Protein C
Encyclopedia
Protein C, also known as autoprothrombin IIA and blood coagulation factor XIV, is a zymogen
Zymogen
A zymogen is an inactive enzyme precursor. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it...

ic (inactive) protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

, the activated form of which plays an important role in regulating blood clotting, inflammation
Inflammation
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process...

, cell death
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

 and maintaining the permeability
Vascular permeability
Vascular permeability, often in the form of capillary permeability, characterizes the capacity of a blood vessel wall to allow for the flow of small molecules or even whole cells in and out of the vessel. Blood vessel walls are lined by a single layer of endothelial cells...

 of blood vessel walls in humans and other animals. Activated protein C (APC) performs these operations primarily by proteolytically
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...

 inactivating proteins Factor Va
Factor V
Factor V is a protein of the coagulation system, rarely referred to as proaccelerin or labile factor. In contrast to most other coagulation factors, it is not enzymatically active but functions as a cofactor...

 and Factor VIIIa
Factor VIII
Factor VIII is an essential blood clotting factor also known as anti-hemophilic factor . In humans, Factor VIII is encoded by the F8 gene...

. APC is classified as a serine protease
Serine protease
Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site.They are found ubiquitously in both eukaryotes and prokaryotes...

, as it contains a residue
Residue (chemistry)
In chemistry, residue is the material remaining after a distillation or an evaporation, or to a portion of a larger molecule, such as a methyl group. It may also refer to the undesired byproducts of a reaction....

 of serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...

 in its active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...

. In humans, protein C is encoded by the PROC gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...

, which is found on chromosome 2
Chromosome 2 (human)
Chromosome 2 is one of the 23 pairs of chromosomes in humans. People normally have two copies of this chromosome. Chromosome 2 is the second largest human chromosome, spanning more than 237 million base pairs and representing almost 8% of the total DNA in cells.Identifying genes on each...

.

The zymogenic form of protein C is a vitamin K
Vitamin K
Vitamin K is a group of structurally similar, fat soluble vitamins that are needed for the posttranslational modification of certain proteins required for blood coagulation and in metabolic pathways in bone and other tissue. They are 2-methyl-1,4-naphthoquinone derivatives...

-dependent glycoprotein
Glycoprotein
Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...

 that circulates in blood plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...

. Its structure is that of a two-chain polypeptide consisting of a light chain and a heavy chain connected by a disulfide bond
Disulfide bond
In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

. The protein C zymogen is activated when it binds to thrombin
Thrombin
Thrombin is a "trypsin-like" serine protease protein that in humans is encoded by the F2 gene. Prothrombin is proteolytically cleaved to form thrombin in the first step of the coagulation cascade, which ultimately results in the stemming of blood loss...

, another protein heavily involved in coagulation, and protein C's activation is greatly promoted by the presence of thrombomodulin
Thrombomodulin
Thrombomodulin, CD141 or BDCA-3 is an integral membrane protein expressed on the surface of endothelial cells. In humans, thrombomodulin is encoded by the THBD gene...

 and endothelial 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 and leukocyte
White blood cell
White blood cells, or leukocytes , are cells of the immune system involved in defending the body against both infectious disease and foreign materials. Five different and diverse types of leukocytes exist, but they are all produced and derived from a multipotent cell in the bone marrow known as a...

s (white blood cells) that APC affects. Because of the crucial role that protein C plays as an anticoagulant
Anticoagulant
An anticoagulant is a substance that prevents coagulation of blood. A group of pharmaceuticals called anticoagulants can be used in vivo as a medication for thrombotic disorders. Some anticoagulants are used in medical equipment, such as test tubes, blood transfusion bags, and renal dialysis...

, those with deficiencies in protein C, or some kind of resistance to APC, suffer from a significantly increased risk of forming dangerous blood clots (thrombosis
Thrombosis
Thrombosis is the formation of a blood clot inside a blood vessel, obstructing the flow of blood through the circulatory system. When a blood vessel is injured, the body uses platelets and fibrin to form a blood clot to prevent blood loss...

).

Research into the clinical use of activated protein C also known as drotrecogin alfa-activated
Drotrecogin alfa
Drotrecogin alfa is a recombinant form of human activated protein C that has anti-thrombotic, anti-inflammatory, and profibrinolytic properties. Drotrecogin alpha belongs to the class of serine proteases. Drotrecogin alfa has not been found to improve outcomes in people with severe sepsis...

 (branded Xigris) has been surrounded by controversy. The manufacturer Eli Lilly and Company
Eli Lilly and Company
Eli Lilly and Company is a global pharmaceutical company. Eli Lilly's global headquarters is located in Indianapolis, Indiana, in the United States...

 ran an aggressive marketing campaign to promote its use in people with severe sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...

 and septic shock including the sponsoring of the 2004
Surviving Sepsis Campaign
Surviving Sepsis Campaign
The Surviving Sepsis Campaign is a global initiative to bring together professional organizations in reducing mortality from sepsis. The purpose of the SSC is to create an international collaborative effort to improve the treatment of sepsis and reduce the high mortality rate associated with the...

 Guidelines. A 2011 Cochrane review however found that its use cannot be recommended as it does not improve survival however does increase bleeding risk.

History

Protein C's anticoagulant
Anticoagulant
An anticoagulant is a substance that prevents coagulation of blood. A group of pharmaceuticals called anticoagulants can be used in vivo as a medication for thrombotic disorders. Some anticoagulants are used in medical equipment, such as test tubes, blood transfusion bags, and renal dialysis...

 role in the human body was first noted by Seegers et al. in 1960, who gave protein C its original name, autoprothrombin II-a. Protein C was first isolated by Johan Stenflo
Johan Stenflo
Johan Stenflo is a Swedish chemist and teacher. He received his M.D. in 1968 and his Ph.D. in 1973 at Lund University, where he later became a professor of clinical chemistry. In 1985, he became a member of the Royal Swedish Academy of Sciences. He is also a member of the Norwegian Academy of...

 from bovine plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...

 in 1976, and Stenflo determined it to be a vitamin K-dependent protein. He named it protein C because it was the third protein ("peak C") that eluted
Elution
Elution is a term used in analytical and organic chemistry to describe the process of extracting one material from another by washing with a solvent ....

 from a DEAE-Sepharose
DEAE-Sepharose
DEAE-Sepharose is a tradename for the anion-exchange reactive group, diethylaminoethyl covalently linked to Sepharose ....

 ion-exchange chromotograph. Seegers was, at the time, searching for vitamin K-dependent coagulation factors undetected by clotting assays, which measure global clotting function. Soon after this, Seegers recognised Stenflo's discovery was identical with his own. Activated protein C was discovered later that year, and in 1977 it was first recognised that APC inactivates Factor Va. In 1980, Vehar and Davie discovered that APC also inactivates Factor VIIIa, and soon after, Protein S
Protein S
Protein S is a vitamin K-dependent plasma glycoprotein synthesized in the endothelium. In the circulation, Protein S exists in two forms: a free form and a complex form bound to complement protein C4b. In humans, protein S is encoded by the PROS1 gene...

 was recognised as a cofactor
Cofactor (biochemistry)
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....

 by Walker. In 1982, a family study by Griffin et al. first associated protein C deficiency with symptoms of venous thrombosis
Venous thrombosis
A venous thrombosis is a blood clot that forms within a vein. A venous thrombosis is a blood clot that forms within a vein. A venous thrombosis is a blood clot that forms within a vein. (Thrombosis is a medical term for blood clotting (Haemostasis) occurring in the wrong place, i.e...

. Homozygous protein C deficiency and the consequent serious health effects were described in 1984 by several scientists. cDNA cloning of protein C was first performed in 1984 by Beckmann et al. which produced a map of the gene responsible for producing protein C in the liver. In 1987 a seminal experiment was performed (Taylor et al.) whereby it was demonstrated that activated protein C prevented coagulopathy
Coagulopathy
Coagulopathy is a condition in which the blood’s ability to clot is impaired. This condition can cause prolonged or excessive bleeding, which may occur spontaneously or following an injury or medical and dental procedures.The normal clotting process depends on the interplay of various proteins in...

 and death in baboons infused
Route of administration
A route of administration in pharmacology and toxicology is the path by which a drug, fluid, poison, or other substance is taken into the body.-Classification:Routes of administration are usually classified by application location...

 with lethal concentrations of E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

.


In 1993, a heritable resistance to APC was detected by Dahlbäck et al. and associated with familial thrombophilia
Thrombophilia
Thrombophilia is an abnormality of blood coagulation that increases the risk of thrombosis . Such abnormalities can be identified in 50% of people who have an episode of thrombosis that was not provoked by other causes...

. In 1994, the relatively common genetic mutation that produces Factor VLeiden
Factor V Leiden
Factor V Leiden is the name given to a variant of human factor V that causes a hypercoagulability disorder. In this disorder the Leiden variant of factor V cannot be inactivated by activated protein C. Factor V Leiden is the most common hereditary hypercoagulability disorder amongst Eurasians...

 was noted (Bertina et al.). Two years later, Gla-domainless APC was imaged at a resolution of 2.8 Ångström
Ångström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....

s. Beginning with the PROWESS clinical trial
Clinical trial
Clinical trials are a set of procedures in medical research and drug development that are conducted to allow safety and efficacy data to be collected for health interventions...

 of 2001, it was recognised that many of the symptoms of sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...

 may be ameliorated by infusion of APC, and mortality rates of septic patients may be significantly decreased. 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
Food and Drug Administration
The Food and Drug Administration is an agency of the United States Department of Health and Human Services, one of the United States federal executive departments...

  for treating severe sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...

. In 2002, Science
Science (journal)
Science is the academic journal of the American Association for the Advancement of Science and is one of the world's top scientific journals....

published an article that first showed protein C activates protease-activated receptor-1
Coagulation factor II receptor
Proteinase-activated receptor 1 also known as the coagulation factor II receptor is a protein that in humans is encoded by the F2R gene. PAR1 is a G protein-coupled receptor involved in the regulation of thrombotic response...

 (PAR-1) and this process accounts for the protein's modulation of the immune system.

Synthesis, structure and activation

Human protein C is a vitamin K-dependent glycoprotein structurally similar to other vitamin K-dependent proteins affecting blood clotting, such as prothrombin
Thrombin
Thrombin is a "trypsin-like" serine protease protein that in humans is encoded by the F2 gene. Prothrombin is proteolytically cleaved to form thrombin in the first step of the coagulation cascade, which ultimately results in the stemming of blood loss...

, Factor VII
Factor VII
Factor VII is one of the proteins that causes blood to clot in the coagulation cascade. It is an enzyme of the serine protease class. A recombinant form of human factor VIIa has U.S. Food and Drug Administration approval for uncontrolled bleeding in hemophilia patients...

, Factor IX
Factor IX
Factor IX is one of the serine proteases of the coagulation system; it belongs to peptidase family S1. Deficiency of this protein causes hemophilia B. It was discovered in 1952 after a young boy named Stephen Christmas was found to be lacking this exact factor, leading to...

 and Factor X
Factor X
Factor X, also known by the eponym Stuart-Prower factor or as prothrombinase, is an enzyme of the coagulation cascade. It is a serine endopeptidase .-Physiology:...

. Protein C synthesis occurs in the liver and begins with a single-chain precursor molecule: a 32 amino acid N-terminus signal peptide
Signal peptide
A signal peptide is a short peptide chain that directs the transport of a protein.Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals....

 preceding a propeptide. Protein C is formed when a dipeptide
Dipeptide
A dipeptide is a molecule consisting of two amino acids joined by a single peptide bond.Dipeptides are produced from polypeptides by the action of the hydrolase enzyme dipeptidyl peptidase. Dietary proteins are digested to dipeptides and amino acids, and the dipeptides are absorbed more rapidly...

 of Lys198 and Arg199 is removed; this causes the transformation into a heterodimer with N-linked carbohydrates on each chain. The protein has one light chain (21 kDa) and one heavy chain (41 kDa) connected by a disulfide bond
Disulfide bond
In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

 between Cys183 and Cys319.
Inactive protein C comprises 419 amino acids in multiple domains
Protein domain
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...

: one Gla domain
Gla domain
Vitamin K-dependent carboxylation/gamma-carboxyglutamic domain is a protein domain that contains post-translational modifications ofmany glutamate residues by vitamin K-dependent carboxylation to form gamma-carboxyglutamate . The Gla residues are responsible for the high-affinity binding of...

 (residues 43–88); a helical aromatic
Aromatic amino acids
Aromatic amino acids are amino acids that include an aromatic ring.Examples include:* Among 20 standard amino acids: phenylalanine, tryptophan, and tyrosine* Others: thyroxine-See also:* Aromatic L-amino acid decarboxylase...

 segment (89–96); two epidermal growth factor (EGF)-like domains
EGF-like domain
EGF-like domain is an evolutionary conserved protein domain, of about thirty to forty amino-acid residues long, which was found in a large number of mostly animal proteins. All these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted...

 (97–132 and 136–176); an activation peptide (200–211); and a trypsin
Trypsin
Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when...

-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 a zymogen
Zymogen
A zymogen is an inactive enzyme precursor. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it...

, waiting to be activated. 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 the N-terminus of the heavy chain.
The active site contains a catalytic triad
Catalytic triad
A catalytic triad refers to the three amino acid residues found inside the active site of certain protease enzymes: serine , aspartate , and histidine . They work together to break peptide bonds on polypeptides. In general terms, catalytic triad can refer to any set of three residues that function...

 typical of serine proteases (His253, Asp299 and Ser402).

The activation of protein C is strongly promoted by thrombomodulin
Thrombomodulin
Thrombomodulin, CD141 or BDCA-3 is an integral membrane protein expressed on the surface of endothelial cells. In humans, thrombomodulin is encoded by the THBD gene...

 and endothelial protein C receptor (EPCR), the latter of which is found primarily on endothelial cells (cells on the inside of blood vessels). The presence of thrombomodulin accelerates activation by several orders of magnitude, and EPCR speeds up activation by a factor of 20. If either of these two proteins is absent in murine
Mouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...

 specimens, the mouse dies from excessive blood-clotting while still in an embryo
Embryo
An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...

nic state. On the endothelium, APC performs a major role in regulating blood clotting, inflammation, and cell death (apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

). 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. 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.

The Gla domain is particularly useful for binding to negatively charged phospholipid
Phospholipid
Phospholipids are a class of lipids that are a major component of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a phosphate group, and a simple organic molecule such as choline; one exception to this rule is sphingomyelin, which is derived from...

s for anticoagulation and to EPCR for cytoprotection
Cytoprotection
Cytoprotection is a process by which chemical compounds provide protection to cells against harmful agents.For example, a gastric cytoprotectant is any medication that combats ulcers not by reducing gastric acid but by increasing mucosal protection. Examples of gastric cyctoprotective agents...

. One particular exosite
Exosite
An exosite is a secondary binding site, remote from the active site, on an enzyme or other protein.This is similar to allosteric sites, but differs in the fact that, in order for an enzyme to be active, its exosite typically must be occupied. Exosites have recently become a hot topic in biomedical...

 augments protein C's ability to inactive Factor Va efficiently. Another is necessary for interacting with thrombomodulin.

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 the HUGO Gene Nomenclature Committee
HUGO Gene Nomenclature Committee
The HUGO Gene Nomenclature Committee approves a unique and meaningful name for every known human gene based on a query of experts. In addition to a long name, the HGNC also assigns an abbreviation to every gene...

 is "PROC" from "protein C". It is located on the second chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...

 (2q13-q14) and comprises nine exon
Exon
An exon is a nucleic acid sequence that is represented in the mature form of an RNA molecule either after portions of a precursor RNA have been removed by cis-splicing or when two or more precursor RNA molecules have been ligated by trans-splicing. The mature RNA molecule can be a messenger RNA...

s. The nucleotide sequence that codes for human protein C is approximately 11,000 bases long.

Protein C in zymogen
Zymogen
A zymogen is an inactive enzyme precursor. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it...

 form is present in normal adult human blood plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...

 at concentrations between 65–135 IU
International unit
In pharmacology, the International Unit is a unit of measurement for the amount of a substance, based on biological activity or effect. It is abbreviated as IU, as UI , or as IE...

/dL. Activated protein C is found at levels approximately 2000 times lower than this. 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 healthy term 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 after adolescence
Adolescence
Adolescence is a transitional stage of physical and mental human development generally occurring between puberty and legal adulthood , but largely characterized as beginning and ending with the teenage stage...

. The half-life
Half-life
Half-life, abbreviated t½, is the period of time it takes for the amount of a substance undergoing decay to decrease by half. The name was originally used to describe a characteristic of unstable atoms , but it may apply to any quantity which follows a set-rate decay.The original term, dating to...

 of activated protein C is around 15 minutes.
Protein C, also known as autoprothrombin IIA and blood coagulation factor XIV, is a zymogen
Zymogen
A zymogen is an inactive enzyme precursor. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it...

ic (inactive) protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

, the activated form of which plays an important role in regulating blood clotting, inflammation
Inflammation
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process...

, cell death
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

 and maintaining the permeability
Vascular permeability
Vascular permeability, often in the form of capillary permeability, characterizes the capacity of a blood vessel wall to allow for the flow of small molecules or even whole cells in and out of the vessel. Blood vessel walls are lined by a single layer of endothelial cells...

 of blood vessel walls in humans and other animals. Activated protein C (APC) performs these operations primarily by proteolytically
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...

 inactivating proteins Factor Va
Factor V
Factor V is a protein of the coagulation system, rarely referred to as proaccelerin or labile factor. In contrast to most other coagulation factors, it is not enzymatically active but functions as a cofactor...

 and Factor VIIIa
Factor VIII
Factor VIII is an essential blood clotting factor also known as anti-hemophilic factor . In humans, Factor VIII is encoded by the F8 gene...

. APC is classified as a serine protease
Serine protease
Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site.They are found ubiquitously in both eukaryotes and prokaryotes...

, as it contains a residue
Residue (chemistry)
In chemistry, residue is the material remaining after a distillation or an evaporation, or to a portion of a larger molecule, such as a methyl group. It may also refer to the undesired byproducts of a reaction....

 of serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...

 in its active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...

. In humans, protein C is encoded by the PROC gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...

, which is found on chromosome 2
Chromosome 2 (human)
Chromosome 2 is one of the 23 pairs of chromosomes in humans. People normally have two copies of this chromosome. Chromosome 2 is the second largest human chromosome, spanning more than 237 million base pairs and representing almost 8% of the total DNA in cells.Identifying genes on each...

.

The zymogenic form of protein C is a vitamin K
Vitamin K
Vitamin K is a group of structurally similar, fat soluble vitamins that are needed for the posttranslational modification of certain proteins required for blood coagulation and in metabolic pathways in bone and other tissue. They are 2-methyl-1,4-naphthoquinone derivatives...

-dependent glycoprotein
Glycoprotein
Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...

 that circulates in blood plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...

. Its structure is that of a two-chain polypeptide consisting of a light chain and a heavy chain connected by a disulfide bond
Disulfide bond
In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

. The protein C zymogen is activated when it binds to thrombin
Thrombin
Thrombin is a "trypsin-like" serine protease protein that in humans is encoded by the F2 gene. Prothrombin is proteolytically cleaved to form thrombin in the first step of the coagulation cascade, which ultimately results in the stemming of blood loss...

, another protein heavily involved in coagulation, and protein C's activation is greatly promoted by the presence of thrombomodulin
Thrombomodulin
Thrombomodulin, CD141 or BDCA-3 is an integral membrane protein expressed on the surface of endothelial cells. In humans, thrombomodulin is encoded by the THBD gene...

 and endothelial 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 and leukocyte
White blood cell
White blood cells, or leukocytes , are cells of the immune system involved in defending the body against both infectious disease and foreign materials. Five different and diverse types of leukocytes exist, but they are all produced and derived from a multipotent cell in the bone marrow known as a...

s (white blood cells) that APC affects. Because of the crucial role that protein C plays as an anticoagulant
Anticoagulant
An anticoagulant is a substance that prevents coagulation of blood. A group of pharmaceuticals called anticoagulants can be used in vivo as a medication for thrombotic disorders. Some anticoagulants are used in medical equipment, such as test tubes, blood transfusion bags, and renal dialysis...

, those with deficiencies in protein C, or some kind of resistance to APC, suffer from a significantly increased risk of forming dangerous blood clots (thrombosis
Thrombosis
Thrombosis is the formation of a blood clot inside a blood vessel, obstructing the flow of blood through the circulatory system. When a blood vessel is injured, the body uses platelets and fibrin to form a blood clot to prevent blood loss...

).

Research into the clinical use of activated protein C also known as drotrecogin alfa-activated
Drotrecogin alfa
Drotrecogin alfa is a recombinant form of human activated protein C that has anti-thrombotic, anti-inflammatory, and profibrinolytic properties. Drotrecogin alpha belongs to the class of serine proteases. Drotrecogin alfa has not been found to improve outcomes in people with severe sepsis...

 (branded Xigris) has been surrounded by controversy. The manufacturer Eli Lilly and Company
Eli Lilly and Company
Eli Lilly and Company is a global pharmaceutical company. Eli Lilly's global headquarters is located in Indianapolis, Indiana, in the United States...

 ran an aggressive marketing campaign to promote its use in people with severe sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...

 and septic shock including the sponsoring of the 2004
Surviving Sepsis Campaign
Surviving Sepsis Campaign
The Surviving Sepsis Campaign is a global initiative to bring together professional organizations in reducing mortality from sepsis. The purpose of the SSC is to create an international collaborative effort to improve the treatment of sepsis and reduce the high mortality rate associated with the...

 Guidelines. A 2011 Cochrane review however found that its use cannot be recommended as it does not improve survival however does increase bleeding risk.

History

Protein C's anticoagulant
Anticoagulant
An anticoagulant is a substance that prevents coagulation of blood. A group of pharmaceuticals called anticoagulants can be used in vivo as a medication for thrombotic disorders. Some anticoagulants are used in medical equipment, such as test tubes, blood transfusion bags, and renal dialysis...

 role in the human body was first noted by Seegers et al. in 1960, who gave protein C its original name, autoprothrombin II-a. Protein C was first isolated by Johan Stenflo
Johan Stenflo
Johan Stenflo is a Swedish chemist and teacher. He received his M.D. in 1968 and his Ph.D. in 1973 at Lund University, where he later became a professor of clinical chemistry. In 1985, he became a member of the Royal Swedish Academy of Sciences. He is also a member of the Norwegian Academy of...

 from bovine plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...

 in 1976, and Stenflo determined it to be a vitamin K-dependent protein. He named it protein C because it was the third protein ("peak C") that eluted
Elution
Elution is a term used in analytical and organic chemistry to describe the process of extracting one material from another by washing with a solvent ....

 from a DEAE-Sepharose
DEAE-Sepharose
DEAE-Sepharose is a tradename for the anion-exchange reactive group, diethylaminoethyl covalently linked to Sepharose ....

 ion-exchange chromotograph. Seegers was, at the time, searching for vitamin K-dependent coagulation factors undetected by clotting assays, which measure global clotting function. Soon after this, Seegers recognised Stenflo's discovery was identical with his own. Activated protein C was discovered later that year, and in 1977 it was first recognised that APC inactivates Factor Va. In 1980, Vehar and Davie discovered that APC also inactivates Factor VIIIa, and soon after, Protein S
Protein S
Protein S is a vitamin K-dependent plasma glycoprotein synthesized in the endothelium. In the circulation, Protein S exists in two forms: a free form and a complex form bound to complement protein C4b. In humans, protein S is encoded by the PROS1 gene...

 was recognised as a cofactor
Cofactor (biochemistry)
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....

 by Walker. In 1982, a family study by Griffin et al. first associated protein C deficiency with symptoms of venous thrombosis
Venous thrombosis
A venous thrombosis is a blood clot that forms within a vein. A venous thrombosis is a blood clot that forms within a vein. A venous thrombosis is a blood clot that forms within a vein. (Thrombosis is a medical term for blood clotting (Haemostasis) occurring in the wrong place, i.e...

. Homozygous protein C deficiency and the consequent serious health effects were described in 1984 by several scientists. cDNA cloning of protein C was first performed in 1984 by Beckmann et al. which produced a map of the gene responsible for producing protein C in the liver. In 1987 a seminal experiment was performed (Taylor et al.) whereby it was demonstrated that activated protein C prevented coagulopathy
Coagulopathy
Coagulopathy is a condition in which the blood’s ability to clot is impaired. This condition can cause prolonged or excessive bleeding, which may occur spontaneously or following an injury or medical and dental procedures.The normal clotting process depends on the interplay of various proteins in...

 and death in baboons infused
Route of administration
A route of administration in pharmacology and toxicology is the path by which a drug, fluid, poison, or other substance is taken into the body.-Classification:Routes of administration are usually classified by application location...

 with lethal concentrations of E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

.


In 1993, a heritable resistance to APC was detected by Dahlbäck et al. and associated with familial thrombophilia
Thrombophilia
Thrombophilia is an abnormality of blood coagulation that increases the risk of thrombosis . Such abnormalities can be identified in 50% of people who have an episode of thrombosis that was not provoked by other causes...

. In 1994, the relatively common genetic mutation that produces Factor VLeiden
Factor V Leiden
Factor V Leiden is the name given to a variant of human factor V that causes a hypercoagulability disorder. In this disorder the Leiden variant of factor V cannot be inactivated by activated protein C. Factor V Leiden is the most common hereditary hypercoagulability disorder amongst Eurasians...

 was noted (Bertina et al.). Two years later, Gla-domainless APC was imaged at a resolution of 2.8 Ångström
Ångström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....

s. Beginning with the PROWESS clinical trial
Clinical trial
Clinical trials are a set of procedures in medical research and drug development that are conducted to allow safety and efficacy data to be collected for health interventions...

 of 2001, it was recognised that many of the symptoms of sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...

 may be ameliorated by infusion of APC, and mortality rates of septic patients may be significantly decreased. 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
Food and Drug Administration
The Food and Drug Administration is an agency of the United States Department of Health and Human Services, one of the United States federal executive departments...

  for treating severe sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...

. In 2002, Science
Science (journal)
Science is the academic journal of the American Association for the Advancement of Science and is one of the world's top scientific journals....

published an article that first showed protein C activates protease-activated receptor-1
Coagulation factor II receptor
Proteinase-activated receptor 1 also known as the coagulation factor II receptor is a protein that in humans is encoded by the F2R gene. PAR1 is a G protein-coupled receptor involved in the regulation of thrombotic response...

 (PAR-1) and this process accounts for the protein's modulation of the immune system.

Synthesis, structure and activation

Human protein C is a vitamin K-dependent glycoprotein structurally similar to other vitamin K-dependent proteins affecting blood clotting, such as prothrombin
Thrombin
Thrombin is a "trypsin-like" serine protease protein that in humans is encoded by the F2 gene. Prothrombin is proteolytically cleaved to form thrombin in the first step of the coagulation cascade, which ultimately results in the stemming of blood loss...

, Factor VII
Factor VII
Factor VII is one of the proteins that causes blood to clot in the coagulation cascade. It is an enzyme of the serine protease class. A recombinant form of human factor VIIa has U.S. Food and Drug Administration approval for uncontrolled bleeding in hemophilia patients...

, Factor IX
Factor IX
Factor IX is one of the serine proteases of the coagulation system; it belongs to peptidase family S1. Deficiency of this protein causes hemophilia B. It was discovered in 1952 after a young boy named Stephen Christmas was found to be lacking this exact factor, leading to...

 and Factor X
Factor X
Factor X, also known by the eponym Stuart-Prower factor or as prothrombinase, is an enzyme of the coagulation cascade. It is a serine endopeptidase .-Physiology:...

. Protein C synthesis occurs in the liver and begins with a single-chain precursor molecule: a 32 amino acid N-terminus signal peptide
Signal peptide
A signal peptide is a short peptide chain that directs the transport of a protein.Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals....

 preceding a propeptide. Protein C is formed when a dipeptide
Dipeptide
A dipeptide is a molecule consisting of two amino acids joined by a single peptide bond.Dipeptides are produced from polypeptides by the action of the hydrolase enzyme dipeptidyl peptidase. Dietary proteins are digested to dipeptides and amino acids, and the dipeptides are absorbed more rapidly...

 of Lys198 and Arg199 is removed; this causes the transformation into a heterodimer with N-linked carbohydrates on each chain. The protein has one light chain (21 kDa) and one heavy chain (41 kDa) connected by a disulfide bond
Disulfide bond
In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

 between Cys183 and Cys319.
Inactive protein C comprises 419 amino acids in multiple domains
Protein domain
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...

: one Gla domain
Gla domain
Vitamin K-dependent carboxylation/gamma-carboxyglutamic domain is a protein domain that contains post-translational modifications ofmany glutamate residues by vitamin K-dependent carboxylation to form gamma-carboxyglutamate . The Gla residues are responsible for the high-affinity binding of...

 (residues 43–88); a helical aromatic
Aromatic amino acids
Aromatic amino acids are amino acids that include an aromatic ring.Examples include:* Among 20 standard amino acids: phenylalanine, tryptophan, and tyrosine* Others: thyroxine-See also:* Aromatic L-amino acid decarboxylase...

 segment (89–96); two epidermal growth factor (EGF)-like domains
EGF-like domain
EGF-like domain is an evolutionary conserved protein domain, of about thirty to forty amino-acid residues long, which was found in a large number of mostly animal proteins. All these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted...

 (97–132 and 136–176); an activation peptide (200–211); and a trypsin
Trypsin
Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when...

-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 a zymogen
Zymogen
A zymogen is an inactive enzyme precursor. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it...

, waiting to be activated. 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 the N-terminus of the heavy chain.
The active site contains a catalytic triad
Catalytic triad
A catalytic triad refers to the three amino acid residues found inside the active site of certain protease enzymes: serine , aspartate , and histidine . They work together to break peptide bonds on polypeptides. In general terms, catalytic triad can refer to any set of three residues that function...

 typical of serine proteases (His253, Asp299 and Ser402).

The activation of protein C is strongly promoted by thrombomodulin
Thrombomodulin
Thrombomodulin, CD141 or BDCA-3 is an integral membrane protein expressed on the surface of endothelial cells. In humans, thrombomodulin is encoded by the THBD gene...

 and endothelial protein C receptor (EPCR), the latter of which is found primarily on endothelial cells (cells on the inside of blood vessels). The presence of thrombomodulin accelerates activation by several orders of magnitude, and EPCR speeds up activation by a factor of 20. If either of these two proteins is absent in murine
Mouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...

 specimens, the mouse dies from excessive blood-clotting while still in an embryo
Embryo
An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...

nic state. On the endothelium, APC performs a major role in regulating blood clotting, inflammation, and cell death (apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

). 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. 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.

The Gla domain is particularly useful for binding to negatively charged phospholipid
Phospholipid
Phospholipids are a class of lipids that are a major component of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a phosphate group, and a simple organic molecule such as choline; one exception to this rule is sphingomyelin, which is derived from...

s for anticoagulation and to EPCR for cytoprotection
Cytoprotection
Cytoprotection is a process by which chemical compounds provide protection to cells against harmful agents.For example, a gastric cytoprotectant is any medication that combats ulcers not by reducing gastric acid but by increasing mucosal protection. Examples of gastric cyctoprotective agents...

. One particular exosite
Exosite
An exosite is a secondary binding site, remote from the active site, on an enzyme or other protein.This is similar to allosteric sites, but differs in the fact that, in order for an enzyme to be active, its exosite typically must be occupied. Exosites have recently become a hot topic in biomedical...

 augments protein C's ability to inactive Factor Va efficiently. Another is necessary for interacting with thrombomodulin.

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 the HUGO Gene Nomenclature Committee
HUGO Gene Nomenclature Committee
The HUGO Gene Nomenclature Committee approves a unique and meaningful name for every known human gene based on a query of experts. In addition to a long name, the HGNC also assigns an abbreviation to every gene...

 is "PROC" from "protein C". It is located on the second chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...

 (2q13-q14) and comprises nine exon
Exon
An exon is a nucleic acid sequence that is represented in the mature form of an RNA molecule either after portions of a precursor RNA have been removed by cis-splicing or when two or more precursor RNA molecules have been ligated by trans-splicing. The mature RNA molecule can be a messenger RNA...

s. The nucleotide sequence that codes for human protein C is approximately 11,000 bases long.

Protein C in zymogen
Zymogen
A zymogen is an inactive enzyme precursor. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it...

 form is present in normal adult human blood plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...

 at concentrations between 65–135 IU
International unit
In pharmacology, the International Unit is a unit of measurement for the amount of a substance, based on biological activity or effect. It is abbreviated as IU, as UI , or as IE...

/dL. Activated protein C is found at levels approximately 2000 times lower than this. 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 healthy term 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 after adolescence
Adolescence
Adolescence is a transitional stage of physical and mental human development generally occurring between puberty and legal adulthood , but largely characterized as beginning and ending with the teenage stage...

. The half-life
Half-life
Half-life, abbreviated t½, is the period of time it takes for the amount of a substance undergoing decay to decrease by half. The name was originally used to describe a characteristic of unstable atoms , but it may apply to any quantity which follows a set-rate decay.The original term, dating to...

 of activated protein C is around 15 minutes.
Protein C, also known as autoprothrombin IIA and blood coagulation factor XIV, is a zymogen
Zymogen
A zymogen is an inactive enzyme precursor. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it...

ic (inactive) protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

, the activated form of which plays an important role in regulating blood clotting, inflammation
Inflammation
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process...

, cell death
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

 and maintaining the permeability
Vascular permeability
Vascular permeability, often in the form of capillary permeability, characterizes the capacity of a blood vessel wall to allow for the flow of small molecules or even whole cells in and out of the vessel. Blood vessel walls are lined by a single layer of endothelial cells...

 of blood vessel walls in humans and other animals. Activated protein C (APC) performs these operations primarily by proteolytically
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...

 inactivating proteins Factor Va
Factor V
Factor V is a protein of the coagulation system, rarely referred to as proaccelerin or labile factor. In contrast to most other coagulation factors, it is not enzymatically active but functions as a cofactor...

 and Factor VIIIa
Factor VIII
Factor VIII is an essential blood clotting factor also known as anti-hemophilic factor . In humans, Factor VIII is encoded by the F8 gene...

. APC is classified as a serine protease
Serine protease
Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site.They are found ubiquitously in both eukaryotes and prokaryotes...

, as it contains a residue
Residue (chemistry)
In chemistry, residue is the material remaining after a distillation or an evaporation, or to a portion of a larger molecule, such as a methyl group. It may also refer to the undesired byproducts of a reaction....

 of serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...

 in its active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...

. In humans, protein C is encoded by the PROC gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...

, which is found on chromosome 2
Chromosome 2 (human)
Chromosome 2 is one of the 23 pairs of chromosomes in humans. People normally have two copies of this chromosome. Chromosome 2 is the second largest human chromosome, spanning more than 237 million base pairs and representing almost 8% of the total DNA in cells.Identifying genes on each...

.

The zymogenic form of protein C is a vitamin K
Vitamin K
Vitamin K is a group of structurally similar, fat soluble vitamins that are needed for the posttranslational modification of certain proteins required for blood coagulation and in metabolic pathways in bone and other tissue. They are 2-methyl-1,4-naphthoquinone derivatives...

-dependent glycoprotein
Glycoprotein
Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...

 that circulates in blood plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...

. Its structure is that of a two-chain polypeptide consisting of a light chain and a heavy chain connected by a disulfide bond
Disulfide bond
In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

. The protein C zymogen is activated when it binds to thrombin
Thrombin
Thrombin is a "trypsin-like" serine protease protein that in humans is encoded by the F2 gene. Prothrombin is proteolytically cleaved to form thrombin in the first step of the coagulation cascade, which ultimately results in the stemming of blood loss...

, another protein heavily involved in coagulation, and protein C's activation is greatly promoted by the presence of thrombomodulin
Thrombomodulin
Thrombomodulin, CD141 or BDCA-3 is an integral membrane protein expressed on the surface of endothelial cells. In humans, thrombomodulin is encoded by the THBD gene...

 and endothelial 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 and leukocyte
White blood cell
White blood cells, or leukocytes , are cells of the immune system involved in defending the body against both infectious disease and foreign materials. Five different and diverse types of leukocytes exist, but they are all produced and derived from a multipotent cell in the bone marrow known as a...

s (white blood cells) that APC affects. Because of the crucial role that protein C plays as an anticoagulant
Anticoagulant
An anticoagulant is a substance that prevents coagulation of blood. A group of pharmaceuticals called anticoagulants can be used in vivo as a medication for thrombotic disorders. Some anticoagulants are used in medical equipment, such as test tubes, blood transfusion bags, and renal dialysis...

, those with deficiencies in protein C, or some kind of resistance to APC, suffer from a significantly increased risk of forming dangerous blood clots (thrombosis
Thrombosis
Thrombosis is the formation of a blood clot inside a blood vessel, obstructing the flow of blood through the circulatory system. When a blood vessel is injured, the body uses platelets and fibrin to form a blood clot to prevent blood loss...

).

Research into the clinical use of activated protein C also known as drotrecogin alfa-activated
Drotrecogin alfa
Drotrecogin alfa is a recombinant form of human activated protein C that has anti-thrombotic, anti-inflammatory, and profibrinolytic properties. Drotrecogin alpha belongs to the class of serine proteases. Drotrecogin alfa has not been found to improve outcomes in people with severe sepsis...

 (branded Xigris) has been surrounded by controversy. The manufacturer Eli Lilly and Company
Eli Lilly and Company
Eli Lilly and Company is a global pharmaceutical company. Eli Lilly's global headquarters is located in Indianapolis, Indiana, in the United States...

 ran an aggressive marketing campaign to promote its use in people with severe sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...

 and septic shock including the sponsoring of the 2004
Surviving Sepsis Campaign
Surviving Sepsis Campaign
The Surviving Sepsis Campaign is a global initiative to bring together professional organizations in reducing mortality from sepsis. The purpose of the SSC is to create an international collaborative effort to improve the treatment of sepsis and reduce the high mortality rate associated with the...

 Guidelines. A 2011 Cochrane review however found that its use cannot be recommended as it does not improve survival however does increase bleeding risk.

History

Protein C's anticoagulant
Anticoagulant
An anticoagulant is a substance that prevents coagulation of blood. A group of pharmaceuticals called anticoagulants can be used in vivo as a medication for thrombotic disorders. Some anticoagulants are used in medical equipment, such as test tubes, blood transfusion bags, and renal dialysis...

 role in the human body was first noted by Seegers et al. in 1960, who gave protein C its original name, autoprothrombin II-a. Protein C was first isolated by Johan Stenflo
Johan Stenflo
Johan Stenflo is a Swedish chemist and teacher. He received his M.D. in 1968 and his Ph.D. in 1973 at Lund University, where he later became a professor of clinical chemistry. In 1985, he became a member of the Royal Swedish Academy of Sciences. He is also a member of the Norwegian Academy of...

 from bovine plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...

 in 1976, and Stenflo determined it to be a vitamin K-dependent protein. He named it protein C because it was the third protein ("peak C") that eluted
Elution
Elution is a term used in analytical and organic chemistry to describe the process of extracting one material from another by washing with a solvent ....

 from a DEAE-Sepharose
DEAE-Sepharose
DEAE-Sepharose is a tradename for the anion-exchange reactive group, diethylaminoethyl covalently linked to Sepharose ....

 ion-exchange chromotograph. Seegers was, at the time, searching for vitamin K-dependent coagulation factors undetected by clotting assays, which measure global clotting function. Soon after this, Seegers recognised Stenflo's discovery was identical with his own. Activated protein C was discovered later that year, and in 1977 it was first recognised that APC inactivates Factor Va. In 1980, Vehar and Davie discovered that APC also inactivates Factor VIIIa, and soon after, Protein S
Protein S
Protein S is a vitamin K-dependent plasma glycoprotein synthesized in the endothelium. In the circulation, Protein S exists in two forms: a free form and a complex form bound to complement protein C4b. In humans, protein S is encoded by the PROS1 gene...

 was recognised as a cofactor
Cofactor (biochemistry)
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....

 by Walker. In 1982, a family study by Griffin et al. first associated protein C deficiency with symptoms of venous thrombosis
Venous thrombosis
A venous thrombosis is a blood clot that forms within a vein. A venous thrombosis is a blood clot that forms within a vein. A venous thrombosis is a blood clot that forms within a vein. (Thrombosis is a medical term for blood clotting (Haemostasis) occurring in the wrong place, i.e...

. Homozygous protein C deficiency and the consequent serious health effects were described in 1984 by several scientists. cDNA cloning of protein C was first performed in 1984 by Beckmann et al. which produced a map of the gene responsible for producing protein C in the liver. In 1987 a seminal experiment was performed (Taylor et al.) whereby it was demonstrated that activated protein C prevented coagulopathy
Coagulopathy
Coagulopathy is a condition in which the blood’s ability to clot is impaired. This condition can cause prolonged or excessive bleeding, which may occur spontaneously or following an injury or medical and dental procedures.The normal clotting process depends on the interplay of various proteins in...

 and death in baboons infused
Route of administration
A route of administration in pharmacology and toxicology is the path by which a drug, fluid, poison, or other substance is taken into the body.-Classification:Routes of administration are usually classified by application location...

 with lethal concentrations of E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

.


In 1993, a heritable resistance to APC was detected by Dahlbäck et al. and associated with familial thrombophilia
Thrombophilia
Thrombophilia is an abnormality of blood coagulation that increases the risk of thrombosis . Such abnormalities can be identified in 50% of people who have an episode of thrombosis that was not provoked by other causes...

. In 1994, the relatively common genetic mutation that produces Factor VLeiden
Factor V Leiden
Factor V Leiden is the name given to a variant of human factor V that causes a hypercoagulability disorder. In this disorder the Leiden variant of factor V cannot be inactivated by activated protein C. Factor V Leiden is the most common hereditary hypercoagulability disorder amongst Eurasians...

 was noted (Bertina et al.). Two years later, Gla-domainless APC was imaged at a resolution of 2.8 Ångström
Ångström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....

s. Beginning with the PROWESS clinical trial
Clinical trial
Clinical trials are a set of procedures in medical research and drug development that are conducted to allow safety and efficacy data to be collected for health interventions...

 of 2001, it was recognised that many of the symptoms of sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...

 may be ameliorated by infusion of APC, and mortality rates of septic patients may be significantly decreased. 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
Food and Drug Administration
The Food and Drug Administration is an agency of the United States Department of Health and Human Services, one of the United States federal executive departments...

  for treating severe sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...

. In 2002, Science
Science (journal)
Science is the academic journal of the American Association for the Advancement of Science and is one of the world's top scientific journals....

published an article that first showed protein C activates protease-activated receptor-1
Coagulation factor II receptor
Proteinase-activated receptor 1 also known as the coagulation factor II receptor is a protein that in humans is encoded by the F2R gene. PAR1 is a G protein-coupled receptor involved in the regulation of thrombotic response...

 (PAR-1) and this process accounts for the protein's modulation of the immune system.

Synthesis, structure and activation

Human protein C is a vitamin K-dependent glycoprotein structurally similar to other vitamin K-dependent proteins affecting blood clotting, such as prothrombin
Thrombin
Thrombin is a "trypsin-like" serine protease protein that in humans is encoded by the F2 gene. Prothrombin is proteolytically cleaved to form thrombin in the first step of the coagulation cascade, which ultimately results in the stemming of blood loss...

, Factor VII
Factor VII
Factor VII is one of the proteins that causes blood to clot in the coagulation cascade. It is an enzyme of the serine protease class. A recombinant form of human factor VIIa has U.S. Food and Drug Administration approval for uncontrolled bleeding in hemophilia patients...

, Factor IX
Factor IX
Factor IX is one of the serine proteases of the coagulation system; it belongs to peptidase family S1. Deficiency of this protein causes hemophilia B. It was discovered in 1952 after a young boy named Stephen Christmas was found to be lacking this exact factor, leading to...

 and Factor X
Factor X
Factor X, also known by the eponym Stuart-Prower factor or as prothrombinase, is an enzyme of the coagulation cascade. It is a serine endopeptidase .-Physiology:...

. Protein C synthesis occurs in the liver and begins with a single-chain precursor molecule: a 32 amino acid N-terminus signal peptide
Signal peptide
A signal peptide is a short peptide chain that directs the transport of a protein.Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals....

 preceding a propeptide. Protein C is formed when a dipeptide
Dipeptide
A dipeptide is a molecule consisting of two amino acids joined by a single peptide bond.Dipeptides are produced from polypeptides by the action of the hydrolase enzyme dipeptidyl peptidase. Dietary proteins are digested to dipeptides and amino acids, and the dipeptides are absorbed more rapidly...

 of Lys198 and Arg199 is removed; this causes the transformation into a heterodimer with N-linked carbohydrates on each chain. The protein has one light chain (21 kDa) and one heavy chain (41 kDa) connected by a disulfide bond
Disulfide bond
In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

 between Cys183 and Cys319.
Inactive protein C comprises 419 amino acids in multiple domains
Protein domain
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...

: one Gla domain
Gla domain
Vitamin K-dependent carboxylation/gamma-carboxyglutamic domain is a protein domain that contains post-translational modifications ofmany glutamate residues by vitamin K-dependent carboxylation to form gamma-carboxyglutamate . The Gla residues are responsible for the high-affinity binding of...

 (residues 43–88); a helical aromatic
Aromatic amino acids
Aromatic amino acids are amino acids that include an aromatic ring.Examples include:* Among 20 standard amino acids: phenylalanine, tryptophan, and tyrosine* Others: thyroxine-See also:* Aromatic L-amino acid decarboxylase...

 segment (89–96); two epidermal growth factor (EGF)-like domains
EGF-like domain
EGF-like domain is an evolutionary conserved protein domain, of about thirty to forty amino-acid residues long, which was found in a large number of mostly animal proteins. All these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted...

 (97–132 and 136–176); an activation peptide (200–211); and a trypsin
Trypsin
Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when...

-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 a zymogen
Zymogen
A zymogen is an inactive enzyme precursor. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it...

, waiting to be activated. 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 the N-terminus of the heavy chain.
The active site contains a catalytic triad
Catalytic triad
A catalytic triad refers to the three amino acid residues found inside the active site of certain protease enzymes: serine , aspartate , and histidine . They work together to break peptide bonds on polypeptides. In general terms, catalytic triad can refer to any set of three residues that function...

 typical of serine proteases (His253, Asp299 and Ser402).

The activation of protein C is strongly promoted by thrombomodulin
Thrombomodulin
Thrombomodulin, CD141 or BDCA-3 is an integral membrane protein expressed on the surface of endothelial cells. In humans, thrombomodulin is encoded by the THBD gene...

 and endothelial protein C receptor (EPCR), the latter of which is found primarily on endothelial cells (cells on the inside of blood vessels). The presence of thrombomodulin accelerates activation by several orders of magnitude, and EPCR speeds up activation by a factor of 20. If either of these two proteins is absent in murine
Mouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...

 specimens, the mouse dies from excessive blood-clotting while still in an embryo
Embryo
An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...

nic state. On the endothelium, APC performs a major role in regulating blood clotting, inflammation, and cell death (apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

). 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. 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.

The Gla domain is particularly useful for binding to negatively charged phospholipid
Phospholipid
Phospholipids are a class of lipids that are a major component of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a phosphate group, and a simple organic molecule such as choline; one exception to this rule is sphingomyelin, which is derived from...

s for anticoagulation and to EPCR for cytoprotection
Cytoprotection
Cytoprotection is a process by which chemical compounds provide protection to cells against harmful agents.For example, a gastric cytoprotectant is any medication that combats ulcers not by reducing gastric acid but by increasing mucosal protection. Examples of gastric cyctoprotective agents...

. One particular exosite
Exosite
An exosite is a secondary binding site, remote from the active site, on an enzyme or other protein.This is similar to allosteric sites, but differs in the fact that, in order for an enzyme to be active, its exosite typically must be occupied. Exosites have recently become a hot topic in biomedical...

 augments protein C's ability to inactive Factor Va efficiently. Another is necessary for interacting with thrombomodulin.

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 the HUGO Gene Nomenclature Committee
HUGO Gene Nomenclature Committee
The HUGO Gene Nomenclature Committee approves a unique and meaningful name for every known human gene based on a query of experts. In addition to a long name, the HGNC also assigns an abbreviation to every gene...

 is "PROC" from "protein C". It is located on the second chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...

 (2q13-q14) and comprises nine exon
Exon
An exon is a nucleic acid sequence that is represented in the mature form of an RNA molecule either after portions of a precursor RNA have been removed by cis-splicing or when two or more precursor RNA molecules have been ligated by trans-splicing. The mature RNA molecule can be a messenger RNA...

s. The nucleotide sequence that codes for human protein C is approximately 11,000 bases long.

Protein C in zymogen
Zymogen
A zymogen is an inactive enzyme precursor. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it...

 form is present in normal adult human blood plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...

 at concentrations between 65–135 IU
International unit
In pharmacology, the International Unit is a unit of measurement for the amount of a substance, based on biological activity or effect. It is abbreviated as IU, as UI , or as IE...

/dL. Activated protein C is found at levels approximately 2000 times lower than this. 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 healthy term 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 after adolescence
Adolescence
Adolescence is a transitional stage of physical and mental human development generally occurring between puberty and legal adulthood , but largely characterized as beginning and ending with the teenage stage...

. The half-life
Half-life
Half-life, abbreviated t½, is the period of time it takes for the amount of a substance undergoing decay to decrease by half. The name was originally used to describe a characteristic of unstable atoms , but it may apply to any quantity which follows a set-rate decay.The original term, dating to...

 of activated protein C is around 15 minutes.

Pathways

The protein C pathways are the specific chemical reactions that control the level of expression of APC and its activity in the body. Protein C is pleiotropic, 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 inactivating Factor Va
Factor V
Factor V is a protein of the coagulation system, rarely referred to as proaccelerin or labile factor. In contrast to most other coagulation factors, it is not enzymatically active but functions as a cofactor...

 and Factor VIIIa
Factor VIII
Factor VIII is an essential blood clotting factor also known as anti-hemophilic factor . In humans, Factor VIII is encoded by the F8 gene...

, turning them into Factor Vi and Factor VIIIi respectively. When still bound to EPCR, activated protein C performs its cytoprotective effects, acting on the effector
Effector (biology)
An effector is a molecule that binds to a protein and thereby alters the activity of that protein...

 substrate PAR-1, protease-activated receptor-1
Coagulation factor II receptor
Proteinase-activated receptor 1 also known as the coagulation factor II receptor is a protein that in humans is encoded by the F2R gene. PAR1 is a G protein-coupled receptor involved in the regulation of thrombotic response...

. 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.

The activity of protein C may be down-regulated
Downregulation and upregulation
Downregulation is the process by which a cell decreases the quantity of a cellular component, such as RNA or protein, in response to an external variable...

 by reducing the amount either of available thrombomodulin or of EPCR. This may be done by inflammatory cytokine
Cytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...

s, such as interleukin-1β
IL1B
Interleukin-1 beta also known as catabolin, is a cytokine protein that in humans is encoded by the IL1B gene. IL-1β precursor is cleaved by caspase 1 . Cytosolic thiol protease cleaves the product to form mature IL-1β.- Function :Interleukin 1 was discovered by Gery in 1972...

  (IL-1β ) and tumor 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 example eosinophil, which may explain thrombosis in hypereosinophilic
Hypereosinophilic syndrome
The hypereosinophilic syndrome is a disease characterized by a persistently elevated eosinophil count in the blood for at least six months without any recognizable cause, with involvement of either the heart, nervous system, or bone marrow.HES is a diagnosis of exclusion, after clonal...

 heart disease. Protein C may be up-regulated
Downregulation and upregulation
Downregulation is the process by which a cell decreases the quantity of a cellular component, such as RNA or protein, in response to an external variable...

 by platelet factor 4
Platelet factor 4
Platelet factor 4 is a small cytokine belonging to the CXC chemokine family that is also known as chemokine ligand 4 . This chemokine is released from alpha-granules of activated platelets during platelet aggregation, and promotes blood coagulation by moderating the effects of heparin-like...

. This cytokine is conjectured to improve activation of protein C by forming an electrostatic bridge from protein C's Gla domain to the glycosaminoglycan
Glycosaminoglycan
Glycosaminoglycans or mucopolysaccharides are long unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating unit consists of a hexose or a hexuronic acid, linked to a hexosamine .-Production:Protein cores made in the rough endoplasmic reticulum are posttranslationally...

 (GAG) domain of thrombomodulin, reducing the Michaelis constant (KM) for their reaction. In addition, Protein C is inhibited by protein C inhibitor.

Anticoagulative effects

Protein C is a major component in anticoagulation in the human body. It acts as a serine protease
Serine protease
Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site.They are found ubiquitously in both eukaryotes and prokaryotes...

 zymogen
Zymogen
A zymogen is an inactive enzyme precursor. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it...

: APC proteolyses peptide bonds in activated Factor V
Factor V
Factor V is a protein of the coagulation system, rarely referred to as proaccelerin or labile factor. In contrast to most other coagulation factors, it is not enzymatically active but functions as a cofactor...

 and Factor VIII
Factor VIII
Factor VIII is an essential blood clotting factor also known as anti-hemophilic factor . In humans, Factor VIII is encoded by the F8 gene...

 (Factor Va and Factor VIIIa), and one of the amino acids in the bond is serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...

. These proteins that APC inactivates, Factor Va and Factor VIIIa, are highly procoagulant cofactors in the generation of thrombin
Thrombin
Thrombin is a "trypsin-like" serine protease protein that in humans is encoded by the F2 gene. Prothrombin is proteolytically cleaved to form thrombin in the first step of the coagulation cascade, which ultimately results in the stemming of blood loss...

, which is a crucial element in blood clotting; together they are part of the prothrombinase complex
Prothrombinase
The prothrombinase complex consists of the serine protease, Factor Xa, and the protein cofactor, Factor Va. The complex assembles on negatively charged phospholipid membranes in the presence of calcium ions. The prothrombinase complex catalyzes the conversion of prothrombin , an inactive zymogen,...

. Cofactors in the inactivation of Factor Va and Factor VIIIa include protein S
Protein S
Protein S is a vitamin K-dependent plasma glycoprotein synthesized in the endothelium. In the circulation, Protein S exists in two forms: a free form and a complex form bound to complement protein C4b. In humans, protein S is encoded by the PROS1 gene...

, Factor V, high-density lipoprotein, anionic phospholipid
Phospholipid
Phospholipids are a class of lipids that are a major component of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a phosphate group, and a simple organic molecule such as choline; one exception to this rule is sphingomyelin, which is derived from...

s and glycosphingolipids.


Factor Va binds to prothrombin and Factor Xa, increasing the rate at which thrombin is produced by four orders of magnitude. 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 cause haemophilia A
Haemophilia A
Haemophilia A is a deficiency in clotting factor VIII.Haemophilia A is inherited as an X-linked recessive trait, and thus occurs in males and in homozygous females. However, mild haemophilia A has been described in heterozygous females, presumably due to extremely unfavourable lyonization...

.

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. Protein S also binds to Factor Xa, inhibiting the latter from diminishing APC's inactivation of Factor Va.

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.

Cytoprotective effects

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.

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 on transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...

 activity. Important proteins that APC up-regulates include Bcl-2
Bcl-2
Bcl-2 is the founding member of the Bcl-2 family of apoptosis regulator proteins encoded by the BCL2 gene. Bcl-2 derives its name from B-cell lymphoma 2, as it is the second member of a range of proteins initially described in chromosomal translocations involving chromosomes 14 and 18 in...

, eNOS
Endothelial NOS
Endothelial NOS , also known as nitric oxide synthase 3 or constitutive NOS , is an enzyme that in humans is encoded by the NOS3 gene....

 and IAP
Inhibitor of apoptosis
The Inhibitors of Apoptosis are a family of functionally- and structurally-related proteins, which serve as endogenous inhibitors of programmed cell death . A common feature of all IAPs is the presence of a BIR in one to three copies...

. APC effects significant down-regulation of p53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...

 and Bax.

APC has anti-inflammatory effects on endothelial
Endothelium
The endothelium is the thin layer of cells that lines the interior surface of blood vessels, forming an interface between circulating blood in the lumen and the rest of the vessel wall. These cells are called endothelial cells. Endothelial cells line the entire circulatory system, from the heart...

 cells and leukocytes
White blood cell
White blood cells, or leukocytes , are cells of the immune system involved in defending the body against both infectious disease and foreign materials. Five different and diverse types of leukocytes exist, but they are all produced and derived from a multipotent cell in the bone marrow known as a...

. APC affects endothelial cells by inhibiting inflammatory mediator release
Inflammation
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process...

 and down-regulating vascular adhesion molecules
Cell adhesion molecule
Cell Adhesion Molecules are proteins located on the cell surface involved with the binding with other cells or with the extracellular matrix in the process called cell adhesion....

. This reduces leukocyte adhesion and infiltration into tissues, while also limiting damage to underlying tissue. APC supports endothelial barrier function and reduces chemotaxis
Chemotaxis
Chemotaxis is the phenomenon in which somatic cells, bacteria, and other single-cell or multicellular organisms direct their movements according to certain chemicals in their environment. This is important for bacteria to find food by swimming towards the highest concentration of food molecules,...

. 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 in sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...

. Studies on both rats and humans have demonstrated that APC reduces endotoxin
Endotoxin
Endotoxins are toxins associated with some Gram-negative bacteria. An "endotoxin" is a toxin that is a structural molecule of the bacteria that is recognized by the immune system.-Gram negative:...

-induced pulmonary injury and inflammation.

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 is neuroprotective
Neuroprotection
Neuroprotection within the nervous system protects neurons from apoptosis or degeneration, for example following a brain injury or as a result of chronic neurodegenerative diseases....

. APC's antiapoptotic effects are part of the reason that APC is effective in treating sepsis, as reduced levels of apoptosis are correlated with higher survival rates in septic patients. Antiapoptosis is achieved with diminished activation of caspase 3
Caspase 3
Caspase 3 is a caspase protein that interacts with caspase 8 and caspase 9. It is encoded by the CASP3 gene. CASP3 orthologs have been identified in numerous mammals for which complete genome data are available...

 and caspase 8
Caspase 8
Caspase 8 is a caspase protein, encoded by the CASP8 gene. It most likely acts upon caspase 3.CASP8 orthologs have been identified in numerous mammals for which complete genome data are available...

, improved Bax/Bcl-2 ratio and down-regulation of p53.

Activated protein C also provides much protection of endothelial barrier function. Endothelial barrier breakdown, and the corresponding increase in endothelial permeability, are associated with swelling
Swelling (medical)
In medical parlance, swelling is the transient enlargement or protuberance in the body and may include tumors. According to cause, it may be congenital, traumatic, inflammatory, neoplastic or miscellaneous....

, hypotension
Hypotension
In physiology and medicine, hypotension is abnormally low blood pressure, especially in the arteries of the systemic circulation. It is best understood as a physiologic state, rather than a disease. It is often associated with shock, though not necessarily indicative of it. Hypotension is the...

 and inflammation, all problems of sepsis. APC protects endothelial barrier function by inducing PAR-1 dependent sphingosine kinase-1
Sphingosine kinase 1
Sphingosine kinase 1 is an enzyme that in humans is encoded by the SPHK1 gene.Sphingosine kinase 1 phosphorylates sphingosine to sphingosine-1-phosphate SK1 is normally a cytosolic protein but is recruited to membranes rich in phosphatidate , a product of Phospholipase D Sphingosine-1-phosphate ...

 activation and up-regulating sphingosine-1-phosphate
Sphingosine-1-phosphate
Sphingosine-1-phosphate is a signaling sphingolipid. It is also referred to as a bioactive lipid mediator. Sphingolipids at large form a class of lipids characterized by a particular aliphatic aminoalcohol, which is sphingosine.-Production:...

 with sphingosine kinase
Sphingosine kinase
Sphingosine kinase is a conserved lipid kinase that catalyzes formation Sphingosine-1-phosphate from the precursor sphingolipid sphingosine. Sphingolipid metabolites, such as ceramide, sphingosine and sphingosine-1-phosphate, are lipid second messengers involved in diverse cellular processes. ...

.

Role in disease

A genetic protein C deficiency, in its mild form associated with simple heterozygosity, causes a significantly increased risk of venous thrombosis
Venous thrombosis
A venous thrombosis is a blood clot that forms within a vein. A venous thrombosis is a blood clot that forms within a vein. A venous thrombosis is a blood clot that forms within a vein. (Thrombosis is a medical term for blood clotting (Haemostasis) occurring in the wrong place, i.e...

 in adults. If a fetus is homozygous or compound heterozygous for the deficiency, there may be a presentation of purpura fulminans
Purpura fulminans
Purpura fulminans is a haemorrhagic condition usually associated with sepsis or previous infection. It occurs mainly in babies and small children.It was first described by Guelliot in 1884.- Presentation :...

, severe disseminated intravascular coagulation
Disseminated intravascular coagulation
Disseminated intravascular coagulation , also known as disseminated intravascular coagulopathy or consumptive coagulopathy, is a pathological activation of coagulation mechanisms that happens in response to a variety of diseases. DIC leads to the formation of small blood clots inside the blood...

 and simultaneous venous thromboembolism in the womb; this is very severe and usually fatal. 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
Coagulopathy
Coagulopathy is a condition in which the blood’s ability to clot is impaired. This condition can cause prolonged or excessive bleeding, which may occur spontaneously or following an injury or medical and dental procedures.The normal clotting process depends on the interplay of various proteins in...

, deposition of fibrin
Fibrin
Fibrin is a fibrous, non-globular protein involved in the clotting of blood. It is a fibrillar protein that is polymerised to form a "mesh" that forms a hemostatic plug or clot over a wound site....

 and necrosis
Necrosis
Necrosis is the premature death of cells in living tissue. Necrosis is caused by factors external to the cell or tissue, such as infection, toxins, or trauma. This is in contrast to apoptosis, which is a naturally occurring cause of cellular death...

 of the liver.

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.

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 whites is at the cleavage site in Factor V for APC. There, Arg506 is replaced with Gln, producing Factor V Leiden
Factor V Leiden
Factor V Leiden is the name given to a variant of human factor V that causes a hypercoagulability disorder. In this disorder the Leiden variant of factor V cannot be inactivated by activated protein C. Factor V Leiden is the most common hereditary hypercoagulability disorder amongst Eurasians...

. This mutation is also called a R506Q. 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. 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. This mutation is also the most common hereditary risk for venous thrombosis among Caucasians.

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 of autoantibodies
Autoantibody
An autoantibody is an antibody manufactured by the immune system that is directed against one or more of the individual's own proteins. It is derived from the Greek "auto" which means "self", "anti" which means "against" and "body"...

 targeting Factor V, and disfunction of any of APC's cofactors. Also, some acquired conditions may reduce the efficacy of APC in performing its anticoagulative functions. Studies suggest that between 20% and 60% of thrombophilic patients suffer from some form of APC resistance.

Warfarin necrosis
Warfarin necrosis
Warfarin-induced skin necrosis is a condition in which skin and subcutaneous tissue necrosis occurs due to acquired protein C deficiency following treatment with anti-vitamin K anticoagulants .Warfarin necrosis is a rare but severe complication of treatment with warfarin or related...

 is an acquired protein C deficiency due to treatment with warfarin
Warfarin
Warfarin is an anticoagulant. It is most likely to be the drug popularly referred to as a "blood thinner," yet this is a misnomer, since it does not affect the thickness or viscosity of blood...

, 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 limb gangrene
Gangrene
Gangrene is a serious and potentially life-threatening condition that arises when a considerable mass of body tissue dies . This may occur after an injury or infection, or in people suffering from any chronic health problem affecting blood circulation. The primary cause of gangrene is reduced blood...

 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.

Role in medicine

rhAPC has been the subject of significant controversy since its approval for clinical use in 2001. A 2011 Cochrane review however concluded that it does not decrease mortality in severe sepsis or septic shock. It has been noted that rates of severe hemorrhages, drug infusion
Route of administration
A route of administration in pharmacology and toxicology is the path by which a drug, fluid, poison, or other substance is taken into the body.-Classification:Routes of administration are usually classified by application location...

-related fatal events and termination of infusion due to adverse reactions are all higher in clinical use and open-label trial
Open-label trial
An open-label trial or open trial is a type of clinical trial in which both the researchers and participants know which treatment is being administered....

s than in controlled trials. There is a dispute as to whether or not studies after PROWESS confirm its results, and if so, for what subgroups.


Protein C levels have long been noted to predict mortality in patients with sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...

. Because of this, and its pleiotropic anticoagulative and cytoprotective effects, protein C has long been suggested, along with many other drugs, for use in treating patients with severe sepsis. In November of that year, the Food and Drug Administration
Food and Drug Administration
The Food and Drug Administration is an agency of the United States Department of Health and Human Services, one of the United States federal executive departments...

 approved drotrecogin alfa-activated (DrotAA) in the clinical treatment of adults suffering from severe sepsis and with a high risk of death. Drotrecogin alfa-activated
Drotrecogin alfa
Drotrecogin alfa is a recombinant form of human activated protein C that has anti-thrombotic, anti-inflammatory, and profibrinolytic properties. Drotrecogin alpha belongs to the class of serine proteases. Drotrecogin alfa has not been found to improve outcomes in people with severe sepsis...

 is a recombinant form of human activated protein C (rhAPC), i.e. it is a protein produced by recombinant DNA
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...

. It is marketed as Xigris by Eli Lilly and Company
Eli Lilly and Company
Eli Lilly and Company is a global pharmaceutical company. Eli Lilly's global headquarters is located in Indianapolis, Indiana, in the United States...

, but recently recalled and taken off the market.

APC has been studied as way of treating lung
Lung
The lung is the essential respiration organ in many air-breathing animals, including most tetrapods, a few fish and a few snails. In mammals and the more complex life forms, the two lungs are located near the backbone on either side of the heart...

 injury, after studies showed that in patients with lung injury, reduced APC levels in specific parts of the lungs correlated with worse outcomes. APC also has been considered for use in improving patient outcome in cases of ischemic 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
Tissue plasminogen activator
Tissue plasminogen activator is a protein involved in the breakdown of blood clots. It is a serine protease found on endothelial cells, the cells that line the blood vessels. As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown...

 (tPA), to protect the brain form tPA's very harmful side effect
Side effect
In medicine, a side effect is an effect, whether therapeutic or adverse, that is secondary to the one intended; although the term is predominantly employed to describe adverse effects, it can also apply to beneficial, but unintended, consequences of the use of a drug.Occasionally, drugs are...

s, in addition to preventing cell death from lack of oxygen (hypoxia
Hypoxia (medical)
Hypoxia, or hypoxiation, is a pathological condition in which the body as a whole or a region of the body is deprived of adequate oxygen supply. Variations in arterial oxygen concentrations can be part of the normal physiology, for example, during strenuous physical exercise...

). Clinical use of APC has also been proposed for improving the outcome of pancreatic islet transplantation
Islet cell transplantation
Islet transplantation is the transplantation of isolated islets from a donor pancreas and into another person. It is an experimental treatment for type 1 diabetes mellitus. Once transplanted, the islets begin to produce insulin, actively regulating the level of glucose in the blood.Islets are...

 in treating type I diabetes.

External links

  • The MEROPS
    Merops
    Merops may refer to:* Merops , a genus of bee-eaters.* MEROPS, an on-line database for peptidases.It may also refer to several figures from Greek mythology:* King of Ethiopia, husband of Clymene, who lay with Helios and bore Phaethon...

     online database for peptidases and their inhibitors: S01.218
  • The Protein C Pathway- John H. Griffin, retired, TSRI, La Jolla, California
  • Diagram of The Blood Coagulation Pathway and Protein C Pathway
The source of this article is wikipedia, the free encyclopedia.  The text of this article is licensed under the GFDL.
 
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