Separation of Factor V Leiden Molecule, a Mutated Form of Factor V, from Plasma of Homozygous Patient

Abstract

Factor V (FV) is a coagulant in plasma. The FV molecule consists of a heavy chain and a light chain, and Factor V Leiden (FVL) is mutated FV at a single amino acid in the heavy chain. FVL patients are in a dangerous hyper-coagulation state in their body. Current FVL diagnosis is done by DNA analysis, which is expensive and time consuming. Our group has been developing a real-time, cost effective immuno-optical biosensor for FVL diagnosis. For the sensor development, pure FVL, which is not currently available, is needed. Here, we have attempted FVL purification from FVL patient’s plasma. Since plasma contains many proteins and some proteins are structurally homologous to FV, the purification must be done by a very specific method, such as immuno-affinity chromatography. However, an antibody that does not react with FV is not currently available. Because the mutation is in the heavy chain and the amino acid sequence of the light chain of FVL is identical to that of FV, antibodies generated against the light chain of FV were tested for purifying FVL. Plasma was obtained from a homozygous FVL patient. First, the plasma was pretreated by barium citrate and polyethylene glycol 6000, to remove the vitamin K-dependent proteins, alpha globulins, and other smaller than 6 kDa molecular weight proteins. The yield in the process was 54%. Immuno-affinity purification of FVL from patient plasma was then performed using an anti-FV light chain antibody immobilized CNBr-Sepharose, and the purification yield was 25%. In summary, the antibody against the light chain of FV was able to purify the single point mutated form of FV (FVL) from plasma with an overall yield of 14%. The same principle can probably be used for purification of the other single point mutated proteins.