Different anticoagulant response to activated protein C (APC test) and to Agkistrodon Contortix venom (ACV test) in a family with FV-R506Q substitution

To identify the defect(s) responsible for the thrombotic condition affecting a 55-year-old male and his family, we have utilized a new methodological approach (ProC Global®, Istituto Behring, Milan, Italy) to screen the global anticoagulant activity of the protein C pathway, a defect that accounts for the majority of inherited thrombophilias. The test is based on the activation of endogenous protein C in plasma by Protac®, derived from Agkistrodon contortix snake venom (ACV test). Nineteen members of the family were investigated, 11 showed low responsiveness to ACV (normalized ACV ratios < 0.66; normal > 1. 12); in these individuals specific assays of protein C (PC) and protein S (PS) levels and normalized activated protein C ratios (n-APC-r) were performed. A second test evaluating response to APC, using the classic commercial APC test (n-APC-r 1), detected only 10 subjects with abnormal responses : the propositus and two members of the family with n-APC-r 1 values < 0.54, indicating the homozygous state for the R506Q factor V gene mutation, and seven with values ranging 0.69-0.83, consistent with the heterozygous condition (normal > 0.85). Although only ten subjects presented with low n-APC-r 1 values, DNA analysis, in agreement with the ACV test, detected 11 individual with factor V-R506Q substitution (two homozygotes and nine heterozygotes). Thus the classical APC test failed to identify the APC resistance phenotype in two heterozygous subjects whose values were clearly normal (1.05) in the first case and homozygous (0.53) in the second. The ACV test, however, and the modified APC test with test plasma 1/5 diluted in factor V-deficient plasma (n-APC-r 2) completely matched the DNA analysis. A phenotype/genotype correlation was observed in dilutions higher than 1/3 test plasma factor V-deficient plasma. The presence of unknown mechanisms that influence plasma response to exogenous preformed APC (normal at high factor V-deficient plasma dilutions) but not endogenous ACV activated PC was suspected. The suspected low levels of proteins C and S found in several R506Q members of the family were excluded by reassaying the anticoagulant activities at higher plasma dilution ; this supports the known influence of factor V Leiden on functional PC and PS clotting activity. We conclude that the ACV test is appropriate to evaluate the APC resistance condition, but for a firm diagnosis DNA analysis together with the modified APC test are strongly advised even in the presence of unquestionable APC-r values.