Protein C

Protein C, also known as autoprothrombin IIA and blood coagulation factor XIV, is a zymogen, that is, an inactive enzyme. The activated form plays an important role in regulating anticoagulation, inflammation, and cell death and maintaining the permeability of blood vessel walls in humans and other animals. Activated protein C performs these operations primarily by proteolytically inactivating proteins Factor V_a and Factor VIII_a. APC is classified as a serine protease since it contains a residue of serine in its active site. In humans, protein C is encoded by the PROC gene, which is found on chromosome 2.
The zymogenic form of protein C is a vitamin K-dependent glycoprotein that circulates in blood plasma. Its structure is that of a two-chain polypeptide consisting of a light chain and a heavy chain connected by a disulfide bond. The protein C zymogen is activated when it binds to thrombin, another protein heavily involved in coagulation, and protein C's activation is greatly promoted by the presence of thrombomodulin and endothelial protein C receptors. Because of EPCR's role, activated protein C is found primarily near endothelial cells, and it is these cells and leukocytes that APC affects. Because of the crucial role that protein C plays as an anticoagulant, those with deficiencies in protein C, or some kind of resistance to APC, suffer from a significantly increased risk of forming dangerous blood clots.
Research into the clinical use of a recombinant form of human Activated Protein C known as Drotrecogin alfa-activated, branded Xigris by Eli Lilly and Company, has been surrounded by controversy. Eli Lilly ran an aggressive marketing campaign to promote its use for people with severe sepsis and septic shock and sponsored the 2004 Surviving Sepsis Campaign Guidelines. However, a 2012 Cochrane review found that its use cannot be recommended since it does not improve survival and increases bleeding risk. In October 2011, Xigris was withdrawn from the market by Eli Lilly due to a higher mortality in a trial among adults.

History

Protein C's anticoagulant 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 from bovine plasma in 1976, and Stenflo determined it to be a vitamin K-dependent protein. He named it protein C because it was the third protein that eluted from a DEAE-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 V_a. In 1980, Vehar and Davie discovered that APC also inactivates Factor VIII_a, and soon after, Protein S was recognised as a cofactor by Walker. In 1982, a family study by Griffin et al. first associated protein C deficiency with symptoms of venous thrombosis. 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 whereby it was demonstrated that activated protein C prevented coagulopathy and death in baboons infused with lethal concentrations of E. coli.
In 1993, a heritable resistance to APC was detected by Dahlbäck et al. and associated with familial thrombophilia. In 1994, the relatively common genetic mutation that produces Factor V_Leiden was noted. Two years later, Gla-domainless APC was imaged at a resolution of 2.8 Ångströms. Beginning with the PROWESS clinical trial of 2001, it was recognised that many of the symptoms of sepsis 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, a recombinant human activated protein C, became the first drug approved by the U.S. FDA for treating severe sepsis. In 2002, Science published an article that first showed protein C activates protease-activated receptor-1 and this process accounts for the protein's modulation of the immune system.

Genetics

The biologic instructions for synthesising protein C in humans are encoded in the gene officially named "protein C ". The gene's symbol approved by the HUGO Gene Nomenclature Committee is "PROC" from "protein C". It is located on the second chromosome and comprises nine exons. The nucleotide sequence that codes for human protein C is approximately 11,000 bases long.

Structure and processing

Human protein C is a vitamin K-dependent glycoprotein structurally similar to other vitamin K-dependent proteins affecting blood clotting, such as prothrombin, Factor VII, Factor IX and Factor X. Protein C synthesis occurs in the liver and begins with a single-chain precursor molecule: a 32 amino acid N-terminus signal peptide preceding a propeptide. Protein C is formed when a dipeptide 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 and one heavy chain connected by a disulfide bond between Cys¹⁸³ and Cys³¹⁹.
Inactive protein C comprises 419 amino acids in multiple domains: one Gla domain ; a helical aromatic segment ; two epidermal growth factor -like domains ; an activation peptide ; and a trypsin-like serine protease domain. 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, 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 typical of serine proteases.
The Gla domain is particularly useful for binding to negatively charged phospholipids for anticoagulation and to EPCR for cytoprotection. One particular exosite augments protein C's ability to inactivate Factor V_a efficiently. Another is necessary for interacting with thrombomodulin.
Post-translational modifications. Human Protein C has at least five types of post-translational modifications: gamma-carboxylation on the first nine glutamic acid residues in the protein sequence. This modification event is performed by a vitamin K-dependent microsomal carboxylase. The full complement of Gla is required to give full activity to protein C. beta-Hydroxylation of Asp71 in one of the two EGF-like domains to give erythro-L-beta-hydroxy-aspartate. The modification is required for functional activity as was demonstrated by mutating Asp71 to Glu. N-linked glycosylation at three possible glycosylation sites. Plasma human Protein C has been reported to be 23% carbohydrate by weight. Disulfide formation. Multiple proteolytic cleavages of the polypeptide backbone to remove an 18 amino acid signal peptide, a 24 amino acid propeptide and then cleavages at amino acids 155-156 and 157-158 to yield the two-chain structure of the circulating zymogen.

Physiology

The activation of protein C is strongly promoted by thrombomodulin and endothelial protein C receptor, the latter of which is found primarily on endothelial cells. 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 specimens, the mouse dies from excessive blood-clotting while still in an embryonic state. On the endothelium, APC performs a major role in regulating blood clotting, inflammation, and cell death. 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 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.
Protein C in zymogen form is present in normal adult human blood plasma at concentrations between 65 and 135 IU/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. The half-life 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. 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 V_a and Factor VIII_a, turning them into Factor V_i and Factor VIII_i respectively. When still bound to EPCR, activated protein C performs its cytoprotective effects, acting on the effector substrate PAR-1, protease-activated receptor-1. To a degree, APC's anticoagulant properties are independent of its cytoprotective ones, in that expression of one pathway is not affected by the existence of the other.
The activity of protein C may be down-regulated by reducing the amount either of available thrombomodulin or of EPCR. This may be done by inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-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 heart disease. Protein C may be up-regulated by platelet factor 4. This cytokine is conjectured to improve activation of protein C by forming an electrostatic bridge from protein C's Gla domain to the glycosaminoglycan domain of thrombomodulin, reducing the Michaelis constant for their reaction. In addition, Protein C is inhibited by protein C inhibitor.