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 aff ecting a 55-year-old male and his family, we have utilized a new metho dological approach (ProC Global(R), 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(R), derived from Agkistrodon contortix snake venom (ACV test). Nineteen members of the family were investigated, 11 showed low respon siveness to ACV (normalized ACV ratios < 0.66; normal > 1.12); in thes e individuals specific assays of protein C (PC) and protein S (PS) lev els and normalized activated protein C ratios (n-APC-r) were performed . A second test evaluating response to APC, using the classic commerci al APC test (n-APC-r 1), detected only 10 subjects with abnormal respo nses: the propositus and two members of the family with n-APC-r 1 valu es < 0.54, indicating the homozygous state for the R506Q factor V gene mutation, and seven with values ranging 0.69-0.83, consistent with th e heterozygous condition (normal > 0.85). Although only ten subjects p resented with low n-APC-r 1 values, DNA analysis, in agreement with th e ACV test, detected 11 individuals with factor V-R506Q substitution ( two homozygotes and nine heterozygotes). Thus the classical APC test f ailed to identify the APC resistance phenotype in two heterozygous sub jects whose values were clearly normal (1.05) in the first case and ho mozygous (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 co rrelation 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-d eficient plasma dilutions) but not endogenous ACV activated PC was sus pected. The suspected low levels of proteins C and S found in several R506Q members of the family were excluded by reassaying the anticoagul ant activities at higher plasma dilution; this supports the known infl uence of factor V Leiden on functional PC and PS clotting activity. We conclude that the ACV test is appropriate to evaluate the APC resista nce condition, but for a firm diagnosis DNA analysis together with the modified APC test are strongly advised even in the presence of unques tionable APC-r values.