BIOCHEMICAL PROTOTYPE FOR FAMILIAL THROMBOSIS - A STUDY COMBINING A FUNCTIONAL PROTEIN-C MUTATION AND FACTOR-V LEIDEN

Resistance to activated protein C (APC) is associated with a single am ino acid substitution in factor V (Arg(506)-->Gln, factor V Leiden) th at results in delayed inactivation of the molecule by APC. The mutatio n is present in 20% of patients with a first episode of deep venous th rombosis. Arterial and venous thromboses are also associated with the type II protein C deficiency (protein C-Vermont). In protein C-Vermont , the substitution Glu(20)-->Ala alone (rpC(gamma 20A)) is responsible for the defective anticoagulant properties of PCVermont. It was recen tly established that a thrombotic episode occurred in 73% of family me mbers who are heterozygous for both a functional protein C gene mutati on and the factor V Leiden mutation. We evaluated the molecular defect that would accrue in the combined deficiency state of factor V-R506Q/ Va(R506Q) and rAPC(gamma 20A) using recombinant APC and natural purifi ed factorV(R506Q) from patients homozygous for the Arg(506)-->Gln subs titution. While wild-type recombinant APC (rAPC) slowly cleaves and in activates factor V-R506Q and factor Va(R506Q), minimal cleavage of mem brane-bound factor V-R506Q and Va(506Q) by rAPC(gamma 20A) at Arg(306) and Arg(679) occurs, and no loss in cofactor activity is observed. Ou r data demonstrate that rAPC(gamma 20A) cannot inactivate either facto r V-R506Q or factor Va(R506Q) at biologically relevant rates because o f impaired cleavage at Arg(306) and Arg(679). The result is a stable p rocofactor and stabilization of an active cofactor in patients possess ing both mutations. Our data provide a prototype of familial thrombosi s that most likely would be manifested in vivo by the occurrence of ma ssive thrombosis.