Protein C and protein S are glycoproteins, predominantly synthesized in the liver, that are important components of the natural anticoagulant system in the body.[1][2] They are Vitamin K dependent and serve as essential components in the maintenance of physiologic hemostasis.[1]

Deficiency of protein C and protein S results in the loss of these natural anticoagulant properties, thereby resulting in unchecked thrombin generation leading to thromboembolism.

Protein C and S deficiencies can be secondary to inherited gene mutations or due to acquired causes.[3][1][4] The majority of the inherited forms are secondary to missense mutations (60% to 70%) followed by smaller percentages (1% to 15%) of nonsense mutations, splice site mutations, large deletions, small deletions/duplications/insertions, and point mutations.[5]

Protein C Deficiency

Epidemiology:

In the healthy general population, asymptomatic protein C deficiency incidence is reported to be 1 in 200 to 500 individuals, while clinically significant venous thromboembolism is estimated to occur in 1 in 20,000 individuals.[6] No clear racial or ethnic predispositions are known.[7]

Types:

1. Inherited form: This is typically an autosomal recessive disorder; however, de novo mutations have been reported as well. Around 160 mutations in the protein C gene (PROC) located on chromosome 2q14.3 have been described in the literature.[8] 

• i) Type I: Low protein C antigen level and activity levels[9]
• ii) Type II: Normal protein C antigen level, but low protein C activity levels[9]

2. Acquired form: Newborns have physiologically low levels of protein C at birth and have been reported to be as low as 35% in healthy full-term infants. This is an age-related acquired form of protein C deficiency, which increases to the lower level of adult reference range by 6 to 12 months of age.[10] Other causes of acquired protein C deficiency include inflammation/infection, liver disease, chemotherapy/malignancy, disseminated intravascular coagulopathy, and vitamin K deficiency or use of vitamin K antagonist medications.[7][11][12] Of note, warfarin results in a transient procoagulant state with a reduction of protein C levels, and results in a small risk of severe warfarin-induced skin necrosis in patients that have an underlying hereditary protein C deficiency.

Protein S Deficiency [13][14]

Epidemiology: The exact prevalence is not known. However, some studies have estimated the prevalence of 0.03% to 0.13% in healthy individuals.[14]

Types:

1. Inherited form: This is typically an autosomal dominant disorder. The PROS1 gene is located on chromosome 3q11.1, and about 200 mutations in this gene have been described in the literature.[15] The defect is quantitative in type I and III, but qualitative in type II.[14] 

• i) Type I: Low total protein S level, low free protein S level, and protein S activity. This is the most common form. 
• ii) Type II: Normal protein S level (both free and total), but low protein S activity levels. This is a rare form. 
• iii) Type III: Normal total protein S level, but low free protein S level and low protein S activity.

2. Acquired form: Newborns have low levels of protein S at birth, which increases to adult reference range by 6 to 10 months of age, typically sooner than protein C levels.[16] Other acquired causes include liver disease, infection, inflammation, nephrotic syndrome, disseminated intravascular coagulopathy, chemotherapy, malignancy, pregnancy, oral contraceptive use, hormone replacement therapy, vitamin K deficiency, and use of vitamin K antagonists.[11][7][17] Though warfarin-induced skin necrosis is typically described in protein C deficiency, rare cases in protein S deficiency have been reported in the literature as well.[18]

Protein C and protein S are primarily synthesized in the liver. Protein S is also synthesized by platelets, endothelial cells, osteoblasts, vascular smooth muscle cells, and circulates in plasma.[7] 

Protein C is activated by the thrombin-thrombomodulin complex, to form activated protein C (APC) on the surface of the vascular endothelial cells.[1] Once protein C is activated, free protein S in the plasma serves as a cofactor along with phospholipids and calcium, to inactivate factor V and factor VIIIa at specific polypeptide arginine cleavage sites.[1] This results in impaired prothrombin activation, thereby exerting their anti-coagulant action by reducing thrombin generation. About 60% to 70% of protein S is in a bound form, noncovalently attached to C4-binding protein (CBP).[19][20] This protein S-CBP complex enhances the cleavage of activated factor Va, but not as effectively as free protein S.[20][21] Protein S also enhances the effects of APC in fibrinolysis. Protein S also exerts APC independent effects as well by direct inhibition of tenase & prothrombinase complex, acting as an important cofactor to tissue factor pathway inhibitor (TFPI) during the inactivation of activated factor X and further inhibiting thrombin generation.[14]  

In the setting of protein C or protein S deficiency, the coagulation cascade continues unchecked with the overactivity of factor V and factor VII, resulting in excessive thrombin production.[1][2][21]

Mutations to factor V(G1691A) in the activated protein C resistance disorder can prevent the deactivation from happening even in the presence of protein C and S, promoting blood clotting.[3][22] The resistance comes from a single nucleotide point mutation of adenine to guanine, further changing the polypeptide arginine to glutamine at the cleavage site of factor V and causing resistance to cleavage.[3]

Protein C  and S - antigen and activity levels are usually done by collecting a venous blood sample in citrate. They are centrifuged in the laboratory to separate plasma. The plasma is frozen in aliquots and stored at -80 °C until analysis. 

The typical volume of plasma required is 0.5 ml per 2.7 mL. The plasma needs to be frozen within 4 hours of collection. 

The patient should be off warfarin for at least 2 weeks before drawing the sample.

It is important to perform the testing several weeks after an acute thrombosis or inflammation to allow the levels to return to baseline.[1]

Protein C Deficiency [23][13]

• Protein C functional assay - This is the preferred assay in the clinical setting, as this can help identify both types I and type II disorders. Available options include factor Xa based, activated partial thromboplastin time (aPTT) based, or a chromogenic assay
• Total protein C - Measured by immunoassay. This helps distinguish type I and type II deficiencies. 
• Mutational analysis - PROC1 mutation testing is done once the initial testing is suggestive of underlying protein C deficiency. This can help provide genetic counseling to patients and to understand the natural history of the disease. 

Protein S Deficiency [23][13]

• Total protein S - Measured by immunoassay. Other detection methods include ligand-based or monoclonal antibody-based methods. 
• Free protein S - Measured by immunoassay. Antibody-based methods are also used in some laboratories. 
• Protein S functional assay - Measured by a clot based assay. The amount of protein S activity is proportional to the time to clot formation. 
• Mutational analysis - PROS1 mutation testing is done once the initial testing is suggestive of underlying protein S deficiency.