Prothrombin Tokushima: characterization of dysfunctional thrombin derived
from a variant of human prothrombin
T Inomoto, A Shirakami, S Kawauchi, T Shigekiyo, S Saito, K Miyoshi, T Morita and S Iwanaga
A mutant prothrombin, designated prothrombin Tokushima, was purified from
plasma of a proband with 12% of normal plasma clotting activity and 42% of
normal prothrombin antigen. The purified preparation gave a single band
with the same mobility as that of "prothrombin" by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed
proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be
identical to that of "prothrombin." Subsequently thrombin Tokushima was
prepared by CM-Sepharose CL-6B column chromatography after prothrombin
activation by factor Xa. The molecular weight of thrombin Tokushima
estimated by SDS-PAGE was identical to that of "thrombin." Thrombin
Tokushima exhibited less than 22% of normal clotting activity, and the
value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of
"thrombin" when Boc-Val- Pro-Arg-4-methylcoumaryl-7-amide was used as a
substrate. However, active site titration using
p-nitrophenyl-p'-guanidinobenzoate failed to detect any difference between
the two. Thrombin Tokushima was 2.5% as effective as "thrombin" in inducing
platelet aggregation. Interaction of thrombin Tokushima with antithrombin
III was much slower than "thrombin" when followed by SDS-PAGE. Based on the
residual thrombin activity, it was 33% as effective as "thrombin" in
forming a complex with antithrombin III. These results indicate that the
molecular defect resides in the thrombin portion of prothrombin Tokushima
and that the binding sites for various substrates appear to be greatly
impaired.
Volume 69,
Issue 2,
pp. 565-569,
02/01/1987
Copyright © 1987 by The American Society of Hematology
