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Protein C Deficiency Evaluation
Protein C (PC) is a vitamin K–dependent, heterodimeric, plasma glycoprotein that is synthesized as a zymogen in the liver. It is activated on the endothelial surface by the thrombin–thrombomodulin (TM) complex. Once produced, the activated protein C (APC) cleaves and inactivates factors Va and VIIIa. Protein S (PS) cofactor activity is required for optimal proteolytic activity of APC in vivo . PC deficiency is rare in healthy populations, with reported incidence of 0.2%–0.4% of unselected subjects. Most commonly it is transmitted as an autosomal dominant trait. It can be demonstrated in ∼3% of younger patients with venous thrombosis. Diagnosis of congenital PC deficiency is usually made by demonstrating reduced PC activity on at least two separate occasions and exclusion of the numerous acquired causes that can also lead to reduced PC activity. At present, amydolic (chromogenic) PC activity test is usually performed first in the context of screening for PC deficiency. Antigenic PC test may be performed to distinguish quantitative (type I) from qualitative (type II) deficiencies.
PC is a vitamin K–dependent, heterodimeric, plasma glycoprotein that is synthesized as a zymogen in the liver. PC activation requires a complex of an endothelial thrombin receptor TM with thrombin. TM binding induces a confirmation change in thrombin. This change leads to suppression of thrombin procoagulant activity and expression of both anticoagulant and antifibrinolytic activity. PC is recruited to the endothelial surface by the endothelial protein C receptor (EPCR). Thrombin–TM complex cleaves the heavy chain of the EPCR-bound PC leading to formation of proteolytically active APC. Once produced, APC cleaves and inactivates factor (F) Va and FVIIIa, leading to a dampening of the clotting cascade. Inactivation of both factors by APC requires cofactor PS. PS serves an important function of directing APC to the phospholipid surface. Additionally, inactivation of FVIIIa requires the presence of intact FV in the multimolecular complex with APC, PS, and FVIIIa. The APC-containing complex cleaves phospholipid bound FVa sequentially at Arginine 506 (site of the mutation in FV Leiden), Arginine 306, and Arginine 679. Arginine 506 proteolysis is kinetically favored and leads to partial inactivation of FVa. The subsequent cleavage at Arginine 306 leads to the complete inactivation of the catalytic activity of FVa. The significance of the Arginine 679 proteolysis site is not entirely clear. APC complex also cleaves FVIIIa at three Arginine residues (336, 562, and 740) leading to the complete loss of FVIIIa cofactor activity toward FIXa.
Epidemiology and Clinical Presentation
PC deficiency is rare in healthy populations, with a reported incidence of 0.2%–0.4% in unselected subjects. PC deficiency is more frequent in thrombophilic patients. Most commonly it is transmitted as an autosomal dominant trait. The inheritance of a single defective allele of the gene encoding PC ( PROC ) is a moderate risk factor for thrombosis. It can be demonstrated in ∼3% of patients with venous thrombosis. Epidemiologic studies suggest that subjects with a defective allele have approximately a sevenfold increased risk of venous thrombosis compared with their nondeficient relatives in symptomatic families. Additional thrombophilia-associated mutations, such as FV Leiden and prothrombin G20210A mutation, appear to substantially increase thrombotic risk in PC-deficient subjects in a more than additive fashion. Overall, 30%–50% of PC-deficient subjects within symptomatic kindreds develop venous thromboembolism by the age of 50. Association with arterial thrombosis is controversial, and if present, appears weak. Coinheritance of two defective PC alleles results in a severe thrombotic disorder in the early neonatal period, usually within hours of birth. Infants with severe PC deficiency usually present with thrombotic purpura (purpura fulminants [PF]) and disseminated intravascular coagulation (DIC). Delayed presentations of PF and DIC have been reported in adolescent and young adult patients with moderately severe PC deficiency (PC levels between 1% and 20% per mL). PC has a relatively short plasma half-life and its levels drop early in the onset of oral anticoagulant therapy with warfarin. Heterozygous PC-deficient patients who are not otherwise anticoagulated may experience warfarin-induced skin necrosis because of rapid drop of PC levels below 20% per mL in settings when coagulation factors other than FVII are not yet substantially decreased.
Testing Recommendations
Routine screening testing for PC deficiency is not recommended. This is due to the low prevalence of the deficiency in the general population, which gives an increased probability of false-positive results. General thrombophilia testing guidelines are provided in the introductory chapter to this section and are applicable to investigation of PC deficiency. However, testing is appropriate for family members of a known PC deficiency case.
Diagnosis of PC deficiency requires two decreased values significantly in separate occasions. In addition, a finding of decreased PC levels in a first-degree relative is helpful for diagnosis of congenital deficiency. A single normal value of PC activity outside the borderline range is usually sufficient to rule out PC deficiency, and follow-up testing is usually not necessary for a normal result.
Protein C Deficiency Testing Overview
The diagnosis is usually made through determination of plasma PC activity. The type of PC deficiency can be determined through follow-up antigenic PC level testing. Approximately three quarters of deficiency-linked mutations are associated with quantitative (type I) deficiencies, resulting in proportionate decrease in both PC antigenic plasma levels and activity. The remainder of mutations results in qualitative (type II) deficiency resulting from disproportionate decline of PC activity compared with antigenic PC levels. Types of deficiency and the expected test results are summarized in Table 150.1 .
Table 150.1
Protein C (PC) Deficiency Types
| Amydolic PC Activity | Clot-Based PC Activity | PC Antigen | |
|---|---|---|---|
| Type I | Low | Low | Low |
| Type IIa | Low | Low | Normal |
| Type IIb (rare) | Normal | Low | Normal |
Over 160 different mutations in the PROC gene, which encodes PC, have been linked to PC deficiency. In addition, genome-wide association studies have demonstrated a number of loci outside PROC that influence PC levels in human subjects. The association between lower PC levels due to loci outside PROC and thrombosis has not been established. Because of the large variety of mutations linked to PC deficiency, DNA-based testing is currently reserved for a low proportion of cases.
Diagnostic Guidelines
Timing of the Test
The timing of the PC test relative to acquired conditions may have a significant impact on its diagnostic performance and the need for repeat testing. In general, testing for PC deficiency should not be performed when the patient is vitamin K–deficient or has been taking warfarin within the past 10 and preferably 30 days. Numerous additional factors, including liver disease, active thrombosis, or L-asparaginase therapy, may lower PC levels. Testing in the immediate aftermath of a thrombotic event and before initiation of warfarin therapy can be used to rule out, but not to rule, in PC deficiency. A low value at a single time point should not be overinterpreted as an indication of PC deficiency, as multiple physiologic, pathologic, and iatrogenic conditions have a strong negative effect on PC activity levels in the plasma. Some, but not all, studies have reported mild increases in PC activity in nephrotic syndrome, ischemic heart disease, and in women who are pregnant, using oral contraceptives or hormone replacement therapy. It may be prudent to repeat borderline normal results in patients falling in this category to avoid false-negative results. For a summary of preanalytical considerations please refer to Table 150.2 .
Table 150.2
Preanalytical Variables That Can Influence Protein C Activity Levels
| Factors reported to decrease protein C (PC) activity | Warfarin ∗ , vitamin K deficiency ∗ , liver disease active thrombosis, disseminated intravascular coagulation, recent surgical procedure, L-asparaginase therapy, breast cancer therapy, acute respiratory distress syndrome, PC inhibitor (rare) | |
| Factors reported to increase PC activity | Nephrotic syndrome † , ischemic heart disease, myocardial infarction, pregnancy † , oral contraceptives, † hormone replacement therapy † |
∗ Early vitamin K deficiency and warfarin therapy have a greater effect on PC clottable assays then on chromogenic and antigenic assays.
† The effect of these factors has not been consistent in different studies and appears mild.
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