Protein C

Overview

Protein C is a major plasma anticoagulant that acts at the surface of endothelial cells. Activated Protein C neutralizes Factors Va and VIIIa, which effectively blocks the amplification and propagation of coagulation. Protein C is a vitamin K-dependent protein, synthesized in the liver with a short plasma half-life. Protein C assays aid in the diagnosis of liver disease and thrombotic disorders. Clinical studies of dogs with congenital hepatoportal vascular anomalies indicate that Protein C is a non-invasive measure of portal blood flow, useful for differentiating portosystemic shunting (PSS) from microvascular dysplasia (MVD).

Protein C Testing

Sample requirements

Citrate plasma is the only acceptable specimen-type for the Protein C activity assay. At least 0.5 mL separated citrate plasma should be shipped on cold packs for overnight delivery. Please see the detailed sampling instructions.

Assay methodology

The Protein C assay performed at the Comparative Coagulation Laboratory for clinical diagnosis is a functional measure of plasma protein C levels (Protein C activity assay). The test plasma is treated with a venom-derived activator and the proteolytic action of Protein C is then measured based on the enzyme’s cleavage of a chromogenic substrate. This is a fluid phase, commercial assay that does not evaluate Protein C’s interaction with cell membrane receptors (thrombomodulin or EPCR) and does not measure circulating APC

Test applications and algorithms

  • Protein C to differentiate MVD from PSVA
    Developmental defects of the hepato/portal vasculature are common in toy and small breed dogs. Puppies with large shunting portosystemic vascular anomalies (PSVA) often require surgical correction, whereas microvascular dysplasia (MVD) is not a surgical problem. High serum bile acids are a sensitive screening test of both PSVA and MVD, the finding of high bile acids does not differentiate between these 2 syndromes. In contrast, low protein C activity (< 70%) is a characteristic finding in dogs with clinical signs of PSVA. In contrast, dogs with MVD typically have protein C values > 70%. A diagnostic algorithm incorporating protein C helps select dogs for PSVA imaging and surgery.
  • Protein C to assess portal blood flow post hepatic shunt occlusion
    While high serum bile acids often persist after PSVA ligation, a rise in Protein C levels can be used as an indicator of restoration of hepatic blood flow.
  • Protein C to evaluate non-shunting liver disease and vitamin K deficiency states
    Toxins, infection, and non-infectious inflammatory disorders are among the most common causes of hepatic synthetic compromise and progressive liver failure in dogs. Marked reduction in protein C (<50%) is an indicator of severe hepatic insult. Protein C deficiency develops early after exposure to hepatoxins such as aflatoxin, and the combined deficiencies of Protein C, antithrombin, and fibrinogen are associated with a poor prognosis in this patient population. Intra-or post-hepatic biliary obstruction may impair vitamin K metabolism, causing inadequate vitamin K to maintain functional levels of Protein C. Due to its short half-life, measurement of Protein C activity is an early indicator of vitamin K deficiency that is detectable before signs of coagulopathy.
  • Protein C testing in non-hepatic disorders
    Protein C deficiency may develop in patients with sepsis, systemic inflammatory syndromes, and disseminated intravascular coagulation. Protein C deficiency has been associated with development of thrombosis in dogs with parvovirus infection and as a poor prognostic indicator in dogs with bacterial sepsis. High Protein C activity has recently been described as a unique abnormality that differentiated dogs with protein losing nephropathy from those with non-protein losing renal failure. Additional studies are needed to clarify the role of Protein C testing in these clinical patient populations.

Protein C in Disease

Hereditary Protein C deficiency states

In people, inactivating Protein C mutations are a rare cause of severe, perinatal thrombosis referred to as “purpura fulminans”. Heterozygous carriers of these mutations typically demonstrate a mild prothrombotic tendency. A functional lack of APC’s anticoagulant effect is among the most common hereditary thrombophilic conditions in people of Northern European ancestry. A mutation in Factor V (Factor V Leiden; OMIM 188055) renders the cofactor resistant to APC’s proteolytic degradation and therefore enhances thrombin generation through dysregulation of the coagulation cascade. In veterinary medicine, a single case report describes congenital Protein C deficiency in a thoroughbred foal.

Acquired Protein C deficiency states

Acquired Protein C deficiency develops much more commonly in veterinary medicine, often in association with liver disease. In particular, severe Protein C deficiency develops in patients with liver failure and congenital portosystemic vascular anomalies (PSVA). Protein C’s short plasma half-life likely contributes to the development of Protein C deficiency secondary to hepatic synthetic failure. The etiopathogenesis of Protein C deficiency secondary to portosystemic shunting is not yet well-defined. In addition to liver disease, vitamin K deficiency of any cause will cause a functional Protein C defect due to impaired post-translational processing and an inability of Protein C to interact with coagulation complexes.

Protein C deficiency attributed to consumption and depletion has also been reported in patients with sepsis, acute inflammatory syndromes, and disseminated intravascular coagulation.

Protein C Synthesis and Actions

Hepatic synthesis and cell surface activation

Protein C is a vitamin K-dependent serine protease anticoagulant that is structurally similar to the vitamin K-dependent coagulation factors (Factors II, VII, IX, X). These proteins are synthesized primarily in the liver and all require post-translational gamma carboxylation to bind Ca++ and attain a fully functional form. Protein C circulates as a zymogen that is activated at the surface of endothelial cells by interacting with thrombin bound to its endothelial cell receptor, thrombomodulin (TM). This Protein C –thrombin interaction is greatly enhanced by Protein C’s binding to endothelial cell protein C receptor (EPCR). Activated Protein C (APC) is released from the EPCR and then combines with its cofactor, Protein S, on the phospholipid surface of endothelial cells and platelets. Protein C zymogen circulates in plasma with a half-life of approximately 6 hours. The protease APC circulates in only trace amounts where it is rapidly inactivated (within minutes) by plasma inhibitors.

Anticoagulant effects

Protein C exerts a potent anticoagulant effect by inhibiting thrombin generation. Activated Protein C, in a reaction accelerated by Protein S, cleaves arginine sites on Factor Va and Factor VIIIa, to produce degradation fragments with no cofactor activity. The lack of these factors prevents rapid production of Factor Xa and thrombin to drive the amplification and propagation phases of coagulation that generate a large burst of thrombin. This proteolytic inactivation of Factor Va and Factor VIIIa is a critical regulatory mechanism that prevents pathologic thrombosis and thereby maintains blood flow.

Cytoprotective effects

When APC is bound to the EPCR on endothelial surfaces it initiates signaling via activation of PAR-1 at the thrombin cleavage site. In contrast to thrombin activation of PAR-1, APC activation initiates signaling pathways that down-regulate inflammatory cytokines, decrease endothelial adhesion molecule expression, inhibit leukocyte migration, and enhance endothelial cell stability. In addition, APC exerts anti-apoptotic effects on endothelial cells including decreased caspase activation and modulation of mitochondrial permeability transition pore formation which downregulates apoptotic processes. The barrier properties of endothelial cells are enhanced via APC-mediated upregulation of cell membrane receptors and its stimulation of smooth muscle cell in migration.

References

  • Bentley AM, Mayhew PD, Culp WT, Otto CM. Alterations in the hemostatic profiles of dogs with naturally occurring septic peritonitis. J Vet Emerg Crit
    Care
    . 2013 Jan;23(1):14-22.
  • Examination of hemostatic parameters to detect hypercoagulability in dogs with protein-losing nephropathy. Donahue SM, Brooks M, Otto CM. J Vet Emerg Crit Care 2011;21:346-355.
  • Serial evaluation of protein C and antithrombin in dogs with sepsis. J Vet Intern Med 2008;22:26-30.
  • Clinical and clinicopathologic features of dogs that consumed foodborne hepatotoxic aflatoxins: 72 cases (2005-2006). Dereszynski DM, Center SA, Randolph JF et al. J Am Vet Med Assoc 2008;232:1329-1337.
  • Evaluation of plasma protein C activity for detection of hepatobiliary disease and portosystemic shunting in dogs. Toulza O, Center SA, Brooks MB, et al. J Am Vet Med Assoc 2006;229:1761-1771
  • Coagulation parameters in dogs with naturally occurring sepsis. de Laforcade AM, Shaw SP, Freeman LM, et al. J Vet Intern Med 2003;17:674-679.
  • Evidence of hypercoagulability in dogs with parvoviral enteritis. Otto CM, Rieser TM, Brooks MB, Russell MW. J Am Vet Med Assoc 2000;217:1500-1504.
  • Hypercoagulable state associated with a deficiency of protein C in a thoroughbred colt. Edens LM, Morris DD, Prasse KW, et al. J Vet Intern Med 1993;7:190–193.

Frequently Asked Questions

What sample is required for Protein C testing?

Separated citrate plasma (plasma from a blue top tube) is the only valid sample. DO NOT SUBMIT SERUM! Ship at least 0.5 mL of plasma in an insulated box with cold packs for overnight delivery. Please see the detailed sampling instructions.

Does the patient have to be fasted?

No, fasting is not required for Protein C testing. However, marked lipemia and hemolysis can interfere with assay endpoint detection. It is best to draw a new sample if your separated plasma is severely hemolyzed (i.e. bright red plasma).

What is the turnaround time and cost for Protein C testing?

Protein C assays are performed daily and results reported within 24 hours of sample receipt. Refer to the AHDC test/fee list for current pricing.

What is the shipping address for Protein C testing?

FedEx/UPS/Courier Address:

Comparative Coagulation Section/AHDC
College of Veterinary Medicine, Cornell University
240 Farrier Road
Ithaca , NY 14853

US Postal Address (PO Box):

Comparative Coagulation Section/AHDC
College of Veterinary Medicine, Cornell University
PO Box 5786
Ithaca , NY 14852-5786

How can I use the Protein C assay to diagnose dogs with portosystemic shunts?

Protein C deficiency is a marker of portosystemic shunting. While clinical signs, breeds at risk, and bile acid elevation are common to portosystemic shunting and microvascular dysplasia, Protein C deficiency develops primarily in dogs with congenital portosystemic shunts. Including Protein C in the diagnostic workup helps identify patients for imaging studies and ultimately shunt repair.

What is the best Protein C cutoff for differentiating shunts from microvascular dysplasia?

Based on the Cornell University caseload, Protein C values below 70% are found in dogs with shunts whereas dogs with MVD typically have values above 70%. The combined findings of Protein C < 70%, low cholesterol, and low MCV (mean corpuscular volume) further support the presence of a shunt.

Are there other diseases that cause Protein C deficiency?

Yes. Protein C deficiency develops in patients with hepatic synthetic compromise, vitamin K deficiency from any cause, and some DIC and sepsis cases. Liver failure causes severe Protein C deficiency (< 50%) and levels may fall early after exposure to hepatotoxins.

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