Functional human
Factor V has been purified using a rapid immunoaffinity method. Following
barium citrate adsorption of plasma,
Factor V was precipitated with
polyethylene glycol at a concentration between 5 and 14%. The resulting preparation was applied to a column containing an immobilized
immunoadsorbent consisting of an
IgG fraction containing a naturally occurring human monoclonal (
IgG(4)lambda) antibody with inhibitory activity against human
Factor V. The solid phase
immunoglobulin quantitatively bound
Factor V from human plasma. The bound
Factor V was effectively eluted with a
Tris buffer pH 7.2 containing 1.2 M NaCl and 1 M alpha-methyl-D-
mannoside. The isolated native
Factor V with high specific activity (92 U/mg) showed a single band (M(r), 350,000) on both reduced and nonreduced
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis.
Factor V was purified 5,100-fold over plasma with an overall yield of 77%. The purified
Factor V when subjected to
thrombin activation exhibited an 18-fold increase in
coagulant activity. The isolated
Factor V neutralized the inhibitory activities of the
monoclonal antibody that was used to purify it, as well as the rabbit
antibodies produced by immunizing the animals with the purified
Factor V. Immunoelectrophoresis of purified
Factor V against the polyclonal rabbit antiserum resulted in a single precipitin
arc of identical mobility to the
Factor V in normal human plasma. Analysis by double immunodiffusion showed a line of identity between plasma and purified
Factor V and crossed immunoelectrophoresis showed a single species in normal plasma.A competitive
enzyme-linked
immunosorbent assay using the rabbit antibody against
Factor V was applied to quantify
Factor V antigen level in human plasma. Reconstitution of congenitally deficient or immunodepleted plasma with normal plasma or purified
Factor V gave parallel dose-response curves. In 14 normal plasma the
coagulant activity was 0.98+/-0.02 U/ml (mean+/-SEM) and
antigen concentration was 11.1+/-0.4 mug/ml. A pool of 14 patients with congenital
Factor V deficiency were studied. 10 patients had
Factor V antigen ranging from 1.0 to 2.4 mug/ml with corresponding
coagulant activities (0-0.17 U/ml) indicating a low concentration of normal
Factor V, presumably due to decreased synthesis or increased degradation. When these patient plasmas and the normal plasmas were analyzed together an excellent correlation (r = 0.97, P < 0.01) was obtained. However, four patients with
coagulant activity (0-0.08 U/ml) had
Factor V antigen concentrations ranging from 4.4 to 6.1 mug/ml, indicating the presence of a reduced concentration of abnormal
Factor V protein. The presence of patients with
antigen similar in concentration to
coagulant activity and
antigen in excess of
Factor V activity indicates the heterogeneity of congenital
Factor V deficiency.