Synthetic Peptide Assays For Monitoring The Defects Of Extrinsic And Intrinsic Pathways Of Coagulation: Selection Of A Suitable Substrate

Abstract

Several assays based on the use of thrombin specific synthetic peptide substrates have been proposed for the amidolytic equivalents of prothrombin time (PT) and partial thromboplastin time (PTT). All of these methods employ the same principle as the clotting assays; the test plasma is activated with either thromboplastin or activator-cephalo-plastin mixture and the generated thrombin activity is measured employing thrombinr specific synthetic substrates such as Bz-Phe-Val-Arg-pNA (S-2160), H-D-Phe-Pip-Arg-pNA (S-2238), Tos-Gly-Pro-Arg-pNA (Chromozym TH), and CH3-Gly- Pro-Arg-pNA. These substrates and their free peptide forms inhibit the amidolytic action of bovine Xa activated human and Xa Russell’s viper venom in the following order: S-2160 > S-2238 > Sarc-Pro-Arg-pNA ≥ Chromozym TH. A new substrate for thrombin Pyro-Glu-Pro-Arg-pNA (S-2366) and a plasminogen activator (tissue) substrate, H-D-Ile-Pro-Arg-pNA (S-2288) have been tested and were found to produce weaker inhibition of bovine and human Xa. Tos-Gly-Pro-Arg-pNA, Sarc-Pro-Arg-pNA and H-D-Ile-Pro-Arg-pNA were found to produce a ≤ 10% inhibition of the activator generated Xa’s amidolytic activity at concentrations which are commonly employed in the PT and PTT assays. Although in the developmental stages the synthetic substrate equivalent assays are more sensitive than the existing PT and PTT assays and provide useful information on the total amount of thrombin generated in each assay, our results suggest that amidolytic equivalent assays for PT and PTT are feasable. However proper selection of a peptide substrate is important as some of the thrombin substrates and their free peptide forms may inhibit Xa and other serine proteases which are generated during the activation step thereby seriously influencing the results.