthrombomodulin andendothelial protein C receptors (EPCRs). Because of EPCR's role, activated protein C is found primarily near endothelial cells (i.e., those that make up the walls of blood vessels), and it is these cells andleukocytes (white blood cells) that APC affects.[7]: 34 [9]: 3162 Because of the crucial role that protein C plays as ananticoagulant, those with deficiencies in protein C, or some kind ofresistance to APC, suffer from a significantly increased risk of forming dangerous blood clots (thrombosis).
Research into the clinical use of arecombinant form of human Activated Protein C (rhAPC) known asDrotrecogin alfa-activated, branded Xigris byEli Lilly and Company, has been surrounded by controversy. Eli Lilly ran an aggressive marketing campaign to promote its use for people with severesepsis and septic shock and sponsored the 2004Surviving Sepsis Campaign Guidelines.[10] However, a 2012Cochrane review found that its use cannot be recommended since it does not improve survival and increases bleeding risk.[11] In October 2011, Xigris was withdrawn from the market by Eli Lilly due to a higher mortality in a trial among adults.[12]
Protein C'santicoagulant role in the human body was first noted by Seegerset al. in 1960,[13] who gave protein C its original name,autoprothrombin II-a.[5]: 6822 Protein C was first isolated byJohan Stenflo frombovineplasma in 1976, and Stenflo determined it to be avitamin K-dependent protein.[14] He named it proteinC because it was the third protein ("peak C") thateluted from aDEAE-Sepharoseion-exchange chromotograph. Seegers was, at the time, searching for vitamin K-dependent coagulation factors undetected byclotting assays, which measure global clotting function. Soon after this, Seegers recognised Stenflo's discovery was identical with his own.[5]: 6822 Activated protein C was discovered later that year,[15] and in 1977 it was first recognised that APC inactivates Factor Va.[16]: 2382 [17] In 1980, Vehar and Davie discovered that APC also inactivates Factor VIIIa,[18] and soon after,Protein S was recognised as acofactor by Walker.[19] In 1982, a family study by Griffinet al. first associatedprotein C deficiency with symptoms ofvenous thrombosis.[20] Homozygous protein C deficiency and the consequent serious health effects were described in 1984 by several scientists.[21]: 1214 cDNA cloning of protein C was first performed in 1984 by Beckmannet al. which produced a map of the gene responsible for producing protein C in the liver.[22] In 1987 a seminal experiment was performed (Tayloret al.) whereby it was demonstrated that activated protein C preventedcoagulopathy and death in baboonsinfused with lethal concentrations ofE. coli.[16]: 2382 [23]
In 1993, a heritable resistance to APC was detected byDahlbäcket al. and associated with familialthrombophilia.[24] In 1994, the relatively common genetic mutation that producesFactor VLeiden was noted (Bertinaet al.).[25] Two years later, Gla-domainless APC was imaged at a resolution of 2.8 Ångströms.[α][5] Beginning with the PROWESSclinical trial of 2001,[26] it was recognised that many of the symptoms ofsepsis may be ameliorated by infusion of APC, and mortality rates of septic patients may be significantly decreased.[9]: 3161, 6 Near the end of that year,Drotrecogin alfa (activated), a recombinant human activated protein C, became the first drug approved by the U.S.FDA for treating severesepsis.[27] In 2002,Science published an article that first showed protein C activatesprotease-activated receptor-1 (PAR-1) and this process accounts for the protein's modulation of the immune system.[16]: 2382 [28]
The biologic instructions for synthesising protein C in humans are encoded in the gene officially named "protein C (inactivator of coagulation factors Va and VIIIa)". The gene's symbol approved by theHUGO Gene Nomenclature Committee is "PROC" from "proteinC". It is located on the secondchromosome (2q13-q14) and comprises nineexons.[8][16]: 2383 The nucleotide sequence that codes for human protein C is approximately 11,000 bases long.[8]: 4675
Human protein C is a vitamin K-dependent glycoprotein structurally similar to other vitamin K-dependent proteins affecting blood clotting,[29] such asprothrombin,Factor VII,Factor IX andFactor X.[21]: 1215 Protein C synthesis occurs in the liver and begins with a single-chain precursor molecule: a 32 amino acidN-terminussignal peptide preceding apropeptide.[30]: S11 Protein C is formed when adipeptide of Lys198 and Arg199 is removed; this causes the transformation into aheterodimer withN-linked carbohydrates on each chain. The protein has one light chain (21 kDa) and one heavy chain (41 kDa) connected by adisulfide bond between Cys183 and Cys319.
Inactive protein C comprises 419 amino acids in multipledomains:[16]: 2383 oneGla domain (residues 43–88); a helicalaromatic segment (89–96); twoepidermal growth factor (EGF)-like domains (97–132 and 136–176); an activation peptide (200–211); and atrypsin-like serine protease domain (212–450). The light chain contains the Gla- and EGF-like domains and the aromatic segment. The heavy chain contains the protease domain and the activation petide. It is in this form that 85–90% of protein C circulates in the plasma as azymogen, waiting to be activated.[5]: 6822 The remaining protein C zymogen comprises slightly modified forms of the protein. Activation of the enzyme occurs when a thrombin molecule cleaves away the activation peptide from theN-terminus of the heavy chain.[8]: 4673 [30]: S11 The active site contains acatalytic triad typical of serine proteases (His253, Asp299 and Ser402).[16]: 2833
The Gla domain is particularly useful for binding to negatively chargedphospholipids for anticoagulation and to EPCR forcytoprotection. One particularexosite augments protein C's ability to inactivate Factor Va efficiently. Another is necessary for interacting with thrombomodulin.[16]: 2833
Post-translational modifications. Human Protein C has at least five types ofpost-translational modifications: (1) gamma-carboxylation on the first nineglutamic acid residues in the protein sequence. This modification event is performed by a vitamin K-dependent microsomal carboxylase. The full complement of Gla is required to give full activity to protein C. (2) beta-Hydroxylation of Asp71 in one of the twoEGF-like domains to give erythro-L-beta-hydroxy-aspartate (bHA). The modification is required for functional activity as was demonstrated by mutating Asp71 to Glu. (3) N-linkedglycosylation at three possible glycosylation sites. Plasma human Protein C has been reported to be 23% carbohydrate by weight. (4) Disulfide formation. (5) Multipleproteolytic cleavages of the polypeptide backbone to remove an 18 amino acidsignal peptide, a 24 amino acid propeptide and then cleavages at amino acids 155-156 and 157-158 to yield the two-chain structure of the circulating zymogen.[31]
The activation of protein C is strongly promoted bythrombomodulin andendothelial protein C receptor (EPCR), the latter of which is found primarily onendothelial cells (cells on the inside of blood vessels). The presence of thrombomodulin accelerates activation by several orders of magnitude,[7]: 34 and EPCR speeds up activation by a factor of 20. If either of these two proteins is absent inmurine specimens, the mouse dies from excessive blood-clotting while still in anembryonic state.[32]: 1983 [33]: 43335 On the endothelium, APC performs a major role in regulating blood clotting, inflammation, and cell death (apoptosis).[34]: 28S Because of the accelerating effect of thrombomodulin on the activation of protein C, the protein may be said to be activated not by thrombin but the thrombin–thrombomodulin (or even thrombin–thrombomodulin–EPCR) complex.[16]: 2381 Once in active form, APC may or may not remain bound to EPCR, to which it has approximately the same affinity as the protein zymogen.[9]: 3162
Protein C inzymogen form is present in normal adult humanblood plasma at concentrations between 65 and 135 IU/dL. Activated protein C is found at levels approximately 2000 times lower than this.[9]: 3161 Mild protein C deficiency corresponds to plasma levels above 20 IU/dL, but below the normal range. Moderately severe deficiencies describe blood concentrations between 1 and 20 IU/dL; severe deficiencies yield levels of protein C that are below 1 IU/dL or are undetectable. Protein C levels in a healthyterm infant average 40 IU/dL. The concentration of protein C increases until six months, when the mean level is 60 IU/dL; the level stays low through childhood until it reaches adult levels afteradolescence.[21]: 1216 Thehalf-life of activated protein C is around 15 minutes.[5]: 6823
The protein C pathways are the specific chemical reactions that control the level of expression of APC and its activity in the body.[7]: 34 Protein C ispleiotropic, with two main classes of functions: anticoagulation and cytoprotection (its direct effect on cells). Which function protein C performs depends on whether or not APC remains bound to EPCR after it is activated; the anticoagulative effects of APC occur when it does not. In this case, protein C functions as an anticoagulant by irreversibly proteolytically inactivatingFactor Va andFactor VIIIa, turning them into Factor Vi and Factor VIIIi respectively. When still bound to EPCR, activated protein C performs its cytoprotective effects, acting on theeffectorsubstrate PAR-1,protease-activated receptor-1. To a degree, APC's anticoagulant properties are independent of its cytoprotective ones, in that expression of one pathway is not affected by the existence of the other.[9]: 3162 [34]: 26S
The activity of protein C may bedown-regulated by reducing the amount either of available thrombomodulin or of EPCR. This may be done by inflammatorycytokines, such asinterleukin-1β (IL-1β ) andtumor necrosis factor-α (TNF-α). Activated leukocytes release these inflammatory mediators during inflammation, inhibiting the creation of both thrombomodulin and EPCR, and inducing their shedding from the endothelial surface. Both of these actions down-regulate protein C activation. Thrombin itself may also have an effect on the levels of EPCR. In addition, proteins released from cells can impede protein C activation, for exampleeosinophil, which may explain thrombosis inhypereosinophilic heart disease.[β] Protein C may beup-regulated byplatelet factor 4. This cytokine is conjectured to improve activation of protein C by forming anelectrostatic bridge from protein C's Gla domain to theglycosaminoglycan (GAG) domain of thrombomodulin, reducing theMichaelis constant (KM) for their reaction.[16]: 2386 [34]: 29S In addition, Protein C is inhibited byprotein C inhibitor.[35]: 369
Protein C is a major component in anticoagulation in the human body. It acts as aserine proteasezymogen: APC proteolyses peptide bonds in activatedFactor V andFactor VIII (Factor Va and Factor VIIIa), and one of the amino acids in the bond isserine.[16]: 2381 These proteins that APC inactivates, Factor Va and Factor VIIIa, are highly procoagulant cofactors in the generation ofthrombin, which is a crucial element in blood clotting; together they are part of theprothrombinase complex.[34]: 26S Cofactors in the inactivation of Factor Va and Factor VIIIa includeprotein S, Factor V,high-density lipoprotein, anionicphospholipids andglycosphingolipids.[9]: 3161
Factor Va binds toprothrombin and Factor Xa, increasing the rate at which thrombin is produced by four orders of magnitude (10,000x). Inactivation of Factor Va thus practically halts the production of thrombin. Factor VIII, on the other hand, is a cofactor in production of activated Factor X, which in turn converts prothrombin into thrombin. Factor VIIIa augments Factor X activation by a factor of around 200,000. Because of its importance in clotting, Factor VIII is also known as anti-haemophilic factor, and deficiencies of Factor VIII causehaemophilia A.[16]: 2382, 3
APC inactivates Factor Va by making three cleavages (Arg306, Arg506, Arg679). The cleavages at both Arg306 and Arg506 diminish the molecule's attraction to Factor Xa, and though the first of these sites is slow to be cleaved, it is entirely necessary to the functioning of Factor V. Protein S aids this process by catalysing the proteolysis at Arg306, in which the A2 domain of Factor V is dissociated from the rest of the protein.[36] Protein S also binds to Factor Xa, inhibiting the latter from diminishing APC's inactivation of Factor Va.[16]: 2386
The inactivation of Factor VIIIa is not as well understood. The half-life of Factor VIIIa is only around two minutes unless Factor IXa is present to stabilise it. Some have questioned the significance of APC's inactivation of Factor VIIIa, and it is unknown to what degree Factor V and protein S are cofactors in its proteolysis. It is known that APC works on Factor VIIIa by cleaving at two sites, Arg336 and Arg562, either of which is sufficient to disable Factor VIIIa and convert it to Factor VIIIi.[16]: 2387
When APC is bound to EPCR, it performs a number of important cytoprotective (i.e. cell-protecting) functions, most of which are known to require EPCR and PAR-1. These include regulating gene expression, anti-inflammatory effects, antiapoptotic effects and protecting endothelial barrier function.[9]: 3162
Treatment of cells with APC demonstrates that its gene expression modulation effectively controls major pathways for inflammatory and apoptotic behaviour. There are about 20 genes that are up-regulated by protein C, and 20 genes that are down-regulated: the former are generally anti-inflammatory and antiapoptotic pathways, while the latter tend to be proinflammatory and proapoptotic. APC's mechanisms for altering gene expression profiles are not well understood, but it is believed that they at least partly involve an inhibitory effect ontranscription factor activity.[9]: 3162, 4 Important proteins that APC up-regulates includeBcl-2,eNOS andIAP. APC effects significant down-regulation ofp53 andBax.[16]: 2388
APC has anti-inflammatory effects onendothelial cells andleukocytes. APC affects endothelial cells by inhibitinginflammatory mediator release and down-regulatingvascular adhesion molecules. This reduces leukocyte adhesion and infiltration into tissues, while also limiting damage to underlying tissue. APC supports endothelial barrier function and reduceschemotaxis. APC inhibits the release of inflammatory-response mediators in leukocytes as well as endothelial cells, by reducing cytokine response, and maybe diminishing systemic inflammatory response, such as is seen insepsis. Studies on both rats and humans have demonstrated that APC reducesendotoxin-induced pulmonary injury and inflammation.[9]: 3164
Scientists recognise activated protein C's antiapoptotic effects, but are unclear as to the exact mechanisms by which apoptosis is inhibited. It is known that APC isneuroprotective. Antiapoptosis is achieved with diminished activation ofcaspase 3 andcaspase 8, improved Bax/Bcl-2 ratio and down-regulation of p53.[16]: 2388
Activated protein C also provides much protection of endothelial barrier function. Endothelial barrier breakdown, and the corresponding increase in endothelial permeability, are associated withswelling,hypotension and inflammation, all problems of sepsis. APC protects endothelial barrier function by inducing PAR-1 dependentsphingosine kinase-1 activation and up-regulatingsphingosine-1-phosphate withsphingosine kinase.[9]: 3165
Several studies have indicated that the proteolytic activity of APC contributes to the observed cytoprotective properties of APC, but variants that are proteolytically inactive also are able to regulate formation of PAR-activators thrombin and factor Xa and express cytoprotective properties in vitro and in vivo.[37][38]
A geneticprotein C deficiency, in its mild form associated withsimple heterozygosity, causes a significantly increased risk ofvenous thrombosis in adults. If a fetus ishomozygous orcompound heterozygous for the deficiency, there may be a presentation ofpurpura fulminans, severedisseminated intravascular coagulation and simultaneousvenous thromboembolism in the womb;[21]: 1214 this is very severe and usually fatal.[39]: 211s Deletion of the protein C gene in mice causes fetal death around the time of birth. Fetal mice with no protein C develop normally at first, but experience severe bleeding,coagulopathy, deposition offibrin andnecrosis of the liver.[9]: 3161
The frequency of protein C deficiency among asymptomatic individuals is between 1 in 200 and 1 in 500. In contrast, significant symptoms of the deficiency are detectable in 1 in 20,000 individuals. No racial nor ethnic biases have been detected.[21]: 1215
At least 177 disease-causing mutations in this gene have been discovered.[40]Activated protein C resistance occurs when APC is unable to perform its functions. This disease has similar symptoms to protein C deficiency. The most common mutation leading to activated protein C resistance among Caucasians is at the cleavage site in Factor V for APC. There, Arg506 is replaced with Gln, producingFactor V Leiden. This mutation is also called a R506Q.[16]: 2382 The mutation leading to the loss of this cleavage site actually stops APC from effectively inactivating both Factor Va and Factor VIIIa. Thus, the person's blood clots too readily, and he is perpetually at an increased risk for thrombosis.[41]: 3 Individuals heterozygous for the Factor VLeiden mutation carry a risk of venous thrombosis 5–7 times higher than in the general population. Homozygous subjects have a risk 80 times higher.[7]: 40 This mutation is also the most common hereditary risk for venous thrombosis amongCaucasians.[16]: 2382
Around 5% of APC resistance are not associated with the above mutation and Factor VLeiden. Other genetic mutations cause APC resistance, but none to the extent that Factor VLeiden does. These mutations include various other versions of Factor V, spontaneous generation ofautoantibodies targeting Factor V, and dysfunction of any of APC's cofactors.[16]: 2387 Also, some acquired conditions may reduce the efficacy of APC in performing its anticoagulative functions.[7]: 33 Studies suggest that between 20% and 60% of thrombophilic patients suffer from some form of APC resistance.[7]: 37
Warfarin necrosis is an acquired protein C deficiency due to treatment withwarfarin, which is a vitamin K antagonist and an anticoagulant itself. However, warfarin treatment may produce paradoxical skin lesions similar to those seen in purpura fulminans. A variant of this response presents as venous limbgangrene when warfarin is used to treat deep vein thrombosis associated with cancer. In these situations, warfarin may be restarted at a low dosage to ensure that the protein C deficiency does not present before the vitamin K coagulation factors II, IX and X are suppressed.[39]: 211s
Activated protein C cleavesPlasmodium falciparumhistones which are released during infection: cleavage of these histones eliminates their pro inflammatory effects.[42]
In November 2001, theFood and Drug Administration approvedDrotrecogin alfa-activated (DrotAA) for the clinical treatment of adults suffering from severe sepsis and with a high risk of death.[43]: 1332 Drotrecogin alfa-activated is arecombinant form of human activated protein C (rhAPC). It is marketed as Xigris byEli Lilly and Company,[27]: 224
Drotrecogin alfa-activated was the subject of significant controversy while it was approved for clinical use as it was found to increase bleeding and not to reduce mortality.[44][needs update] In October 2011 rhAPC (Xigris) was withdrawn from the market by Eli Lilly due to a higher mortality in a trial among adults.[12][44]
APC has been studied as way of treatinglung injury, after studies showed that in patients with lung injury, reduced APC levels in specific parts of the lungs correlated with worse outcomes.[9]: 3167, 8 APC also has been considered for use in improving patient outcome in cases ofischemic stroke, a medical emergency in which arterial blockage deprives a region of brain of oxygen, causing tissue death. Promising studies suggest that APC could be coupled with the only currently approved treatment,tissue plasminogen activator (tPA), to protect the brain from tPA's very harmfulside effects, in addition to preventing cell death from lack of oxygen (hypoxia).[45]: 211 Clinical use of APC has also been proposed for improving the outcome ofpancreatic islet transplantation in treatingtype I diabetes.[16]: 2392
Ceprotin was approved for medical used in the European Union in July 2001.[46] Ceprotin is indicated in purpura fulminans and coumarin-induced skin necrosis in people with severe congenital protein C deficiency.[46]
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