APC RESISTANCE UNIVERSITY
ACTIVATED PROTEIN C RESISTANCE & PROTEIN C FREQUENTLY ASKED QUESTIONS
Protein C is a vitamin-K-dependent glycoprotein and plasma proenzyme of a serine protease that plays a key role in the down-regulation of blood coagulation. It is activated in vivo by the thrombin-thrombomodulin complex on the surface of intact endothelial cells. Activated protein C (APC) functions as a circulating anticoagulant through proteolytic cleavage and inactivation of the coagulation factors Va and VIIIa. The cleavage occurs at three sites in the heavy chain of each protein. The anticoagulant activity of APC is potentiated by the free form of Protein S (about 60% of PS in plasma is bound to C4bBP, and 40% is in free form) and FV. APC Resistance is actually due to a defect in the protein C pathway, in the factor V molecule as opposed to the activated protein C molecule. APC Resistance is an autosomal dominant hereditary defect mainly due to a point mutation resulting in an amino acid change in the FV gene (Ag506 to Gln mutation, or Factor V Leiden mutation). The mutation destroys one of the three cleavage sites, rendering FVa partially resistant to APC-mediated degradation. APC resistance occurs in 3 – 5% of the general population, but varies largely in different parts of the world. Up to 90% of APC resistance cases are due to the Factor V:Q506 gene mutation. The relative risk of DVT for carriers of the FV:Q506 mutation is estimated to be 8-fold for heterozygotes and 80-fold for homozygotes.
The normal range is approximately 25 – 40 seconds, although it will vary slightly depending on instrument. Although the 1:4 dilution with V deficient plasma strongly decreases interferences from oral anticoagulants and heparin therapy, it cannot be excluded that analysis from patients with high inhibitor activity such as those with phospholipid antibodies (i.e. lupus anticoagulant) may give an abnormal APTT. Increasing the dilution factor to 1:9 or 1:19 may correct the results. According to a study by Nowak et al, a 1:10 dilution may also be useful in children less than one year old. This is due to the special properties of the neonatal hemostatic system, such as low vitamin-K dependent coagulation factors and physiological prolongation of the PT and APTT.
Hereditary protein C deficiency is inherited as an autosomal dominant trait. Heterozygotes for protein C deficiency have protein C activity or antigen levels of 30 – 70% normal, whereas homozygotes with a severe defect have levels below 1%. The prevalence of protein C deficiency is 2 – 5% in patients with thromboembolic disease. Two types of protein C deficiency states are recognized. In type I deficiency, which is the most common type of disorder, the plasma concentration of protein C is reduced both in functional and immunological assays. This reflects a genetic defect causing a reduced biosynthesis of protein C. Type II deficiency is characterized by normal protein C antigen levels, but with decreased functional activity. This type of defect reflects synthesis of abnormal molecules with reduced function. The most common clinical manifestation of symptomatic heterozygous protein C deficiency is deep vein thrombosis (DVT) of the lower extremities. Patients with homozygous protein C deficiency usually suffer from severe and fatal thrombosis in the early stage of life.
Protein C deficiency can also be acquired. Protein C level is influenced by various diseases and drugs such as DIC, DVT, liver disease, sepsis, oral anticoagulant therapy, and surgery.
In contrast, elevated Protein C levels have been reported in such cases as diabetic patients and with the use of anabolic steroids and oral contraceptives. Elevated levels of protein C have no known clinical significance.
PRODUCT SPECIFIC QUESTIONS & ANSWERS
A study by Colucci et al. (Thromb. Haemost. 1994, 72:987 – 988) using the classical APTT method (no FV-deficient plasma pre-dilution) showed that changes in FV levels between 12.5 – 100% did not modify the response to APC. Other experience has shown that the Coatest® APC Resistance – V test may provide ratio values approximately 0.3 units below the median level, which can be fairly close to the cut-off value, when FV levels are 0 – 40%. This may be explained by the fact that although there is often no abnormal bleeding tendency in heterzygotes with factor V deficiency, prolonged PT and APTT times are observed (ref: Sartori et al. Familial association of hypoplasminogenemia and heterozygous factor V deficiency. Clin Appl Thromb Hemost. 1999; 5(4): 277 – 281; Salooja N et al. Severe factor V deficiency and neonatal intracranial haemorrhage: a case report. Haemophilia. 2000; 6(1): 44 – 46. One might also see a slightly decreased ratio in patients with severe liver disease.
According to Chromogenix, FVIII samples above 1.8 IU/ml may result in a reduction of the APC ratio of approximately 0.2 units, although the actual correlation between FVIII activity and APC ratio appears to be weak. When sampling, therefore, the patient should be at rest in order to decrease the FVIII level due to stress.
Variations in plasma levels of protein C have no influence on the APC ratio since a standardized amount of exogenous APC is added.
A low protein C activity is expected in aprotinin treated patients because aprotinin is an inhibitor of activated protein C. Oral anticoagulant therapy interferes with the formation of g-carboxyglutamic acid moiety of the protein C molecules during biosynthesis in the liver, which results in a loss of anticoagulant activity. Non-carboxylated forms of protein C molecules that are inactive in vivo can still be activated by snake venom or thrombin-thrombomodulin and retain amidolytic activity in vitro. Assays using chromogenic substrates will therefore over-estimate the true level of protein C activity in plasma from patients receiving OAC’s. Streptokinase also influences the hydrolysis of S-2366. Sample blank activities should be determined with plasma from patients with thrombolytic disorder treated with streptokinase, as well as plasma where contact activation is suspected, should be compared to sample blank activities. A high blank activity may indicate contact activation has occurred. S-2366 is also sensitive to thrombin. This interference can be quenched by the addition of a thrombin inhibitor such as I-2581.
The QC range is 2.0 – 3.1 for the normal range. If the result for a specific batch is toward the low end of this range, customers, especially those using the STA, may have a problem with the values being near or below the cut-off. Make sure that the APTT reagent was vigorously shaken, and that the reagents were allowed to stand at RT as per the package insert directions. If the problem persists, call technical support at (800) 447-3846. Also, it should be noted that with low response instruments like the MLA, in order to get higher resolution, it may help to dilute the APC/CaCl2 in1.5 ml instead of 2.0 ml.
The APTT reagent cannot be frozen. The other reagents can, but should be rapidly thawed at 37°C and cannot be re-frozen.
No. There is more APC in the APC V kit than in the classic kit. This makes the APC ratios twice as high among normals when not using the V def. plasma as a pre-diluent compared to the classic method. Furthermore, using the APC r-V without the pre-diluent will cause a constant change of the cut-off value due to batch-to-batch variations. This effect is not there if you use the V def. plasma, since it normalizes the plasma and ensures that the cut-off value stays the same from one batch to another.
During the last years recent publication have been showing that the APC Resistance phenotype is a risk factor for venous thrombosis, irrespective of the FV Leiden mutation, and hence this illustrates that the Coatest® APC Resistance and the Coatest® APC Resistance V kits measure different entities. For this reason, both Coatest® APC Resistance and the Coatest® APC Resistance V kits are included in the thrombophilia screening panel in some important laboratories. A recent and interesting publication on inherited thrombophilia was done by Uri Seligsohn and Aharon Lubetsky. In this paper, both APCR and APCR V are suggested as part of six high priority tests. The diagnosis for factor V Leiden should be confirmed by genetic test, in order to decide whether the family members should be examined.