Protein C and S

Etiology and Epidemiology

Protein C and S deficiencies can be secondary to inherited gene mutations or due to acquired causes.[1][3][1][4] Most inherited forms are secondary to missense mutations (60%-70%), followed by smaller percentages (1%-15%) of nonsense mutations; splice site mutations; large deletions; small deletions, duplications, or 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, whereas 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 2 general types. Type I deficiency is characterized by low protein C antigen and activity levels. Type II deficiency is characterized by normal protein C antigen levels but low protein C activity levels.[9]

Protein C deficiency can also be acquired through various mechanisms. Newborns may have physiologically low levels of protein C at birth, with levels as low as 35% in otherwise healthy full-term infants. This condition is considered 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

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.[13][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—Type I and Type III represent quantitative defects, whereas Type II is qualitative.[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 protein S deficiency is characterized by a normal free and total protein S level but low protein S activity levels. This type of deficiency is considered a rare form of the disorder.
• 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 through 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] Although warfarin-induced skin necrosis is typically associated with protein C deficiency, rare cases of protein S deficiency have also been reported in the literature.[18]

Pathophysiology

Protein C and 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 on the surface of the vascular endothelial cells. Once activated, protein C requires free protein S in plasma, along with phospholipids and calcium, as a cofactor to inactivate factor V and factor VIIIa at specific polypeptide arginine cleavage sites.[1] This inactivation results in impaired prothrombin activation, thereby exerting its anti-coagulant action by reducing thrombin generation. About 60% to 70% of protein S is noncovalently bound to C4-binding protein.[19][20] This protein S–C4-binding protein 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 activated protein C in fibrinolysis and exerts activated protein C–independent effects by directly inhibiting the tenase and prothrombinase complex. Additionally, it serves as a critical cofactor to tissue factor pathway inhibitor during the inactivation of activated factor X, further inhibiting thrombin generation.[14]

In protein C or 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 protein C and S, promoting blood clotting.[3][22] This resistance results from a single-nucleotide point mutation, where adenine is replaced by 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

Protein C and S antigen and activity levels are typically measured by collecting a venous blood sample in citrate. The sample is centrifuged in the laboratory to separate the plasma. The plasma is frozen in aliquots and stored at −80 °C until analysis. A volume of approximately 0.5 mL plasma per 2.7 mL blood is required, and the plasma should be frozen within 4 hours of collection. 

Patients should discontinue warfarin for at least 2 weeks before testing. Testing should be performed 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 assay is preferred in the clinical setting, as it can help identify both type I and type II deficiencies. Available options include factor Xa–based assay, activated partial thromboplastin time–based assay, or chromogenic assay.
• Total protein C: Measured by immunoassay to distinguish between type I and type II deficiencies. 
• Mutational analysis: PROC1 mutation testing is conducted once initial tests indicate an underlying protein C deficiency. This testing can assist in providing genetic counseling to patients and enhance understanding of the disease's natural history.[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 performed 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, activated protein C resistance, elevated plasma factor VIII levels, and hyperlipidemia.[21]

Functional protein S assays should be used 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 protein C and S are age-dependent.[25]

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 thromboembolism. Patients who inherit heterozygous alleles for protein C or protein S deficiency 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 involves anticoagulation with heparin bridged to warfarin. The medications should overlap for 5 days until the therapeutic range of the international normalized ratio (INR) of 2.0 to 3.0 is reached for 2 consecutive days.[14][28][7] Protein C concentrate can be used as replacement therapy for protein C deficiency. 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 protein C and S. Warfarin-induced skin necrosis occurs due to the relatively short half-life of protein C and S, which are inhibited first when warfarin is administered. This inhibition further promotes the procoagulant effects of other vitamin K–dependent clotting factors, leading to the formation of microthrombi.[29][30]

Monitoring

Patients receiving long-term warfarin therapy require regular monitoring to ensure that anticoagulation remains within the therapeutic range and that the benefits of treatment continue to outweigh potential risks. The medication should be carefully assessed regularly so the INR is in the therapeutic range.[28]

Quality Control and Lab Safety

Ensuring the accuracy and reliability of protein C and protein S testing relies on rigorous internal quality control (IQC) and vigilant monitoring practices. These assays, whether performed using immunoassay or clot-based methods, are highly sensitive to analytical variability, which makes consistent quality oversight essential.[31]

Before analyzing patient samples, laboratories must run at least 2 levels of IQC, typically representing low and high concentrations. The resulting control values should fall within the manufacturer's specified limits or within internally validated ranges established through comprehensive laboratory studies.[32] Only when IQC results meet these criteria can patient testing proceed. Conversely, if IQC results fall outside acceptable limits, patient sample analysis must be halted immediately. The laboratory must then investigate the root cause and implement effective corrective and preventive actions before resuming testing.[33]

IQC performance is routinely tracked using Levey-Jennings charts, with Westgard rules applied to identify deviations from expected behavior. These tools enable the detection of both random errors and systematic biases.[34] In addition to clear control failures, careful attention must be given to shifts or trends in quality control (QC) data. A gradual drift away from the mean may signal reagent instability, calibration drift, or emerging instrument malfunction. Detecting these changes early allows timely corrective action, thereby preventing compromised patient results.[35] At a minimum, 2 levels of IQC should be run at least once every 24 hours, though increased frequency may be necessary depending on workload, method stability, and clinical risk.[36]

In the event of IQC failure or recognition of significant trends, patient testing must be stopped without delay. Results generated since the last acceptable QC run should be reviewed thoroughly, with re-analysis or withdrawal of reported results undertaken where appropriate. Testing can only resume once QC values are back within defined limits, ensuring patients are not placed at risk from inaccurate reporting.[37]

External quality assurance provides an additional safeguard by independently assessing laboratory performance. In these programs, blinded samples are supplied by external providers and processed in the same way as routine patient specimens.[38] The laboratory submits results for comparison against peer group data or assigned values, and any unsatisfactory performance requires prompt investigation and corrective action. External quality assurance thus strengthens confidence in the laboratory's ability to produce accurate and reliable results consistently over time.[39]

Strict adherence to laboratory safety protocols is considered equally important as quality. Laboratory personnel must always use appropriate personal protective equipment, follow safe waste disposal procedures, and comply with biosafety and Occupational Safety and Health Administration regulations. Staff vaccination, ongoing training, and education on the prevention of exposure to bloodborne pathogens and other hazards are essential components of a robust safety culture. Maintaining a safe work environment not only protects laboratory professionals but also ensures uninterrupted delivery of precise and dependable testing services.[40]

Enhancing Healthcare Team Outcomes

Protein 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 individuals of all ages, with onset ranging 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 for treating patients with symptomatic thrombophilias and monitoring those with 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]