Chromogenix Coatest® APC™ Resistance – V

Only the free, native form of PS binds to APC and functions as a cofactor. PS has the highest affinity for negatively charged phospholipids of all the vitamin K-dependent proteins and has been shown to increase by approximately 10-fold, the affinity of APC for membranes or vesicles containing such phospholipids. This may be of physiological importance since APC degrades preferentially membrane-bound FVa and FVIIIa, but not the circulating, inactivated co-factors. In addition to increasing the affinity of APC to membranes, PS also enhances the cleavage of FVa by APC and works in concert with FV to increase the ability of APC to inactivate FVIIIa.

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.

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.

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.

Variations in plasma levels of protein C have no influence on the APC ratio since a standardized amount of exogenous APC is added.

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.

The APTT reagent cannot be frozen. The other reagents can, but should be rapidly thawed at 37°C and cannot be re-frozen.

Only the free, native form of PS binds to APC and functions as a cofactor. PS has the highest affinity for negatively charged phospholipids of all the vitamin K-dependent proteins and has been shown to increase by approximately 10-fold, the affinity of APC for membranes or vesicles containing such phospholipids. This may be of physiological importance since APC degrades preferentially membrane-bound FVa and FVIIIa, but not the circulating, inactivated co-factors. In addition to increasing the affinity of APC to membranes, PS also enhances the cleavage of FVa by APC and works in concert with FV to increase the ability of APC to inactivate FVIIIa.

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.