Problematic Aspects Of Synthetic Peptide Substrate Assays For Coagulation Parameters

Abstract

With the advent of synthetic chromogenic and fluorogenic peptide substrates, coagulation testing has entered a new era. We have evaluated amidolytic assays for prothrombin, factor X, prekallikrein; prothrombin time (PT) and partial thromboplastin time (PTT) equivalent assays. Factor X is quantitated after activating with Russell’s viper venom with Xa specific substrates. In normals a good correlation is obtained between the coagulant and amidolytic assays (r=0.91, n=50), a poor correlation is reported in patients with liver disease and those treated with oral anticoagulants (r=0.76, n=50). Prothrombin is quantitated using ecarin, staphylocoagulase and phospholipid/Ca+2 as activators. The decarboxy glutamic forms of factor X and II should be compared in the coagulant and amidolytic assays to establish their equivalency. Although PT and PTT equivalent assays using amidolytic assays have been proposed, substrate inhibition of Xa generated during activation and other serine protease have not been fully explored, Dextran activated prekallikrein assay requires adequate level (≥ 50%) of Hageman factor. Heparin is quantitated in absolute terms and do not reflect clinical anticoagulation. In the antiplasmin assay, plasmin preparations cannot be used unless standardized in biochemical terms. Thrombins are standardized in terms of NIH U/ml and contain non-clotting molecular variants with amidolytic activity; therefore thrombin be best standardized in amidolytic terms for these assays. Certain serine protease-inhibitor complexes retain the amidolytic activity while losing their biologic activity, therefore a study on the amidolytic properties of these complexes is warranted. Standardization of the amidolytic method is almost nonexistent and claims for their bioequivalency to the existing clotting assays require support data. While synthetic substrate offers certain advantages, their application should be assessed critically and their limitations determined.