Protein C and S

Etiology and Epidemiology

Protein C and S deficiencies can be secondary to inherited gene mutations or due to acquired causes.[3][1][4] Most 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

In the healthy general population, the incidence of asymptomatic protein C deficiency is 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]

Protein C deficiency may be inherited or acquired.

The inherited form of protein C deficiency is typically an autosomal recessive disorder; however, de novo mutations have been reported. Approximately 160 mutations in the protein C gene (PROC) located on chromosome 2q14.3 have been described in the literature.[8] These mutations fall into two general types. Type I deficiency is characterized by a low protein C antigen and activity levels.[9] Type II deficiency is characterized by normal protein C antigen levels but low protein C activity levels.[9]

Protein C deficiency may also be acquired by one of several mechanisms. Newborns may have physiologically low levels of protein C at birth; levels have been reported to be as low as 35% in otherwise healthy full-term infants. This is an age-related acquired form of protein C deficiency; protein C levels increase to the lower level of the adult reference range by 6 to 12 months of age.[10] Other causes of acquired protein C deficiency include inflammatory or infectious processes, liver disease, malignancies, chemotherapeutics, disseminated intravascular coagulopathy, and vitamin K deficiency or the 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; there is a small risk of severe warfarin-induced skin necrosis in patients with an underlying hereditary protein C deficiency.

Protein S Deficiency [13][14]

The exact prevalence of protein S deficiency in the general population is unknown. However, some studies have estimated a prevalence of 0.03% to 0.13% in healthy individuals.[14]

Protein S deficiency may also be inherited or acquired.

The inherited form of protein S deficiency is typically an autosomal dominant disorder. The PROS1 gene is located on chromosome 3q11.1, and approximately 200 mutations in this gene have been described in the literature.[15] Three distinct types of protein S deficiency have been identified; the defect is quantitative in type I and III but qualitative in type II.[14] 

Type I protein S deficiency is the most common type and is characterized by a low total protein S level, low free protein S level, and low protein S activity. Type II deficiency is characterized by a normal free and total protein S level but low protein S activity levels. Type II deficiency is a rare form of the disease. Type III protein S deficiency is characterized by normal total protein S levels but low free protein S levels and low protein S activity.

Protein S deficiency may also be acquired by one of several mechanisms. Newborns may have low levels of protein S at birth; levels increase to the adult reference range by 6 to 10 months of age, typically sooner than protein C levels.[16] Other acquired causes of protein S deficiency include liver disease, infection, inflammation, nephrotic syndrome, disseminated intravascular coagulopathy, chemotherapy, malignancy, pregnancy, combined oral contraceptive use, hormone replacement therapy, vitamin K deficiency, and the 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 also been reported in the literature.[18]

Pathophysiology

Protein C and protein S are primarily synthesized in the liver. Protein S is also synthesized by platelets, endothelial cells, osteoblasts, and 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 is 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 bound, 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 by directly inhibiting the 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 protein C or protein S deficiency, the coagulation cascade continues unchecked with the overactivity of factor V and factor VIII, resulting in excessive thrombin production.[1][2][21]

Mutations to factor V (G1691A) in the activated protein C resistance disorder can prevent deactivation even in the presence of proteins 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]

Specimen Requirements and Procedure

Proteins C- and S-antigen and activity levels are usually performed by collecting a venous blood sample in citrate. These samples are centrifuged in the laboratory to separate the plasma. The plasma is frozen in aliquots and stored at -80^oC until analysis. 

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

The patient should discontinue warfarin for at least two weeks before drawing the sample.

It is crucial to perform protein C and S testing several weeks after an acute thrombosis or inflammatory condition to allow serum levels to return to baseline.[1]

Diagnostic Tests

Protein C Deficiency

• Protein C functional assay - This is the preferred assay in the clinical setting, as this can help identify both type 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 suggests underlying protein C deficiency. This can help provide genetic counseling to patients and to understand the natural history of the disease.[23][13] 

Protein S Deficiency

• 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 suggests underlying protein S deficiency.[23][13]

Interfering Factors

Interfering factors include the presence of the lupus anticoagulant, factor V Leiden mutations, APC resistance, elevated plasma factor VIII levels, and hyperlipidemia.[21]

Functional protein S assays should be employed alongside the free protein S immunoassays due to various interferences during testing.[19] These interferences in laboratory testing may disrupt analysis, resulting in false positive or false negative outcomes.[24]

Results, Reporting, and Critical Findings

Normal reference ranges for proteins C and S are age-dependent.[25] They are as follows:

Protein C [IU/dL, Mean (range)]

• 1-5 years: 66 (40-92)
• 6-10 years: 69 (45-93)
• 11-16 years: 83 (55-110)
• Adult: 96 (64-128)

Total Protein S [IU/dL, mean (range)]

• 1-5 years: 86 (54-118)
• 6-10 years: 78 (41-114)
• 11-16 years: 72 (52-92)
• Adult: 81 (60-113)

Free Protein S [IU/dL, Mean (range)]

• 1-5 years: 45 (21-69)
• 6-10 years: 42 (22-62)
• 11-16 years: 38 (26-55)
• Adult: 45 (27-61)

Clinical Significance

Patients with hereditary defects of the protein C and protein S pathways are prone to thromboembolic events such as deep venous thrombosis, pulmonary embolism, stroke, and organ ischemia.[23][7][26] Venous thromboembolism is more common than arterial. Patients who inherit heterozygous alleles for protein C or protein S deficiency will present with an onset later during adulthood compared to individuals who inherit homozygous alleles; homozygous mutations frequently present with critical blood clotting complexities at birth, such as purpura fulminans.[7][4] Patients are also at risk for thromboembolism during high estrogenic states such as pregnancy and combined oral contraceptive use.[27] Treatment

The long-term treatment for protein C and S deficiencies is anticoagulation with heparin bridged to warfarin. The medications should overlap for five days until the therapeutic range of the international normalized ratio (INR) of 2.0 to 3.0 is reached for two consecutive days.[14][28][7] Protein C concentrate can be used as replacement therapy for protein C deficiency.[7] In homozygous newborns suffering from hemorrhagic and thrombotic complications of purpura fulminans, protein C concentrate in the form of fresh frozen plasma can be given.[7]

The warfarin dose should be carefully assessed and bridged with a therapeutic dose of heparin as it can impose warfarin-induced skin necrosis in protein C and S deficiency. Warfarin inhibits the Vitamin K-dependent clotting factors and proteins C and S. Warfarin-induced skin necrosis transpires due to the relatively short half-life of proteins C and S, which are inhibited first when warfarin is administered. This further promotes the procoagulant effects of other vitamin K-dependent clotting factors and forms microthrombi.[29][30]

Monitoring

Patients prescribed warfarin for long-term use warrant recurrent monitoring to confirm the medication is in the optimal range and that benefits exceed the risk of harm.[28] The medication should be carefully assessed regularly so the INR is in the therapeutic range.[28]

Enhancing Healthcare Team Outcomes

Proteins C and S are glycoproteins synthesized in the liver, which function to maintain the physiologic function of coagulation within the body. When mutated or dysfunctional, they can cause symptoms of blood clotting in all ages, with onset from birth to late adulthood. These thrombophilias prompt care from interprofessional healthcare teams, which include primary care providers, hematologists, nurses, and pharmacists.

This team-based approach provides an integrated, evidence-based strategy to treat patients with symptomatic thrombophilias and monitor asymptomatic thrombophilias. The interprofessional team should be up-to-date with the latest management guidelines for anticoagulation use and regularly monitor the INR to maintain therapeutic ranges. Patients should be educated on their disease, medication compliance, and factors that may interfere with medication to cause sub-therapeutic or toxic levels. Genetic counseling should be offered to at-risk patients with a history of thrombophilia or a family history of the disease. The interprofessional team should be able to inform their patients about the risk and probability of the condition being transmitted to offspring.

The care of protein C and protein S deficiency is most beneficial when managed in an interprofessional team strategy to form a therapeutic alliance and enhance patient-centered care to achieve the desired outcome.[27][13][23]