Activated factor IX-antithrombin III complexes in human blood: quantification by an enzyme-linked differential antibody immunoassay and determination of the in vivo half-life.
An enzyme-linked immunoassay has been developed to quantitate the activated human factor IX-antithrombin III complex (IXa-AT III). Test plasma was absorbed onto barium citrate and eluted to remove free antithrombin III (AT III). The IXa-AT III complex in the eluate was then measured by using polystyrene balls coated with immobilized rabbit anti-AT III antibody-binding fragments and anti-factor IX (anti-FIX) antibody-binding fragments labeled with beta-D-galactosidase. The assay was very sensitive, detecting a concentration as low as 0.15 fmol/assay/100 microliters plasma, which corresponds to as little as 0.002% of activated FIX in plasma. Experiments with purified FIX, IXa-alpha, AT III, IXa-AT III, normal plasma, AT III-depleted plasma, and FIX-deficient plasma demonstrated that the assay is specific. IXa-AT III generation could be detected in normal native whole blood within 2.0 minutes after venipuncture. The complex could not be detected in native whole blood from patients with severe hemophilia B even at 1 hour after venipuncture. The mean plasma level of IXa-AT III in healthy adults (n = 32, ages 23 to 54 years) was 9.67 +/- 2.86 pmol/L (mean +/- SD), whereas in eight patients with disseminated intravascular coagulation levels ranged from 11.7 to 35.0 pmol/L. The IXa-AT III titer was significantly reduced in three patients with severe factor VII deficiency (5.70 +/- 1.99 pmol/L) but was normal in three patients with severe factor XI deficiency (11.2 +/- 1.59 pmol/L). Purified IXa-AT III was cleared in vivo in rabbits with a half-life value of 30 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
I
I
Takahashi
K
K
Kato
I
I
Sugiura
J
J
Takamatsu
T
T
Kamiya
H
H
Saito
D000328:Adult; D000818:Animals; D000991:Antithrombins / Q000032:analysis* / Q000493:pharmacokinetics; D004211:Disseminated Intravascular Coagulation / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D005164:Factor IX / Q000032:analysis* / Q000493:pharmacokinetics; D005168:Factor VII Deficiency / Q000097:blood; D005173:Factor XI Deficiency / Q000097:blood; D006207:Half-Life; D006801:Humans; D007163:Immunosorbent Techniques; D008875:Middle Aged; D011817:Rabbits
D000991:Antithrombins; D005164:Factor IX