Summary
Antithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural
anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was
associated with AT and TM resistance-related thrombophilia. To assess the AT and TM
resistances associated with other missense mutations by single base substitution in
the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro)
and investigated the effects on AT and TM anticoagulant functions. All variants except
596Pro were secreted in amounts comparable to that of the wild-type but exhibited
variable procoagulant activities. After a 30-minute inactivation by AT, the relative
residual activity of wild-type thrombin decreased to 15 ± 4.0%, in contrast to values
of all variants were maintained at above 80%. The thrombin–AT complex formation, as
determined by enzyme-linked immunosorbent assay, was reduced with all tested variants
in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative
fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12%, whereas
that of tested variants was 37%–56%. In a surface plasmon resonance assay, missense
Arg596 mutations reduced thrombin–TM affinity to an extent similar to the reduction
of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like
cells, APC generation was enhanced differently by variant thrombins in a thrombin–TM
affinity- dependent manner. These data indicate that prothrombin Arg596 missense mutations
lead to AT and TM resistance in the variant thrombins and suggest that prothrombin
Arg596 is important for AT- and TM- mediated anticoagulation.
Keywords
Prothrombin - missense variant - Arg596 - antithrombin resistance - thrombomodulin
resistance
