Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance

Abstract

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.

Optimisation of antithrombin resistance assay as a practical clinical laboratory test: Development of prothrombin activator using factors Xa/Va and automation of assay

“…Antithrombin‐resistant prothrombin is a mutant presenting paradoxical features in thrombosis and haemostasis in vivo, that is the mutant thrombin derived from antithrombin‐resistant prothrombin displays a mild decline in the procoagulant activity. However, a substantial impairment in its inactivation by antithrombin results in a prolongation of the procoagulant activity, causing thromboembolism in some patients . In clinical laboratory tests, plasma from the patient with a heterozygous ATR mutation does not exhibit a significant alteration in PT and APTT levels .…”

“…However, a substantial impairment in its inactivation by antithrombin results in a prolongation of the procoagulant activity, causing thromboembolism in some patients. 10,12,15,17 In clinical laboratory tests, plasma from the patient with a heterozygous ATR mutation does not exhibit a significant alteration in PT and APTT levels. 12,15 Miljic et al reported a significant decline in the PT-based prothrombin activity and an increase in the endogenous thrombin potential (ETP) in the thrombin generation assay in heterozygous carriers of prothrombin Belgrade.…”

Venous thrombosis in pregnant women: genetic and epigenetic risk factors

“…The most common of them are (Fig. 8): proteins C and S deficiency; antithrombin III deficiency; anomaly of coagulation factor V (the Leiden mutation); anomaly of prothrombin G 202110A [12]. These congenital disorders lead to disruption of the blood coagulation system.…”

Thrombophilia Screening: Universal, Selected, or Neither?

“…7 For example, it was reported recently that missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance-related thrombophilia. 8 Other genetic risk factors for VTE are dysfibrinogenemia ( FGG , fibrinogen γ′), 11 F11 gene, 9 and non-O blood group. 12 –14 The initial observation that an elevated factor VIII level is a risk factor for venous thrombosis 15 is now supported by robust evidence.…”