Protein C is a serine protease that is produced in the liver and consists of light and heavy chain, linked by a disulfide bond. The protein has a half-life of 8 hours, although intravenously administered activated protein has a half-life <20 minutes (Okijama et al 1990). Protein C has similar structure to other vitamin K-dependent coagulation proteins (factors II, V, VII and X), in that it contains a glutamic acid domain that requires carboxylation to form γ-carboxyglutamatic acid (Gla) in order to bind calcium, which is required for the protein to bind to phospholipids on membrane surfaces. The carboxylation reaction (which is carried out by γ-glutamyl carboxylase, an enzyme within hepatocytes) requires vitamin K, so protein C is considered a vitamin K-dependent enzyme. Protein C is not normally active, but is activated when thrombin cleaves protein C, in the presence of thrombomodulin on endothelial cells. The activation of protein C is modulated by the endothelial protein C receptor, which enhances protein C activation by thrombin, and protein S, which serves as a cofactor for activated protein C. Protein S is found in two forms, free and that bound to C4b-protein. Only the free form of protein S is active as a cofactor for protein C.
Protein C has several functions:
- Anticoagulation: It is an anticoagulant protein that inhibits thrombin formation by binding to and inactivating (through cleavage) FVa and FVIIIa, the cofactors of the “intrinsic” tenase and prothrombinase complexes, respectively. Inactivation of these cofactors markedly slows thrombin generation (thereby thrombin inhibits its own production) and is one of the breaks that stops coagulation. Mutations in FV (FVLeiden), that renders the cofactor resistant to protein C inactivation, are responsible for hereditary thrombophilia, a common risk factor for thrombosis in human beings (van Cott et al 2016).
- Profibrinolytic: Activated protein C inhibits plasminogen activator inhibitor-1, promoting fibrinolysis. The profibrinolytic effects of activated protein C are dependent on calcium, the cofactor Protein S and phospholipid membranes.
- Cytoprotective and anti-inflammatory: Protein C is now known to have cytoprotective and anti-inflammatory responses. The anti-inflammatory responses are thought to be mediated via the endothelial protein C receptor activating protease-activated receptors 1 and 3 on endothelial cells and provides a critical link between hemostasis and inflammation.
- Tissue regeneration: Blood vessels (angiogenesis), wound healing and neurogenesis (Griffin et al 2015).
Protein C has been purified from canine plasma and can be activated by Protac (a venom from the copperhead snake that activates protein C). The activated protein inhibited the ability of FVa and FVIIIa (from diluted pooled canine plasma) to restore the APTT of factor-deficient plasma ( (Wong et al 2014).
Activity is measured directly using a specific chromogenic substrate.
Chromogenic substrate cleaved by activated protein C.
The protein C is first activated by a snake venom (such as Protac) then a protein C-dependent substrate is added to plasma. The rate of cleavage of the substrate is proportional to the activity of protein C (Fry et al 2011).
Units of measurement
Results are expressed as a percentage of a species-specific standard pool, designated as 100%.
Protein C activity was stable for 7 days at -20°C or -80°C storage, but declined after 28 days of storage at both freezer temperatures (Fry et al 2011)
Measurement of protein C activity is usually performed as a single test to facilitate the diagnosis of DIC, hepatic dysfunction (due to hepatic parenchymal disease) and abnormal hepatic blood flow (portosystemic shunts). Reference intervals for protein C have been established at the Comparative Coagulation Laboratory at Cornell University (dogs: 75-135%, cats: 65-120%).
Decreased protein C activity
- Decreased production:
- Abnormal hepatic blood flow: Studies have shown that protein C can act a biomarker of hepatoportal perfusion. This is because protein C concentrations appear to be governed by hepatic portal flow and are not only influenced by synthetic capability of the liver. In one study, 88% of dogs with congenital or acquired shunts had low protein C activity (<70%). In contrast, 30 of 35 dogs with microvascular dysplasia had protein C activity ≥ 70%, making this a potentially useful test to differentiate these two conditions (Toulza et al 2006). Protein C activities increase after resolution of congenital shunts (to a greater degree than normalization of bile acids), suggesting protein C measurement may be useful for assessing the efficacy to surgical ligation of the shunt (Toulza et al 2006).
- Liver dysfunction or failure: A decrease in synthetic functional mass of the liver (>70%) can result in low protein C activity as can hepatic atrophy secondary to abnormal portal blood flow. The lowest protein C activities were seen in dogs with hepatic failure in one study, although activity was also decreased in some dogs with other hepatic diseases, e.g. chronic hepatitis (Toulza et al 2006). Cats with various liver diseases have low protein C activity, however the mechanism is unclear (could be due to decreased synthesis, defective activation, defective vitamin K metabolism or increased consumption) (Dircks et al 2012).
- Vitamin K deficiency/antagonism: Cholestasis, resulting in decreased vitamin K absorption or anticoagulant rodenticide toxicosis, will result in low protein C activity. In one study of 10 dogs with extrahepatic bile duct obstruction, low protein C activity was seen in 3 dogs (Mayhew et al 2013). However, clinical signs generally manifest as hemorrhage versus thrombosis in dogs, due to concurrent inhibitory effects of vitamin K lack/antagonism on the procoagulant proteins (particularly thrombin). Anticoagulant rodenticide toxicosis is also expected to result in low Protein C activity.
- Inherited defects: An inherited or congenital defect of protein C was suspected in a Thoroughbred colt with clinical evidence of recurrent thrombosis (Edens et al 1993).
- Decreased activation
- DIC: Low protein C activity is seen in humans and animals with DIC (Connor et al 2015). This is thought to be due to downregulation or cleavage of thrombomodulin, downregulation or cleavage of the endothelial protein C receptor, or decreased concentrations of free protein S (see below). Increased consumption could also be occurring.
- Inflammation: Inflammatory cytokines can cause downregulation of thrombomodulin (which will decrease protein C activation) and will decrease free protein S (due to increases in C4b-binding protein, a positive acute phase protein, which binds to protein S), the co-factor for protein C. This would explain low protein C activities that can be seen in some dogs with bacterial sepsis (as would concurrent initiation of DIC) (De LaForcade et al 2003, De Laforcade et al 2008) or after infusion of lipopolysaccharide (Madden et al 1989).
Increased protein C activity
This has been associated with a bleeding phenotype in patients with trauma. This is thought to be due to the anticoagulant actions of protein C (limiting clot formation) and its profibrinolytic effects (breaking down clots rapidly as they form) (Chang et al 2016).