Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: An insight into the APC-resistance assay

Human factor V is activated to factor Va by alpha-thrombin after cleavages at Arg(709), Arg(1018), and Arg(1545). Factor Va is inactivated by activated protein C (APC) in the presence of a membrane surface after three sequential cleavages of the heavy chain. Cleavage at Arg(506) provides for efficient exposure of the inactivating cleavages at Arg(306) and Arg(679). Membrane-bound factor V is also inactivated by APC after cleavage at Arg(306). Resistance to APC is associated with a single nucleotide change in the factor V gene (G(169)-->A) corresponding to a single amino acid substitution in the factor V molecule: Arg(506)-->GIn (factor V Leiden). The consequence of this mutation is a delay in factor Va inactivation. Thus, the success of the APC-resistance assay is based on the fortuitous activation of factor V during the assay. Plasmas from normal individuals (1691 GG) and individuals homozygous for the factor V mutation (1691 AA) were diluted in a buffer containing 5 mmol/L CaCl2, phospholipid vesicles (10 mu mol/L). and APC.