Heterogeneity of human factor V deficiency. Evidence for the existence of antigen-positive variants

Abstract

A B S T R A C T 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 (IgG4 X) 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 a-methyl-D-mannoside. The isolated native Factor V with high specific activity (92 U/mg) showed a single band (Mr, 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 ,lg/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 jg/ml with corresponding coagulant activities (0-0.17 U/ml) indicating a low concentration of normal Factor V, presumnably 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 ,ug/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.

Biosynthesis of factor V in isolated guinea pig megakaryocytes.

“…Plasma containing human anti-human Factor V antibody (1 1) was a kind gift of Dr. Helen Glueck, University of Cincinnati Medical School (Cincinnati, OH). Rabbit anti-human Factor V antibodies, a human anti-Factor V immunoaffinity column, and purified human Factor V were prepared in our laboratory as previously described (10).…”

“…A human anti-human Factor V antibody (I1) and a rabbit polyclonal anti-human Factor V antibody (10) were assayed for their ability to inhibit the guinea pig plasma Factor V coagulant activity. In the inhibition assay twofold serial dilutions of antiserum were incubated with equal volumes of guinea pig plasma for 18 h at 4VC.…”

“…In this report, Factor V in isolated guinea pig megakaryocytes was quantified by a competitive enzyme-linked immunosorbent assay (ELISA)' assay by use of a cross-reacting rabbit monospecific antibody to human Factor V (10). Factor V was visualized in megakaryocytes by cytoimmunofluorescence.…”

Coagulant factor Xa inhibits prostacyclin formation in human endothelial cells. Role of factor V.

“…The supernatant of the cell extract was analyzed for Factor V content by a competitive enzyme-linked immunosorbent assay (ELISA) method described earlier. 16 Quantities of Factor V antigen as small as 10 ng could be measured by this procedure. The Factor V antigen content of the spent medium was similarly assayed.…”

Severe coagulation factor V deficiency caused by 2 novel frameshift mutations: 2952delT in exon 13 and 5493insG in exon 16 of factor 5 gene

“…Most factor V-deficient patients have low factor V activity and antigen level; however, discrepancy between functional and antigenic levels has also been described. 8,9 More than 200 factor V-deficient cases have been reported in the literature, but the molecular basis for factor V deficiency has been established in only a few cases. [10][11][12][13] Additional molecular defects in the factor V gene have been identified in patients with "pseudohomozygosity" for factor V Leiden (the Leiden allele plus the null allele).…”