Development of a radioimmunoassay for quantitating prethrombin 2 in human plasma.
We have developed a radioimmunoassay (RIA) for prethrombin 2 (Pr2), a potential intermediate in the transformation of prothrombin to thrombin. Antisera against human Pr2 were raised in rabbits and the respective immunoglobulin G fractions were chromatographed on prothrombin-Sepharose. The specific antibody population obtained was used to construct a double-antibody RIA capable of measuring as little as 0.05 nmol/L of this component. The immunoreactivity of prothrombin was approximately 40,000 times less than that of Pr2 on a molar basis. Because of nonspecific contributions of plasma constituents to the immunoreactive signal, the measurement of Pr2 in this milieu required the use of a titration curve in which Pr2 was added back to Pr2-depleted plasma. This assay was then used to determine the levels of this species in two patient populations with increased prothrombin activation as determined by the prothrombin fragment F1 + 2 RIA, a measure of the in vivo cleavage of prothrombin by factor Xa. The mean Pr2 concentrations in eight patients with disseminated intravascular coagulation and six asymptomatic individuals with congenital antithrombin deficiency not receiving antithrombotic therapy were not significantly elevated as compared with those of normal controls (0.244 nmmol/L and 0.242 nmol/L vs. 0.184 nmol/L, respectively). Our studies show that Pr2 is cleared from the plasma of dogs with a t1/2 of approximately 25 minutes. Given that the t1/2 of F1 + 2 is estimated to be approximately 90 minutes, the low plasma levels of Pr2 observed in patients with thrombophilia cannot result from rapid clearance of this component.(ABSTRACT TRUNCATED AT 250 WORDS)
Conway
E M
EM
Charles A. Dana Research Institute, Beth Israel Hospital, Boston, MA 02215.
Lau
H K
HK
Bauer
K A
KA
Rosenberg
R D
RD
D000818:Animals; D000918:Antibody Specificity; D000991:Antithrombins / Q000172:deficiency; D002846:Chromatography, Affinity; D004211:Disseminated Intravascular Coagulation / Q000097:blood; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000032:analysis* / Q000276:immunology; D006207:Half-Life; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D008970:Molecular Weight; D011516:Prothrombin / Q000032:analysis* / Q000276:immunology; D011863:Radioimmunoassay; D012016:Reference Values; D013924:Thrombophlebitis / Q000097:blood
D000991:Antithrombins; D004792:Enzyme Precursors; D007074:Immunoglobulin G; D011516:Prothrombin; C012007:prethrombins